Document 06MX9BXQ69NoZog8kG3Zz4GXx

s /? // and neurochcmical amiecot. 14:431-444. ig: titered urinary n in mice and men, r pod VhronichetUm i W W i i i Jf:56-fib, i cardiovaxoliri nci I d Liwrie,- T, D. V.t nic lead exposure in .156. 1974. ,h_nonic lead inioxica*55:359-365. 1969. ;iologie*l disposition ihectomued animals. id exposure in lacti,i maternal blood and !, 1975/'Vi*' : \ .**''uJuVHutribtP .e rr. Ftd P n xttd . }H., and Hejtmancik. .dio\iyraU r responses .I 40*01-411,1977. ' ' *e and norepinephrine rrmoeot 79:134, 1977. 'jhronic lead exposure 'response to norepine- level of perinatal lead :*diotoxicity. Ris Comm and Ziegler, M. G.: 20:186. :H, J. H ,, H ejtm ancik, ! poisoning. In Brown, riKt Oxford. Elwrvirr* - `V--.'TVT'l *1 I tl Chapter 5 IMMUNOTOXICOLOGY AND CHEMICAL CARCINOGENESIS J o seph K. P rince ABSTRACT The influence of environmental chemicals on mamma lian systems has been given much attention over the last several years. Bioassays of a chronic duration as well as short-term genetic assays have"b`een deVe1opEd~\^h.an ,eye_toward,the. early . recognition of the occurrence of a lesion in some target organ. PhysiologicalTand biochemical * changes are targeted for.analysis and surveillance in .an effort to develop a screen that will signal the possibility of the occurrence of chronic disease. .. Although the liver and(l;idncy have, been most intensively investigated, other systems have been used in this effort to establish an early warning,system. The majority of the studies carried out have not taken into account the possibil ity that the i mmune-svstem- may be the JdejtLvehide_ for such bioassays. For whatever reasons, investigators have underestimated the importance of the immune system as T h e author, w is h lo acknowledge h elp fu l comment! and auggeitioni contributed to the author by Dr. M . M . Yokohama. U .D . Ph.D,, D irector of C lin ic a l Im m unology, and Dr. John hederka Ph-D.. Chief of Toxicology and Pham ucom etHo. of the U n rve n iiy of Illin o u H o ip iu l and Medical School SufT. ' : T h e author alio w iih rt to acknowledge the very h elp fu l clerical a a iiiu n c c provided by M rt. Debbie Frip t, who did the typing and editing.of ihe m anuacripi. . The author alto acvepu the blam e for a n y 'rrro n or i'nconiiatenrici which may be found, and rrq u c m any reader to pleaae no tify the author of ruch miatakea aa may occur, w ithout in te n t. *" - '> V. r' 71 t i 72 Emnronmenlal Toxicology related to disease states. This author feels that a nucleus of information has been developed by immunological inves tigators to indicate that the immune system organs and tissue (thymus, spleen, bone marrow, lymphocytes, mono cytes) may provide a fertile soil for such investigations that attempt to provide early detection of capacity for chronic disease by environmental chemicals. Immune deficiency or modulation of the immune response has been known for many years. Irradiation, malnutrition, thymectomy and certain anticancer drugs currently in use are proven to cause a change in the immune system. Although the immune system is complex andnot completely understood, sufficient information is available to indicate that various elements in the immune system appear quite _ sensitive to chemicals:-- ----------------------------- It is well established that active cells are the most sensi, tive to chemicals..-A..good:deal'.of.informatidiv~has~been " developecfon the use of amicancer agents'and'their capa bility to induce secondary and tertiary' cancers in patients undergoing amicancer treatment. Some of the changes associated with such treatment at the cellular levelare also seen in vivo and invitro and of course raises the'question of whether this activity can,.be'at work during*exposure to environmental chemicalsPJThis author feels that there are some relationships that make the next decade in immunotoxicological research possibly the most exciting and poten tially the most important. Introduction D uring the last five years, many of us have heard the statement that "a -large, number of cancers may have'their etiology in the environment." This statement helped to propel an already ener getic national 'desire to'seek cancer causes and-cures. Although there are many different opinions as to the actual numbers of cajncer cases contributed by-.the various environmental factors, it has been generally agreed upon,, among biomedically oriented research scientists, that environmental carcinogenesis is a real entity.1 In an effort to seek a cure or to begin a prevention pro- I pram. the cn iisk to humai In order to lias had to use rxira|>olation tiplc specie t studies, and cl cautiously, ha data is lackiu consternation Recent devi a nucleus of c these problem immune mccl hitely, rescarc system cells (s products suda*** body's im muvj-' infection, graf The fact th; tissuepresent potential forjthe study of pt Kpidemioloj various detent environmental provide direct statistical corn disease have b capable of cat among human l>ecn an im por predictive toxic a suspect chem Microbial as the successes c nucleus of ;tcal inves>rgans and les, mono* esligallons for chronic c response inutrition, illy in use e system, ompletely 0 indicate 3ear quite osl sensihas been ieir capat patients changes 1are also "V on-bi^:' *--jr e to * there are mmunoldpoten-J . *e statement logy in the ready ener- Although lumbers of d factors,''' ly oriented is is a real mtion pro-'.. J Immunotoxicology and Chemical Carcinogenesis 73 I gram, the environmental chemicals that present a carcinogenic ' risk to humans, are an ideal target for investigation.1 , f In order i?establish some measure of risk, predictivcloxicology i has had to use the various available tools; epidemiology, statistical : extrapolations of pharmacological dose response studies, the mul tiple specie end target organ bioassays, microbial, biochemical studies, and chemical structure relationships.4-9 These tools, used : cautiously, have served us very well. However, the fact that human . \~diita~l^lacking in a majority of the cases has been a source of consternation. ; Recent developments in the area of immunology have provided ; a nucleus of data and of concepts that may help resolve some of these problems associated with extrapolations to humans. Although immune mechanisms and the system has not been defined abso lutely, research data has been developed that shows that immune system cells (small lymphocytes, T and B cells, macrophages) and t products such as lymphokines play a very important role in the^ ^ body's immune reaction to possible pathology from microbial J j infection, graft rejection, and tumour production. / The fact that these cells are readily accessible and are human . . tissue present in extensive amounts in peripheral blood, and'haye'-i ' potential .'for rap id 'pro liferation makes them an ideal vehicle for . the study of potential toxicity....... : '*"* , Predictive Toxicology in Retrospect* ` Epidemiology is the discipline that integrates'the impact of the .-. various determinants involved in the disease process. In assessing environmental hazards, it has been used as a tool that helps to provide direction toward actual laboratory investigations. T he \ statistical correlations of the distribution of the determinants of *7 disease have been able to relate the possible chemical toxicants j capable of causing pathology to the occasion of disease states I among humans who have been exposed. Thus, epidemiology h as been an important tool in providing the first step in the process of predictive toxicology--helping to determine in many cases whether .. . a suspect chemical-warrants investigation.3-11*12.13 Microbial assays have supported predictive toxicology due to the successes of Ames, and McCanh' in 'establishing aberrations O\J c 74 Environmental Toxicology seen in mutagenesis assays as being related to the process of carcinogenesis.U'15-1GThe philosophical basis for this procedure lay in the fact that a chemical capable of causing DNA related lesions could be delected in microbial systems. If a chemical had the capacity to cause mutations in microbial cells, then genetic toxicity was a reality. Using the strain of Salmonella lyphimunutn, which has a histidine negative mutation, Ames and co-workers were able to show that exposure to chemicals causing genetic lesions did cause a reversion or back mutation. The normal Sal-7 7 monel la cell was modified by introducing membrane defects so a . ? chemical could enter. The excision repair system had to be neu-^ tralized so that any lesion that could occur remained, and a bacterial^ ; plasmid was introduced to insure errors in DNA replication. Final ly, rat liver extract was introduced to provide.a mammalian meta bolic system for activation of the chemical that may not be a direct carcinogen and requires.transformation..v. . Since this System waTintxwiuced, others1,7;.1.8'have validated theri assay system using hundreds of chemicals and have reported th ar' apprnvim:itplv 00 p/vr^Pj-it nf the chemicals tested, which WCfC .reported in animal -system s'to-be carcinogenic,, were in fact mutagenic in the Ames assay system.; A lthough the correlation is lairJy good,- caution and^prudence must ;be exercised.