Document 1yM8gBd4rxggvEv278yGKkdKo

1 1 IN THE UNITED STATES DISTRICT COURT 2 FOR THE WESTERN DISTRICT OF VIRGINIA 3 ROANOKE DIVISION 4 5 BARBARA S. CARTER, Administratrix of the estate 6 of David Howard Carter, 7 Plaintiffs, Civil Action 88-0172-R 8 VS. 9 SHELL OIL COMPANY, et al., 10 Defendants. 11 DREAMA N. RIDDLE, 12 Administratrix of the estate of Larry D. Riddle, et al., 13 Plaintiffs, 14 Civil Action 88-0505-R 15 VS. 16 SHELL OIL COMPANY, et al., 17 Defendants. 18 CATHERINE H. SLUDER, Administratrix of the estate 19 of Edward F. Sluder, et al., 20 Plaintiffs, Civil Action 88-0124-R 21 VS. 22 SHELL OIL COMPANY, et al., 23 Defendants. 24 25 DEPOSITION OF RICHARD D. IRONS, M.D. 2 1 1290 Broadway Suite 700 2 Denver, Colorado 80203 3 August 15, 1990 4 APPEARANCES 5 THE LAW OFFICES OF HERSCHEL L. HOBSON, by HERSCHEL L. HOBSON, ESQ., Beaumont, Texas, 6 appearing for the Plaintiffs. 7 AKIN, GUMP, STRAUSS, HAUER & FELD, by CLIFFORD J. ZATZ, ESQ., Washington, D.C., appearing 8 for the Defendants. 9 Also Present: Daniel Thau Teitelbaum, 10 M.D. 11 12 The deposition of RICHARD D. IRONS, 13 M.D., produced, sworn and examined upon oath on the 14 15th day of August, 1990, at 10:21 a.m., at 1290 15 Broadway, Suite 700, City and County of Denver, State 16 of Colorado, before me, Barbara Wishart, a Certified 17 Shorthand Reporter and a Notary Public within and for 18 the City and County of Denver, State of Colorado, 19 pursuant to the Federal Rules of Civil Procedure, for 20 the examination of the said RICHARD D. IRONS, M.D., a 21 witness called for examination by the plaintiffs 22 herein. 23 24 25 AVERY REPORTING SERVICE, INC. (303) 825-6119 3 1 2INDEX 3 Witnesses: Page No. 4 RICHARD D. IRONS, M.D. 5 Examination by Mr. Hobson 4 6 Exhibit No. Marked 7 Irons Deposition Exh. 1 165 (Diagram) 8 Irons Deposition Exh. 2 165 (CIIT Group Paper) 9 Irons Deposition Exh. 3 165 (Shell Article) 10 Irons Deposition Exh. 4 165 (File) 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 AVERY REPORTING SERVICE, INC. (303) 825-6119 4 1 RICHARD D. IRONS, 2 being first duly sworn to state the truth, the whole 3 truth and nothing but the truth, testified on oath as 4 follows: 5 EXAMINATION 6 BY MR. HOBSON: 7 Q. This will go under the rules. 8 Dr. Irons, do you care to read and sign your 9 deposition? 10 A. Yes. 11 Q. We'll make those arrangements 12 afterwards. 13 Would you introduce yourself, please. 14 A. Richard D. Irons, I-r-o-n-s. 15 Q. How are you employed, please. 16 A. I am director of Molecular Toxicology 17 and Environmental Health Sciences for the University 18 of Colorado. 19 Q. Dr. Irons, I would like to hand you 20 just a sheet of paper here, if I could, and I think 21 you have a pen there with you. Could I ask you to 22 sketch out for me, please, your view of how a human 23 develops leukemia, the steps that the body goes 24 through in the development of leukemia? 25 A. Leukemia is a whole constellation of AVERY REPORTING SERVICE, INC. (303) 825-6119 5 1 different diseases. To discuss every particular 2 disease and what we know about it is not possible 3 within the context of a single sheet of paper. 4 Q. Okay. Let's take a leukemic disease, 5 then, that is of concern in this case. Tell me what 6 you understand the disease process that's involved in 7 this case. 8 A. Well, in the case of chronic 9 myelogenous leukemia, which is the disease that 10 Mr. Riddle had, it has been virtually conclusively 11 demonstrated that it originates in the pluripotential 12 stem cell. 13 Q. Since we have got a disease entity, 14 then, among this constellation of leukemias, let's 15 stick with CML, and if you would start me through 16 this process of a healthy person developing CML. 17 A. To the extent that I can, it is a lot 18 easier to work backwards. 19 Q. Either way, but if you would chart it 20 out for me, please. 21 A. Okay. What I have sketched here is a 22 schematic of normal blood cell differentiation. If 23 you would like I can use that to describe what we 24 know about CML. 25 Q. Yes, sir, please. AVERY REPORTING SERVICE, INC. (303) 825-6119 6 1 A. The principle for virtually all 2 leukemias, not necessarily the lymphomas, but for the 3 leukemias, we know that the cell of origin, the 4 target cells reside in precursor compartments that's 5 generally called the stem cell compartment in the 6 bone marrow. In the case of CML we have a great deal 7 of evidence to indicate that this here is the cell of 8 origin. 9 Q. When you say this here, can we go back 10 and try to figure it out? 11 A. The pluripotential stem cell. 12 Q. All right, sir. 13 A. The evidence for that is really quite 14 impressive. Now the actual cell type that is 15 associated with the disease in the peripheral blood 16 early on in the evolution of the disease is a 17 relatively mature granulocyte, which is going 18 to--this is really a line or continuum of 19 differentiation and maturation that occurs primarily 20 in the bone marrow, the lymphoid series which goes on 21 outside the bone marrow. 22 But in the case of CML, the principal 23 cell type that is associated early on with the 24 disease is an accumulation or an abundance of cells 25 of relatively mature level of differentiation, AVERY REPORTING SERVICE, INC. (303) 825-6119 7 1 relatively mature for leukemia cells, and they would 2 be at the metamyelocyte level usually, or even some 3 mature cells, the granulocyte series. 4 So although the target cell for the 5 development of the disease originating cell is back 6 here, the first7 Q. Back here being the? 8 A. In the pluripotential stem cell 9 compartment, the manifestation that one sees early on 10 in the disease is in the granulocytic series in terms 11 of the cell type that's first described. Now one of 12 the reasons that we know that CML arises at this 13 level14 Q. Being the pluripotential? 15 A. At the pluripotential stem cell level 16 is that at various times during the disease in some 17 patients one can look for what's basically the 18 heterogeneity of the origin of all the cells in the 19 blood in the lymphoid system, and usually the 20 erythrocyte is the most useful for this. A normal 21 person has erythrocytes produced from many different 22 stem cells, so it is a very heterogeneous 23 population. If, however, they are being produced by 24 single cell, then you will see a monoclonal 25 distribution if you use appropriate markers to look AVERY REPORTING SERVICE, INC. (303) 825-6119 8 1 at it. In other words, you would see that cells are 2 being all produced from a single cell as opposed from 3 many. 4 We also have chromosomal markers, and 5 in the case of CML the most prominent is the 6 Philadelphia chromosome. At various times during the 7 disease one can see the Philadelphia chromosome 8 appearing in cells of different lineages, including 9 cells in the lymphoid lineage, primarily the B-cell. 10 The significance of that is there is only one cell 11 type that can give rise to both of these, and that's 12 at the level of the pluripotential stem cell. 13 So the relationship between the cell 14 that causes or is responsible for the development of 15 CML and the actual cells that one sees in the 16 peripheral blood early on is different. In other 17 words, these cells are relatively mature, but the 18 cell of origin is a pluripotential stem cell. 19 Q. Is there a precursor cell to the 20 pluripotential stem cell in an adult? 21 A. In an adult, no. 22 Q. So I guess in laymen's terms we would 23 call the pluripotential cell the father of all the 24 blood cells? 25 A. Yes. Yes. AVERY REPORTING SERVICE, INC. (303) 825-6119 9 1 Q. Now if you would, tell me--and if you 2 need to make a separate chart we will--how the 3 development of CML as you understand it is different 4 from the development of AML, or acute myelogenous 5 leukemia? 6 A. Most of the knowledge that we have on 7 both of these model systems comes from work of 8 Phillip Fialkow and his group in Seattle. Some 9 others contributed to it, but that's the primary 10 group that's done most of the work in this area. The 11 acute--you said AML? 12 Q. That's right. 13 A. The acute myelogenous leukemia is a 14 leukemia that the cell type that one sees is a 15 relatively--is an immature, it is actually a blast 16 cell, an abnormal blastoid cell, so instead of having 17 relatively mature cells as being the manifestation in 18 the peripheral blood, one has an accumulation of 19 blast cells. So differentiation is blocked at an 20 earlier stage. 21 The cell of origin in AML has been more 22 difficult to pin down because we don't have the same 23 definitive characteristics that we can look at such 24 as the Philadelphia chromosome and some of the clonal 25 markers that we do for CML. In general the far AVERY REPORTING SERVICE, INC. (303)825-6119 10 1 majority of cases that have been studied today show 2 an origin either at the multipotential myeloid cell, 3 which is right here, or at a committed level. By 4 multipotential I mean the cell is capable of giving 5 rise to different lineages but has very limited 6 capability for self-renewal. So it is committed to 7 these lineages. It cannot produce B-cells, for 8 instance. 9 We are talking about production of 10 erythrocytes, granulocytes, platelets, the myeloid 11 series, if you will. Some originate back here. And 12 the reason that we know that is because again looking 13 at clonal markers we occasionally can find 14 involvement of normal--essentially normal 15 erythrocytes precursors when we have frank leukemia 16 myeloid cells present. And so it is obvious that 17 this would have to be the cell of origin. 18 Q. This being? 19 A. The myeloid, the committed 20 multipotential myeloid stem cell. 21 Q. Pronouns will kill us, Doctor, if we 22 don't do this. 23 A. This is difficult for me to do from a 24 chart, put the words in. Some arise at a later level 25 in which one apparently is looking at cells that are AVERY REPORTING SERVICE, INC. (303) 825-6119 11 1 either committed only to the myeloid or the monocytic 2 line, or I suspect that there are some instances, 3 although at present I can't remember where, where 4 there might be a rare instance of where erythrocytic 5 leukemia actually came from a committed cell, 6 although I think the evidence is clear for the 7 myeloid multipotential cell being near the origin 8 there. 9 Q. Is there any way that you know to tell 10 whether in the case of AML the stimulating event that 11 actually leads to the production of acute myelogenous 12 leukemia actually occurred in the pluripotential stem 13 cell or the myeloid stem cell, the multipotential 14 myeloid stem cell? 15 A. In those patients that have been 16 examined, I would say somewhere around 95 percent 17 have been demonstrated to arise at this level. There 18 is a small percentage, a few cases that one cannot 19 say definitively that they arose at this level. 20 There is some evidence they may have arisen in the 21 early stage. 22 Q. I may not have communicated my question 23 very well. The changes that you observe that you say 24 lead you to conclude that the leukemia originated in 25 the myeloid stern cell, could that change have AVERY REPORTING SERVICE, INC. (303) 825-6119 12 1 actually occurred in the pluripotential stem cell and 2 yet only manifested itself in the myeloid stem cell 3 and not the lymphoid stem cell? 4 A. To answer that question the way you 5 phrased it would require that one be able to prove a 6 negative, which is really not an appropriate--it's 7 impossible within the context of scientific inquiry. 8 What one can say is that, first of all, in the case 9 of CML, that does arise at pluripotential stem cell, 10 it has been--that has been relatively readily 11 demonstrated and demonstrated, corroborated. 12 In the case of the AMLs there is 13 only--there is very little evidence to suggest that 14 the pluripotential stem cell can in fact be involved 15 in some minor number of cases. And there is 16 considerable evidence linking this as the cell type. 17 Q. This being the myeloid stem cell? 18 A. The myeloid stem cell for the far 19 majority of cases. Independent of that, in terms of 20 mechanisms of causation that we know of, there is 21 fairly demonstrative evidence, I would say, extremely 22 strong evidence to suggest that this is the target 23 cell and not the pluripotential stem cell. 24 Q. You are saying the myeloid stem cell is 25 the target cell, not the pluripotential stem cell? AVERY REPORTING SERVICE, INC. (303) 825-6119 13 1 A. Yes. 2 Q. When you say the target cell for 3 mechanisms, what kind of mechanisms are you including 4 there? 5 A. Virtually anything that involves 6 potential alteration of the genetic information 7 within the cell. For potent alkylating agents you 8 are talking about mutation events. 9 Q. Strong alkylating agents would be like 10 chemicals for chemotherapy? 11 A. Chemotherapy, chemotherapeutic agents. 12 However, I should state that there are no leukemia 13 models that we have that suggest that there is a 14 single initiating type of paradigm or single event 15 that DNA leads to the development of leukemia. 16 Leukemias tend to be multifactorial in their 17 characteristics. It is clear that multiple events 18 have to occur in order for them to evolve. 19 MR. HOBSON: Let's take a couple of 20 minutes, because this is very technical. I suspect 21 that our court reporter has some questions about 22 words, and I would like to get those cleared up right 23 now if we could. 24 (Whereupon, there was a recess.) 25 Q. (BY MR. HOBSON) I would like to come AVERY REPORTING SERVICE, INC. (303) 825-6119 14 1 back now, Dr. Irons, to the chart you have drawn us 2 that we discussed the myeloid leukemias off of. You 3 have drawn out the cell lineages that lead to those 4 diseases, and we talked just briefly about 5 mechanisms. I would like for you now, if you would, 6 to go back and put in here the different mechanisms 7 that you are aware of that lead to the production of 8 CML and AML. 9 A. We do not know ultimately what the 10 mechanism is that leads to the development of CML. 11 The Philadelphia chromosome is a reciprocal 12 translocation between chromosome 9 and 22. That 13 translocation involves two gene or gene areas, one, 14 the able proto-onco gene, and the break cluster 15 region associated with 22 that are put close together 16 where they are normally not found. And there 17 is--it's at least tempting to conclude that that 18 influence on the able proto-onco gene is important 19 with respect to at least the progression and 20 development of the disease. 21 What's not entirely clear is whether 22 the Philadelphia chromosome was causal or not. There 23 is certain evidence to suggest it might not be, even 24 though there is compelling indirect evidence to at 25 least lead one to assume that it plays a role. As to AVERY REPORTING SERVICE, INC. (303) 825-6119 15 1 what other events lead up to that, if in fact there 2 are any, is not clear. 3 With respect to the acute myelogenous 4 leukemia, certainly in the secondary leukemias, those 5 secondary to drug therapy or in the case of benzene, 6 there is considerable evidence to suggest that 7 aneuploidy may play an important role, although again 8 we don't have a definitive mechanism available for 9 any leukemia. Aneuploidy meaning the selective loss 10 of chromosomes and/or the loss of portions of those 11 chromosomes. 12 In secondary acute leukemias, AMLs, 13 there is a very high proportion of those have losses 14 of either chromosome 5, chromosome 7, or 5 Q minus 7 15 Q minus, which would mean a portion of those 16 chromosomes, whether or not initiating events in the 17 form of alkylation mutation play a role in the 18 evolution of these tumors has not been established. 19 I think there is at least equal if not greater 20 evidence to suggest that these nondisjuctional events 21 are implication errors, play as important if not a 22 more important role in the development of leukemias 23 than any hypotheses involving a direct mutational 24 theory. 25 Q. Now I want to stick with my game plan. AVERY REPORTING SERVICE, INC. (303) 825-6119 16 1 Do we need to use any of the charts, and the one you 2 have now created, to talk about the lymphocytic 3 leukemias? 4 A. Not really. 5 Q. Tell me, if you would, your 6 understanding of the development of the lymphocytic 7 leukemias in man. 8 A. Let's take ALL because that is of 9 significance with respect to--I have to apologize, 10 the diseases are straight but the names aren't. I 11 think we are talking about Mr. Sluder. The majority 12 of ALLs arise from a committed lymphoid precursor 13 cell, and that could mean either of these two cell 14 types. 15 Q. Being which ones? 16 A. Either the Pre T lymphocyte precursor, 17 if you will, or the Pre B lymphocyte precursor, or in 18 some cases the lymphoid stem cell multipotential 19 prelymphoid precursor, if you will. The majority of 20 cases arise in the case of a T-cell leukemia, at 21 least there is a presumption they arise here. 22 Q. Here being? 23 A. At the level of the Pre T lymphocyte, 24 committed cell, the T lymphocyte lineage. In the 25 case of Mr. Sluder he presents with the Calla AVERY REPORTING SERVICE, INC. (303) 825-6119 17 1 antigen, or the common lymphoid antigen, which is an 2 antigen expressed on a variety of different cell 3 types not limited to the lymphoid series, but it has 4 been associated with staging those, the degree of 5 differentiation of those tumors. That would lead me 6 to think or suspect that it might have arisen at this 7 level. 8 Q. Being the lymphoid stem cell? 9 A. The committed lymphoid stem cell. It 10 is multipotential in that it likely can give rise to 11 both, either B or T. I don't know of any gene 12 arrangement studies on Mr. Sluder, which really 13 eliminates the possibility of coming up with a 14 definitive answer to that. 15 Q. Now is there a difference in your mind 16 between the chronic lymphocytic leukemia and acute 17 lymphocytic leukemia? 18 A. The evidence for chronic lymphocytic 19 leukemia to the best of my knowledge is it arises 20 primarily at this stage of a committed Pre B cell. 21 Q. What's the indication to you that 22 that's the origination of the CLL? 23 A. Based upon studies using immunoglobulin 24 gene arrangements in those cells. 25 Q. Is there a difference in your mind AVERY REPORTING SERVICE, INC. (303) 825-6119 18 1 between the lymphomas and the lymphocytic leukemias? 2 A. Yes. 3 Q. Explain those differences for me. And 4 if we need to do a similar type of chart to show the 5 development of lymphomas, I would like to do that. 6 A. Why don't we proceed with this, and if 7 you want me to do another chart, we can do another 8 chart. 9 Q. Fine. 10 A. One can get into semantic arguments 11 about a lymphoma or leukemia, especially in animal 12 models where the distinction is often not clear at 13 all. In man one can have lymphomas that may have a 14 leukemic episode and present with cells in the 15 peripheral blood and take on characteristics of a 16 leukemia and vice versa where you get infiltration of 17 tissues from, say, an acute leukemia that begins to 18 invade other tissues which can also include the 19 secondary lymphoid. If it is all right with you, I 20 would just as soon not--I'll try to distinguish 21 between those semantic arguments and issues and 22 lymphomas in general. 23 Unlike the myeloid compartment of the 24 bone marrow, which is associated with the production 25 of granulocytes, macrophages, monocytes, platelets, AVERY REPORTING SERVICE, INC. (303) 825-6119 19 1 and red blood cells, most of the maturation 2 associated with the normal development of the 3 lymphoid system goes on outside the bone marrow. 4 In the case of B-cells you do have some 5 development going on in the bone marrow, and you have 6 final commitment and terminal differentiation 7 occurring in the peripheral lymph nodes and/or spleen 8 in response to stimuli. 9 In the case of the T lymphocytes the 10 precursor migrates from the bone marrow to the thymus 11 where it undergoes a similar, although not by any 12 means identical, differentiation process, again 13 terminal differentiation, a functional activity going 14 on in the secondary peripheral lymphoid organs, not 15 in the bone marrow. 16 In the case of a non-Hodgkin's 17 lymphoma, depending upon whether you are talking 18 about immunoblastic, a B-cell or T-cell, you are 19 talking about different cells, the cell of origin is 20 most probably these cells in the peripheral lymphoid 21 organs. They arise in the lymph nodes or 22 occasionally in the thymus, and they are external to 23 the bone marrow. 24 Q. These cells being the B-cell or T-cell? 25 A. Yes. The B and the T-cell are AVERY REPORTING SERVICE, INC. (303) 825-6119 20 1 different from the other cells of the blood and 2 immune system. Arguably the monocyte falls into this 3 category, but these cells are capable of continued 4 proliferation given appropriate stimuli in the 5 periphery. 6 Q. Again being B-cell and T-cell? 7 A. These cells are committed to responding 8 to appropriate stimuli and to proliferating and 9 affecting either the production of antibody or cell 10 killing in response to infection. There is a very 11 elaborate network that normally keeps these cells and 12 their proliferation in check, and that is what goes 13 on in the peripheral lymphoid organs. The 14 development of various lymphomas occurs in cells 15 within those organs. 16 Q. In a person who presents with this, I 17 guess, multiple diagnosis of lymphoma and lymphocytic 18 leukemia where it appears to go back and forth, how 19 do you express what's going on in the body in terms 20 of what's going on in the cells? In other words, 21 identifying the disease versus what's going on in the 22 body, and if it appears like they have lymphocytic 23 leukemia at one time and then they appear they have 24 lymphoma the next, what is the difference that's 25 really going on with the body as you see it? AVERY REPORTING SERVICE, INC. (303) 825-6119 21 1 A. Well, usually the progression of the 2 disease from diagnosis gives you a handle on at least 3 what the architectural site of origin is. In other 4 words, if it arises in a peripheral lymphoid organ as 5 opposed to the bone marrow and there is no 6 involvement on the bone marrow, that's characteristic 7 of a lymphoma, for certain types of lymphomas, the 8 majority of them. 9 For some types you may have involvement 10 of the bone marrow. Certainly in the case of 11 multiple myeloma it's very likely that the disease 12 arises in the bone marrow, even though it is a 13 disease of committed B-cell precursor. 14 One also looks for antigens that may 15 help you in deciding what particular category you are 16 going to place it in terms of identifying T-cell and 17 the level of differentiation. 18 Q. Is it possible in your view for a 19 person who, say, comes in and presents at one time 20 like they have a lymphoma, later looks like they have 21 a lymphocytic leukemia because of the changes you 22 described, really what happened was you just didn't 23 catch the first time that it was looking like a 24 lymphocytic leukemia? In other words, they tend to 25 go back and forth, don't they? They appear like they AVERY REPORTING SERVICE, INC. (303) 825-6119 22 1 are a lymphoma, then a leukemia, then a lymphoma. 2 How do you know which came first? 3 A. If you have a biopsy of a peripheral 4 lymph node that demonstrates the characteristics of a 5 lymphoma and you don't have any evidence of bone 6 marrow at that time, you are looking at a leukemia, 7 you are looking at initial presentation of a 8 lymphoma. Whether or not that at some time during 9 its natural history involves the peripheral blood or 10 invades the bone marrow is not necessarily indicative 11 of the origin of the disease. 12 Q. You are saying, then, that a person who 13 truly has a lymphocytic leukemia will always have 14 bone marrow involvement? 