Document YjaEGpk9d01ngzv7X9XKdEGK

and ncurochcniical ormarci JV;43J--44-1. i: aJlrrrd urinary n in mice and men. r ped VHronchcjtm I W M J;S6-fiO, cardiovascolari nei d Lawrie.-T. D. V.: mic lead exposure in 156.1974. Tronic lead tnioxica' .2SJ:3S9-*365, 1969. ologidal disposition ihectomued uumals. td exposure in lacii1 maternal blood and n 1975.,'V / \ J&?*1TuTdtnbtP Lxi.en. ltd Proceed H., and Hejtmxncik. diovaacuLar responses 40.407-413,1977' * eand noreplhcpKrine J9;134.1977. Kronic lead exposure response to norepine- levrl of p erin au l lead dioloxicity. J? Comm xnd Ziegler, M. 0 .: "166.1978; >571" ' j. H ,, Hejtmaneik, poisoning. InBm wn, Oxford, Elsevirr- if-If'Vi /? // C hapter 5 IMMUNOTOXICOLOGY AND CHEMICAL CARCINOGENESIS J oseph 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^feen deveidpecTw^h an ,,eye loward the. early . recognition of the occurrence of a lesion in some target organ. PliyriologicalTand biochemical' changes axe 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#kidney have..been most intensively investigated, other systems have been used in this effort to establish an early warning system. T he majority of the studies carrie io u t have not taken into account the possibil ity that the i mmune,system may be the Jdeal_vehicle for such^bioassays. For whatever reasons, investigators have underestimated the importance of the immune system as The tulhor wuhea to acknowledge help/ul comment and lu^pcttioni contributed to the author by Dr. M. M. Vokotimi, M.D. Fh.D- Director of Clinical Immunology, and Dr. John Bedrrb Ph.D, Chief of Toxicology and PharmaeometHo. of the University of Illinois Hospital and Medical School Staff. 7: .The author alao wiihe* to acknowledge the very helpful clerical auiiunc provided by Mnu Debbie Fript, who did the typing and editing of the.manuxnpi. The author alto accept the blame for any error or inconsistencies which may be found, and requests any reader to pleate notify the author of tueh mistakes u may occur, without Intent, " 1 , . . *. \ 71 1 a i1 72 E m i'ronnttntal 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 complex and not 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 sensilive tojchemicals.^A..gpod;deahof inform auoiThss' been "developed oh the use of amticaheer 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 level'are also seen in vivo and in vitro and of course raises the"question of whether this activity can.be at work during'exposure to environmental chemicals?This 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. Incroducdoa D uring the last five years, many of us have heard the statement that **aJarge. num ber of cancan may have their etiology in the environment.1" This statement helped to propel an already ener getic national1desire to1seek cancer causes and* cures.-Although there are many different opinions as to the actual num bers of cancer cases contributed by the various environmental factors, it has been generally agreed upon., among biomedically oriented research scientists, that environmental carcinogenesis is a real emity.1 In an effort loseek a cure or to begin a prevention pro c / gram. the cn ii.sk to humui In order to has had to use extrapolation liplc specie t studies, and cl cautiously, ha data is lackin consternation Recent tlcvt a nucleus of c these problem immune mccl lutcly, rescarc system cells (s products sue body's immi infection, graf The fact th; tissue present |x>icmial for.r the study of p< Kpidemiolo; various detent environmental provide direct statistical corr* disease have b capable of cat among human been an im por predictive toxic a suspect chem Microbial as the successes c c nuclein of ical in v i rgin s and es, mono:sliga(ions or chronic : response nutrition, dy in use 2 system. jmpletelv j indicate ear quite osl sensibas been eir capapatients changes are also:* . J -jre to ' here are nmuno- . dpoten-. t.'i I j j : : =statement ogy in the eady enerAl though umbers of.. 1factors,'it; ` y oriented 'is a real ntion pro-.... Immunotoxicotogy and Chemical CarcinogtntiU 73 pram, the environmental chemicals that present a carcinogenic risk to humans, arc an ideal target for investigation.1 . In order tu establish some measure of risk, predictivctoxicology 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 \~dalFly 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 products such as lymphokines play a very important role in the^ body's immune reaction to possible pathology from microbial j infection, graft rejection, and tum our production. ' The fad that these cells are readily accessible and are hum an ; . tissue present in extensive amounts in peripheral blood and have '-; potential Tor rapid proliferation makes them an ideal vehicle.for the study of potential toxicity.......... ` Predictive Toxicology in Retrospect' Epidemiology is the discipline that intgrales'the impact of t h e : 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 \ disease have been able to relate the possible chemical toxicants j capable of causing pathology to the occasion of disease states / 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 ihc