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1 IN THE CIRCUIT COURT TWENTIETH JUDICIAL CIRCUIT OF ILLINOIS 2 ST. CLAIR COUNTY 3 FRANCES E. KEMNER, et al. 4 Plaintiffs 5 vs. 6 MONSANTO COMPANY, 7 Defendant. ) ) ) ) NO. 80-L-970 ) ) ) ) 8 Before the HON. RICHARD P. GOLDENHERSH, Judge 9 10 11 12 TESTIMONY OF DR. FRANK DOST 13 November 5, 1985 14 15 16 17 APPEARANCES: 18 MR. REX CARR, Attorney at Law, and MR. JEROME SEIGFREID, Attorney at Law 19 Appeared on Behalf of the Plaintiffs 20 MR. KENNETH HEINEMAN, Attorney at Law, and MR. JOSEPH NASSIF, Attorney at Law 21 Appeared on Behalf of the Defendant 22 PATRICIA GANDY, CSR, RPR 23 Official Court Reporter 24 i 1 INDEX 2 DR. FRANK DOST 3 Direct Examination by Mr. Heineman 4 5 EXHIBITS 6 MARKED OFFERED 7 Defendant's 1281 . . . . . . 2 5 8 Defendant's 1282 . . . . . . 27 31 9 Defendant's 1283 . . . . . . 32 82 10 Defendant's 1284 . . . . . . 33 82 11 Defendant's 1285 . . . . . . 70 82 12 Defendant's 1286-89 . . . . 81 82 13 Defendant's 1290 . . . . . . 93 103 14 Defendant1s 1291 . . . . . . 104 15 Defendant's 1292-95 . . . . 107 16 17 18 19 20 21 22 23 24 2 ADMITTED 5 31 83 83 83 83 104 2 1 BE IT REMEMBERED AMD CERTIFIED that heretofore, on 2 to-wit: Tuesday, November 5, 1985, being one of the regular 3 judicial days of this Court, the matter as hereinbefore set 4 forth came on for hearing before the HON. RICHARD P. GOLDEN5 HERSH, Circuit Judge In and for the Twentieth Judicial Circuit, 6 State of Illinois, St. Clair County Building, Belleville, St. 7 Clair County, Illinois, and the following was had of record, 8 to-wit: 9 10 THE COURT: Good morning. 11 CONTINUED DIRECT EXAMINATION 12 BY MR. HEINEMAN: 13 Q Dr. Dost, I'd like to take you briefly back for a 14 minute to the discussions we had yesterday on porphyria and 15 the no observable effect level, the NOEL. Have you caused 16 a chart to be prepared which would assist you in explaining 17 this situation? 18 A Yes. 19 (At this time Defendant's Exhibit 1281 was marked 20 for identification.) 21 Q All right, sir, I'll show you what's been marked 22 as Defendant's Exhibit 1281 and ask you to examine that and 23 Identify it for me, please. 24 A This is a chart prepared from tabular data that was 3 published by Smith, et al., in the journal Biochemical Pharma* 1 2 cology, Line 30 in 1981, and this is a description of the 3 effect of an acute or of acute doses, single doses of TCDD as distinguished from the experiments that you have- mentioned, . 4 5 I believe, yesterday in which lower doses were given on a 6 weekly basis over an extended period of time. 7 Q Now, would you be so kind as to step down here and 8 explain that to the jury please? 9 A This is another dose response curve. It looks a 10 little different from the ones that we talked about the other 11 day.7 You recall'that in the ones that w e 've seen earlier 12 there is kind of an S shaped arrangement. This is really 13 the same, the only difference that the, the change is very, 14 very-rapid. The slope here is extremely steep, and what it shows 15 isthats '.at a dose, and this differentiates between/males 16 and females because there is some difference In the response of males and females to most of the effects of TCDD; It isn't M 18 much>v but it's measurable. And again this is a logarithmic ( ' ).V , - 19 stale and this would be a dose, I believe, of about 6 micro 20 grams per kilogram. And you can see that the diamond and the 21 circle tare superimposed here, they both have got the/same 22 doses, this I think would be 12 mlcrograms per kilogram and 23 this would be 16. And what it shows here is that ttis 24 rather substantial single doses, now this is just a, single 4 1 dose, not a chronic exposure but a single dose, that it requires 2 a very substantial single dose before this kind of response 3 merges. The measurement that is being made is the appearance 4 of porphyrin actually depositing in the liver. You recall 5 yesterday I mentioned that they can appear in the urine and/or 6 in the liver, and here we are talking about hepatic porphyrin, 7 that is these intermediate product in the synthesis of heme 8 accumulating in liver cells and in the response the animals 9 manage a degree of impairment in heme synthesis very, very 10 well until they get up to some substantial dosage, and then, 11 the accumulations in the tissues becomes very, very rapid be 12 cause the -- presumably because the impact at the enzyme 13 level becomes so substantial that the material is not being 14 disposed of very readily. 15 Q Now, Dr. Dost, yesterday you spoke about the chronic 16 level or the no effect level for chronic dosage being at what 17 level? 18 A Well, it is at a level down on the order of a tenth 19 of a microgram per kilogram per week or less. Statistically 20 if you run, if you run the dose response curve all the way 21 down, the no effect level approaches a hundredth of a micro22 gram per kilogram per week. Statistically the no effect level 23 is on the order of a tenth of a microgram, and by that I mean 24 that it comes up a little but it is not enough that statis- 5 1 tlcian would say that it is different from the control. 2 Q And that is what period of time does that chronic 3 test cover? 4 A The two that describe this most effectively, one 5 by Goldstein, it ran for sixteen weeks. And another by 6 Cantoni for forty-five weeks, one injection a week at a 7 variety of doses. They ,ran through a dose response or dose 8 levels'of a hundred to one micrograzns per kilogram. In other 9 words there would be four doses, *01, .1, it went to 10 10 micrograms per kilogram, actually, per week. 11 Q So it would be .01, .1, 1, and 10? 12 A Correct. 13 MR. HEINEMAN: Thank you, your Honor, at this point we would move the admission of Defendant's Exhibit 1281. (Defendant's Exhibit 1281 was offered into evidence.) MR. CARR: No objection. THE COURT: Admitted without objection. Thank you. Q Dr. Dost, at the close of testimony yesterday we were talking about the cardiovascular system and the effects of dioxin on serum lipids, do you recall that, sir? A Yes. Q And what is the -- what do the studies demnstrate with respect tb the effect of TCDD on the production bf serum lipids? 6 1 A In general, at substantial doses, the fats and 2 cholesterol In the serum will rise as the dose Is decreased, 3 and this Is substantial doses experimentally. Single doses 4 at a dose of around 10 micrograms per kilogram in the rat. 5 The serum lipids, the fats will rise, cholesterol esters 6 tend to decrease-, not drastically, but they tend to drop 7 below normal levels. And then if you follow this over a 8 period of time, all of these measurements tend to return 9 toward normal in about, In twenty-one days they are approaching 10 normal. Hie slopes of the data would suggest that by thirty 11 days the!irr measurements would be In the normal ranges. 12 Q Now, are elevated lipids a clear Indicator of 13 disease? How does one interpret elevated lipids? 14 A Well, to determine whether they are an indicator 15 of disease depends on far more than elevation of lipids, 16 because we all have elevated seruia lipids after a meal, 17 depending on the nature of the meal. The Increased lipids 18 may be just typical fats, it could very well be high 19 cholesterol, and that is going to be a ..relatively transient 20 process because this is the normal thing that we do in 21 processing our dietary intake. Any time that we eat some 22 thing there is going to be reflected in the circulation 23 elevated levels of sugar, amino acids, fats, depending on 24 what is in the diet. That Is fairly straight forward. When 7 1 we start dealing with a disease state, again, increased blood 2 lipids all by themselves could not provide a diagnosis. A 3 diabetic will often have elevated blood lipids, but that 4 wouldn't diagnosis diabetes. It depends on other kinds of 5 effects. It is associated with diabetes. Smokers may have 6 elevated blood lipids, but they may not, too. But it seems 7 to be -- there seems to be some association. People with 8 hypertension often have elevated blood lipids, but here we're 9 dealing with a chicken and egg situation in which the condition 10 that led to elevated blood lipids may have caused oyer a long, 11 long period of time a change in the arteries that we would 12 call arteriosclerosis that resulted in the hypertension, so 13 what I am saying here is that a long term, a long sustained 14 elevation in blood lipids and blood cholesterol could very 15 well lead to and In fact the statement is sometimes made that 16 prolonged elevation of blood lipids causes cardiovascular 17 disease. Other people are more conservative and say that it 18 is associated with it. Whether that's a weasel word, X don't 19 know. In any case, there is at least an association with 20 cardiovascular disease, and that's why we're talking about 21 it perhaps in the contest of cardiovascular disease. But 22 there are a variety of other conditions that will cause either 23 a prolonged or a relatively foreshortened period in which blood 24 lipids are elevated. 8 Q And are the results In the animal data dose dependent? 1 2 A Yes, they are dose dependent. The difficulty is that in the case of lipid abnormalities with respect to TCDD at least, 3 the doses have not been carried downward all the way to clear 4 no effect levels. The dose response is very clear, but we 5 6 don't identify a specific no effect level below which ho abnormalities in lipid metabolism appear, tfany of the studies 7 8 use only a single dose because they are trying to understand the mechanism by which this event occurs. But it would appear 9 that the no effect level is substantial upon the order of a 10 'll microgram per kilogram. 12 Q Now, would you say it would appear that it is sub stantial? 13 14 A By that I am just making a judgment on what the dose 15 response information is. You recall that the other day I told 16 you (that if you don't have enough points on a dose response 17 curve to carry all the way to a no effect level, it is possible 18 to project within limits from what data there is where the dose 19 response, where the no effect level might be expected However, 20 one cannot simply say here is where the dose, here ie where the 21 no effect level is. I would judge on the basis of the -existing 22 data that it is on the order of a microgram per kilogram. 23 Q Now, based upon a judgment of a microgram per kilogram t '4 24 and the dose that you calculated with respect to the Sturgeon 9 1 exposure of the 10 grams of soil, would that Sturgeon dose be 2 sufficient to generate elevated blood lipids? 3 A Well, we calculated that a dose of .001 micrograms 4 per kilogram per day would represent a 222nd, if you will -5 X beg your pardon, that is 222 times higher than that exposure 6 that we postulated, and since we are talking about 1 microgram, 7 then that number would be expanded by a thousand. 8 Q So the Sturgeon fiose which you calculated would be 9 222,000 times 2 low? 10 A It would be that much less than the 1 microgram per 11 kilogram that I am postulating. 12 Q Now, I'd like to talk to you this morning about 13 neurological effects of TCDD. Now, briefly describe the 14 neurological system that we are talking about. 15 A Well, the neurological system is all of the 16 it is the central nervous system, the brain and the spinal 17 cord, and all of the peripheral nerves. So the neurological 18 system Is our system for transmitting rapid messages from 19 one part of the body and for Integrating them and acting 20 upon them in the central nervous system. It is all of the 21 nerves of the body. 22 Q Now, had there been neurological effects observed 23 in humans exposed to dioxin? 24 A To my recollection, no. I am not entirely familiar 10 1 with all of the epidemiology, but I do not recall any in 2 humans. 3 Q All right, are you familiar with the examination 4 by Dr. Suskind of workers at the Nitro Plant who were exposed 5 to in the cleanup of an accident there in 1949? 6 A I remember some of that information, yes. I remember 7 one individual that from which a nerve biopsy was taken that 8 was.`found to have suffered some demyelinization. 9 Q Now, do you recall whether or not there were reported 10 neurological effects in those individuals who were examined by 11 Dr. Suskind? 12 A I'm sorry, I don't remember. 13 Q All right, let's deal with the animal data. What did 14 the studies show with respect to animal data and the effect of 15 TCDD In causing neurological effects? 16 A It shows that TCDD is essentially inactive as far as 17 either the central or the peripheral nervous system. There 18 are reports of animals at high doses showing listlessness and 19 effects like that, but that can hardly be described as an 20 effect on the nervous system. These are very, very sick 21 animals and any sick animal is going to be listless, and so 22 I don't think that data like that or statements like that can 23 be interpreted as reflecting neurological change in the 24 experimental animals. What we have to rely on are the 11 1 primarily the pathological examination of animals in which the 2 central and the .peripheral nervous system are examined micro-* 3 scopically in detail to determine whether there has been any 4 change in the architecture of the nerve cells. 5 Q Now, when you say that someone reports that the 6 animals look listless, at what doses have those reports been 7 made? 8 A Well, those have been at high doses that are pro 9 ducing, they are on the verge of a lethal effect. These re 10 animals that are clearly Intoxicated by the TCDD, they have 11 been badly injured. 12 Q All right. And so the list -- to what do you 13 attribute the listlessness? 14 A A very sick animal, and I would not try to ssign 15 any specific -- any sick animal is going to be listless. 16 These animals don't convulse, they don't show signs that would 17 suggest that their central nervous system has been stimulated 18 as far as depression is concerned. There is no way of 19 assigning that kind of effect. However, those animals have 20 suffered a great deal of other kinds of damage, and a sick 21 animal is listless. 22 Q All right. Now, what do the studies demonstrate 23 with respect to neurological effects on animals due to TCDD? 24 A Really, nothing. The only effect that, for example, 12 1 that Kociba reported in his lifetime studies on rats was, and 2 this is at a dose range that was actually Increasing the 3 mortality of the animals, there was a good deal of pathology 4 generally in those animals. There was, and I believe'. I men 5 tioned something about this yesterday, some capillary break 6 down in the central nervous system so that there was bleeding 1, 1 * 7 around nerve cells, around nerves, but that's not a function 8 of effect on the nervous tissue. That's a function of an 9 effect on the circulatory system, 10 Q All right, now, Kociba, he carried on two different 11 sets of studies? 12 A This is the two year studies. In the thirteen week 13 studies in which they ran still higher doses of microgram per 14 kilogram per day, which again caused a good deal of other 15 kinds of pathology, there was no mention, they specifically t *) It 16 describe the parts of the brain that they examined pathologically 17 and they reported no information about any effects oh the brain. r 18 The lexistom is in a pathological study to identify eVery tissue 19 that Is examined, and then to report only on the tissues in 20 which some kind of abnormality has been found, and for this 21 reason there is often a statement in the methods of the paper 22 describing the methods of examination and the tissues exami 23 nation and no further discussion of certain organs, arid that 24 means that there has been nothing Identified as being abnormal. 13 1 Q Have there been any studies done? You mentioned 2 yesterday a Dr. Calder having done some studies? 3 A Yes, well, he was concerned about effects on the 4 peripheral innervation, that is out in the legs and arms and 5 so forth, of course he is studying rats, so they have four 6 legs, and 1 looked specifically for evidence, pathological 7 evidence of Injury to peripheral nerves after substantial 8 doses, of TCDD and found none. 9 Q Now, with respect to myellnlzatlon, we talked briefly 10 yesterday about myelin, what had the studies demonstrated with 11 respect to the effect on the myelin surrounding the nerves 12 after exposure to TCDD? 13 A Demyelinization is one of the easier morphological 14 -- by that I mean anatomical changes to detect in nerves, 15 In nervous tissue, and there have been no reports whatsoever 16 of changes in the myelin sheath of nerves in animals intoxi 17 cated with TCDD. 18 % Q j* This is an effect that can be observed upon exami- ' - v * 19 nation? 20 :rA As it happened I am not a pathologist, you must 21 understand, but I 've done research on hexachlorophene, which is 22 an demyelin agent, and while I had a pathologist direct And 23 analyze;pathological effects on those animals, I was able to 24 identify without any instructions at all just with the pathology 14 1 that I learned as a veterinary student thirty years ago or more, . 2 I was able to Identify the areas of . demyelinization. It is a 3 very easy thing to see. 4 Q Now, Dr. Silbergeld has testified to this jury that 5 there's been work to demonstrate that 2,3,7,8 TCDD causes 6 peripheral neuropathy and she cites the work of animal studies 7 done by, among -- she talks about the Strike Group in the 8 Netherlands, Elovaara's Group in Finland, and workers at 9 NEIHS, are you familiar with Dr. Elovaara's work? 10 A Elovaara was, I believe, a postdoctoral -- in any 11 case, I was a worker in the laboratory of Harry Vanio in Finland 12 who has done a good deal of work on TCDD and phenoxy herbicides. 13 It had nothing to do with myelin. They were working with a 14 strain of rat that has a genetic defect that -- let me see 15 if I can explain this. When hemoglobin or heme proteins are 16 used up, when they break down, the heme has to be, hds to be 17 also destroyed. And one of the products of the heme breakdown 18 is a pigment called bilirubin, and it is excreted in the bile, 19 by and large* That has to be done actually in the cells just 20 as heme is made in each cell that uses it, that breakdown has 21 to take place in the cell when finally that enzyme becomes 22 ineffective and has to be disposed of. But you don't repair 23 proteins, you don't repair enzymes, discard them and;start 24 oyer again. The strain of rat that Elovaara was using, and 15 1 he did this deliberately, was a strain that does not dispose 2 of bilirubin very well, I think I mentioned earlier that in 3 mice, why, there are strains and strains and strains of rats. 4 There are probably a hundred different strains of mouse used 5 in research, and they are almost designed for certain kinds* of 6 investigation, and the Gun rat, which is what Elovaara was 7 using, is a rat with a genetic defect that doesn't permit it 8 to dispose of bilirubin properly, and what they found was in 9 those rats there was in effect, in rats treated with TGDD 10 with that genetic defect, they were able to find a somewhat 11 amplified toxicity, some biochemical changes in the nervous 12 system of the rats with that genetic defect. The doses again 13 were fairly substantial, we are not talking about fractions 14 of microgram as we have been. 15 Q Did those studies demonstrate peripheral neuropathy? 