15 A. Not necessarily always, but it 16 certainly is going to be the predisposing factor you 17 are going to look for. In certain cases that's 18 not--and depending on the specific disease type, the 19 sub-classification, that's not necessarily always 20 true. 21 Q. Now I would like to go back for a 22 moment and talk about the control mechanisms. You 23 raised multiple myeloma, and I don't want to get to 24 that yet, though I will, and some other diseases 25 before we finish today. Once one of these changes in AVERY REPORTING SERVICE, INC. (303) 825-6119 23 1 one of these cells that leads to one of the leukemias 2 occurs, does that automatically mean that that person 3 will have that disease manifest itself? 4 A. No, I don't think that's by any means a 5 given. In fact I would say the general sum knowledge 6 in experimental clinical immunology, that's not the 7 case. 8 Q. And there are reasons biologically that 9 these individual changes don't always lead to 10 disease, correct? 11 A. Certainly. Most of which we really 12 don't have a clear understanding of. But it is clear 13 there are multiple control mechanisms and the system 14 is designed, to put it very crudely, to provide for 15 multiple points of control over the process. 16 Q. It's almost as though God foresaw that 17 these diseases could occur and built in mechanisms to 18 deal with them so you don't get the disease every 19 time this error occurs, if we put it in that 20 perspective? 21 A. That's right. 22 Q. It's those defense mechanisms that I 23 would like to go into next with you. Explain to me 24 the ones that you have a feeling for. I think you 25 said some are unknown, but the ones that you have a AVERY REPORTING SERVICE, INC. (303) 825-6119 24 1 feeling for, knowledge about, tell me about the 2 defense mechanisms that you know, again trying to 3 limit it to the leukemias. 4 A. Okay. 5 Q. Although it may apply to other things 6 as well. 7 A. The first and obvious defense 8 mechanism, we are dealing with a system that is 9 producing huge numbers of cells every day. It is 10 characterized by the most rapid replication or cell 11 division that you are going to find in a million 12 cells normally. In this whole expansive territory on 13 this graft that I have called the differentiating 14 bone marrow and immune cells, we are dealing with 15 cells that are, and again excluding the lymphocyte 16 series, virtually aptotic. .They are programmed on an 17 irrevocable differentiation process that leads to the 18 production of functional end stage cells that can't 19 divide. 20 Q. I think you said earlier the exception 21 to that is the T-cells, B-cells and maybe the 22 monocytes? 23 A. Maybe. There is argument here. There 24 is replication error going on in these cells 25 normally. AVERY REPORTING SERVICE, INC. (303) 825-6119 25 1 Q. These cells being otherwise the myeloid 2 tissues? 3 A. The other myeloid precursor cells, the 4 rapidly dividing cells that are identifiable in the 5 bone marrow. 6 Q. You left some of those out? 7 A. Yes. If you want me to itemize what we 8 know about the morphologically identifiable types, I 9 can do it, but otherwise10 Q. That's fine. 11 A. At least until we get toward the end of 12 this differentiation spectra we are looking at 13 rapidly dividing cells, so we are producing bone 14 marrow cells through a series of amplifying and 15 division steps producing blood cells. These cells 16 are dividing so rapidly, and it's been appreciated 17 for virtually 100 years that there is some error 18 going on in there. If you look hard enough you can 19 find cells that have either imperfect nuclei, have 20 died as a function of the division process, so there 21 is replication error that occurs in these cells. 22 Q. Let's try to put that just kind of in 23 perspective, I guess. You have put here--I'm having 24 a little bit of trouble with your writing there from 25 this distance. What was this cell here called? AVERY REPORTING SERVICE, INC. (303) 825-6119 26 1 A. I called it the committed erythroid 2 precursor. 3 Q. Basically there is a cell with that 4 title that ends up being an erythrocyte in the 5 circulating blood? 6 A. For the erythroid precursor there are a 7 couple of different stages involved in the maturation 8 of this cell, so this as a term really refers to a 9 couple of different potential cell types. But for 10 the purposes of this discussion, that's accurate. 11 Q. What I would like to do is for one of 12 these cells that's the precursor cell at this level 13 of division, how many ultimately circulating 14 erythrocytes in the ball park are we talking about 15 originally from one cell? 16 A. That is the subject of quite a bit of 17 discussion. It depends a lot on influences that 18 occur in terms of differentiation here. 19 Q. The person who is not ill would just 20 ordinarily--just ball park it for me. You are 21 talking a thousand, a million, a billion? 22 A. I suspect we are talking on the order 23 of hundreds to thousands. 24 Q. And what's the time period that it 25 takes to go from that one cell to the hundreds of AVERY REPORTING SERVICE, INC. (303) 825-6119 27 1 thousands, again ball parking it? I know it changes 2 with different conditions. 3 A. Ball parking, somewhere between one and 4 three weeks. 5 Q. And when you say rapidly dividing 6 cells, you mean rapidly dividing7 A. Rapidly dividing cells. 8 Q. There are errors that occur? 9 A. There are errors. But these cells, as 10 I said, are irrevocably programmed to produce end 11 stage cells. They are not capable of immortality. 12 If a replication error occurs, the worst thing that 13 can happen to that cell is it dies, which for the 14 system is of minimal importance. 15 Q. Unless they are all defective? 16 A. Well, in which case then you have a 17 rapidly developing anemia, or pancytopenia if you are 18 talking about everything. 19 Q. I diverted you onto that little rabbit 20 trail, so let's get back to the main subject. You 21 were talking about the defense mechanisms that are 22 built in, and you have gotten us to these rapidly 23 developing cells and why the defense mechanism is 24 needed in even a human that's healthy or apparently 25 healthy. Tell me now more about the defense AVERY REPORTING SERVICE, INC. (303) 825-6119 28 1 mechanisms, please. 2 A. Well, that particular process is in and 3 of itself a defense mechanism because you have--a 4 cell is at maximum state of susceptibility to either 5 spontaneous or external influences that are going to 6 produce the types of errors we are talking about 7 earlier while it is dividing. And in fact for most 8 known chemical leukemogens the replication is the 9 period of time in which the cell is susceptible to 10 that kind of damage. 11 Q. Say that last part again, please. 12 A. For most leukemogens, agents that we 13 know cause leukemia, certainly for chemicals and 14 drugs, the time of susceptibility is the time of 15 replication. It's also the time of susceptibility 16 for the cells spontaneously. 17 Q. I want to be clear here because my 18 first impression when I heard you say that was that 19 the chemical leukemogens only react with cells after 20 they are in this committed stage. That's not what 21 you intended, is it? 22 A. That's not what I mean at all. As a 23 matter of fact, in the case of benzene the 24 manifestations of cytotoxicity are bone marrow 25 suppression that occur from high-level exposure occur AVERY REPORTING SERVICE, INC. (303) 825-6119 29 1 because cells out here that are not targets for the 2 development of leukemia are in fact eliminated or 3 killed off. That is what creates the anemia. That's 4 what creates the suppression in circulating cells. 5 But that is of no consequence with respect to the 6 development of leukemia directly. So that is a 7 protective mechanism, it's the mechanism that 8 protects us daily from developing leukemia as a 9 normal consequence of just living. It's for the 10 large majority of us. 11 Q. Now there are other defense mechanisms 12 as well besides the one you just discussed. Tell me 13 about the others that we know about. 14 A. The other defense mechanisms involve 15 the level of activation within the compartment of 16 cells that is capable or has some capability for 17 immortality, and that is collectively the stem cell 18 compartments. 19 The pluripotential stem cell 20 compartment, this cell is characterized by the21 virtually within the context of the cells of the 22 blood and immune systems. It has virtually limitless 23 horizons with respect to differentiation. It can 24 produce any of these cells. It also can produce 25 itself. We are basically allotted during AVERY REPORTING SERVICE, INC. (303) 825-6119 30 1 embryogenesis a fixed number of these things, and 2 they continue to keep us going for our whole life. 3 It is, however, characterized by an 4 incredibly strong resistance to division. 5 Pluripotential stem cells do not divide very often. 6 And there is a division history, replication history, 7 which is to say that after so many divisions the 8 evidence suggests that the cell progressively loses 9 its pluripotentialness and takes on 10 committed--characteristics of the committed stem cell 11 populations. 12 As we move down this line going from 13 pluripotential stem cell to committed definable 14 hematocritic precursor cells that produce the blood 15 cells, we have a process whereby the cell begins to 16 take on restricted differentiation capabilities. It 17 no longer is capable of becoming everything. It is 18 progressively restricted. And at the same time it 19 has much more limited capability for any kind of 20 self-renewal. That's lost relatively quickly. 21 We are looking here at a spectrum, we 22 are not looking at--we can talk about the isolation 23 of these populations and their discussion as specific 24 entities, but I think it is more realistic to 25 consider it a spectrum of activity. And you can AVERY REPORTING SERVICE, INC. (303) 825-6119 31 1 define a cell type in terms of probability that it 2 will do one thing or another much more readily than 3 you can, say there is this absolute population that 4 has X characteristics. 5 Q. The differentiation depends on stresses 6 and responses to stresses as well, so the population 7 can change depending on different circumstances, 8 correct? 9 A. Yes. Yes. That's how we respond to10 Q. Infection? 11 A. Infection. That's how we respond to 12 insults or anemia. And it's a highly regulated 13 process. 14 These cells, the myeloid stem cell, for 15 example, is a multipotential cell; that is, it has 16 the capability of giving rise to the erythroid, 17 granulocytic, monocytic, and megakaryocytic 18 lineages. It has a greater potential for division. 19 It has much more limited potential for self 20 replication or self-recapitulation of self, and in 21 fact at this stage we are talking about a cell that 22 is basically committed. It cannot go back. 23 These cells, the committed precursor 24 cells, the myeloid, monocytic, or what have you 25 within this committed framework, are the stem cells AVERY REPORTING SERVICE, INC. (303) 825-6119 32 1 that are rapidly dividing. They have very limited 2 differentiation potential. They still have the 3 potential to--they certainly have the capability of 4 becoming immortal under the wrong influences, but 5 once you get past the stem cell compartment, there is 6 no evidence that they have capability to become 7 immortal. They are now irrevocably committed, not 8 only in terms of lineage but in terms of replication 9 history, to end up as a terminally differentiated 10 cell. 11 Q. When you say these cells have the 12 ability to become immortal? 13 A. They are appropriate targets for an 14 influence, the replication error or any other 15 hypothesized mechanism for the development of 16 cancer. They are appropriate cells for the evolution 17 of leukemia. 18 Q. Now your studies that you have done you 19 have looked both at the ability of chemicals to 20 attack these cells and cause these changes and you 21 have looked for the toxic response of these chemicals 22 on the body, two different things, right? 23 A. If I understand you correctly, yes. 24 Q. That's what I would like for you to 25 differentiate for me now. Isn't it true that even AVERY REPORTING SERVICE, INC. (303) 825-6119 33 1 though you may have one of these leukemias caused by 2 one agent, another chemical agent may affect whether 3 or not the disease actually manifests itself because 4 of toxicity and other forms that have to do with this 5 control mechanism? 6 A. That is certainly a reasonable 7 hypothesis, and it's one, in fact, that has been put 8 forward to at least explain the difficulties in 9 producing certain types of leukemias in animals. I 10 should qualify that. Whether or not that in fact 11 proves to be the case is not clear. We don't have 12 enough information on the panoply of regulatory 13 processes that are involved to really say that with 14 authority. It is a logical assumption based on our 15 current knowledge. 16 Q. Your work that you have done, for 17 instance, with benzene has shown that benzene is 18 toxic to the immune system? 19 A. In very high concentrations, yes. 20 Q. At least you have been able to 21 demonstrate the toxicity at high concentrations, but 22 when you lower the concentrations finding effects in 23 the immune system becomes very difficult, does it 24 not? 25 A. Finding significant effects in terms of AVERY REPORTING SERVICE, INC. (303) 825-6119 34 1 an effect on health, yes. 2 Q. It doesn't mean that lower 3 concentrations don't produce an effect, it is just it 4 gets very difficult to measure and say it is a 5 distinctive effect from what might be current by 6 chance alone? 7 A. I'm sorry, could you repeat the 8 question. 9 Q. Yes, sir. You said--where I'm going 10 with this is that something like benzene may well and 11 probably is toxic at lower concentrations. It's 12 whether or not the toxic effect is significant 13 statistically and able of detection by the methods 14 that you have that you have a problem with. 15 MR. ZATZ: There is no question 16 pending. 17 Q. (BY MR. HOBSON) That's what you are 18 saying, isn't it? 19 A. That's an oversimplification. It 20 depends on what you mean by toxicity. If you are 21 talking about a measurable end stage, alteration that 22 holds resistance to infection, yes. If you are 23 talking about effects that occur at a different 24 level, say in terms of the differentiation of cell 25 types, that's not at all clear. AVERY REPORTING SERVICE, INC. (303) 825-6119 35 1 Q. What I'm really asking you is when you 2 say that's not at all clear, it means you haven't 3 been able to develop the techniques or you don't have 4 the capacity to make the differentiation of whether 5 it occurs or not, you just don't know? You can't say 6 it doesn't, and gets back again to proving the 7 negative that you were talking about earlier? 8 A. There is certainly--basically we can't 9 say that it doesn't, but there is no evidence to 10 suggest that it does. 11 Q. Now one issue, of course, I need to 12 address with you is whether or not benzene produces 13 leukemia as a causative agent. And by asking that I 14 want to know if you believe that benzene exposure can 15 cause any of these genetic changes to lead to any of 16 the leukemias. Would you give me your opinion on 17 that, please. 18 A. Are you asking me whether benzene leads 19 to the development of a specific leukemia or the 20 genetic changes in general or what's the question? 21 Q. The question is, is benzene a true 22 carcinogen when we talk about the leukemias, and if 23 it is, in your opinion, which of the leukemias? 24 A. I think there is reasonable evidence to 25 conclude that high-level benzene exposure can lead to AVERY REPORTING SERVICE, INC. (303) 825-6119 36 1 the development of acute myelogenous leukemia and 2 several variants of acute myelogenous leukemia. 3 Q. Variants being? 4 A. Erythroleukemia, metamyelocytic, 5 probably not monoblastic, although I wouldn't rule it 6 out. Virtually the variants situated in the FAB 7 classification with acute leukemia. 8 Q. What is it that you have seen in your 9 opinion in the way of the discussion we have had 10 about these cells that lead you to that conclusion? 11 A. We are talking about two different sets 12 of evidence that lead me to that conclusion. One is 13 my general knowledge of the role of replication in 14 this process, the mechanism of cell differentiation 15 in hematopoiesis, and my knowledge of the 16 epidemiology and clinical literature associated with 17 benzene exposure. 18 Q. Which defect is produced by the benzene 19 exposure that leads to the AML? 20 A. It's going to occur in this population 21 of cells. 22 Q. Are we talking about now the bone 23 marrow compartment after the pluripotential cell? 24 A. Yes. We are talking about committed 25 cells, either multipotential or unipotential stem AVERY REPORTING SERVICE, INC. (303) 825-6119 37 1 cells. 2 Q. Now what is the effect in those cells? 3 Are we talking about a change in the genetic makeup, 4 or what is it? 5 A. This is based on my knowledge of how 6 benzene and its metabolites affect cells most likely 7 to be due to replication error involving 8 nondisjuctional events. 9 MR. HOBSON: It's a good time for a 10 break. 11 (Whereupon, there was a recess.) 12 Q. (BY MR. HOBSON) Dr. Irons, I would 13 like for you to explain to me, if you would, please, 14 how you mean the terminology of the nondisjuctional 15 events, the errors that you just talked about. 16 A. What was the beginning of the 17 question? 18 Q. I would like for you to explain what 19 you just told me about benzene causing the 20 leukemias. I think you said at high concentrations, 21 and you mentioned some errors that were made and 22 there were nondisjuctional events. 23 A. In my opinion the most likely mechanism 24 whereby benzene produces leukemia is through the 25 development of replication errors during division, AVERY REPORTING SERVICE, INC. (303) 825-6119 38 1 replication errors being primarily aneuploidy, the 2 loss of a chromosome, or technically to gain as well, 3 but in this case we are talking loss of a chromosome 4 during division and/or the loss of part of a 5 chromosome. 6 Q. What is the basis of saying that? Have 7 you done work that demonstrates this? 8 A. Benzene metabolites are very effective 9 producers of aneuploidy in dividing cells. 10 Q. And aneuploidy means changes in 11 chromosomes? 12 A. Yes. 13 Q. Which ones of the benzene metabolites 14 are you talking about here now? 15 A. Hydroquinone, p-benzoquinone, 16 benzenetriol, and if in fact transmuconaldehyde is a 17 real metabolite of benzene, its chemistry is 18 consistent with that as well. 19 Q. Have you looked at these metabolites 20 individually to see if they are individually the 21 cause of these replication errors, or do they only 22 have the replication error effect when more than one 23 of the benzene metabolites is present? 24 A. You can produce the effects in isolated 25 cells with hydroquinone and p-benzoquinone or AVERY REPORTING SERVICE, INC. (303) 825-6119 39 1 benzenetriol. That's been demonstrated. Certainly 2 the mechanics of it has been demonstrated in my 3 laboratory. The effects on chromosomes has been 4 demonstrated in the laboratories of Dr. Martin Smith 5 at Berkeley and by David Eastman. Changes consistent 6 with that are produced in whole animals following the 7 administration of metabolites in high concentrations, 8 but you have to use very nonphysiologic doses in 9 order to produce toxicity. 10 Q. Should hydroquinone be considered a 11 carcinogen as benzene is? 12 A. No. 13 Q. That's what I'm trying to get at. What 14 is it about your work that says that benzene, which 15 is metabolized to hydroquinone, and if hydroquinone 16 is one of these things that causes a replication 17 error, what establishes hydroquinone as being--and 18 benzene going through metabolic changes being a 19 carcinogen? 20 A. Although benzene is a very simple 21 molecule if you look at it, has very complex 22 metabolism, it's clear that the effects of the 23 quinone metabolites that I described are potentiated 24 in vivo if they are considered concomitantly with 25 phenol, which is another metabolite of benzene, in AVERY REPORTING SERVICE, INC. (303) 825-6119 40 1 fact it is really the first major metabolite. If you 2 administer phenol and hydroquinone together you can 3 enhance the effect you see with hydroquinone by 4 itself, but it is extremely difficult to produce bone 5 marrow toxicity with the benzene metabolites when you 6 are administering them de novo. 7 Whereas when you administer benzene you 8 have a situation in which the metabolism and 9 pharmacokinetics of the production of the metabolites 10 is such that it predisposes to their concentration in 11 the target tissue, which is the bone marrow. It is 12 very difficult to produce that with the metabolites 13 by themselves because they are pharmacokinetics, and 14 inherent toxicity really preclude creating the 15 situation that is compatible with producing bone 16 marrow damage without incurring other types of 17 toxicity like central nervous system toxicity. 18 Q. Do you have any experience, then, with 19 the result of concomitant exposure to benzene and 20 phenol? 21 A. Benzene itself and phenol? 22 Q. Yes. 23 A. No. 24 Q. What would you have in the way of an 25 opinion as to the results of that kind of exposure? AVERY REPORTING SERVICE, INC. (303) 825-6119 41 1 A. It would be very complicated. I would 2 not want to hazard what would happen. You are 3 looking at certainly different levels. You are 4 looking at primary metabolism in the bone marrow, 5 secondary metabolism--excuse me, not bone marrow, the 6 liver. Secondary conjugation events that are going 7 to be effectively competed for in the liver that's 8 going to change the disposition in the metabolism, 9 secondary metabolism. You have got secondary and 10 tertiary metabolism on the quinone metabolites in the 11 bone marrow. That would be an interesting 12 experiment, it would also be a very, very difficult 13 one to do. 14 Q. What about concomitant exposure to 15 benzene and hydroquinone? 16 A. I think it would be exceedingly 17 difficult to deliver hydroquinone to the bone marrow 18 under any circumstances through exogenous 19 administration that would be compatible with the 20 production of significant bone marrow damage without 21 having very serious CNS effects. I don't think 22 administering benzene is going to change that. It 23 may exacerbate hydroquinone toxicity. 24 Q. Would concomitant exposure to benzene 25 and phenol alter the metabolism of benzene metabolic AVERY REPORTING SERVICE, INC. (303) 825-6119 42 1 pathways? 2 A. It's hard to say. It would depend on 3 the relative--first of all, it is going to depend on 4 the relative affinities of benzene and hydroquinone 5 for the cytochrome P 450 in the liver. I'm not 6 familiar with the details of the kinetics of those 7 two with respect to initial oxidation to really say 8 anything authoritative on that. 9 Q. Just so I make sure I'm asking the 10 question that you are answering, when I say changes 11 in the metabolic pathway, I think you could answer 12 that one of two ways, that the metabolic pathways 13 themselves are changed, or that the relative 14 direction down one of the metabolic pathways is 15 changed? 16 A. I doubt you are going to see any major 17 changes in metabolic pathways with any of these 18 scenarios. You are talking about competing 19 influences that may change the relative production of 20 a given metabolite in a given place, and I think that 21 to hazard an opinion on those influences would be 22 very, very difficult. 