-:Doctor U. Saffiotti of the National Cancer Institute has expressed confidence in the Ames system as having the ability to predict carcinogenic potential, but also added that animal bioassays, using mammalian species are required as conclusive evidence of a chemicals' ability to cause carcinogenesis.18 When an appropriate experimental design is used, the mamma lian bionssav generates the biological data that.has been most sensitive in determining whether a given <-Vnmirl_may have-' carcinogenic potential. :D u c ;lo .the fact^that. animals used for carcinogenesis bioassays are highiy7inbred,^6me strains may express a predisposing genetic tendency 'iowarcftHe occurrence of neoplastic response from chemical exposures. It was therefore recommended by the National Academy of Sciences21 that carcinogenic studies be carried out in multiple species of mammalian laboratory *nimals, so ;haT"Tny expression ol a genetic lesion causing neoplastic response be validated or supported as much as possible. Animals, is do hurr base pres* lat ion win An tiers* vide a Miii lluTapeut. dictors in large anin chemicals burden in Carcino many spec the most s alone is ut tissue, mo. biochcmu reticulum^ in interpre or. such dai... ... In th e irrv icals. the N use of. ransfvccics ide guarantee r it is all ihat Similar a used in care far the mu evidence foi sure to envi The rccei confirmed tl plus mice It: wry. from a that given d Huorcnc, an Mice that \v ihowcd urin c *i -*ss o rdurc lated Ihad nclc riu m. riten nclic Sali so a n cuerial inalTdairccl ihc ihat Yac m is r U. ' :nce ?nc lian lily maio .nI ave for rr\s Stic ded vs niitc *ii. Jmmunooxicotogy and Chemical Careinogen eris 75 is do humans, have varying susceptibilities, so lhat ihe larger data base presents a greater possibility of providing effective extrapo lation when predicting possible toxicity. Anderson and Schein23 have used the dog and monkey to pro vide a suitable overlap in combined studies on anticancer chemothcrapeutics and found that they were in fact very favorable pre. dictors in preclinical studies. However, the size and cost of such large animals for carcinogenesis assay, given the large number of chemicals in the environment, would present an extreme cost burden in dollars and in human resources. Carcinogenic or teratological studies h ave been reported using many*species.2cr5~Clegg26has indicated that the mouse has provided the most success, but he and Jensen17 caution .that this specificity alone is unsuitable for establishing carcinogenic response. Organ tissue morphology is different; rate, of onset of polyploidy, biochemical-enzymic differences, and rough to smooth endoplasmic reticulum change during exposure cause a great deal of difficulty -virt interpretatibrt bf the ^ctuahpatH blo^rThey'do suggest h 6wev;cr, such data Should be used as a warning signal for further study. In their report on principals for evaluating environmental chem icals, the-National'Academy of Sciences^ NAS)21 recommends the use of--randomly; bred hamster, ra t,'a n d mouse' s' the animal species ideal :forJ'carcihdgen" bid assay.20 Once 'again, this is no -.^'giiararitee^of successfully being able'to predi^H urnah toxicity, b"ut" it is all that we have developed up to the present time. Similar arguments exist for any of the animal species that are used in carcinogenic bioassay. The everpresent reality is that thus far the multiple* species bioassay data have provided the best evidence for extrapolating possible chronic pathology from expo sure to environmental chemicals. The recent mcgamousc study reported by Littlefield et al." has confirmed that the dose-response studies performed on somc.26,000 plus mice has validated that such information'is absolutely neces sary, from a standpoint of both dose'and tim e.'H e has'reported that given differcnt'lengths of time of exposure to 2-acelylaminofluorcne, an animal could respond with two'different end points. Mice that wcre*'doscd for* eighteen months and sacrificed later showed urinary bladder neoplasms; they also required continual 76 Environmental Toxicology presence of the carcinogen although the neoplastic response waj induced early in the study. However, in animals that were exposed for thirty-three months, doing serial sacrifices, a liver neoplasm became evident only late in the study, although it was induced early during exposure and did not require the continual presence of the carcinogen. This data provides substantial evidence of the necessity of the long-term bioassay. A product of that study was a confirmation of a principle applied to regulatory function in public health pro- ] grams that "there is no level of exposure greater than zero for a I toxic substance, which can be assumed to be without harmful effect."2 1 In contrast to that principal, risk benefit analyses have been introduced based on the public's right of acceptability of possible adverse effect to a given chemical exposure. The .calculated risk allows some level-.of. a, loxinao .coexist j n ,,the m ln t-o fi human existence,''correlated ;with; the fact th at humanity derives some benefit from that, coexistence. In order to calculate that "level of *acceptability^ it is necessary .to extrapolate from the dose of the experiment, associating some end point.with a given level and length of time o fth e exposure.:, A ^source ;of consternation has been, w hi<A partof,l^q^cur^^i;o.^ dose to exposed humans.19"35 Based on the data of the megamousc study, both lesions are induced, in their respective tissues, early in the exposure, suggesting a_linear no threshold dose-response^ Thus the lower end of the dose curve would be the ideal place to begin extrapolations.** Although,this philosophy is not espoused by all, it is a practical approach^ tfiat-can provide consistency, assuming the data from bioissays,provide sufficient evidence that chronic pathology "is a reaJpossibi lity. Wilson.56with some reser vations, supports.this philosophy, suggesting it is useful for making comparisons with benefits easier to calculate. --^ The point I have tri.ed.to make is that regardless of.-the meth- od(s) being utilized^ problems .exist, that prevent the .unanimous | acceptance of'ahy of the given.assaysystems. Even when taken in i concert, the major stumbling block has been that, we do not have i information as to how this material will react in humans. Jm m unotxicohgy and Chemical Carcinogen esii 77 Cellular Basts of Toxicology The influence of environmental chemicals on the various bio chemical, morphological, and histopalhological parameters has been intensively investigated in various laboratory animals. Epidemiological and statistical analyses have produced more sophis ticated and complex mathematical formulas dealing with the extrapolation of dose-response levels and the prediction of toxic associations. Since our predictive ability and the skill we use in evaluating exposure are dependent upon basic biological processes, it is important and prudent that we should,direct our energies to answer the basic questions describing how toxicity occurs at the cellular level. Disturbances at the membrane, cytoplasmic, or nuclear levels and at micromolecutar structure-activity levels need to be very carefully analyzed to determine the sites and modes of toxicity. Whether for acute or chronic toxicity, these are the fun-^ damental questions that must be answered in order to determine thclevels of chemicals humans mav tolerate without adverse effect. This is not a new or reorganized philosophy, but a reconfirmation of n thconginaTprincipies laid down by o u r toxicological forefathers. As the numbers of chemicals :hterig into theT environment' have increased, the long-term bioassavs for all chemicals becomes less and less possible due to* the'length of time and large costs inyolyedI^Sequentllyj':i bcoms''more important to further ' d^clop cellular bioassky-s as genetic defect predictors. The quicker response time and reduced costs are" obvious benefits, although cxtrapoltion is a difficult constraint. Since the correlation of 7 animal responses to human situations are probIematicalTII=s_uggest wc devote more of our attention and energieTtOffardTise of human Cclls^-Since the protection and well being of human life is our * ultimate end point, data that can be derived from culture and analysis of human cells can provide us with the best evidence for u making predictions and risk assessments. Immune Celt Analyses One area that has not been well investigated but deserves attention ls the human, immune system. Vos10 as well as Silkworth and Loose,57,have found evidence that supports the concept.that envi- 78 Environmental Toxicology ronmental chemicals can produce modifications in the activities of the immune system cells. Antibody mediated resistance, cell modi* aled resistance, as well as increased levels of mortality due to lowered resistance to bacterial infections have been reported in their reviews. Silkworth proposes that cellular and humoral medi ated responses may be the appropriate indicators of environmen tal toxicitv. H e_ indicates they are quite sensitive, they can be conveniently evaluated, and he has had limited success using this lest system in the evaluation of polychlorinated biphenyls (PCBs), 'Aro--c--h-l-or --10-16 \(-A---C--L' R' /)I, --and hexachlorob-e-nz----e-n-We \(HCB)/. T'aylor*4 has analyzed various immune functions in theCgumea pig,<during dietary studies; however, he had conflicting resufisdueTo various other factors that could not be controlled. Luster et