16 A No, they had nothing to do with the peripheral nervous 17 system. They were working with the brain. 18 Q Now, I'd like to deal next with the immune system. 19 I wonder if you would describe the immune system in animals 20 and humans. 21 A Well, the immune system is in animals and tiumans is 22 In essence the same. There are differences among species but 23 the function and the general concept are the same. The immune 24 system first is the protection system that the body has for 16 1 dealing with infection, that is external organisms that can 2 cause damage to the organism. It is also Intended to deal 3 with foreign substances, foreign proteins. One of the reasons 4 that oftentimes an insect bite can cause such a ferocious- response 5 Is because of foreign proteins that the insect injects into the 6 skin and the body Immediately sends cells in to try to deal 7 with that foreign material, the mechanism that rejects organ 8 grafts, and I think everyone is aware of the present medical 9 advances 'in grafting organs, kidneys, heart, etc. ,Aiid one 10 of the problems that they have is preventing rejection; That's 11 an immune response to the foreign protein of the new part. The 12 immune system apparently also responds to cells which are be 13 coming cancer cells because they have become new and strange. 14 So what we have here is a protective system that is primarily 15 circulated in the blood that responds to external assault, 16 primarily Infectious attack. It has two general divisions, 17 it is an exceedingly complex system, and I would never claim 18 to he immunologist, but it is a very complex system that can 19 be divided into two parts. One part is known as the cell 20 mediated immune response, which means that for the most part 21 it consists of cells which attack foreign organisms,; which 22 attack foreign cells. If we have cancer cells starting to 23 develop, it may attack those cells and it in general responds 24 to, if you will, things that are particulate in nature by 17 1 oftentimes foreign inert material, some components of the cell 2 mediated immune response may actually engulf them, may actually 3 surround them and remove them. The other part of the immune 4 system is called the humoral immune system. Humoral arises 5 from the same root as hormone, and what it means is that this 6 is related to materials that are actually in solution in the 7 blood, primarily antibody, primarily antibodies, and antibodies 8 are proteins that have -- that arise from experience. In the 9 immune system there are some proteins that are very nonspecific, 10 the gamma globulin is a nonspecific immune response that will 11 tend to interact with foreign organisms, and it's there all the 12 time. If you have been vaccinated against some disease or if 13 you have had exposure to some disease, you will develop anti 14 bodies and you also develop a memory in the immune System, so 15 that if you are exposed at another time, the body immediately 16 makes more antibodies to respond to the challenge. In other 17 words, if you have had smallpox or chickenpox, almost everyone 18 my age at least had chickenpox at sometime or another. You 19 don't get it a second time,, and the reason you don't get it 20 a second time, it happens to be a viral disease, but the reason 21 you don't get chickenpox a second time is because the body has 22 learned about chickenpox and it is sitting there poised to 23 respond to the next Infection. So the ,humoral immune system 24 has a memory that responds to the second assault, let's say, 18 1 or if you have been vaccinated, what we've done is instruct the 2 body to learn to make those antibodies, and you respond to the 3 first assault. So that in a very general sense is the immune 4 system. Now, the components of it are exceedingly complex. 5 Q Now, you say in a vaccination, what do you inject in 6 a vaccination? How does that work? 7 A Well, what you would probably do is in most cases 8 It is a killed vaccine, so-called, which is essentially a 9 suspension of the organisms that has been killed by some 10 chemical means, usually, and that is all of the proteins of 11 the organism that the immune system learns to respond to, but 12 it doesn't have any of the activity, it can't be infectious. 13 So if you inject it, then the immune system makes antibodies 14 and it also prepares itself for the future production of 15 antibodies. So whenever, after the vaccination has taken, 16 so to speak, you come in contact with those organisms, the 17 body immediately creates a response and fights off the 18 organisms 19 Q What is the role of the thymus gland in all of this? 20 A The thymus gland is critical to the cell mediated 21 immune system, because it is a -- it processes certain kind 22 of cells called lymphocytes, and in some fashion changes their 23 character so that they will then go out into the lymph nodes 24 and they will reproduce by typical cell division, as cells do, 19 1 there Is probably a genetic change in these cells as a result 2 of some interaction with the epithelium of the thymus and that 3 is not clearly understood, the mechanism. However, these cells 4 are given the capability of responding to the kind of challenge 5 that the cell mediated system has to meet. That is, they will 6 go and attack any organisms that are foreign, that don't belong. 7 It will attack cells that don't belong, and these lymphocytes 8 leave the thymus with this change in their genetic instructions. 9 They go to the thymus and they form clones. Now clones are 10 just collections of cells. A cell has divided, made another 11 cell just like it, they divide and make cells just like them, 12 and so we have a colony of cells in each lymph node and they 13 are continually shed into the circulation. There are also 14 cells In reserve, and when some kind of infection that requires 15 their services arises, then they move into the sometimes 16 right out of the blood vessels into the tissues to respond to 17 the challenge. These are the kinds of -- I would imagine 18 that when you have an infection on a skin wound and it gets 19 pusey, that primarily is lympocytes of this sort, as well as 20 certain kindsof cells called macrophages that actually engulf 21 foreign material and they move into the area to clean up the mess. Q Now, what is the relationship between the thymus gland and the age of a person, whether he be in Infancy or 20 1 adulthood? 2 A The thymus has Its greatest importance in early life, 3 very early. And in the human it begins to function, and as a 4 matter of fact has accomplished a major part of its activity 5 probably before birth. It begins to function several months 6 before birth and is going through this process, because the 7 fetus has its own circulation, it is processing lymphocytes 8 for a long time prior to actual birth. So that the human infant 9 is b o m with a fairly effective immune system, it isn't perfect, 10 but it is very nearly in place. And the thymus probably has, 11 will have disappeared in a child of perhaps ten years. In the 12 rat, and most other species In which we do experimental work, 13 the animals are born very very early, I think I mentioned 14 yesterday that the rat pup is b o m at about the same stage of 15 development as a two and a half month human fetus, so ther;rat 16 has got a long way to go in terms of its development before it 17 actually is competent. And this function of the thymus in the 18 rat is not really fully developed, and the thymus as a con sequence is really very sensitive in a very young rat. ^ Q Now, are T cells those cells that are coded or pro grammed In some way in the thymus gland? A Yes. Q Humans are b o m with circulation T cells? A Apparently, yes. 21 1 Q How about rats? 2 A No, they don't seem to be, although at the time of . 3 birth it is a little difficult to examine them. But they appear 4 not to have started the process of T cells until after birth, 5 Q Now, what does that mean then in terms of whether 6 a chemical would have an immune effect on a rat as opposed to 7 a human? Does this difference in development between the rat 8- and the human have any effect on your ability to extrapolate 9 data from one to the other? 10 A In the rat during the earlier stages of development 11 up until weaning and even for a period afterwards, the thymus 12 is extremely important, and if you interfere with thymic 13 functions, then almost certain, well, you're going to probably 14 interfere with development of T cells. The human infant is 15 probably past the stage of maximum dependence, keep i n m i n d 16 that the thymus still functions in a young child. But the 17 initial function of delivering coded, to use your term, in 18 structed lymphocytes has been done, and those lymphocytes 19 once out in the periphery maintain their own population. 20 There are still some being processed by the thymus, but a 21 lot of the effect, a lot of the T cell activity arises from 22 the reproduction of T cells out in the lymph nodes. So a 23 good part of the work has been done, much of the work has been 24 done as far as the thymus is concerned in an infant. 22 1 Q Well, does dioxin have an effect on the immune system 2 in experimental animals? 3 A Oh, yes. 4 Q All right, and what sort of effect does it have? 5 A Well, it has a general depressing effect on the immune 6 system. It has'.a more -- it has a greater effect on cell 7 mediated immunity than it does on humoral immunity. However, 8 there are some effects on humoral immunity. A lot of It 9 depends on what kind of tests are done, and there are a very 10 large number of different kinds of tests available to examine 11 the function of the immune system, and many of them do show 12 effects of TCDD and there are some of course that do not. But 13 1 think that it is, as a general statement, yes, TCDD is able 14 at sufficient doses to have an effect on the immune system. 15 Q Because of the differences between the rat and the 16 human being in this particular parameter, the immune system, 17 how reliable is that data on rats in terms of predicting what's 18 going to occur in a human being? 19 A Well, there are two kinds of Information. One is, 20 of course, what I just mentioned, that the cell mediated system 21 is fairly functional in a newborn human. The other Is that the 22 Impact of maternal treatment, that is treatment of th mother 23 prior to birth in experimental animals does not seem to be as 24 substantial as dosing afterward when the animals are nursing 23 1 because it is well-known that TCDD is able to find its way into 2 milk, that's not at all a secret. And there seems to be a 3 greater effect on the rats after birth during the period when 4 the thymus is actually starting to do its work. So I.would 5 suggest that the human infant is not as vulnerable to infection 6 to failure of immune function, it is protected by the maternal 7 immune system of course prior to birth, just as the rat fetus 8 is, 'ifter birth it is in considerably -- it is much more 9 competent in terms of its immune function. Youhave to keep 10 in mind that the effect on the immune system inthe -- on 11 the cell mediated immunity apparently is not an effect on 12 T lymphocytes per se. They don't seem to be, they don't seem 13 to be injured, even though the thymus is very severely affected, 14 it is one of the most sensitive organs to TCDD. 15 Q So the effect is on the -- is on the thymusgland 16 and not on the lymphocytes themselves? 17 A It would appear so. 18 * Q What happens to the -- what do the studies Indicate 19 happens to the thymus in animals? 20 A Well, if you follow almost any experimental animal, 21 and young animals are usually used because the thymus is 22 functional in young animals, and therefore more vulnerable, 23 you find that after TCDD treatment, assuming that the dose is 24 sufficient, the thymus shrinks, it becomes smaller and smaller. 24 1 One of the reasons it shrinks is because all of the lymphocytes 2 that are contained in it leave. Now, there is a continual, probably it isn't that the lymphocytes leave, it is more than 3 likely that no new lymphocytes are coming in for processing and 4 the ones that were going to leave do. It appears that the 5 6 effect is on the, well, the thymus is a gland that Is comprised primarily of pithelial cells. In other words they are cells 7 8 Interestingly enough that are related back in early development to the cells that form the skin. Those are the cells apparently 9 that are affectd by TCDD. There Is recent work that is 10 11 described In Dr. Greenlee's Group has been working on this for 12 quite some while. So the effect on the thymus is really to 13 stop or interfere with the function of the cells that line the thymus, and the lymphocytes that it processes are simply 14 not brought out anymore and the thymus shrinks way down. 15 Q What are the clinical manifestations of an impairment 16 of the immune system in animals? 17 A Well, there is a, you know, we are exposed constantly 18 to every kind of infection that you can imagine, just about. 19 20 And we occasionally catch cold, we occasionally get gastro 21 enteritis, or we may even get an infection in some tissue. 22 The manifestations of a failed Immune system would be infectious 23 disease. Now, that shouldn't be interpreted to mean that every 24 infectious Illness is a function of a failed Immune system 25 1 because we do get infections. I have been fighting off a cold 2 for three weeks and I haven't fully succeeded yet, but I'm 3 fighting It off. And that cold got a foothold, now maybe If*? 4 my immune system had been perfect X wouldn't have caught cold. 5 But if it requires perfection, then we are all defective be 6 cause I don't know of anyone who never gets colds. But once I 7 got the cold, then all of my immune functions are coming to 8 bear in trying to get rid of it. So if we. are looking at the 9 experimental context, we would do experiments in which we 10 administer an infectious organism to an animal and observe 11 its ability to respond, and lots of experiments like that are 12 done. We continually monitor animal colonies to see whether 13 there is an increased incidence of this. If we are seeing an 14 increased incidence, far more,animals with the sniffles or 15 Other kinds of disease, we know that we've either got a massive 16 infection in the colony or that we imaginably might have some 17 kind of an immune dysfunction. 18 Q Well, when the animals are treated with TCDD, does 19 the immune system just fail completely? 20 A No, it appears that it doesn't fail, because at doses 21 -- there are effects, and again the dose response relationship 22 applies in Immune function just as it applies everywhere else. 23 It appears that there is an upper limit. It is known very clearly 24 that animals that die of TCDD intoxication do not die of infection. 26 1 Animals that are maintained, it seems to me that I made some 2 mention of this earlier, animals that are maintained without 3 any microorganisms in their environment at all are just as 4 sensitive to TCDD as animals maintained in a normal environ 5 ment. Animals that are maintained over a lifetime, we con 6 tinually seem to mention Dr. Kociba's work, but lots of other 7 long term studies as well do not show any increase in Infectious 8 disease, and even though they don't speak specifically of 9 Infectious disease, Infectious disease causes pathology just 10 like chemicals do, and effects in the lungs and so forth would 11 be identified in the pathology reports on these kinds of studies. 12 So there is a clear upper limit to the degree of impairment 13 that TCDD might cause. And we can see this in experiments in 14 which animals are infected and their mortality is measured 15 following TCDD intoxication. 16 Q Well, is there a no observed effect level for immune 17 response? 18 A In every experiment where there has been an adequate 19 range of doses, no observed effect=;level becomes quite apparent,, w 20 Whether there are examinations of specific components of the 21 immune system or whether they are looking at the ability of 22 animals to withstand infection. 23 Q In connection with what you are discussing there, Dr. 24 Dost, have you caused to be prepared a chart dealing with that 27 1 subject? 2 A Yes, I have, 3 Q Would the use of that chart assist you in explaining 4 it to the jury? 5 A Yes, it would. 6 (Defendants Exhibit 1282 was marked for identification.) 7 Q All right, sir, let me show you what's been marked as 8 Defendant's Exhibit 1282 and ask you to examine that "And identify 9 it for me, please. 10 A This is a graph that is prepared from data, tabular 11 data that was reported by a researcher named Thigpen, that's 12 an unfortunate name, in the journal Infection and Immunity, 13 Volume 12, 1975 in which he treated mice with TCDD for four 14 weeks and then infected them with an organism, that is, with 15 a salmonella, the species is burned and this is an organism 16 that when administered to mice at sufficient doses is in fact 17 lethal, and this is one of several experiments that he did, 18 but it does represent the general findings in the studies. 19 Q Now, S-burn? 20 A Salmonella, salmonella burn, salmonella is a very 21 common genus of microorganisms. This particular one is one 22 that is used in the laboratory and it does appear out in the 23 general environment also. This is again a dose response curve, 24 and again it looks a little funny because the dose response is 28 1 so steep. As iisudi.thei dosage is expressed logarithmically, 2 that is .1, 1, and 10 micrograms of' TCDD given weekly. And 3 these are in young mice. And what we see here is that there 4 is an upper limit to the -- this is the mortality, this is 5 the percentage of animals that died given that treatment. 6 This is the control level, the'.number of animals, the percentage 7 of animals that were given no TCDD which succumbs to the infec 8 tion, What we have here is at this highest dose which is 9 clearly an upper limit because you can see at the next highest 10 dose we are up here on somewhat less than 70 percent, and then 11 we are up here to 75 percent, something like that, and that's 12 as high as the mortality would go. And when we're up into 13 doses like this at, let's say 50 or 60 micrograms, some of the 14 animals will simply die of the TCDD at the lowest dose that they 15 use here, which is at about, what's that going to be, something 16 on the order of probably .4 micrograms per kilogram per week * 17 in four weekly doses, there was no effect. In other words, 18 that dose of TCDD did not increase the susceptibility of these 19 animals to infection. And that is a common finding wherever a 20 dose response has been worked through. 