23 Q. Now the information that you just gave 24 me about how benzene is metabolized and the influence 25 of the metabolites, is that based on studies in AVERY REPORTING SERVICE, INC. (303) 825-6119 43 1 animals or in man or both? 2 A. Primarily animals, but also some human 3 tissues, some cell work, but primarily animal work. 4 Q. I think you have published in the past 5 that while these metabolic pathways may be somewhat 6 similar in animals and man, and even similar from 7 animal species to animal species, the relative 8 path--the amount that goes down each one of these 9 paths relative amounts changes from species to 10 species and animal to animal and from animal to man, 11 right? 12 A. As a general statement, yes, but there 13 are specific cases where it is relatively 14 straightforward to determine what those differences 15 may be. And they may be slight, they may be great, 16 it depends on the nature of the species that you are 17 working with. 18 Q. In talking about man, which animal 19 species is the closest animal model for metabolism of 20 benzene in man? I'm talking about now these relative 21 percentages and the pathways. 22 A. That's a very difficult question to 23 answer in a single statement. In terms of overall 24 rate of metabolism, I would say you are probably--you are looking at a greater rate of metabolism, say, in AVERY REPORTING SERVICE, INC. (303) 825-6119 1 the mouse in generic terms than you are going to be 2 in, say, the rat, which is probably similar to or 3 less than you are going to find in the monkey, which 4 is going to be similar to or approximate to that 5 which we see in man. That's very general. You can't 6 really apply that to a specific pathway or a specific 7 compound. 8 Q. Perhaps you have anticipated where I 9 was headed. Why don't we do these studies in 10 monkeys? 11 A. To some extent there have been attempts 12 to do studies in monkeys. I think there have been 13 several attempts to demonstrate bone marrow toxicity, 14 at least one that I think was conducted by the 15 American Petroleum Institute in, I believe, the 16 early '70s. 17 Q. In monkeys? 18 A. I'm not sure whether they used a monkey 19 or not. I think they tried, but I haven't looked at 20 that study in years, so I really couldn't. 21 Q. Tell me which study you are thinking 22 about as best you can describe it. 23 A. It's known as the Zoo Study, if you 24 will. They basically attempted to produce bone 25 marrow toxicity through the acute or subacute, I AVERY REPORTING SERVICE, INC. (303) 825-6119 45 1 forget, administration of benzene to a variety of 2 different species and attempt to look for a better 3 model. 4 Q. Have you considered using monkeys? 5 A. The trouble with using monkeys is they 6 are extremely expensive, there are ethical 7 considerations you have to deal with when approaching 8 using primates, having the appropriate facilities to 9 do them, there aren't very many institutions that can 10 do long-term monkey experiments. I have considered 11 using monkeys, but I have elected to look directly at 12 human cells and to compare metabolic and mechanistic 13 aspects of cell differentiation replication between 14 rodent models and man. 15 Q. Doing the same kind of studies in 16 monkeys, primates, that you have done in mice or 17 rats, though, certainly studying the monkey, all 18 those things being equal, cost and that sort of 19 thing, give you much better estimates of what's going 20 to happen to man, doesn't it? 21 A. I think to conduct similar studies to 22 what I have done in mice is almost impossible if not 23 virtually impossible. It is just not logistically 24 feasible. I could be wrong, but I don't know of a 25 facility in this country that can do studies of that AVERY REPORTING SERVICE, INC. (303) 825-6119 46 1 magnitude with that number of primates. 2 Q. But now a number of the studies that 3 you have done dealt with relatively small numbers of 4 rats and mice, haven't they? 5 A. True. But it depends on what questions 6 you are asking. If you want to talk about 7 comparative metabolism, those could be done in 8 monkeys, yes. 9 Q. Now you mentioned that you have looked 10 at some human cells. 11 A. Yes. 12 Q. What have you done along that line? 13 A. We are developing defined stem cell 14 assays, we are characterizing differentiation and 15 proliferation of human cells in culture systems that 16 are analogous to those that we have developed for 17 mouse cells. 18 Q. So these are not human studies, but 19 they are studies on human tissues? 20 A. They are studies on human tissues, 21 that's correct. And they are developmental. They 22 are in progress, they are not by any means completed 23 studies. 24 Q. I want to come back for a moment now to 25 benzene creating these replication errors that you AVERY REPORTING SERVICE, INC. (303) 825-6119 47 1 talked about earlier. Once I'm in your field it's 2 more difficult for me to phrase my question so it can 3 mean something to you, so bear with me, if you 4 would. Besides benzene being able to cause a change 5 in chromosome, the replication error you told me 6 about, does benzene toxicity or the metabolites of 7 the benzene from a toxicity standpoint also play a 8 part in the development of leukemia? 9 A. I'm sorry, but I'm not following the 10 question. 11 Q. I'm trying to get to, I guess, benzene 12 causes the chromosomal changes which sets in motion 13 these developments of malfunctioned cells? 14 A. Metabolites do. 15 Q. The metabolites of benzene. Does the 16 toxicity of either benzene or benzene metabolites 17 also contribute to the induction of the disease at 18 the end point? In other words, does it affect the 19 immune system so that the policing of these cells is 20 also stopped? 21 A. I think you would have to be at such a 22 high level of exposure that you would have problems 23 associating with host resistance and immune 24 dysfunction long before you would have the 25 development of a frank leukemia. But again, this is AVERY REPORTING SERVICE, INC. (303) 825-6119 48 1 pretty much speculation. Effects of benzene 2 metabolites on isolated cells of the immune system 3 have been well characterized and have a dose response 4 nature. We have done most of that work, a good deal 5 of it at least. 6 But again, we are talking about a 7 system that has numerous fail-safe and networking 8 mechanisms and demonstrates an isolated effect on a 9 group of cells, and showing it has any effect 10 whatsoever on immune surveillance or immune response 11 is totally different. And my impression of benzene 12 is it is immunosuppressant only in high 13 concentrations. 14 Q. Tell me something like--high 15 concentrations is a relative term. How can you 16 quantitate that? 17 A. You see frank evidence of suppression 18 in the bone marrow, frank evidence of toxicity to the 19 immunogens, so you are talking exposures that are 20 consonant with the production of demonstrable 21 pathology in target organs. 22 Q. Are we talking now about exposures that 23 occurred through, say, the inhalation of benzene 24 vapors, or are we talking about some other method of 25 delivering benzene to an animal system? AVERY REPORTING SERVICE, INC. (303) 825-6119 49 1 A. It is a heck of a lot easier to do it 2 when you inject the stuff than it is with 3 inhalation. You can do it with inhalation, but it is 4 far more difficult to get a reproducible effect. 5 Q. What levels are we talking about at the 6 tissue level? 7 A. At the tissue level, in terms of 8 dosing, we are looking at exposures in the area of 9 100 to 300 parts per million for a concentration for 10 inhalation. And injection is a totally artificial 11 game in terms of trying to describe what any relevant 12 exposure is from the standpoint of inhalation. But 13 we are talking nonphysiologic dosing regimen to be 14 sure. 15 Q. You have said several times that we are 16 looking at populations here, populations of cells, 17 populations of animals. When you carry out your 18 animal studies with benzene and you find 19 statistically significant changes, if we talk about 20 for a moment the production of leukemias, have you 21 done that in animals, produced leukemias from benzene 22 exposures? 23 A. I have produced some lesions in the 24 mouse that are certainly equivalent to 25 myelodysplastic changes. Whether or not they are AVERY REPORTING SERVICE, INC. (303) 825-6119 50 1 frank leukemias is an argument that pathologists may 2 carry on for some time. But certainly it is possible 3 to produce myelodysplastic changes in the mouse. The 4 mouse is a very bad clinical model for extrapolation 5 to man in terms of a disease entity, but you can 6 certainly produce changes reminiscent to 7 myelodysplasia in a mouse. 8 Q. What percentage of the population of 9 mice would you find these changes, these 10 myelodysplastic changes in? 11 A. It's going to depend on the dose. I 12 would say to some degree or another concentrations 13 certainly above 300 parts per million or greater you 14 are going to see the majority of the animals showing 15 changes. And you are going to have a significant 16 number showing changes at 100. 17 Q. And is this concentration in the air or 18 what you are injecting? 19 A. No, that's air, inhalation. 20 Q. You say greater than 300 the majority 21 have changes, myelodysplastic changes? 22 A. Somewhere between 1 and 100 you 23 basically plateau out. Between 100 and 300 you 24 plateau out. 25 Q. Below 100 parts per million do you not AVERY REPORTING SERVICE, INC. (303) 825-6119 51 1 find any changes? 2 A. You can see some. You can see some 3 hematopoietic changes associated with transient 4 suppression in and around 50. 5 Q. And when you say you can see some6 A. You by no means have the same--you 7 don't have myelodysplasia in the majority of animals 8 at 50. You may have some at 50 that demonstrate some 9 myelodysplastic changes. 10 Q. Is this true in this system as it is in 11 other systems that below 50 you may see changes 12 similar to the group at 50 and above, but you can't 13 attribute those changes to the exposure because they 14 are infrequent? 15 A. I've never seen any myelodysplastic 16 changes in animals below 50. I have not conducted 17 major exposures of animals below 50 because I have 18 had a devil of a time producing these changes between 19 50 and 300. The problem with benzene has been 20 producing basically persistent lesions in rodents at 21 any concentration, and that was my goal with these 22 experiments. 23 Q. That gets back to the selection of the 24 model? 25 A. It gets back to the selection of the AVERY REPORTING SERVICE, INC. (303) 825-6119 52 1 model, but I think it also is likely to be a function 2 of the exposure regimen as well. 3 Q. And then what regimen were you using? 4 A. Continuous exposure and also exposures 5 involving periods of time where the animals have 6 between, say, four and seven days in between 7 exposures. 8 Q. When you say continuous exposure, are 9 these 24-hour exposures? 10 A. No. We are talking either exposures 11 for six to seven hours a day every day, or exposures 12 for, say, three to four days a week with days off in 13 between. 14 Q. Did you find any difference in those 15 two exposure regimens? 16 A. At the higher concentrations there are 17 some differences. What's remarkable to me is that 18 exposure to transient--exposure for three days a week 19 compared to, say, exposures for seven days a week, 20 that there is not that much difference between the 21 three-day exposures versus the seven for benzene 22 itself. That is to say three days at high level 23 produces almost as much damage as seven days' 24 exposure at high level. You lose it when you go 25 down. At 50 parts per million I don't recall any AVERY REPORTING SERVICE, INC. (303) 825-6119 53 1 definitive resolution to whether there is a 2 difference, but at higher concentrations it is 3 important. 4 Q. Is this work published? 5 A. Some of it is in abstract form, the 6 rest of it is not. 7 Q. Where was that work done? 8 A. CIIT. 9 Q. When, approximately? 10 A. 1987, '88. We are talking now about 11 peripheral blood values, bone marrow cellularity and 12 histopathology. We are not talking about 13 bioassay-type studies. We were looking for evidence 14 of effects that were associated with any kind of 15 damage rather than simply looking for leukemogenesis. 16 Q. You told me how long these exposure 17 sessions took place. Were these over a lifetime of 18 the animal, or were they one set period of time? How 19 did you do that? 20 A. I forget the exact paradigms for all 21 the various combinations. We did do one group that 22 were exposed for, I believe, 12 weeks, 12 to 16 23 weeks, and then held them for, I think, a year. This 24 was similar to a paradigm that was used by Dr. Eugene 25 Cronkite of Brookhaven National Lab in which he AVERY REPORTING SERVICE, INC. (303) 825-6119 54 1 exposed animals for a brief period of time and held 2 them. 3 Those studies demonstrate that there is 4 a very complex dose-duration product that's 5 associated with the production of certainly 6 proliferative lesions in mice. Those appear to be 7 more effective than long-term exposures to very high 8 concentrations that have been done in a classic 9 bioassay type of paradigm. 10 Q. I'm not sure I understand what you have 11 told me there on that series of experiments. You say 12 you took some animals that you exposed for three days 13 and left them off for many days. You took another 14 series, exposed them seven days? 15 A. Six or seven, I forget exactly. 16 Q. And you did this for a long period of 17 time? 18 A. For up to 12 weeks. 19 Q. Both regimens? 20 A. I believe so. I have certainly done 21 both experiments. Whether they were done in the same 22 context or not, I can't remember. But we have done 23 both. 24 Q. Then you held these animals for a year? 25 A. Yes. AVERY REPORTING SERVICE, INC. (303) 825-6119 55 1 Q. And then looked at the peripheral blood 2 to see if there were any changes that you could find? 3 A. Yes, and histologic changes in bone 4 marrow. 5 Q. Were their different exposure levels 6 for these two regimens as well? 7 A. Between 50 and 300, yes. 8 Q. Did you find any persistent changes at 9 the end of this one-year holding period? 10 A. There were some very impressive 11 myelodysplastic changes in the animals certainly at 12 the 300 parts per million level. There were some at 13 the 100 parts per million level. I would be 14 hard-pressed to establish that there were the same 15 types of changes at 50. Between 100 and 300 there 16 were plenty. 17 Q. What was the difference, if any, 18 between the two exposure regimens, the short term 19 versus the longer term? 20 A. That's where I can't remember whether 21 or not we did those. We carried them out that far. 22 As I recall, certainly exposures between 17 and 300 23 under a typical six- or seven-day week paradigm 24 exposed for 12 or 16 weeks and held will produce 25 long-term myelodysplastic changes. The problem with AVERY REPORTING SERVICE, INC. (303) 825-6119 56 1 interpreting that data is we are talking high 2 exposures, we are talking plenty of evidence for 3 infection and distinguishing between a myelo 4 proliferative disorder and response to infection and 5 one associated with the development of a long-term 6 proliferative lesion that might, for instance, be 7 typical of myelodysplastic syndrome in man is very 8 controversial and in fact is one of the reasons why 9 that study has not yet been published. The 10 interpretation of the findings is very controversial. 11 Q. I take it you ran a control group? 12 A. We ran a control group. 13 Q. What did you find in the control group 14 with relation to myelodysplastic syndrome? 15 A. There are some proliferative problems, 16 myeloproliferative disorder and lesions in the spleen 17 of some of those animals that could be associated 18 with infection that was also demonstrated in those 19 animals. 20 Q. Was it less marked in the control 21 group? 22 A. I would say it probably is, but the 23 diagnosis is controversial. 24 Q. The25 A. The history of the mouse as a model for AVERY REPORTING SERVICE, INC. (303) 825-6119 57 1 leukemogenesis has been fraught with difficulty in 2 extrapolating myeloid lesions. It is a very 3 difficult task, and you will not find consensus among 4 pathologists. Q. Would the exposure regimens that you 6 used in these animals, the 300 parts per million 7 benzene vapor or 50 parts per million benzene vapor, 8 would those exposures lead to changes in the animal's 9 blood or lymph system, immune system, that would make 10 them more susceptible to disease, infectious disease? 11 A. In my opinion, yes, but that's an 12 opinion. Yes. And that is a problem with respect to 13 thoroughly defining the model. I suspect there are 14 within that group of animals, and I forget the number 15 we started with, between two and three leukemias, but 16 I would be hard-pressed in a scientific form to 17 defend those against alternative interpretations of 18 the histopathology. 19 Q. Now the exposure regimens that you used 20 that were strictly benzene vapor and air for the 21 series of tests you just talked to me about? 22 A. Right. 23 Q. Have you done any work to distinguish 24 between benzene vapor exposure being delivered to the 25 target organs and skin exposure? AVERY REPORTING SERVICE, INC. (303) 825-6119 58 1 A. Skin in my experience is a very poor 2 route of exposure except under conditions of perhaps 3 high-level exposure prolonged contact with the skin. 4 I have never considered skin as a viable route for a 5 study. It is hard enough to produce toxicity that 6 you can reproduce when you are administering the 7 inhalation. 8 Q. What is the last series of skin studies 9 for benzene that you looked at? 10 A. I have not personally done skin studies 11 with benzene. I have elected to use inhalation. 12 Q. You have seen others, though, who have 13 done skin permeation studies? 14 A. Skin permeation, yes. What's the 15 question? 16 Q. If skin permeation is a viable route of 17 exposure to benzene? 18 A. Not in any experimental system that I 19 know of. 20 Q. What about for man? 21 A. My opinion is that under conditions of 22 prolonged exposure to high levels of benzene, 23 prolonged exposure being contact with liquid benzene 24 in the skin, that it probably is a significant route 25 of exposure. I don't think it constitutes an AVERY REPORTING SERVICE, INC. (303) 825-6119 59 1 important route of exposure relative to the lungs in 2 the gaseous phase. The lungs are much more important 3 if you are exposed to benzene in the air. 4 Q. What about benzene as a component of a 5 solvent, have you seen the studies that have been 6 done showing skin permeation of benzene contained in 7 solvents and how the solvents affect the transport of 8 benzene through the skin barrier? 9 A. I believe I have. I don't recall the 10 specifics, but I do, I have seen those. 11 Q. In those studies the fact that other 12 solvents are present actually makes the skin 13 absorption route for humans much more significant, 14 doesn't it? 15 A. My impression is that skin absorption 16 for benzene was relatively small under most 17 conditions. I would have to look at the data to 18 render any more of an opinion than that. 19 Q. You talked about myelodysplastic 20 syndrome in connection with these mice, I think, a 21 while ago. Tell us what you mean by myelodysplastic 22 syndrome in the context that you used it. 23 A. Myelodysplastic syndrome is basically 24 an abnormality that describes a constellation of 25 potential symptoms that is associated with long-term AVERY REPORTING SERVICE, INC. (303) 825-6119 60 1 bone marrow damage. It encompasses terms that are 2 now archaic such as preleukemia. It can involve one 3 or more multiple alterations in circulating 4 peripheral blood cells in terms of number and 5 behavior, and has a reasonably high rate of 6 transformation into AML. 7 Q. Can you use your chart that you 8 prepared for us to explain myelodysplastic syndrome? 9 A. Only to the extent we are talking about 10 a recurring abnormality in the differentiation and 11 production of the various cell types. We are talking 12 about basically long-term abnormality in 13 differentiation and production, so we are talking 14 about, again, cells in the stem cell compartment. 15 Q. So you are talking about an effect that 16 occurred in the stem cell compartment that is 17 manifested out in the peripheral18 A. Yes. 19 Q. What kinds of changes lead to 20 myelodysplastic syndrome? Are they the same kinds of 21 changes that lead to leukemia, or different? 22 A. In general they are the same kinds of 23 changes. We are talking about bone marrow damages, 24 bone marrow suppression, followed by an abnormal25 what is apparently an abnormal differentiation AVERY REPORTING SERVICE, INC. (303) 825-6119 61 1 pattern for the myeloid cells in the bone marrow 2 primarily. It's consistent with high-level 3 exposure. Certainly chemotherapeutic agents are 4 known to do it. There is in fact to my knowledge no 5 demonstrative evidence that you will get secondary 6 leukemia in the absence of the development of frank 7 bone marrow suppression and/or myelodysplastic 8 changes. 9 Q. Say that last part again, please. 10 A. That secondary leukemias, leukemias 11 arising secondary to chemotherapy or exposure to 12 benzene, the evidence is very strong that you 13 basically have to have significant bone marrow 14 suppression and/or the development of pancytopenia or 15 myelodysplastic syndrome as a prerequisite for the 16 development of AML in those patients. 17 Q. That leads me to two questions. What 18 is the basis for saying that? Is that based on 19 animal models? 20 A. No, that's human data. 21 Q. Which human data? 22 A. Primarily the chemotherapeutic 23 literature, as well as those cases for which one can 24 define high-level exposure to benzene, but primarily 25 the chemotherapeutic literature in which patients are AVERY REPORTING SERVICE, INC. (303) 825-6119 62 1 clearly defined and followed from the beginning of 2 treatment. So you have got a handle on exposure, you 3 know what their exposure was, and you follow the 4 progression of their disease. 5 Q. How soon after the exposure to the 6 chemotherapeutic agents does the myelodysplastic 7 syndrome follow? 8 A. I think it varies considerably because 9 you have to distinguish between transient suppression 10 of the bone marrow which you are going to see during 11 chemotherapy. 12 Q. That's the purpose of the chemotherapy? 13 A. It has that effect, yes. What you are 14 really talking about is persistent changes that occur 15 after cessation of treatment. 16 Q. I need for you to put that in more, I 17 guess, laymen's terminology. What do you mean? 18 A. If you have suppression, if you have 19 thrombocytopenia secondary to chemotherapeutic 20 treatment, that's a relatively frequent event. If it 21 persists after treatment has ended, it is likely to 22 be indicative of a persistent problem. And it's very 23 difficult to predict what you are going to see. You 24 may see thrombocytopenia, you may see 25 lymphocytopenia, a combination there of a variety of AVERY REPORTING SERVICE, INC. (303) 825-6119 63 1 different possibilities exist. But persistent 2 changes after cessation of treatment would at least 3 be indicative of potential myelodysplastic changes. 4 Q. A person has chemotherapy, which is 5 going to lead to myelodysplastic syndrome. Do they 6 always then continue with the myelodysplastic 7 syndrome? 8 A. Not necessarily. It is highly 9 variable. Some may persist with isolated changes 10 such as thrombocytopenia for years, some may 11 spontaneously recover, some recover with further 12 treatment. A high percentage will go on to develop 13 acute myelogenous leukemia, or they can also have 14 myelodysplastic syndrome, there is nothing that 15 prevents them from doing that. 16 Q. Are all of the people who die with 17 leukemia after administration of the chemotherapeutic 18 drugs, have they expressed the myelodysplastic 19 syndrome? 