al.,99 as well as Luster and Faith.110have evaluated the rationale for using immuno comptence a w .form of 1abor^tory.investiga pps..jj^ey. recom: mend using--multiple .parameters^of study to properly' evaluate environmental-chemical.effects, since normal immune responses are dependent upon:m acrophages..and at..Ieast.lhree^sets of. lymphocytes. These cells .interact among themlyes* and with various.cell. products (lymphokines.and/or complement),* so it can* become qu iie^cpmplica ted.; However, the^studi es. th y. perfonn'ed did show that exposure to various chemicals produced immune- cell modulation. Dean et ah41 have .examined cell and humoral mediated im m unocom ptence.and also recommend multiplc__ parameter assays, with most of them dealing with systems designed to study tumour/cancer.10*37"41 It is obvious that since the immune system deals with a large population of cells of human origin that are available from the peripheral blood, it is an ideal group of cells in Which'to study * toxicity. *; ..... . Since these cells are present in the circulating peripheral blood, *77 7 we can correlate their reactions to chemical exposure in a realistic manner, because it is similar to an environmental exposure. Chem icals that are taken in by humans must be absorbed and transported through the circulating blood, which provides the media for close association and residence time. Generally speaking, most chemi cals are not biotransformed within the absorptive area of the intestinal tract. In their contact with the blood elements during tics of triedine (o cc) in Heeli n')cn- iM be j tliis : d s), *lor5fl i-Ing "ious .'ll as unr>:om- uaic nscs s of viih meora! Iplc nc<I : ; /."i**- c". rpc' the ady ` otl. Stic m- ' ted .* nihe :*' Im mu notoxicology and Chemical Carcinogen eris 79 circulation, not only do you get contact, but also a realistic mode 7 ? j of how and/or what may occur given a chemical and a sensitive or 0 susceptible cell. Cellular response may be at the membrane sur face, within the cytoplasmic confines, at the genetic-nuclear level or at all levels. Since immune lymphocytes can be stimulated into differentiation, they can show sensitivity at all levels, making them an ideal vehicle to study cell toxicity modes. With that type of information, im m uno- and predictive-toxicology would greatly benefit. Since immunotoxicology is at its infancy, large amounts of input in developing additional methodologies for detecting cellular determinants and/or receptors and for characterizing pathways and/or molecular constituents are needed. It is a complex task, but these areas must be explored and the quantification of such reactions at the cell level must be identified if we are to gain any further skill in prediction and extrapolating doses to humans. Immunotoxicology: The Link to Humans?. The. imipun^jdefense^ is "a, dual system cornposed of n atu ral/'- `^onTpeETHc-ba^crs"^uch4ls'Skin keratin and pH, of the natural ' mucociliary barriers-as w eH asihe normal bacterial flora that act to prevent bacteria-from penetrating., A third nonspecific mech anism begins operating when that first barrier has been breached. Macrophagej;<and:polymo'rphbnuclear Ieucocytes, together with ,..*P >cific humorahtsoluble' factors, and lymphokmes, act in concert '" t o sequester,"degrade,`an d :neutralize"the material recognized as foreign. - If these natural and nonspecific barriers have been overwhelmed, the second and more specific defense mechanism comes into play. It involves the activation oflymphocytes that are preprogrammed to combat, infection. T hese'lymphocytes are the main element of this system and-compose-the basis of the humoral, and.cell medi- ted im m unet^ferise 'niecfmlnisms.' They are indistinguishable morphologically,; but; because of differences in the programming oqucnces, the. lymphocytes Have different capabilities.. Toti[>otcnt.(or.-pluripotent):stem cells derived from the bone marrow.are.distributed to the peripheral circulation and home in ' on certain -tissue forcducation and processing. Lymphocyte clones 80 Environmental Toxicology are processed by the ihvmus gland and are programmed with cer tain specific surface antigens and cellular memory for use in re sponse to fungi, parasites, intracellular viral infections, tissue grafts and cancer cells. These are tl^eT M ym phocytc^nd arc responsible for cellular mediated response. That is the response whereby the T-cell is mobilized or stimulated to seek out the foreign material for specific reaction and neutralization. Another group of lym phocytes arc processed by gut associated lymphoid tissue (GALT), the supposed equivalent of bursa tissue in the avian models and are then callec$^B<sJ) They respond to bacterial infection by transformation into a plasma cell that secretes antibodies. The .^afftibodies^are located in the peripheral blood, circulating about until theyhtget an antigen and then begin the process of neulrali- ( Z J zation and^Higesnon. Each lymphocyte {T or B) has received specific information during processing and is a clone with a response specificity. T he spcifienty's due tcTchemical markers imbedded in cell surface conformations that are sensitive only to specific anti gens. There are some populations of lymphocytes that although thought to be'end cells, 'have.been .shown to live for many years and can. recirculate from-lymph node to lymph and to blood. Another lymphocyte" population that does not possess either im* munoglubulin (B) or T-cell surface markers is present and they arc called n ull cells. They are considered to be involved in target coll killing termed antibody dependent cell mediated cytotoxicity (AD CC).* Genetic Regulation Immune responses are controlled genetically by a series of linkedgens known in mice as the major histocompatibility comp1ex^M H C /and in humans as th ^ H LA ^compIex. A similar series of genes has. been identified in eachm am m alian species examin ed.^THITpEenomenTH'as discovered when guinea pigs inocu lated with an antigenic complex of Dinitrophenyl conjugated poly l-lysine (PLL) responded by demonstrating antibody production and delayed hypersensitivity. Strain #2 responded well; however, strain #13 responded poorly. This was taken as evidence that the *T h* re a d e r ti uryed to e o m u ll on e of ih e ru n rrn tly iv ilab lc ieU on im m u n o lo g y ^ m ore detailed inform ation. specific r relations Furthc amino ac responde . grouping other han but strair respond were invo respond. Further was link control of .r c o g n i t i f . * mouse complex i regions ha' tissue Iranand the "T The H - 2k ' surface anti reactions a: identifies * recognize a lion of an i altered as lymphocyte proliferate: individuals with demi thymidine v response to be afTccted b and determi membranes fied. c 1 CCT- .n rr- grafts jy the lerial ' lyniALT). Is and on bv . The about Jirali- ceivcd ;ponsc ded in T-~mi1- -gh ' years cr im- ey are *ct cell CC).# ies of cornseries examin ocu*d poly iuction owever, kat the o-y (or ImmunQtoxiculngy and Chemical Carcinogenetis 61 specific response is due to the carrier, poly 1-Iysinc, and its specific relationship to the PLL gene, which has the capacity to respond.45 Further studies were carried out using variations of polymers of amino acids, ant^seperat^genes have been delineated. Strain ff2 responded to a copolymer of glutamic add and alanine {random grouping of AAs) but no response was seen in strain if13. On the other hand, strain ff13 responded well to glutamic acid and tyrosine, but strain ff2 did not. FI generation hybrids (13 x 2) however responded to both immunogens, indicating that two different genes were involved, with both having the gene indicating a capacity to respond. Further observations demonstrated that the ability to respond was linked to the MHC locus. Since that development, genetic control of immune responses and of antigsns^evoking^allograft recognition have been localized to theTH-2 gene complex of mouse chromosome ff17, and the^ m a n j eukocyte antigeiC(HLAp complex in the human on chromosorrf^jjfd^jii the-mouse, rwo regions have been identified: H -2k and H -2d, wHich-.determine tissue transplantation antigens and cellular immune cytotoxicity, and the "I" region, .which controls induction of immune-responses. The H -2k and H -2d regions have been shown to code for the cellsurface antigens that serve, as targets for. cell mediated immune reactions as,well as the antibody response. This median ism, which identifies, some surface factor of, the cell, is used not. only: to recognize allograft differences, but also plays a role in the recogni tion of an individual's own cells, which may become changed or altered.as in.viral infection or neoplastic disease. The .mixed lymphocyte reaction is controlled by these lod , and they will proliferate and take up radio thymidine if cells from two different individuals are mixed together. If the cells come from individuals with-identical M HC loci,` there ,will be no Teaction, *and-no.thymidine will be incorporated..Thus, such an activity as normal response to nonself invasion could be upset should a given gene be affected by a chemical exposure. Since the cell's surface markers and determinants are expressions of genetic activity,-those surface membranes may have unusual conformations that could be identi- fied. c 82 nriVcmminifl/ Toxicology h ImmuncosurYcillancc *07 Cell Surface Recognition System Within the immune defense system there exists a mechanism I I. that has the capacity to recognize the foreign nature of intrusions ' into a host. Bacterial infections occur, and the antibody response It causes neutralization. Foreign tissue graft (allograft) causes the cell mediated response to rejeerthat tissue. Particulate material is sequestered, and phagocytosis causes the degradation of such mate n 0 I. rial till it becomes an innocuous residual body or is discharged from the host. In each case, the immune system recognizes that ( this material is not identified as self, and sets about to rernove thcL^ jim^siomjThe immune system has developed and matured in each I> hosCTwHereby it has learned to recognize the tissue it was associated 11 with during its process- of- maturation and development.