21 Q What is a direct challenge test? 22 A Well, a direct challenge test, if you are familiar 23 with a tuberculin test, what's done in tuberculin tests is to 24 inject into the skin, I think they do it on the forearm if I 29 1 remember correctly, into the skin, not under the skin, the skin 2 is thick, you realize, and so they inject right into the dermal 3 layer of the skin, either -- well, usually it is a killed 4 preparation of tuberculin organisms, and then what we're looking 5 for is to see how the body responds to it. Now if you have 6 never had tuberculosis the response will be minimal because 7 the body has never learned about tuberculosis. If you have had 8 an infection relatively recently and it's been active, why there 9 may be a very vigorous response. And if you had an infection 10 twenty years ago it may be somewhat lesser because there has 11 been a lot of forgetting going on during that period of time. 12 So what we're looking at here are invasion of white cells to 13 attack that presumed invader. 14 Q Now, what does the line on this chart mean, the 15 horizontal line in the middle of the chart? 16 A That just identifies the control level of mortality. 17 In other words, animals that were not given TCDD but were given 18 the Information, and this Is a challenge, too, for that matter, 19 it is an infectious challenge. We are given the microorganisms 20 in 25 percent of the animals that were not treated with TCDD died 21 of the infection. The no effect level is .5, I don't remember 22 what I estimated it at, but it was calculated. 23 Q As .5 micrograms per kilogram? 24 A Yes, it intersects the .5. 30 1 Q With respect to the dose that you calculated from 2 Sturgeon soil, would that dose be sufficient to effect the 3 immune system in the mice that were -- 4 A As reflected here, no, this no effect level is a 5 half a microgram, and this is a thousandth of a microgram, 6 that's a difference of, what, 500 fold. 7 Q So it would be 500 times less than the dose necessary 8 to cause an immune -- 9 A I beg your pardon, I'm looking at this other no 10 effect level. I misstated myself. We have, or have I? No, 11 we have with respect to the dose at, that we calculated at 12 Sturgeon, for the sake of simplicity call this .5 of change 13 it up to 5, and that means that we have a 10,000 fold difference. 14 Q 10,000 fold difference? 15 A Well, the difference between point -- we have the 16 difference between .5 and .00005, essentially. 17 Q So the Sturgeon dose calculated by you would be 18 10,000. times less than that required to begin to show an effect 19 in, the mice? 20 A' In this direct challenge experiment, yes. 21 \Q Now, when the animal's immune system is affected by 22 a sufficient dose of TODD, is this a permanent effect?7 23 ;-VA No, it will recover, but it takes -- it' takes a 24 substantial amount of time in an experimental animalj it will 31 1 take months. 2 Q To recover? 3 A To recover. 4 Q But does recovery occur? 5 A Eventually these return to normal. 6 THE COURT; Before you get into your next subject, 7 is this a good point for a short break? 8 MR, HEINEMAN: Yes, sir, fine. 9 THE COURT: Okay, we will take a short break at this 10 time. I would remind you, and this would go for any other breaks 11 we take during the day, that you are not to discuss this matter 12 among yourselves, with anyone outside the jury panel, or as of 13 yet form any opinions ox conclusions about the matters on trial. 14 The Court is in recess. 15 (At this time Court was in recess.) 16 MR. HEINEMAN: Your Honor, at this time we would move 17 the admission of Defendant's Exhibit 1282, (Defendant18 Exhibit 1282 was offered into evidence.) THE COURT: Any objection? MR, CARR: None, your Honor. THE COURT: Admitted without objection. Thank you. (Defendant's Exhibit 1282 was admitted into evidence.) Q (By Mr. Heineman) Dr. Dost, in your review of the testimony of Dr. Zahalsky on behalf of the plaintiffs, do you 32 1 recall his testifying about a study by a man named Boorman? 2 A Yes. 3 Q Let me show you what's been marked as Plaintiffs' 4 Exhibit 260, yes, it is 260. Have you seen that before, sir? 5 A I've seen a photograph of it. 6 Q All right, is this the photograph that you have seen, 7 sir? 8 A Yes, it is. 9 (Defendant's Exhibit 1283 was marked for identification.) 10 Q What has been marked as Defendant's Exhibit 1283, sir? 11 Is that a photograph of Plaintiffs' Exhibit 260? 12 A Yes, it is. 13 Q Now, are you familiar with the Boorman article itself, 14 sir? 15 A Yes, I am. 16 Q And did you cause a chart to be made of some of the data 17 from some of the data in there -- .18 A There is a table in the paper, and it was copied directly 19 from the, that is, it isn't a photocopy of the page, but the 20 data has been copied in essence in<& table. 21 Q And based upon your review of the testimony of Dr. 22 Zahalsky, is that the table from which Dr. Zahalsky purported 23 to create this exhibit? 24 A Yes, it is. 33 1 (Defendant's Exhibit 1284 was marked for identification.) 2 Q Now, let me show you what's been marked as Defendant's 3 Exhibit 1284, sir. Can you examine that and identify it for me, 4 please? 5 A Yes, it is a copy of a table that was in an article 6 by Boorman that was published in Environmental Health Perspectives, 7 and we do not have a specific reference on here, but it is the same 8 article. 9 Q It is adapted, it's taken directly from this Boorman 10 article? 11 A It is taken from the article and it is the data from 12 which Dr. Zahalsky's chart or table was derived. 13 Q All right. Now, do you agree, sir, with the testimony 14 of Dr. Zahalsky with respect to that chart? 15 A No, I do not. 16 Q All right, I wonder if you would step up here and 17 explain to the jury why you don't agree with this chart. 18 A I'll put this up here where it can be seen. The reason 19 that I don't agree is because some very important data has been 20 left out of the chart and there has been an incorrect represen 21 tation of the dose, and also in reading the testimony, I saw no description of the experiment and the nature of the experiment is of considerable importance because the animals -- and it was necessary to go to the original article, Boorman's article 34 1 is a review, and they reproduced from an earlier paper done by 2 the same group, these data, and In the earlier paper they des 3 cribe more completely the manner of treatment of the animal. 4 What they did was to administer to pregnant mice TCDD seven days 5 prior, I believe, to the birth of the pups, and then at seven and 6 fourteen and twenty-one days following during the period when 7 they are nursing, so that the animal, the pups were presumably 8 exposed while in the uterus, and then exposed through the milk, 9 and this a common way of exposing very young rodents in the 10 experimental context, that is, to treat the mother and deliver 11 the material in milk. There is a very serious error made in 12 representing the dosage. When we speak of parts per trillion 13 or million or billion, we are talking about concentration. We 14 do not use this representation in discussing dosage, because the 15 only thing that dosage tells us is how much went into the animal 16 for unit of body weight. It doesn't tell us anything about the 17 concentration in the liver or in bone or in the kidney or the 18 brain or any other tissue. The concentrations within organs is 19 going to vary with the organ. So this tells nothing about what 20 is, it doesn't represent what's happening in the animal, neither 21 does the dose in milligrams per kilogram, but it is the only 22 information we have when we administer a chemical to an animal. 23 We don't know where it went, we know enough about a chemical we 24 can say, yes, so much of it is in the liver and the kidney and 35 1 so forth. The other error which is extremely critical, even if 2 we accept this idea after parts per trillion as representing the 3 dose, if it were parts per trillion, it would mean that the dose 4 was ,005 micrograms per kilogram and .015 micrograms per kilogram, 5 accepting for the moment that this improper reference is the way 6 to use the language. The fact of the matter is that the doses 7 that were administered were a thousandfold., higher. This is 8 milligrams per kilogram, a thousandth of a milligram per kilogram 9 is one microgram. This is an erasable -- the dose is one micro 10 gram per kilogram per week. So the dosage here has been repre 11 sented as a thousand times lower than it really was, I don't 12 have a great deal of objection*, to changing the terminology 13 because Dr. Zahalsky was trying to explain the nature of these 14 celia. He did use a different convention than the authors did 15 for describing the results. He talks about percentage of in 16 hibition, that is, that means how much of the activity of the ' 17 those cells was decreased. Whereas, the authors speak.to how 18 much activity was left. That's not serious, that's just a 19 convention. But I want to point out that he is talking about 20 in this case the loss of competency and the loss of competency 21 obviously would be greater as the dosage increases* The impor 22 tant thing is however that he left off the lowest dose, he left 23 off the lowest dose. Now, this information, this is the number 24 of cells in the bone marrow. It is not just cells making red 36 1 blood cells, but all cells in the bone marrow. These are cells 2 that will eventually form red blood cells as he states, these 3 are cells that eventually will form macrophages, similar cells 4 that actually,.I mentioned cells that go out and surround, 5 engulf foreign material. The important information in this 6 paper is the dose response relationship. Now, these other 7 chemicals, don't worry about -- we simply wanted to reproduce 8 all .of the material so that you can see what the original table 9 looks like. This is the data that we're concerned with, and 10 surely enough there is a dose response, one microgram per 11 kilogram per week, 5, 15. And as you might expect/ here is an 12 effect, 15 micrograms Is a good size dose for any animal, any 13 rodent, and here only 61 percent of function has been left, 14 and they describe the: -v. statistical validity of that* In 15 Other words, that is a result in which they have great confidence. 16 This is also a result that they have confidence in, but somewhat " 17 less because it isn't that far removed from the control. These 18 numbers are percentage of control, and that's the reason we 19 don't see a separate line for the control information* In this 20 one, they didn't determine the effect for whatever reason. At 21 the lowest dose, but again there is 68 percent of the function 22 left here, 37 percent at this higher dose, which is not surprising. 23 In this case there was a more severe effect even than the others 24 at 15 micrograms per kilogram. Very little effect, at 5, and you 37 1 can see here that they consider, or the statistics tell them 2 that 90 percent is so near to a hundred percent in this experi 3 ment that you can't tell the difference. And here at one micro4 gram per kilogram is actually these animals have more than the 5 normal amount of cells, don't take that as meaning that a low 6 dose of TCDD makes the bone marrow work better. This is the 7 kind of variation that one is going to expect in an experimental 8 context.. What I want to show you here, though, is we have a 9 no effect here at one microgram per kilogram. A plot of a dose 10 response curve which suggests that you are going to have a no 11 effect level at this point, but the data isn't there and all we 12 can do is say that is suggested. Here we clearly have anno 13 effect dose with given the statistics, the animals are even 14 better than controls. Again, when I see information like that 15 it doesn't mean they are better, it means that there is enough 16 statistical variability, but given the location of this f;: \ \ 17 point and this point, it is very clear that there is a no effect 18 dose here. So at the dose level that he did not lhclude in 19 his testimony there is .clearly a dose, a no effect dose. 20 Q Now, what is the no effect level there, Dr. Dost, 21 is one microgram per kilogram? 22 A In this experiment, yes. 23 Q Now, was this an acute exposure? 24 A Well, it took place over a period of, I think, four 38 1 weeks in which the material was administered prior to birth of 2 the pups, and administered periodically after the birth of the 3 pups, so that there were four doses at this level and I think 4 the total period was over four weeks. 5 Q So would that be called a chronic exposure? 6 A No, it would be -- of course when you define those 7 terms, why everybody has his own definition of what is acute, 8 I guess I would call it, it is a short subchronic experiment, 9 but it could even be called a long acute experiment. It is a 10 little difficult to say when you have that kind of a dosing 11 schedule. Their objective was to maximize; at a given level 12 of input they wanted to maximize the exposure to those pups. 13 Q Now, what's the difference between the no,effect 14 levelvof TCDD demonstrated on that dosing of those rat pups 15 and the concentration or the exposure level or the dose, 16 pardon me, the dose computed from the Sturgeon incident? 17 A Well, we're back at microgram per kilogram again, 18 arid if 1 remember that would be, the difference would he 19 2 2 2 , 0 0 0 . 20 Q 222,000 what? 21 A 222,000 fold difference between, that isttheadosethat 22 we've just been speaking of here at one microgram per kilogram 23 per Week, or whatever the period would be, is but one of those 24 doses, one of those four doses is 222:^000 times higher than 39 1 this exposure that we hypothesized. 2 Q From Sturgeon? 3 A From Sturgeon. 4 Q Now, and that was a no effect level when it was 5 administered -- when that dose, 220,000 times more was 6 administered once a week for four weeks? 7 A It is my recollection that that was the schedule, 8 it whs one time prior to the birth of the pups, and then three 9 times afterward. 10 Q Now, Dr. Zahalsky testified about proteases. Do 11 you recall that testimony? 12 A I remember something about it, yes. 13 Q He suggested that proteases 14 HR. CARR: What page, Counsel? 15 MR. HEINEMAN: 135. 16 MR. CARR: What day? 17 MR. HEINEMAN: May 3rd. 18 Q He suggested that proteases act on proteins and attack 19 tissue. They attack protein in tissue, do you recall that? 20 A Yes. 21 Q Do you agree with what Dr. Zahalsky said? 22 A Only in part. 23 Q All right. Would you tell us about your disagreement 24 there? 40 1 A Well, proteases are in fact enzymes that break down 2 protein. They do not act, when you say tissues, I am assuming 3 that is living tissues. 4 Q Yes, sir. 5 A They do not act in living tissues except under extra- 6 ordinary circumstances. They only act, let me explain. Proteases 7 are present in all cells. Now, I'm using the term very generally; 8 it is my recollection he mentioned a couple of proteins in here. 9 But proteases exists in cells for the purpose of degrading 10 proteins, enzymes, other proteins in the cell when they are no 11 longer working and have to be disposed of. In fact, proteases 12 are responsible for regulating the lifetime of certain proteins 13 in the cell, and this is a very effective system. If a protein 14 should not stay there too iong, it will be in the course of time 15 depending on its size be degraded by the proteases that are 16 always in the cell. There are also proteases in certain cells 17 in the circulation and their responsibility, and they are part of the immune response, too, in a sense because their function is to break down. If a cell engulfs another cell, It's got to dp something with it, it can't just continue to hold onto that cell. It's got to break it down, it's got to digest it so the cell eventually, that is picked up eventually is digested. There are a certain amount of free protease in the circulation as well because they will tend to leak out of cells that have failed, 41 1 they will leak out of the macrophages that are in the tissues 2 and in the blood, just in the normal course of attrition of 3 the cells. So there is always a level of a variety of proteases 4 that break down various kind of protein in the circulation. And 5 if a cell is in, a cell is severely damaged, for example, a 6 lung cell, if a heavy smoker, the cell is alive and it is 7 functional but it is not well. And in that circumstance a 8 cell in that marginal zone between function and nonfunction or 9 life and death could very well be attacked by proteases that 10 circulate. But they don't attack healthy tissue. 11 Q Now, what is the purpose of them? As I understand 12 it, a protein is a chain of amino acids, didh't you tell us 13 that the other day? 14 A That's correct. 15 Q Okay, now when the body -- and does the body 16 normally dispose of protein as an everyday occurrence? t 17 A Oh, certainly, because they either wear out or they 18 are no longer needed, and since we do not repair a protein 19 that isn't working properly, the body degrades it, makes use 20 of those amino acids, starts over again, uses them for what 21 ever it needs amino acids for. 22 Q Is it something like using building blocks? 23 A Well, the structure of a protein, yes, is like building 24 blocks. There are a relatively limited number of amino acids. 42 1 probably twenty or so, that are Incorporated into proteins, and 2 the proteins re very, very long, the molecular weights I think 3 I mentioned the P450 enzymes as having the cytochromes as having 4 molecular weights on the order of 50,000, well, the molecular 5 weight of a typical amino acid may be anywhere from 50 to 150 6 perhaps, or somewhat greater. They are small components, but 7 the pattern, the sequence in a given protein is very, very "8 specific. And in fact it is even specific across species. 9 Insulin, for example, which is a complex protein from the pig 10 works perfectly well in the human because it is essentially J. 11 the same structure. There are minor differences, but it is 12 possible to go through and identify every amino acid and 13 establish its sequence in a protein. So we have, in all our 14 clla all of these proteins that keep us functioning, and 15 that are really the structural framework of the cell* If 16 there is failure of a molecule, and every molecule is going 17 to fail sooner or later, something has to be done with it. 18 We can't leave the debris lying around like the trash in my 19 back yard. It is necessary to dispose of it, get it out 20 of the way and make use of the parts. 21 Q And is that what the proteases do? 22 A And that's what the proteases do, and they are within 23 cells, normally. I suppose in a perfect organism we wouldn't 24 find any in the circulation, but no organism is perfect, there 43 1 is always leakage and they appear in the circulation. 2 Q Now, Dr. Zahalsky on about the same page or the next 3 page, I guess 136, talks about some materials called Alpha 1 4 anti-trypsin and Alpha 2 macroglobulin, do you remember? 