20 A. All is a very strong word. I would say 21 that it's my understanding of the literature that 22 there is certainly evidence of bone marrow 23 suppression and/or blood dyscrasias in almost every 24 patient receiving alkylation therapy in large doses. 25 And one would expect that for those going on to AVERY REPORTING SERVICE, INC. (303) 825-6119 64 1 develop leukemia, AML, that virtually all of them are 2 going to have some anlage. 3 Q. Some4 A. Some evidence of bone marrow 5 suppression prior to the development of the leukemia. 6 Q. Of course, prior to the development of 7 leukemia encompasses the whole time period from the 8 administration of the drug until the development of 9 leukemia? 10 A. An expression of leukemia. 11 Q. Isn't it true, though, that some of 12 these people who get their chemotherapy and develop 13 the myelodysplastic syndrome, then they recover so 14 that you don't find an expression of the 15 myelodysplastic syndrome or even any of these changes 16 in the peripheral blood, peripheral blood is 17 essentially normal, they later develop leukemia, 18 acute myelogenous leukemia even? 19 A. I would question whether or not you are 20 talking about completely normal because in studies 21 that have been done of patients receiving 22 chemotherapy and/or radiation therapy, in at least 23 one study done with Michael Dexter in Testa 24 Laboratory in England has demonstrated fairly 25 significant stern cell behavior in patients years AVERY REPORTING SERVICE, INC. (303) 825-6119 65 1 after cessation of therapy with almost normal 2 peripheral blood picture, but I think 3 thrombocytopenia is relatively persistent even in 4 these patients. So I'm not sure I could agree with 5 the statement as you--the question as you phrased it. 6 Q. Doesn't that then argue against what 7 you just told me earlier, that you always have one of 8 these changes in the peripheral blood prior to 9 developing an AML that's caused by benzene? 10 A. No, because you have demonstrated some 11 frank damage to the bone marrow to begin with. 12 Q. But if you have had benzene exposure at 13 even 50 parts per million, you have demonstrated some 14 toxic effect to the bone marrow, have you not? 15 A. Not necessarily. I think the 16 likelihood is that between 50 and 100 parts per 17 million you will in some people. You will in some 18 mice. 19 Q. And if you look at a large enough 20 population of people, there are going to be some 21 people that that expression of toxicity may occur 22 below 50 parts per million? 23 A. It depends where you are on the dose 24 response curve, and I would say you are on the lower 25 end. I am comfortable with the idea that somewhere AVERY REPORTING SERVICE, INC. (303) 825-6119 66 1 between 50 and 100 parts per million is likely to 2 cause bone marrow damage. The evidence between 25 3 and 50 is really slim, and below that is problematic. 4 Q. This takes us really to the concept of 5 individual susceptibility that out there on the tail 6 of the population there are some people who are going 7 to be outside the norm, just like there are some 8 animals outside the norm. 9 A. That's correct. In the case of the 10 model we are talking about, I think we see those 11 people in the relatively low--small number of 12 individuals that will develop AML following very 13 high-level exposure to benzene. The majority of 14 cases of benzene toxicity described in the literature 15 are not leukemia. We are talking about bone marrow 16 suppression, we are talking about anemias, 17 pancytopenia, aplastic anemia, and a portion of those 18 will go on to develop AML. We have seen the 19 susceptible population when we see the incidents of 20 AML following high-level benzene exposure. 21 Q. The term high level is a relative term 22 depending on the individual susceptibility to 23 exposure? 24 A. I'm talking 100 parts per million and 25 greater, and perhaps 50 to 100 parts per million. AVERY REPORTING SERVICE, INC. (303) 825-6119 67 1 Q. When you say 100 parts per million or 2 greater, is that parts per million years? 3 A. 100 parts per million absolute 4 concentration of exposure for some duration of time. 5 Q. That's the next question, what 6 duration? 7 A. I don't think we have sufficient 8 evidence to make a clear statement on that except to 9 say we are talking repeated exposures. Certainly the 10 cases that have appeared in the literature have been 11 individuals that have been exposed for periods of 12 time in excess of six months, and the majority of 13 them exposed for long periods of time. 14 Q. At more than 100 parts per million per 15 eight-hour workday? 16 A. Not necessarily integrated, but 17 certainly exposed to levels between 50 and 100 parts 18 per million or greater. We have some studies, as I'm 19 sure you are well aware, from Turkey and elsewhere 20 where the exposures are horrendous. 21 Q. What about the worker who goes out and 22 gets exposed to benzene on a regular basis for a 23 period of, say, 15 minutes a day one time to 200 or 24 300 parts per million, where does he fit in the 25 pattern? AVERY REPORTING SERVICE, INC. (303) 825-6119 68 1 A. The only experience that I can lend to 2 clarification of that has to do with animal studies 3 in which I have tried to take the dose regimen issue 4 from days exposure down to hours of exposure, in 5 looking at exposures of 45 minutes to an hour and a 6 half, interrupting and looking at different 7 regimens. And I have found it incredibly difficult 8 to produce reproducible bone marrow toxicity in mice 9 at exposures less than an hour and a half to two 10 hours at a time. I know of no data to suggest that 11 you can maximize saturation of metabolism or 12 effectively produce the same biochemical scenario 13 that will exist with prolonged exposure in that kind 14 of a regimen. That is not to say that it might not 15 happen, but I certainly don't have any evidence to 16 support it. 17 It's unlikely that you are going to 18 have efficient metabolism of benzene for the 19 production of--optimal production of metabolites that 20 can get to the marrow and undergo further metabolism 21 unless you saturate benzene metabolism first, and 22 that's a process that takes--that may have some 23 species differences, but one would expect it is going 24 to saturate later in man than it is in a mouse, so we 25 are talking probably greater than two hours. AVERY REPORTING SERVICE, INC. (303) 825-6119 69 1 Q. Of course, you could achieve that by 2 having several lower-level exposures? In other 3 words, an exposure for ten minutes one hour, another 4 ten-minute exposure the next hour, if you are talking 5 about reaching the bone marrow with a certain dose? 6 A. No, only if you abide by an 7 accumulative exposure paradigm, which I don't think 8 is consistent with this particular situation at all. 9 Metabolism requires consistent exposure because you 10 are going to eliminate it as rapidly as well, so that 11 if you are exposed for a few minutes one hour and 12 then a few minutes the next hour it is as though you 13 are starting over again. 14 Q. What is the biological half-life of 15 benzene for humans as you have been able to 16 determine? 17 A. I would have to look that up. I don't 18 have that information at my fingertips. There have 19 been some studies done by Maths Berlin in Sweden that 20 come as close to defining that as any, and they are 21 reasonably--they are certainly consistent with what's 22 been seen or predicted from rat studies. 23 Q. But if you say, though, that an 24 exposure one hour and an exposure the next hour is 25 the same as starting over, the biological half-life AVERY REPORTING SERVICE, INC. (303) 825-6119 70 1 would have to be a matter of a few minutes as opposed 2 to a couple hours? 3 A. I would have to look at that data again 4 to comment. 5 Q. How can you make the statement that one 6 hour to the next hour starting over if you don't know 7 what that data is? 8 A. I'm telling you from my experience with 9 the mouse. It is incredibly difficult to produce 10 bone marrow toxicity with that kind of a regimen. 11 Q. This is measurable toxicity that you 12 see in the peripheral blood? 13 A. Or the bone marrow. 14 Q. Or the bone marrow? 15 A. Yes. 16 Q. And you are looking how? 17 A. Hemologic analyses, histopathology. 18 Q. What specific parameters would you be 19 looking at? 20 A. Peripheral blood counts, lymphocytes, 21 granulocytes, platelets, typical CBC. 22 Q. And you are looking for statistically 23 significant difference in changes? 24 A. Yes. Bone marrow cellularity. 25 MR. HOBSON: Lunch anyone? AVERY REPORTING SERVICE, INC. (303) 825-6119 71 1 MR. ZATZ: Sure. 2 (Whereupon, there was a recess.) 3 (Whereupon, the last question and 4 answer were read.) 5 Q. (BY MR. HOBSON) Dr. Irons, I want to 6 ask you a few questions about some other diseases, if 7 I could. Multiple myeloma, would you tell us what 8 multiple myeloma is in humans to you as a disease 9 condition and how it relates to these blood cells 10 that you have drawn in your chart, if it does? 11 A. Multiple myeloma is, I think, most 12 appropriately classified as a lymphoma, although it 13 arises in the bone marrow and is a malignancy of the 14 B-cell lineage, B lymphocyte lineage, that leads to 15 the production of differentiated immunoglobulin 16 producing B-cells also known as plasma cells. 17 Q. Have you been able to determine what 18 kind of a defect occurs that leads to the production 19 of multiple myeloma in humans? 20 A. I think the prevailing opinion at the 21 present time is that the target cell is a committed B 22 and B lymphocyte lineage. We are talking about a 23 committed B lymphocyte. 24 Q. Can you be more specific? Is this a 25 chromosomal change and do you know where the change AVERY REPORTING SERVICE, INC. (303) 825-6119 72 1 occurs or anything like that? 2 A. The information that I believe has led 3 to that general understanding has to do with the 4 rearrangements that are seen in the immunoglobulin 5 chains, the genes that code for the immunoglobulin 6 chains, and the level of maturation associated with 7 those specific changes. 8 Q. Are you aware of any mechanism or any 9 agent that causes that mechanism to change, at least, 10 and leads to the development of multiple myeloma? 11 A. Definitively, no. 12 Q. Well, do you have an opinion about any 13 agent or causative factors that go into the 14 development of multiple myeloma? 15 A. Multiple myeloma is one of the most 16 frequent types of hematopoietic or lymphoid neoplasms 17 that is seen in man, certainly with old age, and from 18 that standpoint is a relatively frequent occurrence. 19 There has been some question raised as to whether or 20 not multiple myeloma can be caused as a function of 21 chronic benzene exposure. At the present time I 22 don't think that there is certainly compelling 23 evidence to indicate that it is, although it 24 certainly has been suggested. And at this point I 25 have not yet reached an opinion on it. AVERY REPORTING SERVICE, INC. (303) 825-6119 73 1 Q. Another disease, Hodgkin's disease. 2 Would you tell me your understanding of Hodgkin's 3 disease and how it fits into this blood system, if it 4 does? 5 A. Hodgkin's disease is from a cellular 6 standpoint a very complex entity. There are a number 7 of different variations, but it appears to be a 8 disease of deregulation, if you will, of normal 9 lymphocyte behavior, primarily T-cells, and involves 10 constellation of other cells as well in the way the 11 lesion is expressed. It is a disease of the 12 peripheral lymphoid system. It arises from, I think, 13 most likely T-cells in the peripheral. There is 14 still some debate as to causation, although I think 15 most authorities agree we are looking at primarily a 16 T-cell lesion. 17 Q. Is this a lesion that starts out in one 18 of the precursor cells, or is it one that starts out 19 in the ultimate T-cell itself? 20 A. Most likely a T-cell. 21 Q. How is it that so many T-cells can be 22 converted if T-cells are one of these groups of cells 23 that don't reproduce themselves, or are they? 24 A. They are. 25 Q. So in other words, you are saying the AVERY REPORTING SERVICE, INC. (303) 825-6119 74 1 T-cell reproduces itself, it has a genetic defect, 2 and the defect is repeated also? 3 A. The lymphoid cells are distinguished 4 from the other cells that are descended to the 5 myeloid lineage in that they retain the ability under 6 certain circumstances to proliferate. They have, as 7 I said before, very elaborate control mechanisms that 8 are apparently at work to keep them from doing so in 9 an unregulated fashion, but they have that 10 capability. From that standpoint they are not in the 11 same category as the other cells of myeloid lineage. 12 Q. Are you aware of any efforts in the 13 case of Hodgkin's disease to see if the lesion, if 14 that's the right word, occurs further up the cell 15 division line back over into some of these stem 16 cells? 17 A. I would not be surprised at all if in 18 fact there was research under way to delineate the 19 target cells in Hodgkin's. At the present time I'm 20 not familiar with the latest literature in that area. 21 Q. Are you familiar with a disease called 22 myelofibrosis? 23 A. Yes, sir. 24 Q. What does myelofibrosis mean to you in 25 the context of the system? AVERY REPORTING SERVICE, INC. (303) 825-6119 75 1 A. Myelofibrosis is a condition that's 2 associated with the fibrous displacement of normal 3 hematopoietic tissue in the bone marrow and 4 frequently involves enlargement of the spleen and 5 extramudullary hematopoiesis. Myeloid metaplasia is 6 a better way to describe it in what's typically 7 called myelofibrosis. It is, as I understand it, an 8 extremely rare condition. 9 Q. Put that in terms of what is going on 10 in the body structurally, if you would, for 11 myelofibrosis. What happens with the disease with 12 these various entities in the different parts of the 13 blood forming system? 14 A. There is an abnormality that involves 15 the production of fibrous or connected tissue 16 sclerotic activity in the bone marrow, and this is 17 associated with the production of hematopoietic 18 tissue, which is normally located in the bone marrow, 19 to the spleen and perhaps other organs as well, 20 sometimes the liver. The spleen is the principal 21 organ. One sees production of blood in the spleen, 22 one can also see the same types of sclerotic changes 23 in pathology in the spleen as well. 24 Q. Are you saying that myelofibrosis, 25 then, is damage to the house where the blood is built AVERY REPORTING SERVICE, INC. (303) 825-6119 76 1 as opposed to damage to the cells that actually make 2 up the blood and turn it into the different 3 components of the blood? 4 A. I think our knowledge base with respect 5 to myelofibrosis is very weak and sparse. On a very 6 crude level that's certainly one hypothesis that 7 could be explored. As to causation of myelofibrosis, 8 the only--I don't know of anything that causes 9 myelofibrosis. 10 Q. Are you aware that benzene has been 11 implicated as a cause of myelofibrosis? 12 MR. ZATZ: Objection to the form. You 13 can answer. 14 A. I am aware that there are cases, 15 extremely few, that have been included in studies 16 that purport previous benzene exposure. To my 17 understanding there has been no demonstration that 18 myelofibrosis is in fact associated with benzene 19 exposure. 20 Q. (BY MR. HOBSON) Is that an area you 21 have looked at at all, or is this what you have 22 picked up in casual reading on other subjects? 23 A. I have read extensively in the benzene 24 literature associated with both the bone marrow 25 damage caused from acute or prolonged exposure to AVERY REPORTING SERVICE, INC. (303) 825-6119 77 1 high concentrations as well as leukemia and 2 mechanisms of leukemia genesis. I don't see anything 3 that would lead me to conclude that myelofibrosis is 4 a consequence of benzene exposure. 5 Q. Is myelofibrosis a fatal disease in 6 humans? 7 A. It can be, yes. 8 Q. Is it always? 9 A. Always is a harsh word. I 10 doubt--always applies to many things. 11 Q. How about usually, then? 12 A. Usually. I'm not a clinician, but as I 13 recall reading that literature it has a fairly dismal 14 prognosis, although I think it can go on for extended 15 periods of time, and in some cases it may go on for 16 ostensibly normal life span, although again I'm not a 17 clinician, so from that standpoint I probably 18 shouldn't even be commenting on it. 19 Q. Would you know if a person who has been 20 diagnosed with myelofibrosis would go on to develop 21 either leukemia lymphoma or multiple myeloma? 22 A. I believe in some percentage of cases 23 that I could not quote, we could look it up, I have 24 got Jandl right here, some percentage of patients 25 with myelofibrosis may go on to develop AML. With AVERY REPORTING SERVICE, INC. (303) 825-6119 78 1 respect to the other diseases you mentioned, I'm not 2 sure. 3 Q. Can you enlighten us as to a mechanism 4 for going from myelofibrosis to AML? 5 A. No, I can't. 6 Q. You mentioned earlier myelodysplastic 7 syndrome, and I've seen you abbreviate that as MDS? 8 A. Yes. 9 Q. To save my tongue can we refer to 10 myelodysplastic11 A. That's fine. 12 Q. You said, I think, in the beginning of 13 the deposition that MDS is really replacing more 14 archaic terms, I think you said, such as preleukemia, 15 and you mentioned some other things. Tell me how you 16 meant to use MDS as far as what it encompasses. What 17 diseases that are historically recorded would you put 18 in the MDS category? 19 A. Well, the classic one that is in there 20 is refractory anemia, or sideroblastic anemia, but 21 there are several different ones that are put under 22 that heading. When I think of MDS myself in the 23 context of bone marrow damage, I sometimes include 24 the more classic transient phenomenon such as 25 thrombocytopenia or lymphocytopenia. Myelodysplastic AVERY REPORTING SERVICE, INC. (303) 825-6119 79 1 syndrome or the syndromes usually have two or more of 2 those that accompany the disease process, but I think 3 they are all evidence of bone marrow damage either 4 transient or blocked. Myelodysplasia is most 5 appropriately associated with an abnormality in, I 6 think, stem cell differentiation. 7 Q. Aplastic anemia, is that a disease that 8 you would put under the umbrella of MDS? 9 A. It would depend on the context. Most 10 usually, no. But aplastic anemia has variations as 11 well in terms of the severity of the disease. And 12 basically the suppression of various cell lines can 13 vary considerably. Aplastic anemia can be a 14 symptomatology associated with severe MDS. 15 Q. Is MDS different for humans than it is 16 for mice and rats or other test animals? 17 A. Yes. I don't think we have an 18 appropriate model for MDS that certainly is 19 extrapolated from the cross species. We have seen 20 changes in the mouse that are very reminiscent of 21 changes that could be associated with MDS in man, but 22 it is by no means an exact model of the disease in 23 man. I don't know of any other. 24 Q. You don't know of any other what? 25 A. Model for myelodysplastic syndrome. AVERY REPORTING SERVICE, INC. (303) 825-6119 80 1 Q. In man? 2 A. I know of no animal model that 3 completely reproduces the disease as it is seen in 4 man. 5 Q. Doesn't that cause you a great concern 6 in extrapolating your findings from rats and mice to 7 man when we start talking about myelodysplastic 8 syndrome and the development of these different kinds 9 of cancers of the lymph and blood? 10 A. No, not legally. Basically you are 11 working with different pieces of information. You 12 are looking at from a mechanistic standpoint the 13 behavior of isolated murine and human stem cells 14 behave in similar fashion. There do not seem to be 15 any major differences in their behavior. The 16 clinical entities between the clinical entities of a 17 mouse leukemia model or any other animal leukemia 18 model appears to be both strain and species 19 specific. 20 You can't translate directly to man the 21 clinical manifestations of the diseases, they are 22 very difficult to do. One has to rely a great deal 23 on human literature to evaluate what goes on in man, 24 I agree, but certainly you can use the information we 25 have on the behavior of the various cell populations AVERY REPORTING SERVICE, INC. (303) 825-6119 81 1 to interpret and to refine those observations made in 2 man. 3 Q. I think you mentioned earlier today 4 that one of the things that you have relied on in 5 forming your opinions as far as literature is the 6 human epidemiology? 7 A. Yes. 8 Q. What specific studies can you cite me 9 to in human epidemiology that you have relied upon? 10 A. For what question? 11 Q. Start off with the abilities of benzene 12 to cause any leukemia in man. 13 A. I'm sorry, I don't understand the 14 question. Do you want me to cite for you which 15 papers have anything to do with leukemia in man and 16 benzene exposure or specific leukemias? I'm not17 Q. I'm interested in the human 18 epidemiology studies that you rely upon in forming 19 your opinions as you expressed earlier in the day. I 20 think you said that benzene causes acute myelogenous 21 leukemia in your opinion and not other kinds. If you 22 have human epidemiology that supports that, I would 23 like to know what it is that you find significant. 24 You said earlier before you would opine 25 that AML is caused by benzene exposure you would want AVERY REPORTING SERVICE, INC. (303) 825-6119 82 1 to see some precursor situations, and I would like to 2 see the human epidemiology you have that supports 3 that that you rely upon. 4 A. The literature I rely on with respect 5 to the relationship between benzene and AML and not 6 other forms of leukemia are the Rinsky study, which 7 I'm sure you are familiar with. Although I would not 8 in the strict sense call it epidemiology, the 9 collections and case studies of Dr. Viglianni and 10 Dr. Aksoy. The Yin study in China, and to a certain 11 extent the Wong study. 12 Q. Which Wong study? 13 A. I would have to in order to give you 14 specific citation, I don't think I brought that one, 15 so I would have to give that to you. 16 Q. Are you talking about the Wong study 17 that was done for the Chemical Manufacturers 18 Association of Benzene Exposed Chemical Workers? 19 A. I believe so. 20 Q. Now if I remember right for the Wong, 21 Chemical Manufacturers Association Workers, none of 22 these men had AML? 23 A. That study has some problems with 24 respect to interpreting the control group and also 25 with respect to the exposure. That's why when I AVERY REPORTING SERVICE, INC. (303) 825-6119 83 1 mentioned it I qualified it. There is something 2 unique about that population. Even so, I don't think 3 the study demonstrates a relationship with the other 4 types of leukemias, but it does--it is anomalous to 5 the extent that it--there are no AMLs in that study, 6 you are correct. It is definitely a unique study in 7 the benzene literature. 8 Q. You mentioned the Yen study? 9 A. Yes. 10 Q. The Yen study, the Chinese study, they 11 found a statistical significant excess of CMLs, did 12 they not? 13 A. That is what they reported, yes. 14 Q. Do you not believe that? 15 A. No, sir, I don't. 16 Q. Why do you think it is untrue? 17 A. The Chinese, the Yen study was 18 conducted by collecting diagnoses that were made at 19 approximately 27,000 different sites throughout 20 China--2,700 maybe, I think I'm off. The diagnoses 21 vary considerably from one place to another in terms 22 of sophistication and also with respect to accuracy. 