successes-of Ames and McCanh 'in establishing aberrations 74 Environmental Toxicology seen in mutagenesis assays as being related to the process of c a r c i n o g e n e s i s . T h e philosophical basis for this procedure lay in the fact that a chemical capable of causing DNA related lesions could be detected 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 typhimurium, which has a histidine negative mutation, Ames and co-workcn were able to show that exposure to chemicals causing genetic lesions did cause a reversion or back mutation. The normal Sal-A monclla cell was modified by introducing membrane defects so a ; chemical could enter. T he 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. mammalian meta bolic system for activation of the chemical that may not be a direct carcinogen and requires .transform ation.. ,.v ... 'Since tfiis System syasYntroiiuced, others*7;.?have validated thfefI assay system using hundreds of chemicals and have reported that" approximately 90 pgreenf-nf the chemicals tested, which were reported in anim aTsvstem slUadhe_caxciiiogenic. were in fact mutagenic in the Ames assay system. A 1diough the correlation is fairly goon,- 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 chemicals* ability to cause carcinogenesis.11 When an appropriate experimental design is used, the mamma lian bioassav generates the biological data that.has been most sensitive in determining whether a given may-have-' carcinogen ic p o ten tial..D u e.to the fact- that, animals used for carcinogenesis bioassay? are highiy^inbred,.some strains may express a predisposing genetic tendency "towarcftHe occurrence of neoplastic" response from chemical exposures. It was therefore recommended by the National Academy of Sciences*1 that carcinogenic studies be carried out in multiple species or mammalian laboratory ani mals, so iliui Triy~expression"^ a^genciTcTesibncausTng neoplastic response be validated or supported as much as possible. Animals, s do him biiK* pres* lotion wh* Anders* vide a Miii (herapeut. dictors in large amn chemicals burden in Carcino. many spec (he most s alone is ui tissue, mo biochemig^p rrticuIumVib? in inlcrprc cr. such chi. . ... In ihcir-r,,icals. the N use of .rans|xvcics ide guarantee r it is all (hat Similar a used in care far the mu e,v*i*de*nce foi sure to envi The rccet confirmed il plus mice h: wry, from a (hat given cl lluorcnc, an M ic e th a t v, shoved tirin c ss oi dure 'a led i'had nciic num, 'ken nciic Sat. so a ncu- erial :nalictareel the V-i Yact n ' U. nee -nie ian lily na- lOM ave for TM stic led vs nilie Is. Immunotoxicoiogy end Chemical Cercinogtnesis 75 as do humans, have varying susceptibilities, so that the 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 chemo therapeutic* 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 have been reported using many~species.2*-3TIlegg?6has indicated that the mouse has provided the most success, but he and Jensen27 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 *sSi*4h<4*' n i nierpretalioffTo f the a cta l^ a iHolo ^ ^ T h ey'do suggest'hpwev;cr, such data should be used as a^waming signal for further study. In their report on principals for evaluating environmental chem icals, the'National Acdemv of Sciencel'fNAS)21 recommends the ._.,^.'^iirantee,of successfully being able'to pre'cfiSmfoaii_toxicity, bVt ' it is allthat we have developed up to the present time. Similar arguments exist for any of the animal species that are used in carcinogenic bibassay. 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 megamouse study reported by Littlefield et al.^* has confirmed that the dose-response studies performed on some 26,000 plus mice has validated that such information' is absolutely neceswry, from a standpoint of both dose'and time. H e has'reported that given different*lengths of lime of exposure [o 2-acetylaminofluorcne, an animal could respond with two'different end points. M ice that were''doscd for eighteen months and sacrificed laier bowed urinary bladder neoplasms; they also required continual I i 76 Environmental Toxicology presence of the carcinogen although the neoplastic response wai 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 Mthere is no le\-el of exposure greater than zero for a j toxic substance, which can be assumed to be without harmful i effect."51 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* foxinvJto.coexist jn he realm--ofchuman 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,, assoaating some end point,with * given level and length of tim ,f.'the exposure/, A `.source; of consternation has been, w h i & p i t r t ' p f c a l l a . t i n g allowablet dose to exposed humans.5*"35 Based on the data of the megamousc study, both lesions re 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.4that-czn provide consistency, assuming the data from biqassays.proyidesufficient evidence that chronic pathology .* a real'possTbi1it*y.\Vilson,58 with some reser vations, supports .this philosophy, suggesting it is useful for making comparisons with benefits easier to calculate. --- T he point I have tried.to make is that regardless, of.