-. It, can *1 distinguish-.between self and nonself. `` 1 -.The T-Iymphocytcs." responsible for cell mediated immunity, are part of the internal policing system that is supposed to recog I'l nize aberrant cells and remove them from the system. It Is able to i:> recognize these cells by surface determinants that reflect the aber la: rant nature, of the'cell-and would not be found on a normal cell.41 tI It is this change in surface antigen markers that separaies_the~self "7O from nonself recognition scheme. This concept was developed for experimental studies in which "nude" (athvmicl mice lack the y ability to reject tissue grafts because of the defect in the processing in I of lymphocytes into T-cells.*4Thus, the animal cgnnoi respond to l:j the foreign nature of a transplanted tumour cell and provides in acceptable medium for growth of various tumour cells, whereas, if a tum our is transplanted into a normal mouse, there is a rejection of the tu m o u r tissue. It is now generally accepted that jnost_ iti . ip.* O.J I transplanted' tumours have tumour specific antigens that are the I 1 basis for the induction of the immune rejection reaction. They are called tum our specific transplantation antigens (TSTA*s). !}>ii;i: This-concept of tum our specific antigens has had a profound liPti impact oh tu m o u r immunology. Mice that were inoculated with 1..... polyoma virus were later capable of rejecting a polyoma tumour til w that was grafted onto a synegenic mouse (genetically identical). tnu, This was interpreted as an example of the immune system's rec- nt..\ l cI Jmmunotoxicnlogy and Chemical Carcinogenesis 83 ognilicm iii.ii ihc tumour tissue was indeed foreign and that prcimniunuaiion was directed at these surface markers. It was also hypothesized that the immune system was capable of limiting tumour development. A logical extension of that concept held that if there is a condition that exists in the animal thafTnodilies of^IteiTT^Mmmune system's capabilities to respond to a nonself entity (miynunemodulation), then the variant cell or foreign entity can take hofclTcicvelop and mature into perhaps a disease state.^--tr Immune Modulation Immune modulation can take-variable forms of stimulation or suppression, and it can be specific or general. Stimulation (specif ic) of the system dijxctly occurs in immunization with microbial cells or products or indirectly as in passive transfer of serum, cells, dr cell products among histocompatible donors and recipients. _ There are also nonspecific or general responses that can occur as with Bacillus ealmette guerin (BCG), Corynebacterium parvum, levam- >ciso]e, and pokeweed. These cells or products are referred to as Ih ito g ^ ic since'theyare:nohspedfic.and have.the ability to stimu- la te B and T cells] as well as'macrophages: o rih e r complement-*, system.^ *' " ` -- - - Immunosuppression, on th e'o th er hand, is.-the reduaion of available immune elements directly or indirectly. Lymphoid drain age, cytoxic drugs, antilym phocyte serum-are direct, as is thy mectomy, which will reduce T-cell populations and response. Irra diation is generaLsupppession of-thr immune response. Bursectomy in fowl will lead to loss of B-cell response. Indirectly, one can also modify the response by increasing or reducing the levels of corticosteroids or using various known antiinflammatory agents. Based on this ability to modify the immune response, medical science began the use of chemotherapeutic*. In the clinical sense, the goal is to suppress or eliminate the ability-to produce-an immune response to specific antigen while allowing other, anti gens to evoke a response, as in organ transplants.45*46 Such sup pression has been used in the clinical treatment of neoplastic disease. Some cancer cells, as do bone marrow, cells and intestinal mucosa, show an"extremely rapid -growth' rate- and, additionally, may have an abnorrnal'cdmplement "of nucleic-acids. Alkylating Environmental Toxicology agents as well as blocking agents are used to interfere with metabo lism of such cells. Because some neoplastic cells metabolic ^ accelerated rates, they are more likely to pick up higher doses nf anti metabolic agents--However, the immune lymphoid system cells are also more active so they can also be affected to a greater degree. Induction of an immunocompromised individual through the use of chemicals, has. become well known in cases of clinical c h e m o th e ra p y .46 Immune Deficiency and Malignancy One of the most significant advances in, cancer information is the finding that immunodeficient states are associated with an increased incidence of malignancy. Kersey61has shown that patients who have naturally occurring states of immunodeficiency such as Wiskott-Aldrich-disease. Ataxia telangiectasia, or agammaglobu linemia have an ..unusually high incidence of malignant disease. Numerous experimental bioassays are cited in his review, show ing that immune-suppression facilitates transplants of malignant cells, increases a .normally rlote incidence of viral or chemically induced cancensan d accelerates growth of mtastass. " ' Immunosuppressive therapy has been used for some twenty oddyears and has had a significant impact in producing secondary and tertiary-cancers in patients who were under such treatment. P e n n ^ h a s been maintaining a,tum our registry of patients who were on immunosuppressive therapy and classified them into five groups with the following results. Patients with Transplanted Cancers Sixty-one patients who had received organ transplants from donors who were neoplastic or within several months subsequent to donation developed.evidence.of malignancy showed that twenty-one patients or 34-percent had evidence of transmitted cancers. Cessa tion of immunosuppressive ierapy and removal of the graft re sulted in the complete disappearance of the disseminated neoplasms. Transplant Patients with De-novo Malignancies In a long-term follow up of the University of Colorado series of renal homografts, 32 of 564 patients developed cancer, an inci- I iL .'C - lA jtaboizc at SC5 of i cells 'eater ough inical on is h an tienls ch as 'obu- ,nant ally 'eniy dary lent, who >five from nl (o -one essai rcilTU. rs of .n c i- I fmmunotoxicology and Chtmtatl Carcinogtnesii 85 dence of 5.7 perce n t.V he Denver Transplant Tumor Registry has data on 4fU de novo cancers that have occurred in 37B patients who have received kidneys. The average age of the patients were thirtynine years old (range eight to seventy years) and the neoplasms occurred from one month to one hundred fifty four months (1 to 154) after the transplant (average 32 months). After the trans plants, the following immunosuppressive procedures were used: Prednisone. Azof hispring, amj.Iymphocvte globulin (ALG), Actinomvein. cvclophosjihnmide. Radiation, plenectomv. thymectomy and thoracic duct fistula procedures were also in use. In conjunc^ioiuwith^AI-G treatment, 6-mercaptopurine, methotrexate, and azaserine-were also used. te patients receiving irradiation, splenectomy, thymectomy, or thoracic duct drainage treatment accounted for 217 dt novo cancers, while the parents' on pharmarnlogic th eq p y accounted for*the remaining 164 cancers. The development of malignancy could not be relatedto the use of any one agent, but appeared toTBe an effect of the general immunosuppression. A significant finding was that the incidence rate of solid lymphomas among the organ transplant patients was disproportionally higher than the general population. One variety, Reticulum Cell Sarcoma, was calculated to be 350 limes more common. The lymphoma, patients were slightly younger than the other cancer patients (36.5 versus 40 years old), and the tumours appeared earlier than the other cancer patients (twenty-three versus thirty-five months). The solid lym phomas occurred in ninety-five patients with the following break down: Reticulum cell sarcomas.....................................*.....................68 -(One patient also had a Kaposi's sarcoma.) Kaposi's sarcoma.......................................................: .............. 11 Lymphoma.................................................................................. 8 (Including 1 plasma cell lymphoma). Lymphosarcoma. 5 Lymphorcticular malignancy ..................... . .o *'V'. v 2 Hodgkins Disease.............................................. . . . V I . 1 Histiocytic Reticulosis (?).............................................................. 