23 In the case of AML it is not really a 24 major issue. With respect to CML it is very 25 important because they don't have cytogenetic AVERY REPORTING SERVICE, INC. (303) 825-6119 84 1 analogies as part of the diagnostic paradigms that 2 they use in general. From that standpoint it is very 3 likely that cases of myelodysplastic syndrome in one 4 version or another are going to be misdiagnosed as 5 CML because they don't have the sophistication for 6 diagnosis of CML that we commonly require in the 7 west. 8 Q. I thought I understood you to say 9 earlier that if a person has MDS they are more likely 10 to develop AML and not CML? 11 A. That's correct. 12 Q. How is it that you can have a person 13 with MDS and misdiagnose it as CML? 14 A. Because you are looking at a chronic 15 proliferative syndrome that may involve relatively 16 mature granulocytes. 17 Q. So you believe that it is more likely 18 to misdiagnose a leukemia as a CML than it is as an 19 AML? 20 A. It is not more likely to--you are not 21 more likely to misdiagnose an acute--could you repeat 22 the way you said it so that I can answer it--answer 23 the question directly? 24 Q. Right. CML frequently goes into a 25 blast crisis which leads to death? AVERY REPORTING SERVICE, INC. (303) 825-6119 85 1 A. That's correct. 2 Q. If you see it in a blast crisis you are 3 going to call it an AML. if you don't know about the 4 previous history, that's a misdiagnosis? 5 A. It's possible. That's more likely than 6 confusing an AML with the CML. If one is dealing 7 with an immunoblast or a lymphoblast crisis you are 8 not likely to misdiagnose it. And if you have got 9 cytogenetic backup you probably will not misdiagnose 10 it because you can confirm in fact that it was a 11 CML. What I'm saying is that whether you have got 12 chronic protracted abnormalities associated with 13 myelodysplastic syndrome and/or chronic abnormalities 14 resulting from bone marrow damage, that the 15 distinction between myelodysplastic syndrome and CML 16 can be very difficult. In fact there have been 17 studies conducted in the United States that 18 demonstrate that in the absence of that kind of 19 cytogenetic sophistication, well over 90 percent of 20 cases can be misdiagnosed. 21 Q. You are familiar enough with 22 epidemiology to know that the comparison group being 23 a general population comparison group, if errors are 24 made in the study group, the same kind of random 25 errors such as misdiagnosis are going to be made in AVERY REPORTING SERVICE, INC. (303) 825-6119 86 1 the reference population and those should cancel out; 2 isn't that the theory of epidemiology? 3 A. That does not hold in this specific 4 case because I would fully expect that in populations 5 exposed to high levels of benzene you are going to 6 have an increase in myelodysplasia, you are going to 7 have bone marrow lesions and suppression and an 8 increased incidence in myelodysplastic syndrome. 9 Q. Has this been followed up as far as you 10 know in the Yen study to confirm there has been this 11 misdiagnosis? 12 A. The NCI, I believe, is currently 13 conducting a study in China now to clarify issues 14 such as that. I don't know what stage they are. 15 Q. You don't know? 16 A. I don't know what stage they are at the 17 present time. 18 Q. Are you aware that other 19 epidemiological studies have found significantly 20 excessive amounts of CMLs in benzene exposed workers? 21 A. No. 22 Q. Are you aware of statistically 23 significant increases in leukemias in benzene exposed 24 workers besides Wong studies of the CMA workers where 25 no AMLs were found? AVERY REPORTING SERVICE, INC. (303) 825-6119 87 1 A. No. Excuse me, could you repeat that 2 last question. 3 (Whereupon, the last question was 4 read.) 5 A. My answer is the same, although I would 6 qualify that by saying that where the epidemiology 7 studies in the rubber industry show an excess of 8 certainly what appears to be lymphoid neoplasms. But 9 the issue of exposure, I think, is one that is not 10 consonant with benzene. There may be some benzene 11 exposures that are documented in some of those 12 studies, but it's a much more complicated exposure 13 paradigm than where you have got relatively clear-cut 14 exposure to just benzene. Those studies are 15 different. Their findings are different. 16 Q. I don't understand that answer. You 17 need to tell me, if you could, a little bit more 18 about what you mean about these exposure paradigms 19 that are not just benzene in rubber workers. 20 A. Well, any situation where you have 21 potential for exposure to benzene, certainly based 22 upon our knowledge of benzene toxicity of the blood 23 and bone marrow and its relationship to AML, it 24 certainly serves as a point of departure for making 25 assumptions as to potential relationship to causing AVERY REPORTING SERVICE, INC. (303) 825-6119 88 1 leukemias of different types, even though the studies 2 demonstrate fairly clearly a relationship with AML. 3 When you look at the rubber industry 4 where benzene exposure has been hypothesized as 5 playing a role, one sees a different pattern of 6 leukemias and lymphomas, including lymphoid. My 7 initial impression with that literature prior to 8 reviewing it in detail was that I certainly didn't 9 have a strong opinion, but I freely related the 10 opinions of others on whether or not there were other 11 tumor types associated with benzene exposure in those 12 populations. 13 But after reviewing the literature in 14 detail and taking into account the potential 15 exposures, I'm convinced that there is evidence to 16 suggest, fairly strong evidence, that benzene is not 17 the causal agent in those studies. That you are 18 looking at a different exposure situation and 19 different constellation of diseases than you see with 20 benzene. 21 Q. Do you have an opinion as to what the 22 cause of these other disease pat-terns in rubber 23 workers would be if it is not benzene? 24 A. I can relate to you in the case of 25 certainly the Checkoway study that a greater AVERY REPORTING SERVICE, INC. (303) 825-6119 89 1 association was found with carbon tetrachloride and 2 with carbon disulfide than with benzene. My 3 interpretation of that is not that carbon disulfide 4 and carbon tetrachloride have been demonstrated to 5 cause these lesions, but certainly benzene is not an 6 age actor in that exposure scenario. 7 Rubber industry and tire manufacture is 8 an incredibly complicated industrial picture from the 9 standpoint of evaluating exposures. Exposures, I 10 think, are very complicated, and I see no evidence in 11 that literature taking it in its entirety to suggest 12 that benzene is the cause of that particular pattern 13 of lesions. 14 Q. Would you even go so far as to say that 15 those kinds of disease patterns that you found for 16 rubber workers, the increase in, I think you said the 17 lymphomas, are those occupational diseases? 18 A. I think there is reason to be concerned 19 about exposure in the rubber industry. I think that 20 the studies suggest there is an increased incidence 21 of lymphoid neoplasms. I do not know why, and it is 22 inconsistent with my knowledge of the benzene 23 literature where in fact we have reasonably clear 24 cut exposures to benzene. 25 Q. Are you reasonably well satisfied, AVERY REPORTING SERVICE, INC. (303) 825-6119 90 1 though, that you can say no lymphoid neoplasms are 2 occupational related even though you can't say which 3 agent in this complex environment was the cause? 4 A. There is reason to conclude that there 5 are excesses in lymphoid neoplasms. With respect to 6 individual ones I would feel much less certain about 7 speculating on specific causes. 8 Q. I'm not even asking you to do that. 9 I'm saying, look at the group. If you had a study 10 and it reported 15, would you be in a position to 11 opine that any one of the 15 was an occupational 12 disease not even knowing what specific agent was the 13 cause? 14 A. If you had previous studies which 15 demonstrate specific increases in the specific tumor 16 type, yes. I would not want to do it on the basis of 17 a collection of different types of tumors. I don't 18 believe that's biologically plausible. 19 Q. Isn't that where you are, though, on 20 the lymphoid neoplasms of rubber workers? 21 A. To a certain extent, yes. We could 22 look at individual studies, if you want. If we are 23 going to talk about specific types I would like to, 24 but the problem there is that in the conduct of most 25 of those studies the object has been to get enough AVERY REPORTING SERVICE, INC. (303) 825-6119 91 1 power to see something. So there has been a lot of 2 lumping, and the lumping makes it very difficult to 3 figure out exactly what's going on. Is there an 4 increase in the general categories? There appears to 5 be. 6 Q. The lumping of the different kinds of 7 diseases dilutes the ability to remove that 8 confounding factor; is that what you are saying? 9 A. The lumping does not allow you to look 10 at specific causation for specific tumor types. 11 Q. And it is a confounding factor? 12 A. Lumping is a confounding factor, yes. 13 Q. And the same token if we start looking 14 at a specific disease category and looking at 15 exposure levels necessary to produce a given disease, 16 if we lump different exposure categories together, 17 that's another confounding factor that dilutes our 18 ability to answer that question too, isn't it? 19 A. I may not be speaking to your question 20 because I'm not sure I understand it, but analysis of 21 exposure is one of the weakest aspects of 22 epidemiology in general. Very hard, very difficult. 23 Q. Getting back to the diseases for a 24 moment. Do all CMLs demonstrate the Philadelphia 25 chromosome that you talked about earlier? AVERY REPORTING SERVICE, INC. (303) 825-6119 92 1 A. No. There is a very small percentage 2 that do not. 3 Q. Where do we classify the CMLs without 4 causing a Philadelphia chromosome? 5 A. About half of them, or roughly half of 6 them, can be shown to have the same molecular lesion 7 in terms of the rearrangement of genes that occurs in 8 Philadelphia chromosome positive CML. There is 9 another proportion of those that very well are missed 10 diagnoses in the sense that a recent study conducted 11 4 at the University of Chicago demonstrated that in 12 some 27 collected cases in the Philadelphia 13 chromosome negative CML, 26 of them, I think, it was 14 either 25 or 26, were rediagnosed as myelodysplastic 15 syndrome. So without the cytogenetics or the 16 molecular genetics that diagnosis is not simple even 17 in the United States. 18 Q. Explain to me again. You said that the 19 Philadelphia chromosome is a specific chromosome, and 20 yet21 A. It's a translocation. It's not--it's a 22 translocation of bits and parts of 2, 9 to 22. 23 Q. And then you said that there are some 24 of the CMLs that have a translocation that resembles 25 the Philadelphia chromosome? AVERY REPORTING SERVICE, INC. (303) 825-6119 93 1 A. They have a molecular change that's 2 very similar to what's seen in the Philadelphia 3 chromosome associated with the cluster region and the 4 proto-onco gene, but they don't have the 5 demonstrable--we are talking about a very refined 6 molecular change that is not demonstrable physically 7 in the eyes of the Philadelphia chromosome, but they 8 have the same molecular changes that are seen with 9 individuals who have the Philadelphia chromosome. 10 Q. Whose work is that that you are talking 11 about? 12 A. Name that comes to mind right off the 13 bat is Kurzrock at Texas. There are several others, 14 but she's done quite a bit. 15 Q. Do the CMLs that have the Philadelphia 16 chromosome and the CMLs that have the similar 17 translocation manifest themselves in the same way? 18 A. As I recall those studies, they are 19 very similar. They are very similar 20 characteristics. In fact the motivation of doing 21 that type of detailed analysis is from a prognostic 22 standpoint. 23 Q. From a24 A. For prognostic reasons. 25 Q. So the CMLs that either do not have the AVERY REPORTING SERVICE, INC. (303)825-6119 94 1 Philadelphia chromosome or don't have this other 2 translocation similar to it you say are misdiagnosed 3 as MDS? 4 A. Not necessarily all of them, but there 5 is certainly compelling evidence to suggest that 6 that's a major problem for that relatively small 7 proportion of cases. I'm not saying that all of them 8 are misdiagnosed. I'm saying that there is evidence 9 that in the absence of that kind of information it is 10 a very difficult call. 11 Q. Do humans who develop MDS later develop 12 CML? 13 A. Not to my knowledge, no. 14 Q. What about other leukemias besides CML? 15 A. AML? Certainly myelodysplastic 16 syndrome secondary to bone marrow damage. 17 Chemotherapy has a very high predisposition to 18 transform into AML. 19 Q. Are you not aware of any studies that 20 show people who have received chemotherapy go on to 21 develop CML? 22 A. Not in the absence of combined 23 radiation therapy. 24 Q. Requires both? 25 A. Yes. And there have been--that AVERY REPORTING SERVICE, INC. (303) 825-6119 95 1 literature base is very large. 2 Q. Does radiation produce CML in humans? 3 A. Yes, there is evidence for that. 4 Q. How do you explain that biologically on 5 your chart here that you have prepared? 6 A. In terms of what mechanistic 7 understanding we have of it. First of all, it is a 8 finding, it's a fact, it's an observation that 9 appears to be very reproducible throughout the 10 literature. Taking that as fact, if one looks at the 11 processes that are involved in damaging cells, 12 chemotherapeutic agents tend to--cells are more 13 susceptible to most chemotherapeutic agents during 14 cycle when they are dividing. 15 Radiation, on the other hand, tends 16 to--cells are actually more susceptible when they are 17 at rest. So radiation has a much greater potential, 18 if you will, for targeting a predominantly resting 19 population, and that's the best explanation that I 20 can give for it. Radiation can also affect dividing 21 cells, but there are no chemicals that I know of that 22 are associated with the production of CML. 23 Q. What other arrays of blood and lymph 24 diseases are you aware of associated with radiation 25 exposure, ionizing radiation? AVERY REPORTING SERVICE, INC. (303) 825-6119 96 1 A. Ionizing radiation causes a large 2 variety of different tumor types within the 3 hematopoietic system in the lymphoid system. The 4 only disease process, the only malignant disease 5 associated with the hematopoietic and lymphoid system 6 that I would say categorically can't be caused by 7 radiation is chronic lymphocytic leukemia, CLL. 8 There is no evidence that anything causes CLL. 9 Q. Can you explain mechanistically why 10 ionizing radiation can have such a broad effect, 11 whereas other agents, chemical agents, cannot? 12 A. Not with any--it would be wild 13 speculation. Radiation doesn't follow 14 pharmacokinetic or metabolic pathways. You are not 15 looking at activation, you are not looking at 16 distribution that can be compartmentalized in any 17 number of different ways. You are looking at 18 potentially different combinations of molecular 19 mechanisms that could be involved. With radiation 20 these things are pretty well simplified, and that may 21 or may not impact on why radiation has such a broad 22 spectrum. I would say the fact that radiation with 23 target resting cells is fairly important probably 24 plays an important role. 25 Q. How would you design an experiment to AVERY REPORTING SERVICE, INC. (303) 825-6119 97 1 show that a chemotherapeutic agent attacks dividing 2 cells as opposed to resting cells or vice versa? 3 A. Well, most of the changes that are seen 4 associated with chromosomal abnormalities require 5 cell division for those abnormalities to occur. 6 T-cells are more susceptible in terms of cytotoxicity 7 during that period, and I think you will find it is 8 generally--it's a generally accepted concept in the 9 medical scientific community that most 10 chemotherapeutic agents act on cytocells. There are 11 some exceptions, but by the same token these appear 12 to be T-cells that are most susceptible to the 13 effects. 14 Q. Isn't that looking at the result as 15 opposed to the effect, the kind of studies you just 16 described? 17 A. You are faced again with the--you can't 18 have it both ways. You are faced again with the 19 problems, just the innate problems of the field, you 20 can't prove a negative. It is very difficult to 21 prove a negative. You can only do so if you have got 22 evidence to demonstrate that all your other 23 technology and techniques are sufficiently sensitive 24 and specific to see if it was there. 25 That's where I think we are with CML. AVERY REPORTING SERVICE, INC. (303)825-6119 98 1 It is really clear what T-cell type CML is. It is 2 not proved to be anywhere near as easy to demonstrate 3 a relationship for other target cells for other 4 diseases. 5 Q. What evidence do you have that 6 radiation affects cells at rest? 7 A. I would have to go back in the 8 literature and find that for you. That comes out of 9 the radiation biology work that was done subsequent 10 to surrounding the studies done following the bombing 11 in Japan during World War II. Some of Gene 12 Cronkite's work looking at radiation sensitivities. 13 I can find that for you, but I don't have it at my 14 fingertips now. 15 Q. Don't all those studies require at 16 least one cell division to occur before you can find 17 a result? 18 A. I can tell you for a fact that if you 19 don't have replication you are never going to see 20 malignancy because if it doesn't divide you are not 21 going to have it. 22 Q. Doesn't that in and of itself say that 23 you don't really know that radiation effect is coming 24 from attacking a cell at rest? 25 A. The susceptibility of the cell AVERY REPORTING SERVICE, INC. (303) 825-6119 99 1 populations to the cytotoxic effects of the agent can 2 be demonstrated in the case of radiation resting 3 cells are as sensitive, if not more, slightly more 4 sensitive than dividing cells. For chemotherapeutic 5 agents dividing cells are clearly the most sensitive 6 population. 7 Q. That's what I was asking earlier is how 8 would you design a study to demonstrate that? 9 A. By looking at the relative dosimetry of 10 the effects of whatever agents you want on 11 predominantly resting or dividing cell populations. 12 Q. Isn't that just answering in a circle? 13 A. That is how you would go about 14 designing an experiment to look at it. I'm not quite 15 sure what you are driving at. 16 Q. Earlier you listed for me some of the 17 metabolites of benzene, and I didn't hear you say 18 catechol. 19 A. Catechol produces some effects on the 20 lymphoid system when you look at it in various 21 tests. It doesn't appear to be as effective in 22 interfering with cell replication as the others, 23 which is why I didn't discuss it. We were talking 24 about replication effects, and in that context 25 catechol, although it has some activity, it's much AVERY REPORTING SERVICE, INC. (303) 825-6119 100 1 less potent than the others, than the quinones. Q. If I understood what you told me 3 earlier, the toxicity of the benzene metabolites in 4 humans to you is not of consequence? 5 A. Independently? 6 Q. Yes. 7 A. No, I think you would be hard-pressed 8 to produce the same effects that you get when you 9 expose someone to high levels of benzene. 10 Q. What about a person who already has an 11 immunological disease, would the toxicologic effects 12 of the benzene metabolites have an effect on a person 13 who already has leukemia or lymphoma? 14 A. It might be beneficial if it had any 15 effect at all, and I doubt it would be easy to 16 demonstrate it. You are much more likely to have--in 17 order to get high enough concentrations that could 18 possibly deliver sufficient material to a relevant 19 target organ like the bone marrow, you would have 20 acute and subacute toxicity in other organ systems. 21 Q. What role does the benzene metabolite, 22 or I should say the benzene metabolites, what role do 23 they play in the regulation of the development of 24 these different cell lines of the blood and the 25 lymph? AVERY REPORTING SERVICE, INC. (303) 825-6119 101 1 A. If I knew the answer to that I could 2 probably get a free ride to Stockholm. That's one 3 thing we are definitely interested in determining. 4 It looks as though some hydroquinone has effects on 5 one particular stroma population in the bone marrow 6 known as the bone marrow macrophages that alters 7 lympho production. How, we don't know. That appears 8 to have effects on in turn development of certain 9 other cell types, but I don't think there is a 10 scenario that paradigms what I just described to you 11 that would impact on the development, the initiation 12 of a leukemia. I think you have to look for a direct 13 effect on cell population. So I would say that 14 exactly what's going on, the jury is still out. 15 Q. Maybe I have been using some bad 16 terminology in trying to communicate. If you have a 17 material that affects either the rate of cell 18 production or the direction of cell development, 19 would you be calling that effect something other than 20 a toxic effect? 21 A. Yes. 22 Q. What do you call that effect? 23 A. Well, I can give you--there are 24 examples. There are a number of endogenous materials 25 that regulate what direction these things go, or at AVERY REPORTING SERVICE, INC. (303) 825-6119 102 1 least their rate of--their proliferation in response 2 to different stimuli that are involved in the 3 physiology of controlling the regulating 4 hematopoiesis. 5 Q. What do we call that change? What 6 shall we call it if not toxic effects, what7 A. That goes on in normal everyday 8 physiology. That's how the process adjusts to any 9 kind of insult we may have, whatever it is. Given 10 that, why don't you rephrase the question so I 11 understand what you are looking for. 12 Q. Some treatments of disease attempt to 13 regulate this cell division and cell development 14 direction, do they not? 15 A. Are you talking in terms of 16 chemotherapeutic agents or new experimental therapy 17 in terms of altering regulation of hematopoiesis? 18 Q. Either one. 19 A. They are totally different. 20 Q. Tell me how they are different and what 21 we call the mechanism in each case so we can 22 communicate. 23 A. Chemotherapeutic agents target dividing 24 cells, certainly those that we are talking about for 25 the most part with respect to the hematologic AVERY REPORTING SERVICE, INC. (303) 825-6119 103 1 abnormalities. They kill them basically. The 2 cytotoxicity. Some are cycle specific, some are 3 phase specific, some are not. Those are agents that 4 are used in the treatment of cancers. Cancers tend 5 to be fairly rapidly proliferating so the bone marrow 6 cells, ergo, you have a problem in preserving and 7 protecting the bone marrow while attempting to kill 8 off tumor cells. 9 There are newer therapies that are 10 experimental at this stage that attempt to either 11 redirect or alter the progression of diseases such as 12 myelodysplastic syndrome and some of the leukemias by 13 using growth factors which had been purified and 14 isolated that are known to affect the normal 15 differentiation processes in the bone marrow. These 16 are highly complicated systems that we don't fully 17 understand, and they are largely experimental. 18 Q. Do those kinds of effects have a name 19 that you use? The second example you gave me of 20 giving direction to cell development. 21 A. I'm not exactly sure what you mean, so 22 I would hesitate to throw out a word because I'm not 23 quite sure what you are24 Q. What I'm getting to is does exposure to 25 a chemical such as benzene play any part in this AVERY REPORTING SERVICE, INC. (303) 825-6119 104 1 direction of cell development, cell proliferation, 2 and indeed the development of defense mechanisms that 3 are involved with the fighting of leukemia? Not in 4 fighting a person who is diseased with leukemia, but 5 a person who has had the effect and how the body's 6 defenses are brought into play? 7 A. I see about three questions in there. 