5 A Yes. 6 Q Those terms in that testimony? 7 A Yes. 8 Q Now, do you agree with what he said about those 9 molecules in that testimony? 10 A It was a little difficult to understand, but basically 11 n o , I don't agree. 12 Q All right. Point out to the jury if you would what 13 Dr. Zahalsky said about these materials and the area of your 14 disagreement. 15 A Those two substances are inhibitors of proteases and 16 they exist in the circulation. They are very important com 17 ponents of the plasma protein profile. In fact, they're even 18 greater quantities than, let's say, the immunoglobulins, which 19- we depend on for immune response. Their function is to inhibit 20 the activity of these proteases that we have been talking about 21 that inevitably escape from cells. In other words, they protect 22 us from proteases. And there are a number of genetic diseases 23 In which these protease inhibitors, particularly the Alpha 1 24 anti-trypsin, the reason it is called anti-trypsin is because 44 1 it is very efficient of the enzyme trypsin which is found in 2 cells and that is also an enzyme that is secreted into the 3 digestive tract to digest meat. But there are genetic defects 4 in which there is a deficiency of the anti-trypsin and people 5 with that deficiency- seem to have a greater incident of' 6 pulmonary emphysema and probably the reason for that is in the 7 normal course of assaults that our lungs take, whether we're 8 smokers or not, there are injured cells and those cells are 9 vulnerable or if trypsin and other enzymes have access to those 10 cells, there's going to be some other kind of break down and 11 that results in emphysema. The main thing about his statement 12 is that he describes anti-trypsin more or less effectively, but 13 then he talks about the Alpha 2 macroglobulin which is just 14 another kind of inhibitor as being some kind of a regulator 15 for anti-trypsin, and it is not. He also makes a peculiar 16 -statement that if you increase the amount of Alpha 2 macro 17 globulin, and I don't know how that is going to come to pass, 18 but if you increase the amount of Alpha 2 macroglobulin you 19 increase the activity of the proteases, and it is JAist the 20 other way around, it is just the other way around. 21 Q What do you mean "just the other way around"? 22 A Well, if you increase the amount of Alpha 2 macro 23 globulin, its function is to inhibit those proteases, not to 24 increase them. It protects the body against the proteases. 45 1 Q Well now, if proteases don't attack healthy tissue, 2 why does the body have inhibitors, protease inhibitors? 3 A Well, 1 already mentioned that even in th best of 4 circumstances cells lose, they break up, they^re going to lose 5 their -- the proteases, the enzymes that break down protein 6 that are inside the cell, that are there for normally to dispose 7 of unwanted or nonfunctional proteins in cells, and I think I 8 mentioned the other day about when the liver is injured, it 9 spills some of its enzymes into the circulation. The cells are 10 not broken, but they've become leaky. The same thing can happen, 11 a leaky cell presumably can lose its proteases, its trypsin, etc., 12 or a cell that completely breaks up. So there's always going to 13 be some level, even though in an ideal state there wouldn't be 14 any, there's going to be some, and we have evolved that kind of 15 protection against that activity because we, always have cells 16 in our body that are perhaps not perfectly healthy, and a cell 17 that is marginally healthy and has been injured in some way 18 would quite imaginably be affected by an enzyme that will break 19 down protein. And so we need to protect against that. 20 Q Now, Dr. Dost, is there any evidence from animal 21 studies or from anywhere else that you are aware of that TCDD 22 affects proteases or the protease inhibitors Alpha 1 anti-trypsin 23 and Alpha 2 macroglobulin? 24 A I know of no evidence, X don't think the question has 46 1 even been examined. 2 MR. HEINEMAN: Your Honor, it's noon, I notice, if 3 it's all right with the Court. 4 THE COURT: Okay, fine. We will break for lunch at 5 this time. We will resume again at 1:15, and the admonishments 6 that I have given you earlier will apply during this break also. 7 The Court is in recess.for lunch. 8 (At this time Court recessed for the noon hour.) 9 Q (By Mr. Heineman) Dr. Dost, when we broke for lunch 10 we had just finished talking about the Boorman study, and I 11 wanted to ask you a little more about that. Do you recall 12 that Dr. Zahalsky testified that the effect of dioxin on the 13 immune system could be initiated by one molecule of dioxin? 14 A Yes, I recall that. 15 Q All right. Now, do you agree with that statement? 16 A No. 17 Q Now, what would be the possibility of an exposure 18 to or a dose of one molecule causing an adverse reaction in 19 an organism? 20 A No possibility at all. 21 Q Why is that, sir? 22 A There are a number of reasons, even if the-substance 23 was highly reactive it is necessary for a reaction to take place 24 that there be a population of molecules. There would have to be 47 1 a lot of molecules present, a reaction that involves a single 2 molecule out of a population, a large population might imagin 3 ably take place, but there are time factors. Molecules are 4 - very, very lively things. They don't just sit in one place 5 all by themselves. And for a reaction to take place, time is 6 necessary. Chemical reactiois are not just instantaneous. It 7 may seem like it when we look at them in a toy chemistry set 8 and pour two clear liquids together and they suddenly turn 9 purple, but there is a great deal of time involved. A molecule 10 has to get into a position where it can react, and then it has U to be held there for some finite period in order for whatever 12 it is going to interact with to react, for the two of them to 13 interact together. Otherwise, it may come into contact and be 14 gone again in an instant and more than likely never to return 15 again to that site. 16 Q Well, a molecule upon entering the body, what happens 17 to it? Where would it go? 18 A Anywhere. One molecule, we are not accustomed to 19 thinking in terms of one molecule. We are accustomed to thinking 20 in terms of large populations of molecules, and even a dose of 21 a microgram or a thousandth of a microgram Is a very, very 22 large number of molecules. 23 Q Well, how does the fact that the Boorman study establishes 24 a no effect level affect Dr. Zahalsky's theory that one molecule 48 1 can initiate this immune function difficulty? 2 A Well, in terms of this particular study, we see a no 3 effect level as a microgram per kilogram. There are other studies 4 looking at other parameters that show somewhat lower no effect 5 levels. We are still talking about enormous numbers of molecules. 6 Q Now, Dr. Zahalsky has said, sir, that when this one 7 molecule would initiate this immune function problem, then other 8 dioxin molecules would be required to deal with the additional 9 messenger RNA and receptors, do you recall that? 10 A I recall something of that sort, yes. 11 Q Now, would a small number of molecules, a handful of 12 molecules have any possibility of causing the immune function 13 effect that is reflected in the Boorman study? 14 A Not really. Maybe X should illustrate w h a t I talk 15 about when I say there would have to be an enormous number of 16 molecules, because 17 Q All right. 18 . A It happens that we know how many molecules are in a 19 given amount of a chemical. It may seem like an awfully esoteric" 20 kind of thing to know, but I guess what I have to do is give a 21 brief lesson in chemistry. But we are talking about TCDD, and 22 I will draw the molecule and show you what I mean* Every atom 23 has a relative mass or relative weight. It isn't very much, but 24 it exists. And we've got a molecule here with chlorine and 49 1 oxygen and carbon and hydrogen. Now, you remember yesterday 2 I pointed out that at each of these comers there is a carbon 3 atom and there is also a hydrogen atom. And we rarely show 4 these in a structure unless they.have some kind of meaning. 5 It happens that the atomic weight of hydrogen is 1 and the 6 atomic weight of carbon is 12 and of chlorine is 35 and a 7 fraction and oxygen is 16. And if we were to add those all 8 up, we would have the molecular weight, so to speak, well, 9 it is the molecular weight of 2,3,7,8 TCDD. We have four 10 hydrogens, and the atomic weight is 1, so that means we have 11 a total of 4, and we have two oxygens, and the atomic weight 12 is 16. So that means 32. And we have four chlorines, and 13 we will call that 35. It is a little more than 35, there is 14 a decimal in there, but let's keep the decimals out of it. 15 So w e 've got four chlorines, and then we've got twelve,carbons 16 and they are each 12. So this comes out 4 times 35 is 140 and 17 12 times 12 is 144, and that adds up to 320. Now, that's the 18 molecular weight of TCDD. And a man named Avigodril back in 19 the last century somehow determined that for every chemical 20 compound, if we take its molecular weight in grams, in other 21 words, the gram molecular weight, if we take 320 grams of TCDD, 22 it has in it a very, very large number of molecules, as you 23 might imagine, the number is 6.023 times 10^3. What that 24 means is then since 1 0 ^ is a trillion, this means that there 50 1 is 600 billion trillion molecules in this much TCDD. Now, if 2 we were talking about, if we were talking about phenol, phenol 3 has a molecular weight of 94 and we would arrive at this in the 4 same way, 94 grams of phenol has the same number of molecules 5 in it, 180 grams of glucose has this many molecules. This is 6 a constant for all molecules, and it is a very useful number 7 as you can imagine. I guess to make use of that 8 Q Dr. Dost, let me ask you a question. Originally when 9 Avigodril came up with that number, there couldnft have been 10 any way for him to count that. How in the world did he arrive 11 at it? 12 A I'm not exactly sure. He certainly didn't have 13 instruments to count that. He deduced it from the Table of 14 Elements and other information known at that time to chemists. 15 It was a rather astonishing intellectual feat, really. 16 Q Has it been established now as being accurate? 17 A It's still valid, and apparently now will remain that 18 way. In other words, his calcualtion was correct. So if we O*1 19 want, we have 360 grams equal 6.023 times 10 . 20 Q Excuse, was that 360 or 320? 21 A I'm sorry, 320. Then 320 milligrams which, is a 22 thousand less would have times 10^0 , 300 micrograms is 1 0^. 23 Well, if we wanted to find out how many molecules are in one 24 microgram, well obviously we would take l/320th, and I don't 51 1 do arithmetic like that in my head. What we really do is divide 2 this by 320. I've got the wrong exponent here, I'm sorry, I 3 would be giving you a million more than you really want. Okay, 4 we come up with a number that is 1.8822, we don't care too much 5 about these late decimals, times 10 molecules in one microgram. 6 Now, we're talking about a microgram per kilogram, so we are 7 talking here about the number of molecules in a kilogramvof 8 mouse, there are about forty mice to the kilogram. So if we 9 wanted to know how many molecules were in each of these mice, 10 then we would divide that number by approximately 40, a mouse 11 weighs about 25 grams. So since we are talking in this experi 12 ment about a dosage per. kilogram, we can leave it at that. 13 Here is the number of ^molecules per kilogram in this experiment 14 which did not produce an effect. Now, if we wanted, let's say, 15 to consider how many molecules, if our no effect level had been 16 a thousand times lower, then the number would be 1.9, I'm just 17 rounding this off, times 1 0 ^ , which is 1.9 trillion molecules 18 in a thousandth of a microgram, which is more than I can count 19 in an afternoon. 20 , Q Well, how does that relate to the Boorman study? 21 I'm not sure I understand. 22 A I'm sorry, the Boorman study found a no effect level 23 at one microgram per kilogram. So the number that we are con 24 cerned about is 1.9 times 1 0 ^ molecules. 52 1 Q So at no effect at all in the animal there was nearly 2 2 trillion molecules of dioxin. 3 A Per kilogram? 4 Q Per kilogram. 5 A Of body weight. 6 Q All right. Well, what does that number, how does that 7 effect what Dr. Zahalsky testified to as to the causation of this 8 immune effect? 9 A Well, it says that one molecule or even a few or eveh 10 a thousand are not likely to have any effect or will not have an 11 effect. 12 Q All right, now I believe you've testified the other 13 day that the Food and Drug Administration has arrived at a no 14 effect level in a human being, in a 70 kilogram man, is that 15 right? 16 A They have arrived at a conclusion. This Is hot measured 17 in human toxicity studios, this is information derived from animal 18 experiments and they have determined or decided that the no effect 19 level in man -- that they can establish a no effect level in man*. 20 t; Q All right, what is that no effect level? 21 A It is one nanogram per kilogram. They use a figure of 22 70 grams just on a general assumption of a 70 kilogram person. 23 Q So that in a 70 kilogram man, which is what; you say,, 24 150 some pounds? 53 1 A 154 pounds. 2 Q There Is a 70 nanogram no effect level? 3 A That's correct. 4 Q All right, how many molecules would that be? 5 A Well, we will multiply this figure by 70, and that's 6 for a 70 kilogram individual, it would be 1.31 -- well, let's 7 call it 1.32 times 1 0 ^ molecules, which would be to put it 8 In manageable form, a trillion is 10^, so we've got 1,32 times 9 10^ or 1*32 thousand trillion. 10 Q Molecules? 11 A Molecules. 12 Q Of TCDD? 13 A Of TCDD. 14 Q At a no effect level? 15 A If we consider this to be a no effect level, yes. 16 Q Now, in terms of the immune system, sir, when you say 17 that an adverse effect on the immune system will cause additional 18 infections, Is that correct? 19 -A Yes. 20 * Q What kind of infections? Are you talking about a cold 21 or a runny nose or what kind of infection is evidenced in an 22 immune suppressed organism? 23 A Well, an organism that has.a significant -- well, 24 to start with there is a lot of excess capacity. We're always 54 1 fighting off infections of one kind or another. An individual 2 that has a noticeably compromised immune system is suffering 3 probably continuous assaults, wounds don't heal, probably con 4 tinuous respiratory ailments. The other problem is that these 5 tend to progress, not only are they allowed to take root or 6 take foothold, but it would seem to me that they would progress. 7 And I think that the, once the barrier, so to speak, had been S overcome of our excess capacity for immune protection, it would 9 seem that the individual animal,human or any animal presumably, 10 or for that matter any organism that relies on an immune system 11 would, I think, suffer fairly substantial infectious disease. 12 Q Let me start, first of all, with your statement about 13 excess capacity. What are you talking about? 14 A Well, the immune system has far more capability^than 15 is necessary to help us get over a simple cold or to get over 16 a case of pneumonia. Now, to be sure, if the infection is 17 massive enough, if it just absolutely overwhelming or if the 18 individual has a protein deficiency and is unable to synthesize 19 protein, and this is a significant problem with heavy protein 20 malnutrition, people cannot manufacture antibodies. It seems 21 to me that if the failure of immune function Is really signi 22 ficant it is going to mean pretty nearly a collapse of the 23 immune system. There i3 a great deal of excess capability :: 24 available. If we interfere with part of the immune system we 55 1 do not necessarily cause animals at least to succumb to infection. 2 We can show a lot of different parameters of immune function, we 3 can look at the ability of the rat, you can inject a challenge 4 into the rat paw and to see whether the paw swells as it ought 5 to because cells are being brought into the paw, or we can look 6 at titers of antibodies. 7 Q Come again now? 8 A We can look at the amount of antibodies available in 9 the blood. 10 Q What is a titer? n A A titer is a measurement of the amount that is avail 12 able for reaction with an infectious system. So there is lots 13 of ways of quantifying depressed immune function, and we know 14 that TCDD at substantial doses depresses immune function, there 15 isn't any question about that. Not all immune function, there 16 doesn't seem to be any impairment of the response to viruses, 17 for example, to the extent that laboratory experiments have 18 demonstrated, but animals do not die of infection that are 19 heavily attacked by TCDD, and we know that their immune function 20 must be at least partially impaired, you see, so 21 Q Now, when you say that there is excess capacity, why 22 then if the immune function has more capacity than is affected 23 by dioxin exposure, why is it that takes so long to fight off 24 a cold? Why is it that an infection can last a while in an 56 1 organism? 2 A If I knew why it took so long to fight off a cold, 3 I would have probably become famous long since. That's a problem 4 that has yet to be solved. The fact remains, though, is that 5 colds, the cold that I have didn't just sweep in and wipe me 6 out. There were a couple of days where I was worried about that, 7 perhaps, and then there was a day or two perhaps that I was worried 8 that I wouldn't die. But nonetheless, that interaction took 9 place, and it has protected me, and I am having a little trouble 10 getting the last of it resolved, but If I didn't have an immune 11 system, I would have dropped very quickly. Why it takes so long 12 is a good question. Certainly these, the antibodies are continuing 13 to be produced. The cells that are fighting off specific organisms, 14 lymphocytes and so forth, are continuing to be produced. Why 15 they can't just step in and mop up instantaneously is something 16 that I don't understand. 17 Q If the type of disease for a quenched immune system 18 would be something more than just a cold -- 19 A Not necessarily. I guess what I am saying is that If 20 you were to reduce the function of some component of the immune 21 system by half, let's say, by intoxication, that doesn't mean 22 that you have eliminated capacity sufficient to cause that 23 animal to succumb to some kind of an infection. There is 24 reserve capacity. We have impaired the animal, but we haven't 57 1 impaired it to the point where his ability to fight off this 2 infection is really affected, and that's really very clear 3 in all of the experimental work, because when animals are 4 challenged with a lethal dose of, well, I showed a dose response 5 relationship earlier. At the highest dose of TCDD, there is 6 still survival. A quarter of the animals survived this impact -7 that in untreated animals killed a quarter of the animals, and 8 we see in animal colonies where TCDD is being administered to 9 animals, there is no change in their response to the host of 10 infectious diseases which exist in most animal colonies^ 'll Q Well, if the immune system were devastated, were 12 quenched, as I think Dr. Zahalsky used the term, what would 13 happen? 