1 1 r 1t I I l { t I I ( t f i 86 Tnihronincntal Toxicology Non Transplant Patients Treated with Immunosuppressives (Antiinflammatory therapy) Data has been collected that indicates that of seventy paiiomc who have been under antiinflammatory therapy, seventy-two ra n . cers developed during treatment with various agents. Disease No. Cases Therapy No. No. Cases Cancer Type Cases Psoriasis 24 Methotrexate Aminopterin Other 23 Lymphoma 4 3 Leukemia I 8 Skin 5 Misc. 16 Renal Disease ;; : - 13 Azathioprine 7 *. , Cyclophosphamide 7 Other 10 Skin Lymphoma Misc. 5 2 6 Rheumatoid Arthritis 10; Cyclophosphamide 8 Other. 8 iN'rr: ; Lymphoma Leukemia Lymphoma : Misc. 5 3 2 4 Systemic Lupus Erythematosus 7 ' Cyclophosphamide Aiotheoprine Other 3 6 6 Kaposi's Sarcoma 1 Other Inflamatory Diseases 16 Agents Used 31 Cancers 16 (as above, alone or (Lymphomas, Leu- as combined) kemias, Hodgkins. and cancer of skin, bladder, colon, bronchus.) Neoplastic Diseased Patients Without Transplants In nontransplant patients with' neoplasms receiving immuno suppressive cancer therapy, Penn4* and Kersey81 showed that sec ond and even third tumours have arisen during treatment with chemothcrapcutics. Of 185 patients with tumours treated with either Mclphalan, cyclophosphamide, busulfun, 6-mercaptf,purine. Immunotoxicology and Chemical Carcinogenesis S7 chlornaphazine. chlorambucil, ihiotepa, methotrexate, and pred nisone, alone or in combination. 19-1 new malignancies developed. Various leukemias accounted for eighty-two ol me new malignancies: thirty-five lymphomas, twenty bladder carcinomas, two cases of cancer of the cervix, and one Hodgkins disease occurred. The remainder of the cases were various and miscellaneous forms of malignancy and totalled fifty-five. Acute leukemia occurred in thirty-nine cases where the patients originally had multiple myeloma. Twenty-seven solid lymphomas developed in cases where the patient's original neoplasm was chronic granulocytic leukemia. These unusual occurrences would tend to dispel any ideas that these cancers were transition forms of_ the existing malignancy. Thus, one can cautiously extrapolate the data and identify a significant association between the develop ment of malignancy of lymphoid elements and the use of chemi cals thaV have* th 'capability of suppressing elements in the ~immune/hos't.defense system. (Chlornaphazine is the one agent capable of directly causing cancer in man since i^'metabolizes to betanaphthylaminc. It has caused bladder cancer in,aniline dye workers.) * - - . ' ______ - ,, -. Leukemias and Chemical Immune Mod Of the four major types of cvtological leukemia,-myeloid and lymphatic constitute the greatest percentage. Myeloid cases peak out at about 60 percent of all leukemias at age thirty to forty, then declines thereafter. The lymphatic type has the highest preva lence in children peaking at about 50 percent and then declines till age thirty to forty. It then rises to 60 percent peak between ages eighty to ninety. On the way up to its peak, the lymphatic type passes myeloid type leukemia at about seventy years of age.*7' An epidemiological study of solvent exposure and leukemia was conducted among rubber workers by McMichael et ai.4*indicating that an association between leukemia and jobs that involved using solventsexisteri. Prior to this lime, Kessler and Lillianfold (1969^) ` as well as Vigliani and Saita49had prepared papers indicating they thought that the current evidence supported benzene, phcnylbutazonc. and chloramphenicol as being luefcemogens: however, they ha'dTound little epidemiological evidence to support this c 88 Enxnron mental Toxicology posture. McMichacl ct al.48 had analyzed some 6600 co-workers (male rubber workers) who were working during the years 1964 to 1972, but had an employment duration of twenty-five years. They were followed for nine years with a 1 percent loss. Matched conA trols were also analyzed with standard mortality ratios and proportional mortality ratios calculated. Of the various relationships that were analyzed, the association of death from lymphatic leukemia with a history of having worked in solvent exposure environment stood out. It is of interest that th^vi^hati^euicTmfa~Ttands out / as associated with solvent exposureTobsTTfiXs leukemia tends to be the m^eld&lasrc^or the stem cell typ.e. McMichael had indicated that.theriguEemogenic effects may have been the result of concom itant exposure to other solvents that were used in the rubber industry. However, In fan teet al.50 studied a population of workers who were occupationally exposed to only benzene during. 1940 to 1949 and who were followed until 1975. Comparisons with two control groups show a significant excess of observed leukemia (p less than 0.002). A five fold excess risk of all leukemias and.a ten fold excess risk of death from mveloid and monocytic leukemias combined were demonstrated-in the "comparison 'between populations. The environments: of those workers were analyzed, and records have shown that the benzene levels were generally lower than the recommended limits at the time they were measured. The observations are in agreement with Vigliani and Saila,'" whereby a specific type of leukemia is associated with the expo sure to benzene. The myelogenous or monocytic leukemia has been shown to correlate very, well with the exposure and-CQlLfirm* the suspicion that benzene is a powerful bong-marrow poison^ Goldstein51has written an excellent review of the toxic hemopoeitic effects of benzene exposure. These effects, although somewhat complicated by concomitant exposure in some cases, are fairly well demonstrated. The mechanism by which this toxicity occurs is not known; however, th e ralteration -of stem cell function is apparent.51-52-M Occupationally^exposed persons as well as laboratory animal stud ies have shown chromosomal abnormalities, and correlates wejj with the known clinical picture of benzene toxicity.54*15-1*6 Goldstein : ' 1., b* iUM*l or kn ilii clil CYl cti: 1 eh: cy I *i op] lirw enc. clei inv and Men (m i V that to 1 cult M .l ml (o b >lru occu Jmm Ai as w who J W | c F t e T J - . V 1T r . f n f l S I i g y M V . r J L M r Z . S Immvnotoxicology and Chemical Carnnogcnctit 89 has reported-that in mice chronically exposed to 100 ppm of f i benzene, leukopenia, due primarily to lymphocytopenia, was clearly cicmonstrated. These manifestations of pancytopenia may repre sent a destruction of the stem cells, failure of the cells to mature, or prevention oi differentiation at some critical stage. Other agents known to produce pancytopenia act in this fashion through mod ification of nuclear material. They include ionizing radiation l Chloromycetin, vinblastine, mitomycin, puromycin, colchicine and cvtochalasin B. Many of these latter compounds have been used in . i clinical treatment of malignancy. Fomi el al.Mcarried out cvtogenic studies and have confirmed chromosomal aberrations occurring in peripheral blood Ivmpho- cytes, due to benzene exposure. " I f one examines the ontogeny of lymphocytes, it is readily apparent that bone marrow stem cells develop into two different lines of cells. The one line produces hemopoietic precursors, and the second line produces the lymphoid cells. Logically, interfer ence at. the -stem', cell level is bound to affect both the blood elements\-*svwell as -the.immune-.system; elements...Laboratory investigations have observed pancytopenias such as monocytic, and myelogenousileukemias due to benzene exposure also show- stem cells that have produced abnormal mature eiythrqcytcs, which further supports-'stera cell nuclear function interference.. ' AVohnan54 has indicated.that ample eyidence is present to show that chromosomal aberrations can be induced through exposure lo benzene. (Japs-andiunction breaks have been observed in cultured human cells (leukocytes and HeLa cells) at 1.1 or 2.2 x 10" 3 M. benzene. H igher doses caused inhibited DNA synthesis. Periph eral lymphocytes stimulated by phytohemaglulinin (PHA) exposed to benzene for sevemy-two hours revealed both numerical and structural-alterations. Aneuploidy. and chromosome breakage occurred seven and eight times more frequently in treated cells. Immunochemicals and Cancer Advances in pharmacological therapy^aTproduccd problems ** well as remissions in.the treatment oPdseasc states.' Patients -Srho haw -been-treated with'antihypertensive, antiarrythmic agents ** well as antubcrcular or anticonvulsivc agents have shown to be V `i i r / K T j i 1 I 9 i i II 1 ( ! j 90 Environmental Toxicology susceptible to a syndrome related to the immune disease railed systemic lupus erythematosus (SLE).4K4i In SLE, the patient develops antibodies stimulated by the patient's own cellular material, especially thcjruifleacelemenis. It affects various tissues, and has a fatal form. One of the^najor findings in diagnosis has been the presence of an LE cell/polymorphonuclear leucocyte-- PMNJ that contains pK^ o c y tjzed~nuclear material. This cell is formed as a consequence of reaction between an antibody present that can bind cellular nucleoproleins. Presumably the mechanism involves the lymphocytic nuclei that reads with the antibody. Lymphocytes become saturated on their adive sites, and PMN \ cells come to engulf the swollen lymphocytes. Digesting away (phagocytosis) the remainder of the lymphocyte, the PMN- pac/ . [ ages the nuclecrprotein matenaT^gs^aT^residual body calied LE. ) Although this disease is' usually^virallv