8 Let me answer the one that I can focus on right 9 away. Anything that affects cells here, takes them 10 out. 11 Q. Here being? 12 A. In the differentiating blood and immune 13 cell compartment where we are producing this rapid 14 division committed cells, anything that destroys a 15 significant number of cells in any one of these 16 pathways, be it the lymphoid, erythrocyte, 17 granulocyte, platelet, what have you, it's going to 18 have an impact on the regulation of the entry of 19 cells into this compartment. 20 That's physiologic response. That's 21 how we can respond to any kind of damage, whether it 22 is loss of blood cells, damage to precursor cells, 23 that's at some point--the system can absorb major 24 challenges with respect to production of these cells 25 and still effectively produce normal cells. It does AVERY REPORTING SERVICE, INC. (303) 825-6119 105 1 so through a number of mechanisms. Ultimately if 2 there is enough damage out here you are going to see 3 a reorientation of the number of dividing precursor 4 and stem cells in this early compartment. And 5 benzene will do that just like any number of other 6 agents will. 7 Q. Now that's not really the question that 8 I was asking, although I accept that as an answer to 9 a question that was in there. What I want to know is 10 now assume that we have a major insult to one of 11 these cell systems. To communicate that information 12 back to the stem cells to tell them to produce, to 13 tell them which way to produce, where the need is, 14 that's a different mechanism than the destruction of 15 the cells out here in the peripheral blood? 16 A. Yes. Yes. 17 Q. So what I'm asking you is does benzene 18 or its metabolites play any role in interfering with 19 or directing that message sending back to the stem 20 cells and giving direction as to what to do next? 21 A. Based on the one system that we have 22 that's been studied which involves hydroquinone and 23 its effects on the macrophages that I indicated, I 24 would say that's a plausible scenario. It is 25 certainly one we are investigating. I can't give you AVERY REPORTING SERVICE, INC. (303) 825-6119 106 1 a definitive answer to it. 2 Q. In the level that you are talking about 3 for hydroquinone being able to affect this message 4 transfer, this is at a level that is-different from a 5 toxic response level? 6 A. No, it's not. Well, depends on your 7 definition. It's not cytotoxic to this individual 8 cell because the cell is still alive, but it's at a 9 functional level within the cell. Now if you have a 10 profound effect on that process in the bone marrow in 11 a number of cells, you are going to have a toxic 12 phenomenon at the level of the organism, you are 13 going to have myeloid suppression, you are going to 14 have decrease in circulating cells. 15 I know of no evidence that would 16 indicate that this goes on at a level that would not 17 be associated with measurable alterations in one or 18 more cell types within the immune or the 19 hematopoietic system. 20 Q. I need for you to explain that last 21 part. I didn't follow the last part. 22 A. I think the changes as we know them, 23 and we are at the very beginnings of this field, are 24 at concentrations that are most likely--the effects 25 are most likely to be seen immediately in terms of AVERY REPORTING SERVICE, INC. (303) 825-6119 107 1 suppression of cell types, either in the marrow or 2 the peripheral blood. I don't think that there is 3 any--there is no evidence to suggest they go on at a 4 level that would not be measurable in terms of the 5 effects on the cells because we are still talking 6 about support for proliferating cells, and it is 7 manifested in a decrease in the production of cell 8 types or an alteration in the direction they go in. 9 So you are going to see it. It will be associated 10 with toxicity at the whole animal level, if that may 11 be what you were looking for. 12 Q. I think you answered my question. Have 13 you gone even so far as to look at one cause of the 14 change out here in the peripheral blood that calls 15 for the message being sent and seeing what level of 16 quinone involvement there is in interrupting a 17 message or altering the message? In other words, the 18 quinone or a benzene, for instance, with a quinone as 19 a metabolite was not the cause of the problem, but 20 the quinone coming in interferes? 21 A. You know, I'm sorry, I thought I 22 understood you before you clarified it. We know what 23 concentrations of hydroquinone will result in 24 isolated cell systems in blocking division, okay? 25 That's relatively well worked out. That is going to AVERY REPORTING SERVICE, INC. (303) 825-6119 108 1 result in the loss of cells here, there is no doubt 2 about it. Cycling cells are more susceptible to the 3 cytotoxic characteristics of these metabolites than 4 toxic cells, no doubt about that. I don't understand 5 what you were asking with respect to the feedback 6 there. 7 Q. There are other agents that can cause 8 the circulating blood cells that are cytotoxic to the 9 circulating blood cells besides benzene? 10 A. Oh, lord, yes. 11 Q. What I'm asking is if you have one of 12 these other agents; that is, the agent that knocks 13 down the peripheral blood, have you looked at what 14 level of quinone involvement there would have to be 15 necessary to interfere with the transmission of the 16 message back to the parent cells? 17 A. I do not understand the question. 18 Other agents. I don't understand how you get other 19 agents and quinone in the same sentence. I don't 20 understand what--the paradigm I don't understand. 21 Q. Take concomitant exposures, there are 22 chemotherapy agents that are able to affect the 23 peripheral blood to knock down the different elements 24 on the peripheral bloods, right? 25 A. Yes. AVERY REPORTING SERVICE, INC. (303) 825-6119 109 1 Q. They have nothing to do with the 2 production of hydroquinone in the body? 3 A. Yes, that's true. 4 Q. So now if you have a concomitant 5 exposure to benzene, which does give you 6 hydroquinone? 7 A. You are looking now exposed to a 8 therapeutic agent and the benzene? 9 Q. Yes. 10 A. Okay. 11 Q. Does the resulting metabolite, the 12 hydroquinone from the metabolism of benzene, 13 interfere with the transmission of information back 14 to these parent cells to do more? 15 A. There is absolutely no information on 16 that whatsoever. 17 Q. Just don't know one way or the other, 18 has not been looked at? 19 A. Has not been looked at. 20 Q. That's what I was trying to ask, have 21 you looked at22 A. No, we are still trying to figure out 23 what hydroquinone does. As soon as you start 24 dropping in multiple agents, you have a very complex 25 system, if it wasn't complex enough to begin with. AVERY REPORTING SERVICE, INC. (303) 825-6119 110 1 Q. Is hydroquinone involved in this 2 process of communications back and forth to the cells 3 in a person that is not exposed to benzene or another 4 chemical agent that will result in a metabolic result 5 of hydroquinone? 6 A. You mean is hydroquinone normally 7 involved in the process? 8 Q. Yes. 9 A. Not hydroquinone. There may be other 10 quinones or alpha beta and saturated carbonyl or keto 11 structures that play a role in regulating this 12 process, but to my knowledge not hydroquinone. 13 Q. Is the reason you focused on 14 hydroquinone because of your interest in benzene 15 metabolism and its effects? 16 A. To begin with, yes, hydroquinone has 17 taken on a life of its own for different reasons 18 related to understanding how cells divide and are 19 regulated. But, yes, if you are looking for a 20 hypothesis from me, I think hydroquinone plays 21 a--probably plays a fairly important role in the 22 evolution of bone marrow damage associated with 23 benzene, which you can get from reading my papers. 24 That's nothing new. 25 Q. We have yet to address another area of AVERY REPORTING SERVICE, INC. (303) 825-6119 111 1 your work that I wanted to go into, and that's work 2 with butadiene. Tell me how butadiene and the 3 metabolites of butadiene, if they do, come into the 4 development of the blood system. 5 (Whereupon, there was discussion 6 outside the record between the deponent and his 7 counsel.) 8 MR. ZATZ: I assume you have some wild 9 theory of what this has to do with this case or how 10 it is going to lead to relevant evidence in the cases 11 of Carter and Riddle, or are we taking Dr. Irons' 12 deposition for some other case simply because we have 13 him here? 14 MR. HOBSON: I don't think we are doing 15 that at all. Any time you depose a man who is named 16 as a witness in many other cases, obviously you can 17 say it has an impact on other cases, so I won't tell 18 you it doesn't. But at the same time I think 19 Dr. Irons' bibliography will show he has published a 20 fair amount of work on butadiene, and I think how all 21 of these chemical exposures--we sat here and we have 22 talked about chemotherapeutic agents and how they 23 develop into different diseases, butadiene is another 24 agent that affects the blood, and how all of these 25 different effects from different chemicals and the AVERY REPORTING SERVICE, INC. (303) 825-6119 112 1 mechanisms fit together I think are important to 2 understanding how he thinks. 3 MR. ZATZ: Well4 MR. HOBSON: I think that's why I'm 5 here. 6 MR. ZATZ: I understand that, and I 7 didn't cut you off in quizzing him on Hodgkin's 8 disease and multiple myelomas and that sort of thing, 9 but it seems to me at some point we go beyond a 10 hematology quiz, which I think is fair game, and we 11 get into subject matter of other cases. If that's 12 what we are doing now, I think there are probably 13 other defense counsel in those cases who would 14 certainly prefer to be here when you are doing it. 15 MR. HOBSON: You going to tell him not 16 to answer, or what? 17 MR. ZATZ: No, I'm not instructing him 18 not to answer. 19 MR. HOBSON: I'm going to ask him the 20 questions. 21 MR. ZATZ: My objection is simply this, 22 just so it is absolutely clear on the record, if 23 there is some way in which all this is going to lead 24 to evidence relevant to these cases, I'll allow him 25 to answer, although I don't particulary see the AVERY REPORTING SERVICE, INC. (303) 825-6119 113 1 relevance. If what you are doing is simply taking 2 Dr. Irons' deposition for some other cases, then I 3 feel constrained on behalf of other counsel to 4 instruct him not to answer. 5 MR. HOBSON: Well, I guess you have to 6 make a choice. 7 MR. ZATZ: Which are you doing? 8 MR. HOBSON: I'm taking his deposition 9 in this case, but I'm not going to tell you it won't 10 impact others because he has been named as an expert 11 in other cases involving butadiene too. But as I 12 said a moment ago, you said I was going through a 13 lecture on hematology, I don't think Dr. Irons holds 14 himself out as a hematologist, he is a toxicologist 15 as I understand his credentials, and we have been 16 talking about the effects of blood--of chemicals on 17 the blood, and that's where I'm going. I think 18 that's where his work has been, how do chemicals 19 affect the blood, and I want to know what his 20 opinions are and how he got there. 21 MR. ZATZ: We can call it 22 hematotoxicology if you think that's fair. At some 23 point if this appears to be simply an endeavor to do 24 discovery in some other case, I may change my mind. 25 MR. HOBSON: Just like a woman you can AVERY REPORTING SERVICE, INC. (303) 825-6119 114 1 have that prerogative. 2 Q. (BY MR. HOBSON) I have forgotten where 3 we were, Dr. Irons, so let's start over. Would you 4 tell me based on your knowledge how butadiene fits 5 into the development of diseases of the blood as you 6 understand it. 7 A. I don't have any evidence to conclude, 8 nor do I believe, that butadiene acts in any way, 9 shape, or form in a manner similar to benzene. It is 10 a totally different agent and it acts through 11 different mechanisms. Unless you want me to just 12 outline the various studies I have done and what I 13 know about butadiene, that's the best answer I can 14 give you. Where we could spend all day talking about 15 benzene and what it does and probably not run out of 16 things I could tell you, with butadiene I'm going to 17 run out of things very quickly. 18 Q. Then we won't take much time. Are 19 there diseases that arise through this pluripotential 20 stem cell? That is, either diseases of the blood or 21 the lymph that butadiene is associated with in your 22 mind? 23 A. The number of diseases that I think 24 arise from the pluripotential stem cell in any event 25 are few and far between. In the case of butadiene, AVERY REPORTING SERVICE, INC. (303) 825-6119 115 1 in certainly the one model I am intimately familiar 2 with, the mouse, that I have a suspicion we are 3 looking in here. 4 Q. In here being the lymphoid stem cell 5 area? 6 A. The lymphoid committed stem cell area, 7 primarily down the T-cell line, but again we are 8 talking about a totally different model in which 9 there are very likely a number of other confounding 10 factors. Huge strain specificity and species 11 specificity with respect to the behavior of butadiene 12 involving any number of different things, not the 13 least of which in the mouse is probably retrovirus, 14 and now we're getting into very deep soil. The 15 mechanisms are very, very complicated. 16 Q. My reading of some of your recent work 17 on butadiene says it wasn't just the retrovirus, that 18 there are other mechanisms involved too? 19 A. There are multiple mechanisms involved 20 with butadiene, I suspect. Do I know that for a 21 fact? No. Butadiene in the mouse, butadiene is a 22 very complicated model. 23 Q. What is the extent of your knowledge 24 about butadiene effects in a human related to the 25 blood and lymph system? AVERY REPORTING SERVICE, INC. (303) 825-6119 116 1 A. Would you mind if I preface that? 2 Q. Surely, go ahead. 3 A. I consider epidemiology in general to 4 be a fairly weak discipline. The methodologies and 5 the power of the tests that are available to us are 6 very difficult, and it is a very difficult field to 7 work in. When I look at epi studies, therefore, I 8 like to look for patterns, not individual studies. 9 Individual study may give you an effect if it is 10 reproducible, then it is more likely to be an 11 association that you need to look at. If you have 12 mechanistic understanding of what's going on, it 13 helps in interpreting that. 14 In the case of butadiene I see a 15 tremendous amount of inconsistency with respect to 16 the available epidemiology studies. Some have 17 suggested a potential relationship with lymphoid 18 neoplasms, others have not. Others find different 19 neoplasms, and I don't believe that the same agent 20 under the same conditions is going to produce 21 different tumor types in different groups. I see no 22 reason to believe that. So at this point I think the 23 epi studies are of very limited value in establishing 24 and understanding the butadiene toxicity in man. Did 25 that answer your question? AVERY REPORTING SERVICE, INC. (303) 825-6119 117 1 Q. It's getting there, yes. Based on the 2 work that you have done with butadiene, if you look 3 at the mechanisms, can you tell me which mechanism 4 seems to be involved in the development of the 5 lymphomas in mice? In other words, is it at the 6 genetic level? Is it some other effect? I know you 7 talked a little about the retrovirus. 8 A. I think the retrovirus influences that 9 disease. I'm not sure whether it is causal, but it 10 certainly plays a role in causation based on the 11 indirect evidence we have. A whole lot of very 12 brilliant scientists have donated their careers to 13 the trash heap of murine leukemogenesis associated 14 with retrovirus. 15 Retrovirology in the mouse is a maze. 16 Based on the chemistry of butadiene and some of the 17 metabolites we know are formed, I suspect we are 18 probably having some effects at the level of the DNA 19 or the Genome, but we are talking wild speculation. 20 It alters differentiation. I have demonstrated that 21 certainly for the hematopoietic system. 22 That doesn't seem to be--it doesn't 23 produce a very pronounced anemia, and the stuff is 24 just not very toxic from the standpoint of acute or 25 subacute effects, but it causes profound activation AVERY REPORTING SERVICE, INC. (303) 825-6119 118 1 in the endogenous ecotropic. Retrovirus is just 2 retro and virus. If you are a mouse you don't want 3 to be exposed to butadiene. 4 Q. Where are you on knowing how butadiene 5 is metabolized in humans? 6 A. I would say we are not very close to 7 knowing how butadiene is metabolized in mice. 8 Initial studies I don't think are complete, and I 9 think the metabolism of the compounds is far more 10 complex than people have originally thought.. 11 Q. So you wouldn't be able to say if the 12 same family, chemical families of metabolites exist 13 for butadiene as for benzene? 14 A. That I can tell you. No. That's easy. 15 Q. It is different? 16 A. It's different. That's probably the 17 only yes, no answer. 18 Q. Tell me what function in the human body 19 the T-cell lineage plays. I know it may be many, 20 many but give me the major roles as you understand 21 them. 22 A. The T-cell is the cell classically 23 associated with what we call cell mediated immunity, 24 cell mediated immune response, and that was 25 appreciated probably as early as we had a clear AVERY REPORTING SERVICE, INC. (303) 825-6119 119 1 understanding of T-cells and B-cells. Over the last 2 ten to 15 years we have come to appreciate that there 3 are multiple different subpopulations of T-cells, and 4 they play an integral role in regulating and 5 mediating immune response. They are very, very 6 active in regulating their own behavior. They 7 regulate the behavior of B-cells. They are the 8 pivotal cell in regulating specific immune response. 9 Now I could go into detail. I teach 10 the subject. We could spend the next several hours 11 if you want. 12 Q. Maybe not several hours, but several 13 minutes. How in disease does the T-cell line get 14 involved? In other words, bring it down, if you can, 15 to more of a layman's language in explaining what a 16 T-cell does in playing its part in fighting off 17 disease or in warring against cancer. 18 MR.. ZATZ: I'm going to object to the 19 form of that question on this because I think it is 20 asking you two very different things. 21 A. It is. 22 Q. (BY MR. HOBSON) There are two 23 questions there, I agree. 24 A. Cell mediated immune response is immune 25 response that's targeted at diseases or organisms AVERY REPORTING SERVICE, INC. (303) 825-6119 120 1 that are not effectively removed simply by producing 2 antibodies to them. It may take up residence inside 3 cells. So cell mediated means basically that the 4 T-cell and the cell response is targeted towards 5 destroying cells that are infected with a foreign 6 agent as a means of stopping the spread of the 7 agent. 8 So T-cells are involved in recognition 9 of self and recognizing whether there is something 10 nasty inside the cell like a virus that needs to be 11 eliminated. And it does so by recognizing some 12 aspect of the virus on the surface of a cell that it 13 otherwise recognizes as part of self. It also helps 14 regulate antibody production and has several 15 different classes of T-cells that help regulate their 16 own response and antibody response. 17 So it goes up and it comes down and you 18 have some appropriate control over the process. It's 19 a very complicated network involved in providing us 20 with immunity to infectious agents that are basically 21 intracellular. 22 Q. You have answered my question as to the 23 disease aspects, primarily, I guess, infectious 24 disease. 25 A. Right. AVERY REPORTING SERVICE, INC. (303) 825-6119 121 1 Q. Does the T-cell play any part in 2 warding off cancer in the human body? 3 A. There are animal models that suggest 4 that profound immunosuppression plays a role in the 5 incidence or development of certain types of 6 neoplasms, various specific mouse models, so 7 certainly some aspects of T-cells, primarily the 8 cytotoxic T-cells and perhaps another cell type 9 that's not really within the T-cell lineage, the NK 10 cell, play a role in mediating against the 11 development of tumors, certain types of tumors for 12 sure. 13 The concept of immune surveillance has 14 been around for a long time. There are very few 15 systems, I can't think of a single model where I 16 would say immune surveillance has been absolutely 17 demonstrated in that context where a single cell is 18 acted upon by a system. Certainly the immune system 19 plays a role in animal models in susceptibility to 20 tumor development, certain types of tumor 21 development. 22 In man the evidence is, of course, a 23 little bit different because we can't go around 24 setting up these nice control groups and exposing 25 people to things. There is certainly evidence that AVERY REPORTING SERVICE, INC. (303)825-6119 122 1 immunosuppression involved in treating various 2 autoimmune diseases and in transplantation cases can 3 lead to the development of certain types of tumors, 4 primarily those of lymphoid origin, it certainly 5 predisposes to that. 6 The problem is in almost all the 7 studies that have been conducted you are not looking 8 at something that is selectively immunosuppressive, 9 you are usually dealing with the administration of 10 alkylating agents as well, which gets back to the 11 whole issue of secondary tumor types, and it is a 12 very complex picture. But certainly for cells of the 13 immune system or lymphoid tumors there is some 14 evidence that immune surveillance plays a role, if 15 only inferentially from the studies of 16 immunosuppressed patients. 17 Q. Do you know if there is butadiene 18 exposure in a tire plant? 19 A. I am not an industrial hygienist. I 20 rely on industrial hygiene data to render an 21 opinion. If you want to know whether I personally 22 know if there has been a butadiene exposure in a tire 23 plant, my answer is no. 24 Q. Do you know of a compound called 25 tertiary butacatechol? AVERY REPORTING SERVICE, INC. (303) 825-6119 123 1 A. Tert butacatechol, I have heard of it. 2 Q. Do you know of any of its uses? 3 A. No. 4 Q. Do you know if tert butacatechol, as 5 you have referred to it, how it structurally compares 6 to the catechol that is the metabolite of benzene? 7 A. No. Any substitution to that ring is 8 going to alter the chemistry of the catechol 9 dramatically. 10 Q. Have you seen any information about 11 metabolism of tertiary butacatechol? 12 A. No. 13 Q. I would like to get, if I could, your 14 understanding of some different terminology so that I 15 can hopefully understand how you might have used it 16 or might use it in the future. A mutagen. How would 17 you use the term mutagen? 18 A. The way it is most popularly used is 19 the way I refer to it, and that is something that 20 actively causes directly a mutation. Mutagenic event 21 at the level of the DNA. 22 Q. And to go further, what do you mean by 23 a mutation at the. level of DNA? 24 A. A specific change associated with the 25 alkylation or deletion of a base para sequence that AVERY REPORTING SERVICE, INC. (303) 825-6119 124 1 leads to the misreading of the genetic code. 2 Q. Clastogenic? 3 A. Something that changes chromosomes. 4 Q. Is there a difference between a 5 mutagenic agent and a clastogenic agent? 6 A. Yes. By the definitions I just gave 7 you, yes. 8 Q. Explain clearly the difference in the 9 use of those two terms for me. 10 A. Shall we cut to it? Benzene metabolite 11 such as hydroquinone are clastogenic, they are not 12 mutagenic in the context of studies aimed at looking 13 at mutagenic mutagens, they are negative. 