14 A We would have no, we Would have no protective response 15 to infectious disease, and this is the kind of problem that . 16 happened. People who have leukemia, who has a bone marrow 17 transplant, have to go through a process by which all of the 18 bone marrow is destroyed, usually by radiation or by chemotherapy, 19 and then new bone marrow is transplanted or, yes, new bone marrow 20 is transplanted into those bones, which is a very painful process 21 but interestingly enough, and that new bone marrow will then 22 proliferate and will form these kind of cells that we are 23 talking about here and repopulate with normal white cells. A 24 person who is in that stage where they have had bone marrow ' 58 1 destroyed have had virtually all of their other immune system 2 destroyed. They are very vulnerable to infection and it is 3 necessary to protect them with great care. People who have 4 organ transplants are usually maintained on drugs that depress 5 the immune system very, very sharply. There are many such drugs, 6 and they depress the immune system to the point where that heart 7 is not being rejected or that kidney is not being rejected. If 8 a person is not fortunate enough to find an organ that is com 9 patible, they are in a continuous state of depressed immune 10 function, continuously vulnerable to major infection and have 11 to live arvery special kind of life in order to make that 12 possible, a great deal of protection, antibiotics a lot, and 13 so forth. And of course we occasionally read about the children 14 b o m completely without an immune system, and no one has solved 15 that problem yet, and they live in a bubble and not all that 16 long. They have to be completely protected from any contact 17 with other humans or any kinds of organisms, and their food is 18 sterilized before It is sent into them and so forth. They have 19 no protection. 20 V Q So that if an organism, an animal does not have a 21 catastrophic illness or if a scratch doesn't become blood 22 poisoning or if a minor infection doesn't advance to a very 23 serious infection, does that indicate that the immune system 24 is working? 59 1 A Yes. 2 Q Let me go next, Dr. Dost, to the kidney as an orgari, 3 if I may. Dr. Silbergeld testified on April the 12th, 1984, 4 Page 97, that the animal demonstrates that TCDD destroys kidney 5 cells. Do you recall that testimony? 6 A Yes. 7 Q Do you agreewith that? 8 A No. 9 Q Why not? 10 A Well, theanimal data show really just the opposite. 11 TCDD at survivable levels does not have an appreciable effect 12 on the kidney even over long periods of time. If I may refer 13 again to Kociba's work and other chronic studies, long term 14 studies, the kidney is not significantly affected. One of the 15 Interesting aspects of the work that Dr. Kociba did was that 16 he found that the normally occurring degenerative kidney 17 disease that occurs in all old animals was significantly 18 diminished at the high dose of TCDD. What that means, I don't 19 know, but it certainly does mean that there was no increased 20 incidence of kidney toxicity in those animals, nor was there 21 any at the lower doses. 22 Q Now, how exactly, I want to get back to these Kociba's 23 studies In just a minute, but how exactly does the kidney work? 24 You were talking before about organs in which -- that handle 60 1 large volume of blood and organs that have a lot of fat so that 2 TCDD can be stored in them. Where does the kidney fall in that 3 spectrum of types of organs in the body? 4 A Well, the kidney is our primary excretory organ for 5 soluble substances, and it would -- I think I'll draw a picture 6 if I might. This by no means is a precise rendition of a kidney 7 tubule, but the kidney is comprised of a large number of individual 8 units, kidney tubules, and the way they work is there is a filter, 9 any fat. The blood breaks up into small vessels and it comes in 10 and there is just a big clump of vessels here surrounding, that 11 is a kind of a cup, and it Is really a filter, and as the blood _ 12 goes through there, probably as much as ten percent of all of 13 the fluid in the blood actually goes through that filter. The 14 cells don't come through, the large proteins like the plasma, 15 albumin, which is a normal part of the plasma, and the immuno 16 proteins that we were talking about, all those large proteins 17 don't go through. This is a rather convoluted tube, but I'm 18 just going to draw it straight. And it is surrounded by cir 19 culation. And what happens is that all of the water, most all 20 of the water, probably 99.9 percent of all of the fluid that 21 went through the filter is brought back out. But in the process, 22 many of the materials that we don't want to bring back are left 23 in that fluid. And most of the materials that we don't want 24 to get rid of like glucose, you know, we have lots of glucose 61 1 in the blood. Normally about 100 million grams per 100 milliliters, 2 and that's going to come through here, too, because it is a 3 small molecule. So it goes down here and we reabsorb it back 4 out again if we are normal. Some people can't do that very well, 5 but we recover all of that glucose, unless we've just eaten a 6 candy bar or something like that, there may be too much to 7 recover, and some of it will escape. And then there are some 8 other cells that actually have the function of secreting certain 9 things into the urine, ammonia and so forth. But anyway, in 10 essence, it is a filter followed by a scavenging unit which 11 brings most of the fluid and valuable stuff back in, and those 12 thousands of units all converge in the center of the kidney. 13 And then from the kidney there is a tube that goes to the 14 bladder. So what we have here is a filter, and it deals -- 15 if we are talking about a toxic substance or any kind of a 16 foreign substance, if it is at all soluble in water, it is 17 delivered to this point and hopefully left in the urine and 18 disposed of. 19 Q Now, the thing that you have drawn, the mass of 20 blood vessels at the top is called what? 21 A It is called the glomerulus. I had nothing to do 22 with naming these. 23 Q All right, and the tube is called the tubule? 24 A This is the kidney tubule, and it has a proximal 62 1 segment, actually it comes in kind of an S shape, and this is 2 the distal segment and there are different functions in dif 3 ferent lengths of that tube, but -4 Q All right. Now, how many of those operating units, 5 let's say, the glomerulus and the tubule with its reabsorption 6 collar, and that sort of thing, how many of those are in the 7 kidney? 8 A Quite a few million. 9 Q So the blood is coursing through this thing? 10 A These are little bitty vessels here, you see, the 11 kidney circulation has divided into an enormous number of 12 vessels. This thing is only one cell in thickness, you know, 13 the wall of this tubule is only one cell. So there is really 14 a collar of cells, you might say. In other words, if you were 15 to -- took a tube from the end, it would look something like 16 this, you see. So it is a very, very small structure. 17 Q And so the blood courses through the kidney, does 18 the kidney receive Its own blood supply for energy and life 19 for the cells? 20 A Well, that's right. It happens, you see, all of 21 these tubular cells receive their circulation from the same 22 blood flow that is bringing, it is a very efficient system, 23 we don't have to have a whole separate plumbing system in 24 order to supply nutrient to the kidney Itself. The same 63 1 circulation that brings fluids to the kidney and carries wastes 2 in here and comes back down here, the same circulation it 3 brings out the fluid that we don't want to dispose of, provides 4 the energy resources for those cells. 5 Q All right. Now, when Dr. Kociba studied the kidney, 6 how many occasions did he do it? 7 A I'm not sure what you mean, how many. 8 Q How may separate studies were there in which this was 9 observed? 10 A Well, they didn't find -- he had done two studies, 11 one thirteen week study that I think I mentioned yesterday, 12 and then the long term, lifetime study that they did. 13 Q Was one of them in 1976? 14 A The short one was reported in 1976. The longer one 15 was reported in 1978, and they had a substantial number of 16 animals ahaeach experimental level, of course. 17 Q Now, what did the 1976 study show with respect to 18 kidney effects in animals? 19 A It didn't show any kidney pathology that I recall. 20 Q Were there any blood chemistry results? 21 A Oh, yes. They did a variety of blood chemistry, 22 and they didn't find, except -- I believe at the higher 23 dose they found some elevations in levels that would suggest 24 that might be interpreted upon the kidney, but there could 64 1 also have been effects upon the liver, too. 2 Q Now, you say the higher doses? 3 A They used a maximum dose in that study, in the 1976 4 study, of one microgram per kilogram per day. 5 Q All right, was that high enough to cause death in 6 some of the animals? 7 A It certainly did, 8 Q And at that high dose, there were some blood chemistry 9 results, is that right? 10 A Yes. 11 Q Did they do any kidney pathology on that occasion? 12 A Yes. 13 Q Did they find any pathological? 14 A To my recollection they found no pathology at all. 15 They found no change in the architecture or the shape of the 16 kidney cells. 17 Q All right, so that in 1976 Kociba did not show that i8 TCDD destroys kidney cells? 19 A That's right, and the same outcome emerged in the 20 study reported in 1978 where they had maintained it for two 21 years. 22 Q All right, now the 1978 was a chronic study for 23 the lifetime of the animal? 24 A Yes. 65 1 Q And how did the kidney effect In the 1978 study 2 relate between the dosed and the undosed animal? 3 A Well, I think I mentioned a short time ago that the 4 normal Incidence of degenerative kidney ailments, all, parti 5 cularly rats, as they age, have a substantial incidence of 6 kidney damage. The nephron here, this unit that we have been 7 talking about will just simply fail, and there will be areas 8 in the kidney where is scar tissue and a lot of the number 9 of functional units decreases with age. 10 Q That naturally occurs? 11 A That occurs in any, it occurs in all of us, of 12 course, but it is quite visible and easily quantifiable in 13 experimental animals because we are examining their pathology 14 all of the time. And the interesting thing in that particular 15 study was that the incidence of that kind of degenerative 16 image decreased at the highest dose, and decreased to an 17 extent that was statistically significant. 18 Q Decreased in the animal that had received the highest 19 doses of TCDD? 20 A Yeah, I'm afraid so. 21 Q Well, is there any evidence in the animal data that 22 the kidney is adversely affected by the 2,3,7,8 TCDD exposure? 23 A Well, really only at doses that have caused the 24 animal to collapse, and sometimes not then. That's where it is 66 1 seen, is at very, very high doses where the animal is probably 2 mortally affected. 3 Q At below that high dose? 4 A It appears that there isn't an appreciable impact on 5 the kidney. 6 Q All right, sir. Now, Dr. Dost, I'd like to talk next 7 about cancer. I wonder if you would define cancer for us. 8 A Cancer is a condition in which cells have lost their 9 regulation, usually have regressed in their differentiation. 10 By that, I mean that in all of our cells begin the same, and 11 as we develop, cells become, we use the term differentiation, 12 and that I think is self-explanatory. Eventually we have to 13 have a liver and we have to have bones and we have to have 14 kidneys, and as the animal develops, cells begin to take 15 direction toward the formation of the various organs, and so 16 as the embryo develops, the cells become more and more and 17 more different until finally we have a complete organism 18 with arms and fingernails and eyes and all of the structures, 19 but they all started with similar cells. In the case of tumor 20 cells, there is a tendency to go back part way down that 21 ladder and lose the differentiation. Now, they don't go all 22 the way back to the point where they are just like initial 23 embryonic cells, but cancer is a collection then of cells 24 that is reproducing by itself and forming usually a tumor 67 I that has some form, and it has been removed pretty much from 2 the formal regulation that the body exerts on Itself, These 3 cells have become autonomous. They are self-governing to a 4 large extent, so that they are no longer controllable and they 5 grow and grow and grow at a slow rate. But they grow all by 6 themselves, without regulation by the rest of the body* 7 Q Now, when you say regulation, how is it that the body 8 regulates a cell's growth? 9 A Well, we are really a very well-organized system 10 and mot only does the body regulate each cell's growth; but 11 the companions, all of the cells in the liver have a common 12 .mission, all of the cells of a given type, and they exert 13 regulation on one another. When they lose that regulation, 14 well, cells that have become a cancer cell may starts to .Lit! 15 multiply, and it's going to form a larger and larger colony 16 of cells that eventually it is Independent of the cel Id that 17 are around it. But we regulate our cells, we have horthones 18 that turn cells on and off. If we want to cause sugar to be 19 absorbed by cells, we release Insulin and that tells cells 20 to absorb the sugar. And that is a form of regulation that 21 arises from a central point. That particular phenomenaodoesn11 2? have anything to do with cancer except when you have a cancer 23 of the organ that produces Insulin, and there might very well 24 be for a while a great deal of insulin produced so that we have 68 1 caused a problem elsewhere in the body. Generally* the problem 2 with cancer is it reaches a point where it invades and inter 3 feres with the function or some of those cells may break off 4 and go to other parts of the body and continue to reproduce 5 by themselves without regulation. 6 Q What's the name of the latter condition you just 7 described? 8 A That's a phenomena known as metastasis, m-e-t-a-s-t-a-s-i-s, 9 and it simply means the movement of the cells from their origin 10 to some other location. 11 Q All right. Now, when you are talking about cells 12 developing, each cell nucleus contains, you mentioned a library 13 of material? 14 A Yes. 15 Q Called what? 16 A DNA. 17 Q Now, but you say it starts, the human embryo starts 18 from the joining of two cells, right? 19 A That's right. It starts from the fertilization of an 20 ^gg, and we have one cell, it divides Into two identical cells, 21 and they divide. Early on, you have a structure that looks 22 rather like a raspberry that's comprised of a substantial 23 number of cells that ate Alinidentical. 24 Q Now, is the DNA in the nucleus in each of these cells 69 ^1 the same? 2 A Yes. 3 Q Now, what about when the differentiation begins to 4 occur, when cells start to become part of the bone r part of 5 the foot or skin or hair or whatever it is? 6 All of the information is there, but by sme process 7 that isn't really well understood, cells only utilize part of 8 the Information, the part that's important to that:function. 9 So if we have a liver cell, that is all the same information 10 that kidney cells have, but they don't use the part that's 11 related strictly to the kidney cells, you see. 12 Q And the kidney cell doesn't use the part of the DNA 13 cell that relates to what the liver parts use? 14 A Exactly. 15 ^ Q So the DNA is the same in every cell? 16 A Yes, it is a question of which book we take off the 17 shelf * 18 --.Q Now, what does DNA have to do with cancer? -' 19 ; A Cancer is generally believed to start with some kind ,20 of ah injury or a lesion on DNA, so that DNA is changed and 21 not repaired, and that that change remains and somehow it 22 remains part of the genetic equipment in the cell, ah<d ultimately 23 is triggered to develop Into a tumor cell. It is a very long, 24 complex process. It ddesn't just happen. 70 1 Q Have you develop a chart which would assist you In 2 explaining this? 3 A I have a chart I use in lectures for that very 4 purpose. 5 (Defendant's Exhibit 1285 was marked for Identifi 6 cation 7 Q Dr. Dost, let me show you what's been marked as 8 Defendant's Exhibit 1285 and ask you to examine that and 9 identify it for me, please. 10 A It is a -- it is the chart that I brought with 11 me that I use in describing the process. It is a general 12 description of the process by which a cell eventually can 13 become a tumor. And as it points out, it is hypothetical 14 steps, this is generated not from a specific body of research 15 data, but it represents the present understanding of the 16 process. All right -- 17 Q Would you step up and use it, please? I am interested 18 in the term "hypothetical." 19 A Well, no one has determined fully the process by 20 which cancer is caused. That probably is the most, prbbably 21 it is the most heavily investigated problem in biology at the 22 prsent time is to try to establish just how cancer Is caused. 23 There is a great deal known about how it is caused, there is 24 a great deal known about intermediate steps. But unfortunately 71 1 the process seems to vary with certain kinds of cancer, and it 2 also, and also there are some very specific and important pieces 3 of information that simply are not yet known, so it would be 4 presumptuous to say that this Is the way cancer happens. It 5 does, I think, represent quite properly the general scope and 6 sequence of the process. So these are all, these are generali 7 zations. They do not relate to some specific tumor type, nor S do they relate to any specific pattern that someone has laid 9 out. But the first thing that happens, you spoke of DNA, and 10 the first thing that happens is apparently a mutation of some 11 other damage to DNA in the cell, and that affects some part 12 of the genetic apparatus. Now, there is a concept of the 13 oncogene that has crept into the popular press, and it is a 14 very important issue of discussion among cancer researchers, 15 and that is the idea that there are segments of DNA, genes, 16 that by some mischance with very minor changes can be converted 17 into a gene that actually leads to cancer. In other words, the 18 Idea is that we have in all of our cells genes that require 19 very little modification to touch them off and cause them to 20 take control of the cell, so to speak, and cause cancer. In 21 any case, there still has to be some kind of a change in the 22 DNA to start this whole process. And that step probably is 23 irreversible. But other things have to happen for it to be 24 expressed, and when I say, by express, I mean other things have 72 1 to happen for it to take effect. Now, one of the things that 2 apparently have to happen is that the cell has to divide, and 3 In dividing it, copies of its DNA for the second generation ' 4 cell. So that there is two identical sets of DNA. And if 5 that happens , the speculation is that probably it takes two 6 cell divisions in order to do that for that to be coaled. So 7 at the same tim X want to step back for just a moment. We 8 have talked about a DNA lesion, that is irreversible There 9 Is damage occurring to DNA all of the time in the cell, naturally, 10 and as a result of everything that we do in our environment. 