induced, therapeutic agents7-' ^uch_as diphenylhydantoiny isoniazid. hydra 1axine. and procainamide have induced the syndrome that generally disappears upon with- 1 draws1 of the drug. These mechanismsthat induce-the SLE syn drome are not yet well'-understood;'ho wever,-chemical* influence on the immune system-has produced antibodies that include and- DNA, antinucleoprotein, amihistones,. aminucleolar RNA, and antibodies to fibrous or particulate nucleoproteins..The impor tance of these data reflect that exogenous chemicals, which modify or influence biological systems, have unusual effects that are not always readily apparent. H ow this important defense mechanism has been modulated by interplay with chemicals is unknown, but any real capacity to effect nuclear material creates greater possi bilities of biologicaldys function. ' Zarrabi el al.37 studied four groups of humans under treatment for some two and one-half years, with chlorpromazine and various other antipsychotic drugs. T he'm ain observation was the preva lence of immunologic and thromboplaslic coagulative disorders. In patients on long-term chlorpromazine, the authors found that patients had increased levels of serum IgM. 405 a 55 fin chlorpro 5 mazine treated patients), whereas controls had 157 23. and nor- mal is considered to range from 60 to 250: patients who were given combined therapy, chlorpromazine plus another d rug, for the same length of lime showed levels of scrum IgM at 493 db 75. Other AJVr-* c Med fill ;il j r IK'S, has, te ll is .sent lism <xly. MN wav ack- l.K. rnt.s licit* ilh- yn- W and >or- lif> not ism but ssi- ent 3US va* :ri ha,` ro or* cn he ir r *' mmunotoxicoiogy and Chetnicat Carcinogenesis 91 antipsychotic* used were thioridizinc, trifluoperazine, thiothixene, perphenazine, haloperidol. lithium, and amitryptilene. This group alone did not provide any significant differences when compari sons were made between the groups studied. Also noted was an increase in length of partial thromboplastin time. In both instances, dgriificant correlation between increased levels of IgM in serum along with thromboplastin time were noted in relation to dose and duration of therapy. Both groups had a positive antinuclear anti body test f63%). both had nucleoprolein antibodies (58%), and- hoth~gr5ups~Hadantibodies to_naiwe-4JNA- {4Q%). __ ~ It is interesting to note the authors'conclusion, "the IgM was the coagulation inhibitor," and was identified through immune neutral ization and immunoglobulin isolation techniques. A product of the /immunological analyses was the finding that the percentage nf T- ' lymphocytes were below normal in thirteen out of fortv-one patients treated with Chlorpromazine and twenty out of forty-two patients under single or combined treatment developed splenomegaly. Recently, Doctor I. Fidler5at the Detrich Maryland Cancer Research C enter,w orkingw ith macrophages from mouse peri toneum, found that for some odd reason .the, macrophages began losing their tumoricidal activity during in vitro studies.. Ordinari- ly, such macrophages in the peritoneum are not cytotoxic to tumour cells in vitro;:however,- a* lymphoidne released by an, activated lymphocyte, referred to-as'macrophage activating factor (MAF) has the ability to stimulate the macrophage to become tumoricidal. Such peritoneal exudate macrophages (PEM) can also be stimu lated to become cytotoxic by bacterial products (lipopolysacchar- ide--LPS), endotoxins, pyran copolymers, double stranded RNA, or during chronic infection with obligate bacteria. The m vitro studies were slopped and. in certain cases, the procedure modified b lo u se the PEMs were found to have lost the cytotoxic capability. A careful examination of the occurrence of lost activity seemed to correlate; with a. minor change in the drinking water used for the mouse colonies. Water.fed to the mice had a chlorine level of about twelve to sixteen parts-.per `million (ppm). This high level (is necessary to reduce the'rate1:of early death syndrome in the colonies due to fctudomonas infection since the mice had been lethally irradiated* ^1 5 ' a 5$ nn n o6 A 92 Environmental Toxicology J 1 Due to an unusually high incidence of such early death jyn. dromes, the chlorine level was raised to 25 to JO ppm, and ih#. experimental studies were carried on as usual. At the start of the experiment and just before treatment, the mean number of PEMs per mouse was 21 -4 x 106. One week later the level of PEMs in the mice receiving hype^cklo^mated water had decreased to 13 2 x 106 per mouse, dontrolv were" "7a yielding 25 3 x 106 per mouse. On consecutive weeks, PEM yield increased from the control mice, but mice on hyperchlormaud water had reduced PEMs or remained lower than controls. Mice that were receiving tap water yielded macrophages that when stimulated were tumorcidal in vitro to B16 melanoma cells or to UV 112 fibrosarcoma when activated by Concanavalin A-MAF, as measured by release of radioactivity. The mice drinking hyperchlorinated water exhibited lowered 4 levels of cytotoxic ability for the first two weeksTand. by the end of the third week of treatment, the cells, although stimulated by Con A-MAF, were not tumoricidal. These studies show that hyperchlorinated water produces pro found alterations in the numbers and tumoricidal capacity of 7 PEMs. Lower levels [10-15 ppm) may also exert such influence although over a longer period of time. ' Supporting these effects was a report by Fidler that showed that patients on long-term hemodialysis developed acute hemolytic anemia when treated with water that, although filtered by reverse osmosis, has 2 to 4 ppm chlorine. Chlorine compunds brought 4 4 \ L about a denaturation of the hemoglobin by direct oxidation and also by inhibition of the direct oxidative pathway (Hexose mono \J phosphate shunt) of red blood cells (RBCs). T he damage to RBCs was found to be cumulative over several periods of dialysis. / Although the mechanism for this depression of macrophage tumoricidal activity is unknown, Fidler suggests several possibili ties. The vacuoles of the macrophage system are probably involved in the cytotoxic mechanism. This has been shown by Hibbs and Weinberg19 whereby inhibition of the lysosomal enzymes of the macrophages occurs by addition of trypan blue, and stabilization of the lysosomal membranes occurs with the addition of hydro cortisone, and the cytotoxic activity is suppressed. t syn: the . the veek aled were ?EM lated Mice *hen r lo -F, as ered -dof jn proty o .enee i ihal dytic verse njght a and tono-dBCs ihegc ibiliulvcd s and >f the alion ytlro- mX.S-1K?Z-^W SnCX / Immunotoxicology and Chemical Carcinogenesis 93 Macrophages that have been activated by Ivmphokincs have enhanced bactericidal activity, and mctabolically are shown to have a four to eight times increase in the uptake of glucose and its oxidation as compared to controls. Since the chlorine {compounds) inhibits the HM I'S pathway5'*(that is the major path for glucose oxidation in the RllC's), there is a possibility that chlorine and/or its compounds may sufficiently inhibit glucose oxidation to the point whereby macrophages that have reduced tumouricidal capac ity will allow aberrant cells to continue growing and relocalizingl Concurrently, indirect pathology also occurs! Chlorine levels affect erythrocytes' glucose metabolism, and large amounts of hemoglo bin degradation products and/or large numbers of damaged RBCs can suppress macrophage tumoridical activity. Regardless of the mechanism by which the chlorinated water or chlorine compounds may exert their influence, the important fact remains thatTinacrophagc activity is compromised. Since if carries a major role-in host defense against neoplastic disease, the possi bility exists that.a host who is exposed io such compounds may in fact become immunocompromised. Silica, carageenan, and trypan blue are substancesthatalso have the ability, to suppress macro phages .in lab'.studies'havc been reported to decrease host'resistance against transplantable tumours. Dandliker et al.