14 Q. What about the other metabolites of 15 benzene? 16 A. Same. Para-benzoquinone is going to be 17 very similar to hydroquinone. To my knowledge none 18 of them have been shown to be mutagenic. Catechol is 19 more cytotoxic than hydroquinone but less effective 20 at clastogenic events at least related to 21 nondisjunctional events. Phenol just kills cells. 22 It is not effective in either category. 23 Q. Promote or promoter in terms of in the 24 context of carcinogenicity? 25 A. If we talk in the context of AVERY REPORTING SERVICE, INC. (303)825-6119 125 1 carcinogenicity it means one thing. If we talk in 2 context of diseases of the blood and bone marrow it 3 is entirely different, usually being that they have 4 never been established to be relevant to the 5 hematopoietic system. 6 Q. I have heard you say that before, 7 that's why I asked in terms of carcinogenicity. 8 A. Usually they refer to studies done in 9 the skin. There are some liver models in which one 10 is looking at a paradigm in which an agent 11 administered after an initiating event can enhance or 12 exacerbate the expression of tumor phenotype in one 13 model or another. 14 Q. When you say there is no model that has 15 been found for the hematopoietic system, has it been 16 looked for? 17 A. Certainly the same agents that have 18 been shown to be effective in at least the paradigms 19 that have been used in the skin models and liver 20 models have been looked at in terms of the 21 hematopoiesis. There is none. Certainly the 22 compounds have been looked at in the same context, 23 but I don't know of anyone who has specifically done 24 initiation promotion studies because we don't have a 25 system that is amenable to that. AVERY REPORTING SERVICE, INC. (303) 825-6119 126 1 The problem with promotion paradigms is 2 they aren't definitive in terms of what you are 3 looking for--they are a paradigm. They are an 4 elaborate hypothesis, if you will, and if it doesn't 5 fit the algorithm, then the biology is just as 6 relevant as any other biology, but it doesn't fit 7 your model so you don't know what you have got. 8 Therefore, I would say they have been 9 looked at in the context that the same compounds have 10 been administered to the animals that develop tumors 11 of the hematopoietic system, but there is no clear 12 paradigm that can be associated with initiation and 13 promotion. 14 Q. You have used many times today the term 15 paradigm. 16 A. Model, pattern. 17 Q. I just want to make sure I don't miss 18 that one too. The term immune system depressant, 19 what would that mean to you? 20 A. Something that suppresses immune 21 response. In general I mean something that 22 demonstrably alters host resistance to the animal. 23 You can say, for example, that hydroquinone as an 24 immune system suppresses certain aspects of immune 25 function in isolated systems. That doesn't mean AVERY REPORTING SERVICE, INC. (303) 825-6119 127 1 automatically that hydroquinone administered to the 2 animal at tolerable concentrations in some way is 3 going to automatically lead to immunosuppression, 4 altered physiologic resistance to infection, or what 5 have you as a function of that. So I distinguish 6 between something that suppresses immune function and 7 something that alters an isolated cell function. 8 Q. How broad would you be using the term 9 immune system in that context? 10 A. The immune system is anything and 11 everything that includes lymphocytes, macrophages, 12 dendritic cells, a variety of effecter cells, and 13 accessory cells, the whole immune system. 14 Q. Something like hydroquinone, does it 15 affect the whole of the immune system, or a very 16 narrow part? 17 A. You can show isolated effects in a 18 variety of different cell types that are interesting 19 that probably are important with respect to 20 understanding what hydroquinone can and can't do. 21 They are excellent models for studying the behavior 22 of that compound. The consequence of high-level 23 exposure to hydroquinone in terms of its effects on 24 the immune system is a separate issue, and I do not 25 know of any evidence to suggest that you can AVERY REPORTING SERVICE, INC. (303) 825-6119 128 1 administer hydroquinone even to animals and produce 2 effects on immune response that don't also result in 3 reduction in cell types, damage to the bone marrow, 4 and a variety of other findings as well. 5 Q. If we talk about benzene exposure, how 6 long after exposure ceases will you expect to find 7 immunologic effects? 8 A. Immunologic effects? 9 Q. Yes. 10 A. What do you mean by immunologic 11 effects? 12 Q. I'll start there. Give me a list again 13 of the immunologic system, and then we can talk about 14 the different effects, maybe that's a better way of 15 doing it. 16 A. You mean cell types? We were talking 17 about B-cells, T-lymphocytes, macrophages, monocytes. 18 Q. All of them? I think you see where I 19 want to go. 20 A. If I did, this would be a little 21 easier. 22 Q. I would like to know the effects that 23 you have seen benzene produce in either animals or 24 man, and which of those effects, if any, persist 25 after exposure ceases, and if you know at what level AVERY REPORTING SERVICE, INC. (303) 825-6119 129 1 of exposure? 2 A. In experimental animals, most of the 3 studies we have done looking at specific effects on 4 immune response have been done with metabolites 5 because it is very difficult to produce reproducible 6 effects with benzene itself. With the metabolites I 7 have looked at short-term scenarios, and we see 8 recovery, we see recovery, it has been difficult to 9 produce changes that go out for any great length of 10 time, it's the devil to work with if you want to 11 produce something that you can look at for any great 12 length of time. 13 Q. May I interrupt you for just a second. 14 With your hands in front of your mouth it is hard for 15 me to understand you, and probably her too. 16 A. Sorry. I don't know of any studies 17 that have looked at the prolonged effects of benzene 18 exposure on immune response in the absence of looking 19 at other things such as peripheral blood counts, 20 damage to the bone marrow, what have you, that have 21 looked at long-term. There may in fact be some and 22 that I have read them, I just can't remember--they 23 certainly didn't leave an indelible stain on my 24 memory. 25 My own impression of benzene, as I AVERY REPORTING SERVICE, INC. (303) 825-6119 130 1 said, it is not a very potent immune suppressant, you 2 have to get up there in the concentrations that are 3 knocking cells off all over before you are going to 4 see an effect. I do recall early in almost the turn 5 of the century Selling and Weiskotten and a few 6 others looked at the effects of inhalation of benzene 7 on rabbits. 8 I believe there is at least one what I 9 would call highly uninformative allusion to it 10 decreasing host resistance to either TB or to 11 pneumococcus or something like that in rabbits. 12 Again I think we are talking acute exposures, or 13 subacute exposures. We are not talking about 14 something that's happening or the exposures were 15 ceased and you looked at immune function. 16 Q. Those were exposures in the rabbits? 17 A. As I recall they were dosing the stuff 18 on gauze or cotton and just holding them under, which 19 is not very quantitative but certainly effective. 20 Q. What about other effects on the blood 21 and lymph systems from benzene, can you tell me what 22 effects that you have found that are long lasting 23 after exposure ceases? 24 A. No. All of ours have been transient 25 with the exception of continued exposure to AVERY REPORTING SERVICE, INC. (303) 825-6119 131 1 hydroquinone in an interrupted regimen in which we 2 got prolonged suppression. But that was with 3 continued exposure. I was trying to produce an 4 equivalent of an aplastic anemia, and came as close 5 as I think I'm ever going to with hydroquinone and 6 phenol together. We have never done it with benzene. 7 Q. Are there any effects that you know of 8 where you have followed a benzene exposure regimen 9 for a period of time? You told me earlier about some 10 studies that you did where you tried to quantitate or 11 part of your opinion about the quantitative 12 relationship of exposure to benzene and certain 13 effects. Have you done similar-type studies where 14 you have exposed animals over a period of time, not 15 exposed them, and looked at any effect some months 16 after? 17 A. On immune response? 18 Q. No, for anything, not just immune 19 response. 20 A. We have looked at--basically that was 21 the paradigm that we were using. Expose them for 12 22 weeks, stopped exposure and held them for a year or 23 longer, and we get mild proliferative changes in the 24 spleen. 25 Q. And your recollection was there was no AVERY REPORTING SERVICE, INC. (303) 825-6119 132 1 effect below 50 parts per million; is that what you 2 said? 3 A. We didn't look below 50 because we have 4 had a devil of a time historically producing 5 reproducible toxicity at higher concentrations, so we 6 were looking at 50. At 50 it was not very 7 impressive. We are certainly not talking about the 8 same magnitude changes. Given a dose response 9 whereas you know you are having effects at higher 10 concentrations, you could look at some of those 11 spleens and say there are some changes here. But in 12 the absence of a dose response you would be 13 hard-pressed to defend that there was a great deal of 14 effect. 15 Q. I had one paper this morning that we 16 brought over at the lunch hour that goes into 17 something--I have both here now. It's not your 18 paper. I'll give you the published one here. Is 19 that a paper that you have seen before? 20 A. Yes. 21 Q. And just for the record here, unless 22 somebody wants to attach it, would you read the title 23 so we can identify later where it was published? 24 A. Induction of Cytogenetic Damage in 25 Rodents After Short-Term Inhalation of Benzene. AVERY REPORTING SERVICE, INC. (303) 825-6119 133 1 Q. It was published? 2 A. Environmental Mutagenesis, pages 29 to 3 40, 1986. 4 Q. Explain for me how what is presented in 5 this paper fits into the context of the earlier work 6 you described for me? 7 A. You are going to have to clarify that. 8 Q. This paper, I think, presents findings 9 from exposures at less than 50 parts per million? 10 A. Yes. 11 Q. And it presents findings that are, I 12 think, a year later; is that right, after exposure 13 ceases? 14 A. I don't see where they were exposed a 15 year later or they were examined a year later. It is 16 18 hours after exposure. 17 Q. It may be my memory has failed me. Why 18 don't you say what the procedure was for dosing the 19 animals and looking and tell me what it was, that way 20 we won't have to guess. 21 A. Rats were--first of all, mice were 22 exposed to concentrations ranging from zero to 1,000 23 parts per million, zero, 10, 100 and 1,000 every six 24 hours. Rats were exposed to target concentrations of 25 zero, 1, .3, 13, 10 and 30 for six hours, and then AVERY REPORTING SERVICE, INC. (303) 825-6119 134 1 they were sampled or tested at 18 hours after 2 exposure. 3 Q. 18 hours after exposure ceased? 4 A. Yes. 5 Q. What levels does this study report that 6 there was significant finding, and what findings were 7 significant at those levels? 8 A. It reports significant findings at 10 9 parts per million for sister chromatidic exchanges 10 and micronuclei in the rats--wait a minute, excuse 11 me--for the mice and at one part per million in the 12 rat experiment. 13 Q. Is that again for sister chromatidic 14 exchange in rats? 15 A. Yes, that's what it reports. 16 Q. Is this any of your work? 17 A. No. 18 Q. This was done at CIIT, but by someone 19 else besides you or your group? 20 A. That's correct. 21 Q. Can you fit in for me the results of 22 this study into your previous statements about not 23 finding convincing any data that shows effects below 24 50 parts per million? 25 A. What we are talking about, when I talk AVERY REPORTING SERVICE, INC. (303) 825-6119 135 1 about 50 parts per million I'm talking about 2 demonstrable effects to hematopoiesis that can be 3 characterized as being significant with respect to 4 bone marrow damage and/or hematopoiesis. Sister 5 chromatidic exchange is a very sensitive technique 6 for looking at potential effects of a compound on 7 either DNA replication or on occasion it correlates 8 with damage, although it is an error-free process and 9 therefore is not necessarily indicative of damage. 10 It's often misrepresented as that, but it is not 11 really an indicator of cytogenetic damage. 12 Q. Tell us in laymen's terms as best you 13 can what sister chromatid exchange means in the 14 context of doing these evaluations, and then if you 15 can relate that to what it means to a cell. 16 A. The last one is easy. It's not really 17 clear that it means anything necessarily with respect 18 to damage, it's an error-free process, or ostensibly 19 an error-free process. It involves the exchange of 20 sister chromatids on the chromosome, and it occurs 21 naturally, spontaneously. So what you are looking at 22 here are differences in rates of what is naturally 23 occurring or spontaneous process. 24 Q. This process that's being changed rate 25 wise is a change in the chromosomes of the cell? AVERY REPORTING SERVICE, INC. (303) 825-6119 136 1 A. It is a change in--yes. 2 Q. And tell us again in laymen's terms, if 3 you can, what you mean by chromosome? 4 A. You have two--you basically are 5 exchanging parts of the chromosome that are the 6 same. You have two and you are basically exchanging 7 parts of them. 8 Q. I don't know that that communicates 9 much to me. Can you do it again in more detail to 10 tell me what sister chromatidic exchange means a 11 little more elaborately but simple terms? 12 A. Chromosomes consist of chromatids which 13 are basically matching complimentary sets of the 14 information. When the cell divides, on occasion you 15 get information or material from one chromosome, one 16 chromatid exchanged with a piece of another, and if 17 you have appropriate staining techniques that allow 18 you to look at multiple generations of dividing cells 19 you can measure that. 20 Q. Now in the context of this study that 21 we are examining, which cells was the change in 22 sister chromatid exchange noted? 23 A. It was measured in vitro; that is, in 24 culture, in lymphocytes taken from animals that were 25 previously either controls or exposed to benzene. AVERY REPORTING SERVICE, INC. (303) 825-6119 137 1 Q. And which cells were being cultured? 2 A. Lymphocytes. 3 Q. And where in the body of these animals 4 would the lymphocytes be found? 5 A. Peripheral blood or spleen, I forget. 6 Cardiac blood. 7 Q. Out of the heart? 8 A. Uh-huh. 9 Q. Would this be blood that's being 10 circulated, then? 11 A. Yes. 12 Q. The heart is a nice reservoir in the 13 animal to-pull the blood from? 14 A. In the mouse it is one of the few you 15 have got. 16 Q. And you say that these cells were then 17 cultured. By that I guess you mean grown or allowed 18 to reproduce? 19 A. They are stimulated with a highly toxic 20 material called lipopolysaccharide or concanavalin, 21 either one which stimulates the cells to divide. 22 Q. And this effect of sister chromatid 23 exchange that you are looking for would be one 24 produced by the benzene exposure? 25 A. That's ostensibly the case. AVERY REPORTING SERVICE, INC. (303) 825-6119 138 1 Q. And the conclusion that it was the 2 benzene comes from comparing the exposed animals and 3 what you find in them to the unexposed animals and 4 what you found in the unexposed group; is that right? 5 A. That's the process. 6 Q. Is this a real finding in your view? 7 A. I think it is highly questionable. 8 Q. Is there anything in the published 9 report that says that this is a highly questionable 10 finding? 11 A. I don't know whether it says that or 12 not. They do qualify the nature of the dose 13 concentration response curves, especially at the 14 lower end. 15 Q. And where do you see that indication? 16 A. On page 38. 17 Q. Would you read the part that you think 18 says that? 19 A. It should be noted that the shape of 20 the concentration response curves for both rats and 21 mice from both end points are nonlinear with the 22 greatest slopes at the lower concentrations. 23 Q. Is there any other indication that you 24 can see in the paper that would say that this is a 25 highly questionable result? AVERY REPORTING SERVICE, INC. (303) 825-6119 139 1 A. One of the reasons why I think you have 2 got the frequency distribution of the raw data which 3 is presented which is not usually done is that you 4 don't have a normal distribution of the effects 5 that--of the results that go into arriving at their 6 conclusion with respect to the dose response. The 7 positive findings are restricted to a very few number 8 of cells, and a great many cells show no findings 9 outside the norm. That is certainly from my 10 perspective a major question with respect to the 11 validity of the results, especially at lower 12 concentrations. It is not a normally distributed 13 population. That suggests potential sources of error 14 or bias may be involved in influencing that type of 15 result. 16 Q. Are these sister chromatid exchange 17 type studies that are reported here so they fall in 18 the category of chromosomal aberrations? 19 A. I think it depends on your semantics. 20 It's a non--when we talk about chromosomal 21 aberrations--first of all, chromosomal means whole 22 chromosome, not chromatid. Second of all, we are 23 talking about--generally when we are talking about 24 cytogenetic analysis we are talking about permanent 25 error, we are talking about error in the DNA message AVERY REPORTING SERVICE, INC. (303) 825-6119 140 1 that is a result of a break, a deletion, a gap, a 2 translocation. 3 In the case of sister chromatid 4 exchange, you have none of those. It is ostensibly 5 error free. So its significance with respect to 6 cytogenetic damage or an aberration is questionable 7 from the physiological standpoint. Where you have a 8 clear-cut demonstration of an increase in sister 9 chromatid exchange it suggests some response to 10 whatever the treatment paradigm was, or whatever the 11 stress was, whether it is compound related or 12 handling related or what have you, it does not 13 automatically connote an error in the DNA message. 14 Q. So the furthest you would be willing to 15 go is to say that exposures at one part per million 16 produced a measurable change? 17 A. As defined by this study. 18 Q. You, of course, are familiar with 19 studies of the peripheral blood of humans exposed to 20 benzene and the findings of changes in chromosomal 21 information at exposures below 10 parts per million, 22 are you not? 23 A. If you show them to me I will be happy 24 to comment on them. 25 Q. With that description you are not able AVERY REPORTING SERVICE, INC. (303) 825-6119 141 1 to2 A. No. 3 Q. What would be the mechanism for these 4 test animals that are reported here to have breathed 5 benzene that would end up showing an effect that's 6 manifested by the sister chromatid exchange test? 7 A. I don't know of a mechanism that would 8 account for sister chromatid exchange. I know of no 9 mechanism for that. I don't know why it happens 10 spontaneously. I don't know why it increases 11 following stress or exposure to various agents. Some 12 agents that can produce it correlate with their 13 immunogens or clastogens, others aren't. 14 Q. What kind of work are you doing now in 15 the way of research? 16 A. Doing studies to characterize stem cell 17 differentiation in the hematopoiesis in defined 18 culture systems. 19 Q. Which systems? 20 A. We are developing them. By defined I 21 mean we are not using--we are using defined growth 22 factors in very precise conditions for examination of 23 the influence of those growth factors on 24 hematopoiesis. 25 Q. Who is we? AVERY REPORTING SERVICE, INC. (303) 825-6119 142 1 A. My laboratory. 2 Q. At the University of Colorado; is that 3 where it is? 4 A. Yes. 5 Q. Which department are you in there? 6 A. I'm in the Molecular Toxicology and 7 Environmental Health Sciences program. I have 8 appointments in the school pharmacy and in the 9 department of pathology in the medical school. 10 Q. Has this group at the University of 11 Colorado done similar-type research in the past? 12 A. It's basically my group, and I have 13 done similar research in the past. And certainly 14 those that are collaborating with me on various 15 aspects of metabolism and/or biochemistry have done 16 similar studies. 17 Q. That wasn't what I was trying to ask.. 18 Prior to you coming to the University of Colorado and 19 joining this department, had any similar-type work 20 been under way? 21 A. Yes. 22 Q. Who was doing it then? 23 A. Dr. William Robinson in hematology and 24 medical oncology. 25 Q. What kind of work was he doing that was AVERY REPORTING SERVICE, INC. (303) 825-6119 143 1 similar to what you are now doing? 2 A. Looking at stem cell regulation 3 primarily associated with leukemias and dysplasias. 4 Q. And which group is he? 5 A. Oncology. 6 Q. What is the funding source for your 7 present work? 8 A. I have several different funding 9 sources. The American Petroleum Institute I have a 10 grant from. I have a grant from the Chemical 11 Manufacturers Association. I have funds provided to 12 me by the University of Colorado. And I have grants 13 pending at numerous agencies. 14 Q. Right now there are three sources of 15 funds? 16 A. Yes. 17 Q. And how long have you been at CU? 18 A. One and a half years. 19 Q. Have those been the only three sources 20 of funds you have had in the past one and a half 21 years for your work? 22 A. Yes. 23 Q. Did you do an RFP to the API and CMA 24 for your funds? 25 A. RFP? AVERY REPORTING SERVICE, INC. (303) 825-6119 144 1 Q. Did you make a proposal for the work 2 that you anticipated doing? 3 A. Yes. Yes. 4 Q. You used to be at CIIT? 5 A. Yes. 6 Q. May I ask why you left? 7 A. I had an opportunity to develop a 8 toxicology and environmental health program in a 9 state that sorely needs one and the independence to 10 do so, and I wanted to take a crack at developing an 11 educational program in toxicology before my career 12 was completed, and this was an opportunity to do 13 that. 14 Q. Were you restricted at what you could 15 do at CIIT? 16 A. In the sense that CIIT is a research 17 institute and my ties with academia were at the level 18 of adjunct participation at surrounding colleges, 19 yes, I certainly couldn't direct a training program 20 or be involved in education, higher education. This 21 has given me the opportunity to have the best of both 22 worlds. 23 Q. CIIT kept you from being involved in 24 higher education? 25 A. No, I lectured a lot, but I certainly AVERY REPORTING SERVICE, INC. (303) 825-6119 145 1 couldn't take on the primary responsibilities of 2 directing an educational program. 3 Q. In other words, you couldn't have two 4 full-time jobs? 5 A. That's right. 6 Q. May I ask if your income went up or 7 went down? 8 A. It went up. 9 Q. When you left CIIT? 10 A. Yes, sir. 11 Q. There has been discussion about your 12 being paid to be here today. Where does the money 13 that you receive go? Does it go to the University of 14 Colorado or to you? 15 A. It goes to me. I have a contractual 16 arrangement with the university that allows for 15 17 percent of my time to be spent consulting, and in 18 fact I spend about 10 percent of my time doing that. 19 It is both a requirement as far as the administration 20 is concerned and pay. It is something they allow me 21 to do. If I didn't do it I would probably hear about 22 it as well. 23 Q. Is there direction given to the kind of 24 consulting you must spend your 15 percent for? 25 A. No. AVERY REPORTING SERVICE, INC. (303) 825-6119 146 1 Q. The funds that you receive consulting, 2 does a portion of those go to the university? 