11 And this lesion has to survive this repair process, and I 12 probably will have an opportunity to discuss the ide of DNA 13 repair a little later. 14 Q Dr. Dost, excuse me, when you talk about our environ 15 ment/ what are you referring to specifically? 16 ;A Diet, everything we do, our ultraviolet radiation 17 from the sun, our environment is kind of hostile plac, and 18 we are taking in all of the time a lot of genetlcally ;active 19 material in our diet. If you were to go through everything 20 that you eat and identify all of the substances that are 21 known to have some kind of genetic activity as determined 22 In microbial mutation tests and so forth, you would soon be 23 searching trying to find out what in the world you cbuld eat 24 that will sustain you without creating a-problem. Apparently, ; t 73 1 we are not creating problems by eating our daily meals because 2 our life span is increasing, but the fact remains that there 3 are lots and lots and lots of materials in the natural environ 4 ment, both physical and chemical, that has capacity to at least 5 theoretically cause injury. Not only that, within the cell, 6 the intercellular environment is also very violent and, there 7 are lots of very reactive molecular species created in the 8 norifcal metabolism in the cell. Perhaps I can talk about that 9 a little later, too. 10 Q r 11 mean? When you talk about genetically active, what do you 12 A I mean it has the ability to interact with DNA. 13 Q Now, we've heard talk here about DNA and RNA, and 14 messengers and all that sort of thing. What does all that 15 mean? Is DNA a ribbon or is it a piece of -- 16 A DNA is, okay, DNA is the primary source of Information, 17 The RNA that we have talked about, there are a number of kinds 18 of>RNA, but the only number of RNA that we have particular 19 concern about is that is made as a copy of a segment of DNA 20 and goes out into the cell to the ribosone or to endoplasmic, 21 reticulum to form a pattern for the synthesis of some protein. 22 DNA is a very, very, very long molecule. There are probably 23 -- it is comprised of pairs. I may have to stop and go back 24 now and then because this is a little complex, but DNA, you 74 1 may have heard of the double helix .which was a book that 2 Watson and Crick wrote about thirty years ago when they 3 finally discovered what the configuration of DNA was. 4 Q And won a Nobel prize? 5 A And they won a Nobel prize. The DNA, X guess the 6 best way to describe it is to think of a zipper, an ordinary 7 zipper. And you know that a zipper has teeth and they come 8 together like so. Well, DNA is set up as pairs of bases, 9 and I don't need to describe what these bases are, but there 10 are four kinds in DNA. And I guess we could say that those 11 four bases ar the letters of the genetic alphabet, and you 12 can imagine that there is an unimaginable -- that's not very 13 good language -- there is an enormous number of possible 14 combinations because these molecules are very, very long. 15 They carry an enormous amount of information. And if we think 16 of this as being, as three, four letters in this alphabet, and 17 there are words in the DNA and they are all three letters long 18 and each of those words codes for an amino add. Now, /when we 19 go through this process of Inducing, which we've talked about, 20 the DNA opens up and we form SNA as I've just mentioned. And 21 that RNA carries the same code, this series of three letter 22 words. It Is very long and it codes for these thousand or 23 two thousand amino acids that come together to make this protein. 24 Q So it unzips it? 75 1 A Unzips, half of it comes apart and the RNA is copied 2 off the other half, or it comes apart and RNA is copied off of 3 one half. But if you think about this zipper, for every 10 4 teeth it has taken one twist, this thing is twisted, and I 5 mention the number of bases. There are in the cell about, 6 between 10^ and 10^ base pairs. In other words, in each cell 7 there are, let's say, 10 words or letters, how would I put 8 that? No, we will divide it into three and make words. We 9 will say there are 3 times 10^ words of information in each 10 cell. That's 30 million, probably more than that, but the 11 point is that in these rather interesting three letter words 12 are the codes for all of the amino acids. And so it is the 13 sequence on the DNA that governs the sequence of synthesis 14 out in the cell. All right, if there is an error, if there 15 is an error in that sequence, then incorrect information is 16 going to be transmitted. Now, that may not make any difference, 17 it may be a serious error or a big chunk of DNA is lost and -18 the: cell dies. That, fortunately, is not an unhappy1circum 19 stance except in the nervous system, but it is happening all 20 the time. In this situation we have Inverted some kind of an 21 Incorrect message. All right, it may stay that way forever 22 and nothing ever happen, but if the cell divides bbfdre it is 23 repaired, that lesion may be thick in the gene so that:;it stays 24 there and the cell then is hearing that message. Well then. 76 1 there are a number of processes that go forward that come into 2 the category of promotion and enhancement. Some of these words 3 are used interchangeably, sometimes you find progression used 4 here and sometimes translation. This question of cell trans 5 formation falls up here aways, but let me read this to you. 6 "It is assumed that specific physiological or biochemical 7 phenomena are required to facilitate the expression of the 8 initiating event." Now, that may be cell injury, there are 9 certain chemicals that are known to promote this effect. I've 10 mentioned the phorbol esters the other day when we were showing 11 that chart on the difference between promotion and initiation. 12 TCDD, phenolbarbitol, smoke condensates having been suggested 13 as promoters. And the promotion process appears to be reversible 14 at least in its early stages, in other words,if it does take 15 place before some of the other events going on, why then that 16 cell may just revert back to normal. It also is clearly dose 17 dependent and clearly there is argument about whether it is 18 a threshold or not, but it Is very clearly dose dependent. 19 Then, the next step Is what is called cell transformation, and 20 the cell changes its whole genetic makeup in some fashion and 21 it does different things than it used to do. It becomes 22 juvenile, if you will. It reverts back to a less developed 23 form. And one of the problems that It may or may not be 24 recognizable as a strange cell, as a different cell, and if it 77 X is recognizable, then the immune system will clean it up. And 2 this is probably going on in our bodies all the time. From 3 this point forward, those cells when they reproduce they carry 4 all of these bad characteristics with them, and they begin to 5 reproduce. If they do evade the immune surveillance and they 6 could very well repeat characteristics of the normal cell that 7 are sufficient that the immune, the cellular immune cells, the 8 killer cells don't recognize them as being tumor cells or nearly 9 tumor cells. Well, so there is a progression here where we go 10 from a genetically injured cell to a cell that has to be further 11 stimulated in some fashion. And then from that we go to cells 12 that are producing, changing their character and eventually 13 becoming a sufficient population of cells that either they 14 form a tumor that can be seen or they may form a tumor in the 15 bone marrow that is recognized as, say, leukemia or something 16 like that, which is a circulating tumor in effect. There is no 17 mass, no hard tumor. I don't know whether I have made that 18 sufficiently clear, it is a very muddy process, and there a 19 lot of different -- there is a lot. of alternatives descrip 20 tions that can be Inserted. 21 Q I'm interested in the initiating event that you've 22 got there at the top. I'm not sure I understand how this 23 occurs. Is this something that has to come in from the 24 outside, or is it something that naturally occurs in the cell 78 1 because you talked about DNA repair and errors in the cell before. 2 A Well, DNA is, to start with, we have an enormous 3 amount of material that has to be manufactured, and we considered 4 that the manufacture of DNA in the cell is a very accurate process. 5 But the error rate is about one in every million base pairs. Well, 6 I've already talked about a situation in which we've got what, 7 10, that's 100 billion, I beg your pardon, a 100 million base 8 pairs in the cell. And if we are, actually the number is higher 9 than that, it is probably about 300 million. And if we have, 10 if we have an error rate of one in a million, that means that 11 for the growth of a single cell there will have been 300 errors 12 just in that cell alone. Just in manufacturing DNA. And that 13 has to be corrected. There is a repair process that Inspects 14 DNA. It is fascinating. It literally Inspects it, like going 15 over a string of beads and find defective spots, slices them 16 out and fixes them. Now, that is not a perfect process. Some 17 repair may have been missed or there might be a mistake made in 18 the repair process, but if you consider that there are 300 19 errors in a given cell and if, oh, in the liver there is some 20 thing in excess of one billion cells per gram, so let's say 21 10^ cells per gram for a gram of liver, which is maybe would 22 be a piece that size, perhaps, there would be 300 billion errors 23 committed in the process of making the DNA for those cells. ii . 24 So this process is going on all the^ time of error in synthesis. 79 1 The other thing that is happening is that, I 111 just write that 2 down, the other thing that's happening is, as I said, the cell 3 is a very violent place and there are some very violent reactions 4 going on in the cell in the process of generating energy. There 5 are some very reactive oxygen atoms that are generated in the 6 cell, and most of them are trapped and are, don't cause any 7 harm, but enough of them get away that there is a Very signi 8 ficant and measurable amount of DNA damage by some of these very 9 reactive oxygen species inside the cell. And peroxide, hydrogen 10 peroxide is made inside the cell, and there are enzymes that get 11 rid of that stuff. There are traps that collect it, but some of 12 it inevitably escapes and the rate of injury of cellular DNA 13 can be measured by the collection of some of the products, be 14 cause as I said, they slice out pieces of DNA and bring them, 15 they are excreted into the blood and into the urine add they 16 are disposed of. People in Bruce Ames' lab at Berkley have 17 done some interesting work on this, 'and what they find is those 18 products emerge at a rate that Indicates cell damage to the 19 extent of 320 DNA damaging events per cell per day* ihid when 20 you multiply that by the number of cells in the body, which has 21 been estimated, there are various estimates, but the one I 22 generally use is 6 times 1 0 ^ cells, which is 60 trillion cells 23 in the body. Then we have an enormous number of events taking 24 place in the cell every day in the body, every day that can 80 1 be potentailly leading to genetic damage of some kind. Virtually 2 all are repaired. Now, if the system was absolutely perfect, 3 we might not have cancer. But unfortunately the system is not 4 absolutely perfect and there are inevitably some errors^in DNA, 5 some damage to DNA that persists. Now, also, external functions. 6 Irve talked about some of the mutogens that are in natural sub 7 stances, black pepper, broccoli, and when I say broccoli, I don't 8 mean to isolate it, I think we can say that about most vegetables, 9 don't worry about it. Carcinogens, bena'a'pyrene in automobile ^ 10 exhaust, or gasoline, benzene, lots of chemical carcinogens 11 that can also result in this DNA damage that serves as the 12 initiating step. If the concentration is high enough it can 13 cause these kinds of events, too. So we have all kinds of 14 things happening, very high repair rate that presumably is 15 not absolutely perfect, so you can see, there is an incredible 16 amount of activity going on in the cell all of the time that 17 relates to the potential for DNA damage in this initiating 18 event. 19 THE COURT: Mr. Heineman, is this a good point for 20 S short break? 21 MR. HEINEMAN: Yes, sir, it will be fine. 22 THE COURT: We will take a short break at this time. 23 The admonishments that I gave you earlier will apply during 24 this break also. (At this time Court was in recess.) 81 1 (Defendant's Exhibit 1286 through 2 1289 were marked for identification.) 3 BY MR. HEINEMAN: 4 Q Dr. Dost, for the record, I have had these sheets 5 marked. Defendant's Exhibit 1286 is the calculation of the gram 6 molecular weight of dioxin. 7 A That's correct. 8 Q Showing of Avlgodrll's number at the bottom? 9 A Yes. 10 THE COURT: What was that, 1286? 11 MR. HEINEMAN: 1286, your Honor. 12 Q 1287 Is the calculation of thetotal number of cells. 13 A Total number of molecules. 14 Q I'm sorry, total number of molecules, andwhat? 15 A Well, I made an error when I calculated the number. 16 First, we are talking about the number of molecules In one 17 mlcrogram, and then the number If we are talking about a 18 thousandth of a mlcrogram, and- then I carried that calculation to 19 the number of molecules that there would be In a 70 kilogram person 20 and I had made an error in the exponent and 1 had a thousand times 21 more than It should have been, so it should have been 132 trillion 22 rather than 132 thousand trillion. This is not like Senator 23 Dirksen's famous saying, "A billion here and a billion there and 24 you are talking real money." We need to be a little more precise. 82 1 So the number Is 132 trillion, not 132 thousand trillion. 2 Q Okay, and that's exhibit 1287? 3 A If you want, 1 will put per 70 kilogram person. 4 Q All right. Then .1 have marked as Defendant's Exhibit 5 1288 the drawing you did of the-- 6 A Kidney tubule. 7 Q Kidney tubule. You referred toit as a nephron? 8 A A nephron, yes, that's the word for the whole unit. 9 Q And 1289 Is the calculation you did for what, sir? 10 A For the number of natural errors In the formation of 11 the DNA in cells as cells grow, as tissue grows, and then a 12 statement of Dr. Bruce Ames' finding of about 320 DNA or finding 13 the residue that has arlsed from the repair of about 320 damaging 14 events per cell per day, and the number below Is the number of 15 cells in the estimated In the human body, 60 trillion. Perhaps I 16 should identify that number so in the future -- 17 Q All right, thank you. 18 HR. HEINEMAN: Your Honor, at this time we would 19 offer into evidence Defendant's Exhibits 1283 through 1289. 20 (Defendant's Exhibits No. 1283 to 1289 were 21 offered into evidence.) 22 HR. CARR: The Plaintiffs have no objection to these 23 exhibits, your Honor. 24 THE COURT: Fine, they are all admitted without 83 1 objection. Thank you. 2 (Defendant's Exhibits 1283 to 1289 3 were admitted into evidence.) 4 Q Dr. Dost, if you would resume your place there by 5 your chart, discussing;.the mechanisms of cancer, you did some 6 detail on the initiation, and to make sure 1 understand it, the 7 initiation can be an internal event7 8 A It is an Internal event in the cell. 9 Q All right. But it can be caused perfectly naturally 10 without any outside intervention at all? 11 A It can be caused, well, we assume that all of the 12 events that take place in the cell that damage DNA also have the 13 capacity to cause a DNA mutogen. Now, how does that relate to the 14 event of initiation that you are describing? 15 A Well, first I should point out that a cell has 16 initiated does not behave any differently in the organism than a 17 cell that has not been initiated. So its function has not 18 changed, its function hasn't changed, and it appears that, even 19 when it divides, while that Initiating lesion has stayed In the 20 cell and then carried to the next cell, the daughter cells also 21 will not function any differently than the normal cell. So 22 we have got a cell here whose nature hasniCteallybeehychariged in 23 terms of what it does in the organism. Other events ate necessary 24 to make that happen. 84 1 You asked about mutation. Mutations are changes In the genetic material of a cell such that the cell In either 2 Its next generation or possibly some subsequent generation will 3 be altered. Generally when we are speaking at the cellular level, 4 we are talking about a change that appears In the next generation. 5 6 If we are speaking about the whole animal, then we would be discussing a change that is Induced In germinal tissue In a 7 8 reproductive organ, and then again there Is some complex genetics 9 Involved here. It could very well be that offspring of the parent 10 with damage might be similarly affected or if it Is a recessive 11 change of some kind, It may appear several generations down the 12 family tree, In an adult organism. Now, we use, It Is thought 13 and again pretty universally thought, that mutation, the event 14 that leads to mutation are rather similar to the events that 15 lead to the initiation of cancer. And It is possible for us 16 to study those kinds of events In the. laboratory. Microorganisms 17 are very handy for this sort of thing, cultured cells. So 18 there are a variety of laboratory, of fruit flies, there are a 19 variety of systems available in the laboratory for learning 20 whether a chemical has the capacity to interact with IfNA, It 21 happens that there Is a good correlation between the ability of 22 a chemical to Interact with bacteria and other bench-top kinds 23 of tests and the ability of chemical to cause cancer. Generally 24 chemicals that do not cause mutation does not cause the initiating 85 1 kinds of events* 2 Q Well then, as I understand mutation or mutogenlclty 3 le not the same thing that you are talking about here? 4 A The initial event Is probably very similar, just 5 the initial event. The only difference Is that it's likely 6 that in mutogenosls there is sufficient change that the next 7 generation of the cell la changed, you see, and Imaginably we 8 could have cells in, let's say, the liver that had a minor 9 mutation, and they functioned a little differently, and we 10 may accumulate a few cells of that sort In the liver, but It 11 wouldn't matter because It Isn't transmitted to the next 12 generation, and those cells don't necessarily make a tumor, 13 they are just a little bit different from the cells, the normal 14 cells of the liver, and that presumably happens frequently. 