* recently reported their studies of the effects of pesticides on the immune response. Hamsters (LHC/LAK) five to eight weeks old and weighing about 100 grams were given a dose of pesticide equal to one-half-the LDWdissolved in 1 ml of corn oil. Arochlor 1260, Dinoseb, Paralhion, penlachloronitrobenzcne, piperonyl buloxidc, -mixed pvrelhrins, and resmithrin were administered im ragastricallyT he animals were examined for an end point of "redness and swelling" (inflammatory response) and change,-in? tempera lure of the. foot pads, as well as histological exam, after an antigenic challenge. Serum antibody titer, binding affinity, andiheicrogcncily .were determined by fluorescent polari- " The'animals; werc`:firsl. given an injection of fluorescein labeled, ovalbumin, allowed-only water ad lib for twenty-four hours, and then given a bolus of. food with the pesticide by imra gastrie feed ing tube twenty-four ham? afltrr~thc~immunization. The most 94 Environmental Toxicology striking feature reported by the authors after immunanalysis inchcatcd marked humoral and cellular immunosuppression to single doses TDinoscb and Parathion, and a marked stimulation of the cellular response by resmeihrin. The other pesticides showed little or no effect under those conditions. In another study61 using the pesticides Ametryne, Carbaryl, Chlodimcform. DDT, Malathion, Mirex, and parathion, a single - dose of pesticide-'waa-given orally at the -LD^-oirthc-fht days before, two days before, or two davs after immunizatioaAdth sheep erythrocytes. Assays were then conducted using antibody plaque forming cells four days later. (Plaque forming cells-- antibody producing cells that can form a hemolytic plaque in the presence of complement and erythrocytes.) All animals receiving the higher dose exhibited significant depressions in splenic plaque forming cell numbers. Low dose animals receiving-the dose for either eight or twenty-eight days prior to immunizationexhibited no significant reduction in the antibody plaque forming cell num bers. The author indicates that a lack of information prevents a conclusion as to the efficacy'of-these compounds on modulating,' the immune system. Since the methodology only uses one test for the .erythrocyte receptor, little can be concluded. ;Tbis is.why m ultiple parameter assays are necessary. (^FaTth and L u s te r^ have performed extensive investigations on the pre- and postnatal effects of Tetrachlorodibenzodioxin (TCDD) on the immune system and have found that TCDD appears a relatively excellent immunosuppressive in the FischerAVIstar rat strains. The Fischer strain is reputed to be less of a responder than its Fscher/Wistar cousin. However, dosing of nursing females has shown that thgJT_CDD has the capability of causing immunosuppression in litterm ai*- The ffects have lasted as long as 270 days, from three doses to the mother at days zero; seven, and fourteen, applied at five micrograms per kilogram (ug/kg) body weight. At days eighteen and thirty-five, both female and male* littermalcs showed depressed body weight as well as depressedjhymiewgights. These depressed values were evident at day 128 postdosing. The weight of the spleens were also found to be affected. Effects of TCDD exposure on the homing patterns of lymphocytes were also studied in these same rat strains. Splenic cells taken from c the * the I crlU TCL horn in ri that alter C I tinn Tt abili mon exj>c SUXX rest prti hype imm La phen Kidle was I l-dejl Kuril mixti p-tcr come Oe, phen Ivu a rates, to sii *plee IVALl il wee c fmmunotoxicofogy and Chemical Carcinot'vncui 95 the TCDD exposed rais were injected into noncxposed rats, and! I the thymus was found to significantly increase the uptake oT such! cells. Thymic cells taken from nonexposed rats were injected into TCDD exposed rats, and it was found that there was decreased; homing ability to the thymus. T he authors proposed that a change in cellular metabolism occurred altering the cell membrane, or that insertion of the TCDD into the membrane caused surface alterations, and this change in the cell modified its normal hom ing patterns. Various investigators have shown such alterations in immune function due to TCDD exposure.63,M.61 has shown that subritnicahjosesnof'-TCB D ' had the ability to affect host responsc7 when subsequent exposure to^o7monella infection resulted in reduced time to mortality. Thus far, exposure to TCDD has been shown to cause an increase in the susceptibility to bacterial infections (suppression of immune response) as well as suppression of mitogen responsiveness, suppression of the skin graft rejection, and depression of the delayed' ^hypejDLeiisUivity-respons^.-(Suppression~of the T-cell dependent immune functions appears to occur as an.isolated response.). P h e n o l -* (''T avivet al.66 also had occasion to come upon the effects of 1 phenolon immune function, through a case^o'f serendipity, as did ) Fidler. Phenol was being used to:disinfect yaVcages of mice, and 1 was found to be causing depression of the immune response to ^dependent antigens in the time period of four to six weeks. Further analysis of the phenol showed the disinfectant to be a mixture of o-phenylphcnol (5.0%), o-benzvl-p-phenol (4.5%), and p-tert-amylphenol (1.07). OPP, OBP, and PTA, respectively. Such compounds are used in biocides throughout the world. )ehme67J>as studied the metabolism of one compound, o-phenvlphenol (0PP) in the cat and dog and has found that although the two animals metabolize OPP according to two different routes and rates, excessive tissue levels are found in the spleen. Lavia66,decided to study this phenomena on immune function knowing.that the spleen plays a major role in the .immune response. Groups of BALB/c'fcmale mice were dosed with the"phenol .derivatives (all three derivatives were used, but each group of mice received only 4 96 Environmental Toxicology a single compound) at levels of 0.46 milligrams per kilogram (mg/kg) of OPP, 0.41 m r/k g ODP, and 0.09 mg/kg PTA in their drinking waTcr. At weekly intervals, three mice were immunized intraperitoneally with 1.0 x 10sheep erythrocytes (SRBC). Four days later the number of IgM plague forming cells (PFC) was determined. Before the end of the second week of exposure, 45 percent of the mice showed immunodepressive effects. 1ne response after lour weeks showed depression to be occurring in 77 percent of the dosed mice, comparison of OFF with a mixture Of phenol compounds showed the immune depression to be the same, indi cating that OPP appears to have the same capacity for immune suppression as the mixture and must be exercising dominance in producing the response. Monocytes (Macrophages) were also ana lyzed for their capacity to phagocytize yeast cells. This capacity was significantly reduced as compared to controls. Measurement of T and B lymphocyte numbers in_control and OPP rtp n w l mice showed no significant differences. T he net effect was that the macrophages appear to have sustained some defect that reduced their ability to present antigen for phagocytosis or may have just resulted in a reduced number of circulating macrophages that could affect the cooperative cell-cell activation upon'B^lymphocytes. Archer6* has also studied the suppression of the immune system using gallic acid, a phenolic derivative,'and has concluded that the mouse spleen cells that were studied showed a marked depression of the immune system from such exposure probably at the macrophage level. These data are very important, because it shows that immune suppression has occurred at low dose levels and with short periods of exposure, which situation mimics many of the environmental exposures. This is true especially with drinking water. To extrapolate this evidence without asking other pertinent questions clearly is not yet justified, but when taken in concert with the studies done by others, it can be seen^thal the immune system is in fact a very sensitive system'that-triay~5g anddeal vehicle lor indicating potential, toxicity to humans.'Although ultrasiructural morphology or DNA damage studies have been used prior to this time for indications of toxicity, it appears that immune cell biochemistry-^ay~be more sensitive to such low level doses that are prevalTnom the envi- ( ronme Extc tin con Scinen lion of thc-spltionshi of thyn pronoi periph reverse exposu tin chic showee. n-dod|? tri-n-c; atrophy data dc cstablis; in feetio is also s conclue B-Iymp modifie rabbits million priinan depress kilogram in their '.rnuniicd )C). Four PFC) was osure, 45 response 7 percent ol phenol me. indi-immune inancc in also ana; capacity surement exposed have susoti^en for er of ; cell--cell adied the phenolic cells that' n c system ~hese data ession has exposure, exposures, olate this .rly is not s done byact a very. ' potential yorD N A cations of ' , be more:the envt-' [mmunotoxcotogy and Chemical Carcinogcneiii 97 ! ron me nt and w o u ld co m p le m e n t--smrh- h uaassays. Metallo Oreancs Extensive investigations have been made of the effects of oigano tin compounds on the immune system and it has been shown by Sclncn and Penninks69 that Di-n-octvl-tin chloride causes a deple tion of lymphocytes in the_thymus and thymus dependent areas in the spleen and lymph nodes. There was a dose dependent relalionship showing a decrease in the number as well as the viability of thymocytes. Spleen cell numbers and viability were slightly less pronounced, and no-eifecu was-found on bone marrow and/or peripheral lymphocytes and/or monocytes. Thymic atrophy reversed upon discontinuation of the exposure. The results of exposure to Di-n-butyl tin chloride was identical to the Di-n-ootyl tin chloride. Di-n-ethyl-, and Di-n-propyl tin chloride compounds showed less pronounced effects. In contrast, Di-n-methyl-, Din-dodecyl-, and Di-n-octadecyl tin chlorides as well as monooctvl-, tri-n-octyl-, and tetraoctyltinchlorides did not show any thymic atrophy. In this excellent review,; they examined a large body of. data dealing with lead and cadmium on the immune system and established that the major effe<n `is'an increased susceptibility to infection by gram negative bacteria that con tain endotoxin. Lead is also shown to decrease the resistance to viral disease, so one can ,conclude. Lhat the humoral response is somehow alfected d u sin g B-lymphocyte defects in* antibody generation/'or perhaps in*lhe> modification of circulating antibody molecule itself: Koller,70using rabbits dosed with lead acetate, 2200 m g/liter (equal parts per million) in water showed that after ten weeks of exposure, both the primary and secondary response to pseudorabies virus had been depressed. Koller and Kovacic71 showed that mice dosed with lead