3 A. No. 4 Q. When you were at CIIT and you acted as 5 an expert witness in cases, did the moneys you 6 received then go to CIIT or to you? 7 A. First of all, to my knowledge, and I'm 8 not an attorney, I never acted as an expert witness 9 while I was at CIIT. I gave one deposition at CIIT 10 while I was there. I received no compensation for it 11 in any way, shape, or form, and neither did the 12 institute. 13 Q. What was that deposition about? 14 A. It was in Skeen two. 15 Q. You testified in that? 16 A. I ultimately testified in Skeen two, 17 yes. 18 Q. Were you away from CIIT when you 19 testified? 20 A. Yes. 21 Q. You were paid directly for your 22 services then? 23 A. That's correct. 24 Q. Was one of your activities at CIIT to 25 slow down the development of OSHA regulations AVERY REPORTING SERVICE, INC. (303) 825-6119 147 1 regarding chemicals? 2 A. That's nonsense. That's utter 3 nonsense. 4 Q. Was that a purpose of work at the 5 institute? 6 A. No. 7 Q. Well, I need to show you something. Do 8 you know of a Mr. F.B. Thomas, Dr. Thomas at Shell? 9 A. Yes, I do. He is no longer at Shell, I 10 believe, but, yes, I do know him. 11 Q. Were you at CIIT in 1987? 12 A. Yes. 13 Q. Did you attend a joint meeting of the 14 IISRP Industrial Hygiene Committee, and the IISRP 15 Butadiene Steering Committee in Edinburgh, Scotland, 16 in 1987? 17 A. I gave a talk at that meeting. I 18 didn't attend other than to present a talk. 19 Q. Let me show you a letter which purports 20 to be from Dr. Thomas, and attached to it is a 21 summary of that meeting. Let me direct you to the 22 last page, although feel free to read the whole thing 23 if you like. Frankly I'm not convinced that all of 24 it is accurate, but I want to ask you about the last 25 item. AVERY REPORTING SERVICE, INC. (303) 825-6119 148 1 A. What's the question? 2 Q. Would you read the last item out loud 3 so we can put that in the record. 4 A. Irons believes the data from CIIT to 5 date have without doubt slowed OSHA regulatory 6 procedures down and have also set some limits on 7 product tort liability. 8 Q. Did you say that? 9 A. No. 10 Q. So that's just incorrect? 11 A. I suspect that what he was doing is 12 extrapolating from my comments on the mouse model in 13 arriving at those conclusions. I have been in 14 contact with OSHA people during my studies with 15 butadiene and have discussed the mouse model 16 continually. My scientific opinion is the mouse 17 model has a number of very complicated and species 18 specific findings that may or may not be of relevance 19 to man. From that standpoint I don't think the 20 mouse--original mouse data should be assumed to be 21 necessarily relevant to man. 22 I can see where someone in industry 23 might draw this conclusion from those comments, but 24 it is certainly not an object or a motivation for my 25 work. My object for studying butadiene is to AVERY REPORTING SERVICE, INC. (303) 825-6119 149 1 determine what it does. 2 Q. Would you say that the institute work 3 accomplished this, if not your work, then? 4 A. No. No. If our work slowed down 5 regulatory procedures, it's in the context of 6 evaluating the data they had, they elected to review 7 it further. That would be the only way in which it 8 could possibly have any effect. 9 Q. I think the top paragraph there on the 10 same page, doesn't that deal with contacts with OSHA 11 and the mouse model? 12 A. Yes. 13 Q. What was the answer to that question? 14 You might read it into the record so later we'll have 15 it all together. 16 A. Irons noted that he has been asked by 17 OSHA scientists about whether a chemical viral 18 interaction can be modeled by a multi-hit mechanism, 19 suggesting there is some concern at OSHA about 20 defending their mouse lymphoma risk assessment. It 21 should be recognized, however, that dismissing the 22 thymic lymphoma still leaves all of the other tumors 23 seen in the NTP bioassay. 24 Q. Do you recall that discussion with 25 OSHA? AVERY REPORTING SERVICE, INC. (303) 825-6119 150 1 A. Yes. I have discussed with them 2 whether or not it would be appropriate to 3 characterize chemical viral interactions in various 4 models. 5 Q. And I would like to know your response 6 to that, whether you can or not. 7 A. We are talking very complicated here. 8 And at the present time I think we are either talking 9 about concomitant models, interactive models, the 10 virus may act as a cocarcinogen, it may act 11 independently, there is just no way to reliably model 12 it in that context given our knowledge base at that 13 time. 14 Q. Do you have an opinion as to whether or 15 not butadiene should be regulated as a carcinogen? 16 MR. ZATZ: Objection to the form. 17 A. Butadiene is a potent mouse 18 carcinogen. It certainly deserves regulation as one 19 would deal with any other potent mouse carcinogen or 20 compound found to cause cancer in experimental 21 animals, no doubt about that. 22 Q. (BY MR. HOBSON) Are you aware that the 23 American Conference of Governmental Industrial 24 Hygienists has recommended lowering the TLB for 25 benzene? AVERY REPORTING SERVICE, INC. (303) 825-6119 151 1 A. For benzene or Betadiene? 2 Q. Benzene. 3 A. I have heard that. I have not seen it 4 directly, but I have heard that. 5 Q. It's in the latest Archives of 6 Industrial Hygiene, the notice of intended 7 changes--the notice may not be there, but the 8 documentation is there, and they recommend, as I 9 recall, going to a tenth of a part per million times 10 average TLB. Would you have a position one way or 11 the other on that? 12 A. As a policy? 13 Q. Yes, sir. 14 A. No. 15 Q. You have no opinion? 16 A. I don't think it relates to causation. 17 It's a policy issue. It doesn't impact one way or 18 the other on causation. 19 Q. What would be your view, then, about 20 regulatory activities and causation? 21 A. I think they are two separate 22 exercises. They have totally different criteria and 23 totally different--they are functionally totally 24 separate. I don't think that a regulatory standard 25 says anything about causation. AVERY REPORTING SERVICE, INC. (303) 825-6119 152 1 Q. Why wouldn't it say anything about 2 causation? 3 A. Because there are paradigms that are 4 used de facto in establishing regulatory standards 5 based upon risk assessments for which there is no 6 evidence to support or refute the assumptions made in 7 the risk assessments. The one thing you can say 8 about a regulatory standard is the standard itself is 9 probably--you can be virtually certain within 10 reasonable scientific certainty that the exposure 11 level that's set is far below that that's going to be 12 related to causation just by nature of the 13 assumptions that go into the risk assessments. 14 Q. Have you read Mancuso's second edition 15 of his epidemiology book Occupational Epidemiology? 16 A. No, I haven't. 17 Q. Would you have any opinion as to 18 whether regulated exposure levels should have a 19 certain margin of safety built into them? 20 MR. ZATZ: I'm going to object on two 21 grounds. Number one, I think you are asking him 22 opinions about policy matters as opposed to 23 toxicology matters, and we have not proffered him as 24 an expert on what American regulatory policy ought to 25 be; and secondly, based on the use of the word should AVERY REPORTING SERVICE, INC. (303) 825-6119 153 1 in that question. You can answer. 2 A. Given the lack of scientific basis for 3 many of the assumptions that go into setting 4 standards, I think that margins of safety are 5 certainly called for. 6 Q. (BY MR. HOBSON) I think earlier today 7 you told me that in your work that you have done you 8 have seen replication errors, chromosomal damage in 9 laboratory animals at 50 parts per million? 10 A. I have seen damage to bone marrow and 11 to hematocritic organs at those concentrations. 12 Certainly mitotic abnormalities at 100 parts per 13 million and probably at 50. I can't remember 14 precisely. I suspect I have probably seen them at 15 50. 16 Q. Given the difficulties of this animal 17 model and extrapolation to man, do you feel 18 comfortable, then, that the same exposure levels for 19 similar-type effects are appropriate for man? 20 A. I think that exposure--first of all, 21 the types of changes we are talking about, which 22 involve bone marrow damage, are related indirectly to 23 the metabolism of benzene, which is a requirement. 24 And although I can't cite you the specific kinetic 25 studies that I have been involved with or have been AVERY REPORTING SERVICE, INC. (303) 825-6119 154 1 done, because I can name the studies actually but I 2 can't remember the data right now, it's been years 3 since I looked at it, my conclusion would be the 4 mouse is a much more sensitive model with respect to 5 bioactivation and short-term or subacute toxicity 6 than the rat or man is likely to be. So I would say 7 the mouse is likely to be a more susceptible model 8 for subacute damage bone marrow toxicity than either 9 the rat or man. 10 Q. What about for chronic? 11 A. I don't think that you get chronic 12 changes in the absence of subacute changes. 13 Q. That's where I wanted to go with you 14 next. Earlier today you said that the acute 15 myelogenous leukemia in man must have certain 16 precursors before you believe it would be related to 17 benzene exposure? 18 A. In secondary leukemias, yes. 19 Q. Explain to me what you mean by that in 20 secondary leukemias. 21 A. I think, first of all, the evidence, 22 the epidemiologic evidence provides very strong 23 rationale as a basis for that conclusion. 24 Q. Which conclusion? 25 A. That in fact one has to have AVERY REPORTING SERVICE, INC. (303) 825-6119 155 1 significant alterations in the stem cell compartment 2 related to bone marrow toxicity before you incur the 3 risk of developing AML secondary to exposure. I 4 think it requires a physiologic response on the 5 organism that alters stem cell regulation, and I 6 presented today in the context of other questions 7 several lines of evidence to support that. 8 Q. That raises two questions I would like 9 to ask you. Number one is, can benzene produce 10 leukemia in man as a primary result? You said in 11 your answer to my question a moment ago leukemia was 12 a secondary leukemia. 13 A. It can't be primary if you are exposed 14 to benzene. By definition it is a secondary. 15 Secondary means not spontaneous. It is secondary to 16 some exposure, be it a drug, be it benzene, be it 17 radiation, what have you. 18 Q. I didn't understand how you were using 19 secondary there. 20 A. That's what I mean by secondary. 21 Q. Secondly, what are the markers for 22 effects on the stem cell regulation that you would 23 put in the category of a precursor to the development 24 of AML? 25 A. Well, if I had an absolute marker that AVERY REPORTING SERVICE, INC. (303) 825-6119 156 1 I could point to in stem cell population and say this 2 meets to AML, this would be a very prognostic tool. 3 We don't have anything like that at the current 4 time. One of the things that I would want to see 5 first, and in fact as part and parcel of what I'm 6 doing now, is alterations in the way stem cells are 7 regulated that would in fact be consonant with the 8 physiologic response to the damage caused by an agent 9 such as benzene. 10 Q. Let me ask the question this way: You 11 have got an AML, you have got a worker exposed to 12 benzene, list for me the factors that you would want 13 to see before you would say that the AML was caused 14 by the benzene exposure. 15 A. I would want to see accurate exposure 16 data that demonstrates exposures that are significant 17 in terms of potential bone marrow toxicity. If you 18 have exposures at 100 parts per million or greater 19 repeatedly, I'm going to be satisfied with the 20 exposure scenario. Between 50 and 100 I'm going to 21 be concerned about it. If it's below 50 you get into 22 real foggy territory with respect to evidence, and 23 somewhere lower--somewhere between 25 and lower there 24 is just no evidence to support it. 25 I would want to have some indication of AVERY REPORTING SERVICE, INC. (303) 825-6119 157 1 latency. If the disease develops I'm going to be 2 much more happy or confident about the latency issue 3 if we are dealing in the area around ten years from 4 initial exposure, it's going to be in a typical 5 Gawssian fashion becomes less and less clear as you 6 get closer to initial exposure, and at about three 7 years I think it is impossible to draw a conclusion 8 as to whether or not the exposure could be related. 9 That's also way off the end of the Gawssian curve. I 10 believe Rinsky in his study used 5, and this is 11 certainly following up of five years. 12 The type of leukemia, if it is an AML, 13 to me that's important obviously. if in fact the 14 cytogenetic abnormalities that are associated with 15 what classically have been referred to the C-type 16 chromosomes that I have referred to initially, 5 Q 17 minus 7 Q minus 5 or 7, missing that is consistent 18 with secondary leukemias, it is inconsistent with 19 spontaneous leukemias, that would be a factor that 20 would weigh heavily in terms of me reaching a 21 conclusion. 22 Q. Would you only find those abnormalities 23 in AMLs? 24 A. 5 Q minus and 7, to my knowledge they 25 are heavily weighted towards myelodysplastic AVERY REPORTING SERVICE, INC. (303)825-6119 158 1 syndromes and AMLs. It is because of the growth 2 factors on those chromosomes are associated with 3 regulation, specifically the myeloid lineages. Sub 4 type can be useful empirically for reasons which 5 nobody knows. 6 If it is an erythroid leukemic variant 7 that heavily predisposes towards benzene if in fact 8 you have a reliable exposure history. I don't mean a 9 simple erythroid hypoplasia in the presence of a 10 myeloblastic AML, I'm talking about a clear-cut 11 erythro leukemic. 12 But I would not exclude virtually any 13 type of AML as being possible, with one exception, 14 and I wouldn't rule it out entirely, but there is 15 certainly no evidence of monoblastic type. 16 Q. Anything else? The reason I ask that 17 is for some reason in my mind I got the feeling 18 earlier you were telling me that you would not 19 attribute benzene as a cause of AML unless the person 20 had aplastic anemia. 21 A. You are talking about the presentation 22 of the patient at the time of diagnosis. If you had 23 had the ability to clinically monitor a patient from 24 the beginning of exposure to the onset of leukemia, 25 it is my opinion you would very likely and almost AVERY REPORTING SERVICE, INC. (303) 825-6119 159 1 always, if not always, see some evidence of bone 2 marrow suppression. We are not talking exposure to 3 trace amounts, we are talking about real life 4 response to bone marrow suppression incurred as a 5 function of exposure to benzene concentrations that 6 will do that. That's consistent with the literature 7 on chemotherapeutic agents, and I think it is 8 consistent with certainly the case histories that 9 were presented in the early benzene literature. 10 Q. Would the person with AML know they 11 were sick from what you would consider a significant 12 enough bone marrow suppression to be a link to 13 benzene exposure in the past? 14 A. I would say there is a fairly high 15 probability that there would be some evidence of bone 16 marrow suppression, but it is not absolute--I don't 17 think it is absolutely a requirement because the 18 effects on stem cell, if in fact you had a clinically 19 nonsignificant effect that resulted in an altered 20 physiologic regulation of stem cell activity, you 21 could then continue to have prolonged--you could have 22 a lesion at the level of stem cell differentiation 23 that would persist, and it is conceivable that an 24 individual might not seek treatment for the initial 25 episode. I think it is conceivable. AVERY REPORTING SERVICE, INC. (303) 825-6119 160 1 I don't think that if you did bone 2 marrow taps and looked at stem cell regulation using 3 even existing assay procedures that are relatively 4 crude that you would have any trouble seeing them 5 because certainly the chemotherapeutic literature 6 suggests that to be the case. But that's not normal 7 routine diagnostic procedure. 8 I will not tell you that you absolutely 9 have to have a clinically significant presentation. 10 I'm not sure. That may be the case, but I'm not 11 prepared to say that as an absolute. I do think if 12 you look for it with all the means at our disposal 13 you would see. 14 Q. And the patient might or might not know 15 that they have some outward symptoms? 16 A. For some proportion of patients I 17 suspect that's the case. Certainly the prevailing 18 opinion in the medical community is the majority of 19 these patients are surely going to present. 20 Q. When you talk about the bone marrow 21 suppression that you would need to see for an AML, 22 are you talking about suppression of all the 23 different cells or a particular line? 24 A. It is highly variable, highly 25 variable. It is even variable in animals, but it is AVERY REPORTING SERVICE, INC. (303) 825-6119 161 1 more variable in man. 2 Q. It could be the pancytopenia, 3 thrombocytopenia? 4 A. Yes. 5 Q. Would it have to be long lasting? 6 A. This is rank speculation. I certainly 7 would want to see long-standing changes at the level 8 of the stem cell, I expect to see them. I suspect 9 that the clinical presentation could be relatively 10 short. 11 Q. What about if you have a person who has 12 had a history of exposure to benzene at a level 13 several hundred parts per million for some time 14 period and a person who presents then with AML, same 15 person presents with AML some years later? 16 A. Several hundred parts per million? 17 Q. Yes. An exposure that you would accept 18 of being capable of causing the disease. 19 A. Okay. 20 Q. And the person presents with AML some 21 years later after the exposure has ceased, say ten 22 years again to fit into your exposure profile, and at 23 some point in between presented at a physician's 24 office for, say, thrombocytopenia which was not long 25 lasting, or pancytopenia which was not long lasting; AVERY REPORTING SERVICE, INC. (303) 825-6119 162 1 in other words, one of these markers occurred and 2 then disappeared, does that tell you anything about 3 whether or not benzene played a part in the causing 4 of the AML? 5 A. If you show me an individual who has 6 had documented exposure to several hundred parts per 7 million for any significant period of time that meets 8 criteria that I'm comfortable with and develops AML, 9 as far as I'm concerned there is no reason to assume 10 that it was not due to that exposure. 11 I would say you would have some 12 qualifying concerns if it was totally--if it was a 13 cytogenetic presentation that was typical or 14 spontaneous, but nevertheless you have got 15 predisposing evidence to suggest that there could be 16 a relationship there. I would fully expect to see 17 some presentation. You can't be exposed to several 18 hundred parts per million benzene and not develop 19 some hematologic abnormalities. I don't believe it 20 is possible at those levels. 21 Q. I forgot to ask you about another 22 disease earlier. 23 A. Can we do this very quickly? 24 Q. Hairy cell leukemia. How does hairy 25 cell leukemia fit into these different diseases? AVERY REPORTING SERVICE, INC. (303) 825-6119 163 1 A. It is a totally different disease 2 entity existing ostensibly at the level of in a 3 quasi-world with the monocyte and the macrophages and 4 the so-called reticulum cell. It doesn't fit into 5 this paradigm. 6 Q. Are you familiar with any 7 epidemiological literature that links Hairy cell 8 leukemia and exposure to benzene? 9 A. No. I'm familiar with literature that 10 links it to HVL2, the human T-cell varium. 11 Q. You have to go? 12 A. I have to go. 13 Q. Before we go I would like to get the 14 court reporter to mark as Exhibit 1 your chart that 15 you drew, that's this legal sheet. This is the chart 16 that you drew which we are marking as Exhibit 1; is 17 that right, Dr. Irons? 18 A. Yes. 19 Q. I would like to mark as Exhibit 2 the 20 paper that's from the CIIT group that we discussed, 21 Induction of Cytogenetic Damage in Rodents After 22 Short-Term Inhalation of Benzene. That's the paper 23 we discussed earlier, right? 24 A. Yes. 25 Q. Then as Exhibit 3 I would like to mark AVERY REPORTING SERVICE, INC. (303) 825-6119 164 1 the Shell document that I questioned you about. And 2 Exhibit 3 is the Shell paper I questioned you about, 3 correct? 4 A. Yes. 5 Q. Anything else that's handwritten, or 6 just the articles? 7 A. One handwritten note. 8 Q. Could I get those two notes and mark 9 those. Anything else that's handwritten that you 10 have? 11 A. That's it. 12 Q. And then these are just articles? 13 A. Yes. 14 Q. Let me just ask if you would leave this 15 with the court reporter and let her copy this and 16 then return the original to you, would that be all 17 right? 18 MR. ZATZ: Is that all right? 19 Q. (BY MR. HOBSON) As long as you agree 20 to maintain the originals? 21 A. Okay. 22 Q. We'll mark these as the next attachment 23 as a group exhibit. And you brought Jandl's book 24 with you? 25 A. Yes. AVERY REPORTING SERVICE, INC. (303) 825-6119 165 1 Q. Blood Toxicology Textbook of 2 Hematology. And which edition is this? 3 A. The only one. 4 Q. Let's see, '87, copyright date, 5 correct? 6 A. Yes. 7 Q. Bring anything else about the case with 8 you? 9 A. No. 10 (Whereupon, documents were marked Irons 11 Deposition Exhibits 1 through 4 for identification by 12 the reporter.) 13 (Whereupon, at 4:05 p.m., the 14 deposition was concluded.) 15 16 17 18 19 20 21 22 23 24 25 AVERY REPORTING SERVICE, INC. (303) 825-6119 166 1 I, RICHARD D. IRONS, Ph.D., do hereby 2 state under oath that I have read the above and 3 foregoing deposition, and that the above transcript 4 and accompanying correction sheets, if any, 5 constitute a true and correct record of my testimony. 6 7 8 RICHARD D. IRONS, Ph.-D. 9 STATE OF COLORADO ss. 10 CITY & COUNTY OF DENVER 11 Subscribed and sworn to before me by 12 the said RICHARD D. IRONS, Ph.-D,, this- day of 13 1990. 14 My commission expires_ 15 16 17 18 19 20 21 22 23 24 25 AVERY REPORTING SERVICE, INC. (303) 825-6119 167 1 CERTIFICATE 2 STATE OF COLORADO ss. 3 CITY & COUNTY OF DENVER 4 I, BARBARA WISHART, a Certified Shorthand Reporter, a Registered Professional 5 Reporter, and a Notary Public within and for the City and County of Denver, State of Colorado, do hereby 6 certify that previous to the commencement of the examination of the said RICHARD D. IRONS, M.D., a 7 witness called for examination by the plaintiffs herein in the said suit in the said District Court, 8 was duly sworn by me to testify the truth in relation to the matters in controversy now pending and 9 undetermined between the said parties so far as he should be interrogated concerning the same; 10 That the said deposition was taken in 11 shorthand by me at 1290 Broadway, Suite 700, City and County of Denver, State of Colorado, on the 15th day 12 of August, 1990, at 10:21 a.m., and was reduced to typewritten form under my supervision; 13 That the foregoing is a true transcript 14 of the questions asked, the testimony given, and the proceedings had; 15 That I am neither attorney nor counsel, 16 nor in any way connected with any attorney or counsel for any of the parties to said action, or otherwise 17 interested in its event. 18 IN WITNESS WHEREOF, I have hereunto set my hand and affixed my notarial seal this 28th day of 19 August, 1990. My commission expires November 26, 1990. 20 21 22 23 24 25 AVERY REPORTING SERVICE, INC. (303) 825-6119