15 But the carcinogenic event may be sufficiently 16 specific that it only can be transmitted In this fashion to the 17 next generation of cells. The point about mutogenlclty, though, 18 Is that apparently the damage in the cell is about the sam, 19 and there Is an empirical correlation, by that I mean the 20 experimental evidence shows that chemicals that are mutogens 21 are frequently carcinogens. Okay, now, precisely what is 22 happening at the level of DNA In the two cases to link them 23 is not clearly known, but we know that DNA damage takes place 24 In both instances, and that there is a correlation. Chemicals 86 that cause one generally cause the other. 1 2 Q But Is the opposite always true, or equally true? For example, when you talk about whether or not TCDD is a mutogen, 3 as affecting the determination of whether It Initiates cancer, 4 what do the two have to do with one another? 5 6 A Well, It is interesting, because the likelihood that a chemical that Is not mutogenlc In modern test systems or 7 8 a chemical that Is not mutogenlc Is not likely to be a carcinogen. There Is a better correlation from negative information than to 9 10 positive, because not all chemicals that are known to be 11 mutogens are In fact carcinogenic. Many are, and if we 12 find a chemical that is mutogenlc, knowing nothing more about 13 It, we have to assume that that is a potential for carcinogenic 14 activity until It Is proven otherwise, and this is one of the 15 bases by which chemicals are selected for a carcinogenicity 16 assay, because It Is so expensive to do those kinds of tests 17 that we simply can't put every chemical that we have through 18 this very long, elaborate carcinogenicity test that costs several 19 million dollars. It Is necessary to select chemicals which are 20 likely to be positive, 21 Q All right. Mow, if you would, please, I am a little 22 confused about, a little confused, I am confused about, this 23 fixation stage. What happens between the initiating eveht 24 and fixation? 87 1 A Fixation Is just a term that Is used to represent 2 that after, If damage occurs In the cell to the genetic 3 material In the cell, and Is not repaired before the cell 4 reproduces. Our cells all are reproducing at some rate. We 5 have cell division going on In the body all of the time. Nerve 6 cells do not. But cells everywhere else are making new cells 7 at a slow rate. This is not going on at an enormous rate, but 8 It is going on all of the time. If the cell divides before 9 that lesion is repaired and some biologists believe that it 10 has to go through two sequential divisions, but we will just say 11 if the cell divides before that lesion Is repaired. The lesion 12 then becomes locked into the genetic structure of the cell 13 and can't be repaired, and so it Is there. 14 Q And the comment beside the term fixation `uses the 15 term genome, fix the genome of the cell? 16 A What we have done is put information into the genome, 17 in other words the genome Is just the total genetic pattern of 18 the animal and of the cell. And what has happened is that 19 information has been put into the genetic structure of the cell 20 that changes, potentially changes the way it behaves. It Isn't 21 such severe damage that it alters the nature of the cell and 22 causes It to die, It is still functional, but it is sitting 23 there as a potential tumor cell. Now, It isn't a tumor cell, 24 probably won't be one, but there is always the probability some 88 1 slight probability that It can. So we have cells In the body 2 that are, that have been changed so that they are able to be 3 transformed or promoted and changed Into active tumor cells. 4 Q And this again happens whether or not there has been 5 some Influence from the outside. It can happen purely naturally? 6 A Yes. 7 Q And cells like this can be in the body and nothing 8 ever happen? 9 A Yes, that's correct. It appears that In mice there 10 Is a very high population of Induced or Initiated cells In the 11 liver and mice make a very poor model for determining the 12 ability of chemicals to cause liver cancer because they,have a 13. very, very high incidence of liver cancer anyway, Just they are 14 Very easy to throw into that biological state. 15 Q Because they have so many-16 A They've got many initiated cells Just by virtue of 17 being alive. Fart of this is because the metabolic rate in 18 those very small rodents is very, very high and so there is a 19 high frequency of damage and there Is a, they have a relatively 20 inefficient DNA repair system, much less efficient thap human. 21 That's a parameter that can be measured. 22 Q Now, are all of these steps necessary before a 23 malignant tumor results? 24 A Yes. 89 1 Q All of them are necessary? 2 A All of them are necessary. 3 Q All right. 4 A And all of them, there Is, all of them presumably 5 can be Interrupted at some point until we get down here 6 where <we have cells that have actually evaded all of the defense 7 systems. 8 Q And now, can there be a material that Is a carcinogen, 9 for example, that Initiates cancer that will lead to a tumor 10 without the need for a separate promoter? 11 A There are some chemicals that are considered to be 12 complete carcinogens. That is that they will not only cause 13 this kind of event, but If they are sustained in the organism 14 will also apparently carry through the promotion function and 15 will by themselves result in a certain tumor Incidence* V;" 16 Q Well, I want to get back to that In a minute * The 17 promotion aspect, what Is it Chat happens in promotion?. 18 A There Is still aiiibt of questions to be 7&ii0ified 19 about promotion, but it appears that one of the chatadteristlcs 20 of promotion is that cells ate stimulated to proliferate. Cells 21 become more active, they become-~they in some cases dell ^division 22 takes place, the kinds of response that hakes place lh response 23 to injury* As a matter of fact, there are certain kinds Of skin 24 tumors that can be promoted simply by cutting the skin th the 90 region of the application of the carcinogen, and by applying the 1 carcinogen and leaving It alone, nothing happens. If you wound 2 the skin, you get tumor formation. 3 Q Okay. So it la something to enhance? 4 A In some fashion, It enhances. Now, In the simplest 5 6 sense, maybe It is simply something that Increases the rate of 7 multiplication of the cells and sort of magnifies this error that's 8 already shown up. The biochemical mechanism of promotion la 9 not clearly worked out yet. ' Q Okay. Now then, what Is transformation? 10 11 A , Well, transformation Is a process by which these 12 cells change their character. Up until this point, you probably 13 wouldn't be able to identify the cells as being all that much 14 different from the cells from which they arose. But In the 15 process of transformation they change their shape. They change 16 the way they carry out their business, they do things differently 17 and they become less, they become more primitive compared to 18 their very highly specialized form of the cells from which they 19 arose. 20 Q Now, is there a dividing going on all during this 21 period of time? 22 A Well, yes, and probably the process of cell division 23 becomes accelerated at this point to some extent. 24 Q And the new cells that begin at that point,; do they 91 have to go through the process or do they begin that transformation? 1 A Well the new cells, you mean cells that start up 2 here? 3 Q No. 4 A At here, at this point? 5 Q If they are dividing at the point of transformation. 6 A They are dividing, and the cells that arise from 7 that division are also cells that are more primitive. In other 8 words, if we have a population of cells here some place that 9 don't really resemble the original cells, and they are 10 reproducing on their own and they are making new cells just like 11 12 themselves. Q All right. And then the tumor arises as a result 13 14 of that? 15 A Eventually there are enough cells that we would 16 call it a tumor. 17 Q Now, eventually? 18 A Eventually. 19 Q How long does thattake? 20 A Often a long tim. We talk about the latency period 21 for tumors. Fart of that is just .simply the time it takes for 22 a tumor to reach a size that it can be seen. But it ale means 23 the time that these events take, which are not instantaneous. 24 But If we just consider the population of cells, tumors have a-- 92 generally tumors have a doubling time, so called. That Is 1 the number of cells double In a period of-- In most cases say 2 from 30 to 90 days. Okay, In other words, if there are two 3 cells at 30 days, there will be four at 60 and eight at 90, et 4 cetera, et cetera. If it was a 90 day doubling time, we would 5 6 have two cells, let's say the first division took place at the first of the year. There would be four cells in March and eight 7 cells in June and.16 cells in September and by the end of the 8 year there would be 32 cells. Now, that's not very fast. 9 Thirty-two cells are not very many when you consider, you know, 10 I mentioned that something may be a cell mass this big weighs 11 12 probably less than that, weighs about a gram, and there are a. billion cells in that. Sometimes tumor cells are larger, so 13 14 there would be fewer cells. But you can see that It would take 15 a long time to build up a mass at a rate, at a doubling rate of 16 say 60 days. It would probably take five years before you could 17 find that tumor if you were looking for it, you see, and to 18 have a tumor suddenly be noticed with a doubling time like that, 19 it has probably been there for quite a long while before somebody 20 detected It. 21 Q Now, I'd like for you to talk about the difference 22 between an initiator and a promoter, and is there a chart that 23 would help you explain that that we brought with us here today? 24 A We showed one, did we not, the other day? There was 93 1 a chart, maybe not. We have a chart that described the difference 2 between initiation and promotion. 3 (Defendant's Exhibit 1290 was marked 4 for identification.) 5 Q All right, sir, let me show you what's been marked 6 as Defendant's Exhibit 1290 and ask you to examine that and 7 identify it for me, please. 8 A That's a chart that has been taken out of a paper 9 by Tom Slaga that was published in Environmental Health 10 Perspectives, Volume 12, in 1985. It is a really standard 11 representation of the difference between Initiation and 12 promotion. 13 Q And Dr. Slaga is with what organization? 14 A He was at one time with the University of Washington, 15 and I believe at the present time he is at the National 16 Institute of Environmental Health Sciences, although X am not 17 absolutely certain. I haven't visited with him for a while. 18 Q Now, would this chart help you explain .this \ 19 process? 20 A Yes, it would. The black square, and the black 21 squares of Various sizes represent an initiator, a. chemical 22 that can cause this, an early lesion in the cell, the eat*ly 23 lesion in the DNA. These arrows represent doses o f a p r b m o t e r . 24 Now, in the first situation we have a very high dose of the 94 1 initiator, and that very high dose of the initiator, even though 2 nothing has happened, nothing has been administered after that, 3 does cause some tumors. A much, much smaller dose, however, 4 causes no tumors If we have a small dose, and then follow it 5 with several smaller doses of the initiator, we may very well 6 get tumors. Most initiators have some capacity to carry this 7 process on if the initiator id continued in the system. Now, 8 if we take a still smaller dose of an initiator, we get no effect. 9 Now, we have a promoter, and we give that promoter that substance 10 that, and many, many chemicals will do this, that cause this 11 series of events in here between the time the lesion Is locked 12 into the cell and the time that the cell goes out of control, we 13 give that by itself,inothing happens. They won't cause tumors 14 by themselves: If we administer a small dose of the initiator 15 and follow it with a promoter, and these are representing several 16 doses over a period of time, then we get a substantial number 17 of tumors,, compared, for example, with this same dose of 18 Initiator, and no tumors at all without a promoter. Now the 19 effect wears off a little bit with time. If we do the/same 20 experiment and wait for a period of time, perhaps a year or so, 21 to give the promoter, we still will get tumors. Th reason for 22 that is because those initiator cells stay there. There\hs 23 been nothing to remove them, they are there, they are ihctioriing 24 as part of the liver or whatever organ and they are still In that 95 1 state of, I suppose you would say genetic readiness to become 2 a tumor cell. If we should give the promoter first and then 3 the initiator, nothing happens, nothing happens. If we give 4 the initiator and we give less frequent doses of the prohlblter, 5 you may get a response. If you give still less frequent doses 6 of the promoter, no response. Now, what this shows Is just 7 simply, it is a generalization that describes the relationship 8 between substances that initiate the tumor process and those that 9 promote it and they are separate functions. 10 Q Now, Dr. Dost, do you recall what Dr. Zahalsky 11 testified on the 3rd of May, 1984, that exposure to TCDD as a 12 promoter prior to initiation would cause cncer? 1 13 A Tes. 14 Q All right. Do you agree with that statement? 15 A No . 16 Q All right, why not, sir? 17 A Well, he has stated it upside down. If this Is 18 the situation that he is talking about, the promotet; $ $ xself 19 or prior to an initiator doesn't do anything. The definition 20 of the terms makes it very clear which is the first step in the 21 process afad which is a subsequent step. Promotion, initiation 22 is an action of some kind that starts a process. Promotion 23 is some kind of activity that causes a process to gvfrWrd. 24 Q And so if you put the promoter on first before the 96 1 initiator? 2 A Nothing happens. 3 Q And is that demonstrated by the data upon which that 4 table is based? 5 A That's demonstrated by a great deal of data. This table 6 does not represent specific data from a given experiment, it 7 is a representation that has been generated from a great deal 8 of experimentation in this general area. This is intended as 9 a generalization, but this is the situation that represents 10 the application or administration of a promoter prior to an 11 initiator, and there isn't going to be a carcinogenic result. 12 Q Now, what does that chart dmonstrate with respect to 13 a dose response relationship for initiation? 14 A Well, there is a dose response relationship for 15 initiation. We think of it, though, not in terms of the 16 intensity of the response as we would in other kinds of events, 17 but rather the frequency with which the response will take place 18 because cancer is in effect an all or none situation in which 19 if the cell undertakes this.process that we've described, a 20 tumor will at least potentially eventually form. So a tumor 21 can eventually form from a single cell, at least in theory, 22 and probabjty In fact, because It has to start someplace* There 23 is nothing, we don't deal with the issue of whether i.is a 24 big tumor or a little tumor, it is either a tumor or it is not. 97 1 The cancer has either been caused or It has not So we talk 2 about the incidence or the frequency with which cancer occurs 3 in a population so that,we relate that to the number of animals 4 that have been exposed and the number of animals that;have gotten, \ 5 that show tumors. And yes, there is a dose response. As the 6 dose increases, more animals in a population, if it is a carcinogen, 7 more animals in the population will show tumors. Now, it is 8 possible with certain kinds of tumors that occur in large 9 numbers in an animal to also deal with this in terms of number 10 of tumors per animal, et cetera, but that's really another form 11 of frequency. So we are talking about the frequency with which 12 this event occurs and that is dose related. 13 Q- The first two items on the chart, you show the 14 big dose and the small dose? 15 A And I think we can say, yes, this in itself is a crude 16 dose response relationship in that the big dose caused tumors 17 and the little dose didn't. However, we wouldn't know the shape 18 of the dose response curve, all we would know is where the upper 19 end is. 20 Q Now, what do the studies show with respect to a 21 no observed effect level for promotion? 22 A For promotion, there appears to be a no effect level. 23 Q All right, and are there any particular Studies 24 upon which that conclusion is based? i 98 1 A There Is a substantial amount of research on the 2 question of promotion. The data show very clearly the dose 3 response clearly Is shown and there are no effect levels shown. 4 Now, the difficulty Is that there has to be some assurance that 5 there were not some Initiated cells there In the first place, 6 and so most people who discuss this Issue believe that there Is 7 a threshold, but they are not prepared to prove It, okay, and 8 I think that's the best way to describe It. It Is very clear, 9 however, that the dose response Is there and In experiments In 10 which the doses that show no effect are a long ways away from 11 doses which show an effect. It Is fair to say that there Is 12 In fact a threshold. 13 Q Well, what Is the consensus with respect to TCDD? 14 Is It a promoter or Initiator, or what? 15 A The consensus with respect to TCDD Is that It Is a 16 promoter. 17 Q And whose work demonstrates that? 18 A Allen Poland has worked In this area and has shown 19 with various strains of mice that TCDD Is a promoter. Henry 20 Pitot at the University of Wisconsin, In fact, they are both 21 at the University of Wisconsin, has also worked very extensively 22 In this area. In fact, that's his primary area of research. 23 Slaga has worked In this area and has also shown that TCDD Is 24 a promoter. The other evidence arises from the nature of the 99 1 carcinogenicity studies on TCDD In that the nature of the 2 tumor pattern, the behavior of the systems Indicate quite 3 strongly that TCDD Is In fact a promoter and Is probably not 4 an Initiator. 5 Q Now, what Is the evidence that demonstrates that? 6 Is TCDD a mutogen? 7 A TCDD Is not a mutogen. There were some studies 8 In the early 70s In which TCDD had some mutogenlc effect. No 9 one has been able to reproduce that Information, Including In 10 one case one of the people who had done the work, J.P. Seller 11 had made these kinds of findings In the early 70s and himself, 12 he himself has not been able to find that kind of evidence again, 13 and there Is a good reason for this because our systems for 14 detecting, for managing the microorganisms, our system for 15 detecting mutogenlc events Is much more precise than It was 16 even ten years ago. 17 Q You referred to microorganisms, were these early 18 tests in bacteria or something? 