acetate had a significantly increased number of IgM plague forming cells to 5RBC. This further adds'to the complexity of-the situa tion, since the effect may be occurring in the lymphoidorgjm(s) associated with development of B-cells. Additional celj7 may)be stimulated without proper maturation and/or they may have defec tive antibody response capability. Chronic low level dosing of lead to rats pre- and postnatally by' Faith et al.7* showed the thymus ^eights to be suppressed along with decreased responsiveness to 7 7 - aO 98 Enm'rvnmcntal Toxicology mitogen stimulation of lymphocytes and reduced delayed hyper sensitivity response. Of greater importance is the fact that the offspring of females dosed with 25 or 50 pput of lead in drinking water showed no inhibition of growth, as exhibited by body weight gain, nor overt signs of toxicity. However, the analysis of the immune system functions of offspring did show decreased mitogen responsiveness, as well as the depressed delayed hypersensitivity reaction. The inescapable fact is that some lacet ol me immune function has been altered. The doses of 25, 50 ppm lead used for the mice produced blood levels of 29.3 and 52.8 micrograms per 100 ml blood, which are comparable to blood levels found in human children, makes currently allowable lead levels somewhat undesirable. In light of the relationship of humans to lab animals, *\ and to this evidence, supplied by the more sensitive immunological 1 indicator, covert toxicity may in fact be occurring72' 74 even at low j levels. `' Hoffman and Niyogi74have studied metal carcinogens and have indicated that lead and the.other metal salts were able to interfere with the fidelity.of DNA. synthesis. Such interference would, if occurring in the. B-cells, have a .profound effect on resistance since the ability to differentiate in lymphocytes is absolutely necessary for host defense. Should it interfere with the DNA at a small lymphocyte blast stage'perhaps a major clonal species of lymphocyte "coulTbg permanently impaired! " Additional studies by Vos el al.75 using hexachlorobenzene on \ rats has shown the immune system to be stimulated, which con- [ trasts other data presented. Sharma76 has exposed mice to vinyl chloride and found the immune system lymphocytes to be pro foundly stimulated. M iller77 and Kagan and M iller71 also have observed immuno- stimulation in patients who have asbesiosis. Some disturbance, may have occurred in the immune regulatory mechanism, since they have shown hyperactivity in the humoral immune response, increased reproduction in the scrum globulins, secretory IgA, and a variety of auloanlibodics. T he reason for this activity is not clear; however, asbestos studies done on the lung suggest that macrophages trying to digcst_the mineral fibers are damaged, spilling out lysosomal enzymes, which may bring on autoimmune paihnhn. caiiM's n the* puli im pairs that mac there is but who Itccxu--- n i\m . lb of the it c nyperiat ihe inking weight of the `logen sitivity Timune ~ed for is per nd in iwhat mals, Jgical it low. have terfere >uld, if since issary small locytc on convinyl pro- unoiance since onse, and not lhat gcd. nine I Jmtnunotoxicology and Chemical Carcinogenesis 99 pathology. Drath et al. have established lhat smoke from tobacco causes morphological biochemical and functional alterations in the pulmonary alveolar macrophages, which are functionally impaired with respect to phagocytosis. Since it is well established that macrophages are an integral unit of the immune responses,79 there is a significant modulation occurring that cannot be denied, but whose total impact is yet to be discovered. -- Because of a known roie in the immunosurveillance mecha nism, the possibility of modulation by environmental chemicals / of the immune cells certainly seems an excising speculation. n Summary The influence of environmental chemicals upon the public health is of considerable importance. Determining how such chem icals may..cause advers effects upon-hum ans is a continuing problem that* perplexes-all phases oTsaentiQcrinquiry.-Genetic chronic, toxicity-and dose responses are" defined from animal bioassays and microbial DNA studies for human use, and consti tute a major source of controversy'among predictive toxicologists. A major., step .forwards in' resolving such problems ^could _be^ attained with the use of.human lymphoid cells/which are readily * available from the peripheral circulating blood. The lymphoid cells of the immune system are an integral part of the defense mechanism known as immune surveillance and are very impor- j tant in the.recognition and renjoval of abberant or malignant j cells. It is this relationship that inay t>e disturbed by environmen tal chemicals and allows carcinogenesis to proceed in susceptible individuals.. T he use of immunosuppressive therapy has shown there is an increased frequency of various types of malignant disease associated with continued use oTsuch agents. Secondary as well as tertiary cancers have been produced by therapeutic suppression' of the immune system. Concurrently, investigators have-also shown .that occurrence of malignant, disease is also found in individuals with genetic immunodeficiencies at a higher frequency than in immune normal hosts. So it has become apparent that.malignant response\ m a given host may in fact be very seriously dependent upon an immune system that has been somehow compromised. 100 Environmental Toxicology In vitro and vivo studies using pesticides, metallo-organics and various pharmacological agents, have shown that certain activities of lymphoid cells are modulated by the presence of many of these compounds. Lymphocytes have been either suppressed or stimulated, and either condition may be affecting immune response. The blood f monocytes, which play a vital role-in-`the~~B-cell/T-ceIl in te r a r tio'ris, are shown to be very severely disturbed by excessive amounts of chlorine or hemoglobin degradation products. So, direct or , indirect eifects can modulate the immune response, showing the sensitivity of the lymphoid population to environmental influ* ence. The cells themselves may become defective through direct action, or the tissue in which they mature and differentiate may be modified, producing an impotent cell. The cell ^mav/be, affected at the surface, within the cytoplasm where antigenic determinants are synthesized, or within the nuclear protein and/or chromosomal levels. Thus there exists a cell for all seasons morphologists, biochemists, immunologists, pure chem ists, pure biologists, all will find an abundance of suitable material to investigate. The most rewarding.portion of our work may well be that.we will be closer to effective extrapolation for human exposure. Conclusion The mechanisms whereby chemicals influence the immunological surveillance system are not understood. In fact, there are many who have asked questions that may^3ull-th''exd temjmL-bver the im m unosurveillance theory. HoweverT they have-not been suf ficiently substantiated. What has been elucidated in this presenta tion is that we cannot deny the influences external chemicals have on the cells and products of tissue from the lymphoid-immune system. The role of chemically induced malignancy in the immune suppressed patients receiving therapy or the excess occurrences of malignant disease in fimmunodcficient individuals is significant and cannot be ignored. The complexity of the immune system, details of mechanisms, in fact, whether cells or products may be influencing this system is in most cases not known. However, it can be stated that many of the substances mentioned here today ; anti vuics lliCSC , and Dlood tcracounts :ct or g the in Au di reel .ay be slasm jelear >'-'r all v .lerial y well uman ogical many cr the n sufscnias have imunc imuiic ices of ificani ystom, aay be %vcr. it today Immunotoxicology and Chemical Carcinogenesis 101 are in ihe environment and they do have immunomodulaling effects. How does this role of immunomodulalion affect interpre tation of previous studies that discerned that a mouse, rat, guinea pig, or some other lab animal has or has not' responded with malignancy to a carcinogenic chemical? Have those modulating effects been taken into account in concluding that some chemical is or is not a carcinogen? Clearly such questions can establish a compromised position when making conclusions as to whether any chemical should be allowed in the human environment. Mice, rats, hamsters, and guinea pigs have variable systems. Has the chemical tested caused a depression of the system that allowed some'virus to induce a cancer? Or has the animal perhaps a / depressed immune system, due to repression of genetic expression due to inbreeding that nowjdlows^jneoplastic nssponse to occur? Immunotoxicology is the new kid oh the blockand is able to ask some very difficult questionSrQuestioiis foi^wHich we have not all the answers; In the area of predictive toxicology', however, I would suggest the following. Because immunotoxicology may bring about an additional dimension in extrapolation. I would suggest that future studies be directed toward examining these immune system ele ments and how they respond to mutagenic or carcinogenic agents. Not only in the lab animal species, but by using the peripheral blood elements from humans. 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