19 A Yes, these are the so-called Ames tests In which 20 an organism, usually species of salmonella, these are often 21 found In the digestive tract, they are common organisms, but a 22 mutant has been derived that cannot live In the presence of or 23 without one of the essential amino acids. Okay, It Is a mutant 24 and It Is susceptible to being mutated back to an organism that 100 1 can live without the particular amino acid, In this case 2 histidine. Now, there are a number of strains of organisms, 3 well, the trick Is very simple. If you have organisms that 4 have to have histidine to live and you cause a mutation, you 5 treat them and you transfer them to a medium that has no histidine. 6 Organisms that have been mutated back and can use histidine or 7 can make their own histidine will survive on the new media 8 and the ones on the old media, the ones that didn't mutate , 9 can't survive. The concept Is very, very simple and there are 10 a large number of microorganisms that have been modified In 11 this way so that they are very sensitive to chemical mutogen. 12 It Is a very fortunate circumstance, because It means that we 13 can at least make preliminary screens of the ability of a 14 chemical to Interact with genetic material without going to the 15 enormous expense of doing a whole cancer study. We can do a 16 bacterial mutogenosls study In a matter of days. 17 Q All right. So that what has been found in these 18 studies Is that TCDD Is not a mutogen? 19 A TCDD does not cause DNA damage. There are some 20 systems where some cell preparations where It can cause some 21 chromosome rearrangement. This Is not a mutation. 22 Q Well, does TCDD Interact with DNA? 23 A No, not at all. 24 Q And has that been demonstrated In studies as well? ' 101 1 A That has been demonstrated by again, Dr. Poland 2 and Dr. Gunther. Poland has used, has done experiments in 3 which he has used labeled TCDD and has found no evidence of 4 binding to DNA, much evidence of binding to' protein and other 5 structures, but none to DNA. 6 Q Now, Dr. Sllbergeld In her testimony here 7 stated that TCDD-- 8 MR. CARR: What page, counsel, and day? 9 MR. HEINEMAN: April the 12th, Pages 120 and 149. 10 Q States that TCDD causes cancer Itself. It is the 11 most powerful chemical carcinogen Identified, and Is a complete 12 carcinogen. Do you recall her testimony In that regard? 13 A Yes, I do. 14 Q Do you agree with that, sir? 15 A No, I don't agree that It Is a complete carcinogen. 16 Q And on what basis do you disagree? 17 A I've already discussed really the evidence that 18 shows that TCDD is in fact a promoter and it does not 19 Interact with DNA either by directJchemistry or by any ability 20 to cause a significant mutation Incidence, and also by studies 21 that have been done that show that In organisms that re treated 22 with TCDD there Is no Increase In the DNA repair activity, 23 which Is a measurable, a measurable factor In cells. 24 Q Now, at the same time In her testimony she suggested 102 1 that the Koclba studies show quite clearly that exposure to 2 TCDD alone causes cancer, and that's why It Is a complete carcinogen* Do you agree with that statement? 3 No, 1 don't. Vhat the Koclba studies show in 4 that light is that at high doses, at very high doses up at a 5 6 point where the.'animal is suffering really substantial injury, 7 there is a lot of effects on the liver, particularly in those 8 animals, that the Incidence of a variety of tumors changes, a 9 .variety of tumors, and when I say change, I mean just that. 10 There were Increases In the number of hepatocellular carcinomas 11 and also nodules In the liver that probably are reversible by 12 the definitions of most pathologists. There were many kinds 13 of tumors. Now, you must Understand that in any population of 14 animals there is going to be a high population of tumors, a 15 high Incidence of tumors of any kind. Ve all know unfortunately 16 In our population that most cancers appear in older people. 17 The same Is true In lboratory animals. It Is Interesting that 18 In those experiments the Incidence of many kinds of tumors, 19 particularly those of the endocrine system of the glands, 20 actually It decreased statistically. In other words, It Wasn't 21 so much an Increase In total numbers of tumors, but an 22 alteration in the whole tumor pattern. In another study that 23 was done in the National Toxicology Program, there was an 24 Increase In some tumors H< the thyroid at the highest dos. 103 1 When Che National Toxicology Program study was done, Instead of 2 feeding TCDD In the diet., ,t;hey introduced it by stomach tube 3 once a week. Long term .experimentthey gave the rats their 4 treatment once a week on. an ,exact basis with a stomach tube. 5 Those rats did not get the somewhat-- the slight increase In 6 tumors of the palate and., the ,nasal passages and the upper 7 respiratory tract that Kpctha'a rats did. The reason for that 8 Is because the material vaa.-OQt In contact with those areas. 9 Rats when they eat tend .to.Inhale.quite a bit of food, particularly 10 when It Is in that powder ,,orm .as Is used In these kinds of 11 experiments. The fact that these tumors occurred at the sltie of 12 contact Is suggestive of. promotion, particularly when we compare v 13 with the studies that we.re .done .on the skin, .TCDD has also been 14 tested as a skin carcinogen, again,In the National Toxicology 15 Program, and the only tumors, that they found were at high doses 16 again, and they were a tumpt,,called ,a fibrosarcoma of the skin. 17 It Is a connective tissue .tumo^, and there were no other tumors 18 anywhere in the animals ,,That, ..top, Is suggestive of a promoting 19 response rather- than initiationand promoting at the site of 20 application. . .. . . . . . . 21 MR. HEINEMAN: At this point, we would move the admission 22 of Defendant's Exhibit L25Q._. 23 (Defendant1s_Exhibit ,,129Q was offered Into 24 evidence.) ,, 104 1 MR. CARR: No objection, ,,your Honor. 2 THE COURT: Okay., 129.0 ia .admitted without objection. 3 Thank you. ,, 4 (Defendant's.tExhlbit ,,129Q was .admitted 5 into evidence.,) ,, 6 Q Now, Dr. Dost, l',d like to. talk to you again about some 7 of Dr. Zahalsky's testimony, and in that connection, I'd like to 8 show you what's been marked .as Plaintiff's Exhibit 253. Have 9 you seen that before, sir?, .. 10 A I've seen pictures ol it^ 11 Q All right. Or a. picture jof it7 12 A Yes. 13 (Plaintiff 1s^^Exhlbit ^1291 was marked 14 for identification.). 15 Q I'll now show yo,,u wh%t1s Jaeen marked as Defendant's 16 Exhibit No, 1291 and ask, if that's .a blow-up of a photograph of 17 Plaintiff's Exhibit 253.... . , 18 A Yes, it appears o represent it. 19 Q Now, Dr. Dost, you have have you not, sir, the 20 testimony of Dr, Zahalsky,,in .which ,this chart was discussed? 21 A Yes, I have. r, * . * . 22 Q All right. Do you agree,,with that testimony and this 23 chart? ....... A No, I don't, really.^ 105 1 Q All right. Would you please come up and explain to the 2 Jury why you disagree with .the.chart and the testimony relating 3 to it. 4 A This as I recall, appeared in Or, Zahalsky's testimony at 5 a time when he was discussing effects on the Immune system, and 6 if I remember correctly began talking.about this representation, 7 and'I guess that I would, ignore that add take for granted his 8 representation that this, active .represents the way that dioxin 9 causes cancer, and it is.clearly incorrect. There is, the 10 schematic that we're dealing With here-- 11 MR. CARR: Mr. H&lnetian, ,,what would be the date the 12 testimony was taken and ,,thetpage number that he is referring to? 13 MR. HEINEMAN: It starts .at 101, I think, and following 14 that, I think is where he ..starts talking about it. 15 MR, CARR: What .date^ 16 MR. HEINEMAN: May the `; 17 A Well, the scheme, that* s represented here is clearly a 18. scheme that is apparently .related .through the .induction of 19 microsomal enzymes, the P.45Q enzymes. And what we have here, 20 this looks very much like ,,a diagram that Or. Eisen published at 21 one time or a part of that ..diagram, but I fm not prepared to say 22 that it was adapted from. it .directly. When TCDD or any of <a 23 number of other polyaromatft,,hydrocarbons, and we've discussed 24 this earlier, 1 believe,whenitmoves into the cell In order to 106 1 cause the induction process;, .it^as to have a way of starting 2 the process, and just by. itself_in the cell it can't do that. 3 It attaches to a recepto.r. I don't like the term recognizer, 4 because that implies thajt .this is SQ .specific that it identifies 5 each molecule that comes.,into the cell. The fact is that this 6 receptor will bind a ver.y .,,large variety of substances. It happens 7 that it binds TCDD more .intensely than it does any other, and I S think that's just an accident of .the.very flat structure of TCDD. 9 It turns out to have provided us with an excellent tool, however, 10 for studying this process_Qf induction, and It has led to an 11 understanding of the way. that all .of these complex chemicals cause 12 induction. The two join, and .they form a complex between TCDD and 13 the receptor, and that receptor moves Into the nucleus of the cell. 14 Now, this receptor is no.t .out dn .the .surface of the cell, it is 15 free within the cytosol jpt .the body of the cell. That's why they 16 call it a cytosolic receptor. It is in the cytosol of the cell. 17 And it is correct that i.t ..does m^ye from the cell body into ^the 18 nucleus of the cell. Bui-it,,Starts from.in here. The TCDDtcomes 19 into the cell and joins .yitH .the .receptor to form this complex, 20 and the complex then moves .into .the nucleus. . There is a whole 21 series of these, and I suppose..thatye should bring them all 22 up one by one. 23 Q Let me hand you,, sir K wh^t1s previously been marked as 24 Plaintiff's Exhibit 254,., 255, .256, and 257, and ask you if you have 107 1 seen those documents before 2 A These represent he photographs that I have seen and 3 apparently they are In he ,sequence hat they originally appeared. 4 (Defendant1s.,Exhibit^ 129,2 to 1295 were marked 5 for identification.),. 6 Q Now, sir, would ..you compare these photographs by number 7 with the Plaintiff's exhibit ,to .be sure I've got them right? 8 Defendant's Exhibit 1292, is .a .phonograph of what, sir? 9 A Of Plaintiff's Exhibit 2^4. 10 Q All right. ,,. 11 A And this is the .next ,,step. , 12 Q And Defendant's .Exhibit 1293 Is a copy of what, sir? 13 A Let's see , Is a .copy ,,of Plaintiff 1s 255 . 14 Q All right, sir. ,, And ,,Plainfiff's Exhibit 12-- I'm sorry, 15 Defendant's Exhibit 1294, is a .copy .of-- 16 A Plaintiff's 256.,. . . . . 17 Q And Defendant's Exhibit 1295? 18 A Plaintiff's 257.,. _ ....... 19 Q Now, would you like o gq through these one at a time? 20 A Maybe In order to maintain sequence I will put this over 21 on this easel. This complex does In .fact ,go Into the nucleus 22 and It Interacts with the,genetic material In the nucleus. I 23 don't know what really Is meant by a critical target here, because 24 what is Interacted with is a segment of DNA, and this complex, I 108 presume when he says nuclear,,complex .does not act as a message. 1 The message, what It does.it.cause In some fashion, causes the 2 DNA In a particular part.of the .cell, In a particular part of the 3 genome to go through the, process of opening up and making a 4 copy of messenger RNA. JJq v , It is a fact, yes, that It does 5 6 result In the making of .messenger* .We are talking here about a form of nucleic acid called.RNA, and It Is very similar to the 7 8 DNA* Then the messengers ,..the messenger RNA goes to the structures In the cell that manufacture protein,.the endoplasmic reticulum, 9 10 and they provide instructlous .tq make .certain protein, and In a 11 sense, 1 suppose we could .say, .this is correct, except that as he 12 has It depicted here and,.S3-he .states .It here, they are making 13 new receptors. If I understand .t^hat,, he means recognizer aqu 14 representing the receptor .or TCDD * _..That does not happen. What 15 Is made are the group o enzymes .that we have discussed at various 16" times, the P450 enzymes,, the ,other.non-P450 enzymes that we saw 17 Illustrated on that charjt .of .Dr * Eisen's', .1 think yesterday , but 18 ve do not, the cell does, not -make additional receptors, they do 19 not go to the outside lining .of .the cell or the cell wall. I am 20 not sure what Is meant, ,and-they don*.t react with dioxin except 21 that those are the very enzymes -that are responsible for changing 22 dioxin, metabolizing it j3Q that ^t..can be disposed of. They are 23 not a new set of receptor3 tt^at ate here In the cell body which 24 Interact with TCDD. There ,,is\a .clear limit to the number of those 109 1 receptors. There are a few compounds that are known to be able 2 to cause the formation of .additional receptors to a limited 3 extent, TCDD Is apparently ,,not one of them. And these proteins 4 that we are talking about .that .ate ,,formed do not go to the 5 outside of the cell. They .gorr-they. are attached to this 6 endoplasmic reticulum, where.they are synthesized. They stay 7 there and are held in place. . These .are what are known as 8 membrane bound enzymes an,,differentiated from some of the 9 enzymes that I mentioned, qu .another occasion that are soluble 10 that can actually leak o.ut ,,of the .cell. These P450 enzymes 11 can't leak out of the cell.because they are stuck on larger 12 structures that can't le.ave ..Now, apparently Dr. Zahalsky 13 believes that these TCDD*, ,these, .new ,,TCDD receptor complexes go 14 back through the same cycle again, ,,according to this diagram, 15 but then they do something.different According to. his 16 description, they react .and .form ,,reactive intermediates./ There 17 are some real problems with .that because to begin with .he metabolism 18 of chemicals that might .nr .might; ,,not; take .place and might proceed 19 to "reactive intermediatjee'1..akes place put in the cell body on 20 that endoplasmic reticukuifr. ,,,.Tl^ete^are no such reactions taking 21 place, in the nucleus. The .nucleus ,,has other kinds of; work/to do. 22 So somehow or other we have ,two cycles described here that- start 23 out exactly alike and then.are presumed to end up differently with 24 a different function in he ,ecoi^d cycle. So this part of the 110 1 description is really completely.incorrect. We don't have 2 formation of new receptors. There is a great deal of binding 3 of TCDD to the proteins Jta the .liver cell, particularly the 4 endoplasmic reticulum, b.ut it is nonspecific. It is where most 5 of the TCDD that is stored, in the liver ends up and it is 6 just stuck there, it is ,,not really ,doing anything in particular, 7 and it can be taken back, out,,in a .biochemistry lab so we can 8 tell where It is. There., is-lust a/very small amount of receptor. 9 Measurements that have keen made Indicate that no more than one 10 percent, and probably mu,ch .less ,,than that, of the TCDD in the 11 liver cell actually has .bound to a receptor. The rest of it is 12 all bound nonspecifically .to ,,other proteins. So we don't have 13 reactive intermediates formed In the nucleus, and this complex 14 between the receptor and*. dioxin .has .nothing to do with the 15 metabolism of dioxin except as that receptor causes the formation 16 of the enzymes that eventually .can .break dioxin or it can alter 17 dioxin so that it can bn disposed of. 18 The last chart stays that ^now .the .reactive intermediates 19 bind to critical targets,. , We know that TCDD and its metabolites 20 do not bind to DNA. The, implication here is that it has bound to 21 DNA inside the nucleus o.f,the cell.. This has been demonstrated, 22 hot to occur. It has al^so .been demonstrated that we do not have 23 a problem of reactive intermediates that are thrashing around in 24 the cell, which is 3-met.hylcholanthrene or dimethyl bez anthracene 111 1 the reactive intermediates of those substances do a great deal 2 of harm and they do bind...anywhere they can find a point in their 3 path. The implication ijs ,,this .is what results In toxicity and 4 we've discussed this I think sufficiently to show that there isn't 5 a relationship between reactive..intermediates and their binding 6 to toxicity because we d.on't have reactive Intermediates as 7 part of the system. T h e .implication is also that this initiates 8 the process by which cancer cells, cancer is caused, starting 9 with the initiation in the .nucleus ,of the cell, and it also 10 results in transformation ,,,,And I am .not sure what the relationship 11 of these two events is. , We would have to concede that TCDD is 12 capable of the promotlon.^process which, depending on definition, 13 may or may not Include transf;or^ation. . But it has nothing to 14 do with the initiation process, .So, the representation that this 15 scheme is a description of the .way that TCDD causes cancer is,I 16 think, erroneous, entirely erroneous.., I don't know where it arose. 17 THE COURT: Gentlemen g q u 14 .1 see you at the bench for 18 a minute, please? A 19 (At this time an ,,off-the-record conference was 20 held at the ..bencl}*) ,, ,, , . 21 THE COURT: Ladies and gentlemen, we are going Vto break 22 for the day at this time.. ,.We .will .resume again tomorrow morning 23 at 9:30. I would remind you ,,as .1 do for any overnight break that 24 you are not to read, listen to, .or .watch anything about this case 112 1 In particular or subject matter- in.general in any of the media, 2 srint or electronic. Thank you for your attention and cooperation. 3 2ourt is adjourned. We .will start .at .9:30 tomorrow morning. 4 (At this tiiae Court ^as adjourned for the day.) 5 6 7 8 9 10 11 12 13 1 STATE OF ILLINOIS 2 COUNTY OF ST. CLAIR ) ) SS. ) ........... ' 3 4 5 6 7 8 I, Patricia A. Gandy* CS^,.RPR* Official Court Reporter 9 in and for the Twentieth. .Judicial .Circuit, and the Official Court 10 Reporter who transcribed, the above-styled cause had on November 5, 11 1985, do hereby certify ,,that .the foregoing transcript of proceedings 12 is a true, correct and complete transcript of the proceedings had 13 on said date. 14 DATED this 10th day qf Nqyember, 1985. 15 16 17 18 19 20 21 22 23 24 1 STATE OF ILLINOIS 2 COUNTY OF ST. CLAIR SS. 3 4 5 6 7 8 I, RICHARD P, GOLDENIJERSH, Circuit Judge In and for the 9 Twentieth Judicial Circuit, .hereby certify that the above is a 10 true and correct transcript; ,<jf .tl\e proceedings had in the case 11 captioned: FRANCES E. KJ3MNER, ,et a l v . MONSANTO COMPANY, 12 Cause. No. 80-T-L-970, heard .on November 5, 19 85. 13 DATED this ____ _ day of .November , 1985. 14 15 ,, EiJTER: 16 17 18 ,, RICHARD JP. QOLDENHERSH, Circuit Judge 19 20 21 22 23 24