-lhe meth- odfs) being utilized,, problems exist, that prevent the .unanimous acceptance of'a'ny of the given .assay systems. Even when taken in concert, the major stumbling block has been that, we do not have information as to how this material will react in humans. Jmmunotoxicotogy end Chemical Carcinogenesu 77 * Cellular Basil of Toxicology The influence of environmental chemicals on the various bio chemical, morphological, and histopalhological parameters has been intensively investigated in various laboratory animals. Kpidcmiological and statistical analyses have produced more sophis^ licated 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 micromolecular structure-activity levels need to be very carefully analyzed to determine the sites and modes of toxicity. W hether for acute or chronic toxicity, these are the fun damental questions that must be answered in order to determine the levels of chemicals humans mav tolerate without adverse effect. , This is not a new or reorganized philosophy, but a reconflrmation of ihc ongibahprinciplcs laid down b v o u r toxicological forefathers. 1 As the_numbers of chemicals entering into tHcTenvironment have increased, the long-term bioassavs for all chemicals becomes . * .t , , i * *( fi ,, # *m ,, less and less possible due to`the'length of time and large costs inyolveityS^uentiallyi Ti'becomes 'm ore im portant to further ' deyelopcellular bioassa^' as gerietic defect predictors. T he quicker response time and reduced costs are obvious benefits, although * extrapolation is a difficult constraint.' Since the correlation of animal responses to human situations are problemaiicaLJUsuggest wc devote more of our attention and energiesltywardnse of human ceiis^Since the protection and well being of human hie is our~ i f ultimate end point, data that can be derived from culture and i : analysis of human cells can provide us with the best evidence for ^ making predictions and risk assessments. Immune Cell Analyses One area that has not been well investigated but deserves attention 15 ihe human, immune system. Vos10 as well as Silkworth and Loose,57 have found evidence that supports the concept that envi- c 78 E nvironm ental Toxicology 1 ronmcntal chemicals can produce modifications in the activities of ri the immune system cells. Antibody mediated resistance, cell medi 0 ated resistance, as well as increased levels of mortality due to si lowered resistance to bacterial infections have been reported in Li their reviews. Silkworth proposes that cellular and humoral medi 01 ated responsernay be the appropriate indicators of environmen (I: tal toxicitv. H e. indicates they are quite sensitive, they can be & < conveniently evaluated, and he has had limited success using this >1 test system in the evaluation of polychlorinated biphenyls (PCBx), tx Arochlor 1016 (ACLR), and hexachlorobcnzene fHCB). Taylor** has analyzed various immune functions in th e ^ n e a pig during in dietary studies; however, he had conflicting resuItTdireTo various dt other factors that could not be controlled. Luster et al.fMas well as at Luster and Faith.*0have evaluated the rationale for using immuno bi comptence as a .forra of labpr^tory.ip.vestigatiops..^They reepm: . mend u sing-multiple pararaetersvof.study to 'properly' evaluate' K- en vironmental. chemical, ellects, since norm al immune responses a7e dependent upon:m acrophages..and at., least", three^s els of. lymphocytes. These cells .interact among themselves `and with" vario us.cell, products (Iymphokxnesand/or complement), soit can' n< ; I become quite^om plicat^.'H ow ever, the^studies. tHey perionned .:ni did show that exposure to various chemicals produced immune- tn cell modulation. Dean et al.41 have .examined cell and humoral an mediated immunocomptence .and also recommend m ultiple- M parameter assays, with most of them dealing with systems designed sp to study tumour/cancer.KU7-U (o It is obvious that since the immune system deals with a large lot i population of cells of hum an 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, we can correlate their reactions to chemical exposure in a realistic ^?7 L. * manner, because it is similar to n environmental exposure. Chem icals that are taken in by humans must be absorbed and transported through the circulating blood, which provides the media for dose till hi Ui tin air me wq association and residence time. Generally speaking, most chemi i< cals are not biotransformed within the absorptive area of the intestinal tract. In their contact with the blood elements during ma Oil c ies of HfdiIC lo tl in H:dincnn bo this : bsj. Iors< ring ious II as jnnom- jate TSCS : of ilh nc* , irai he 1. tic Ti ed *c liw lK Jm mu notoxicology and Chemical Cam 'noge nerii 79 circulation, not only do you gel contact, but also a realistic mode / ' of how and/or what may occur given a chemical and a sensitive or ^ susceptible cell. Cellular response may be at ihe 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 immuno toxicology 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. ) Immunotoxicoloer: The Link to Humans? The..imi^un^jdefense^is a;dual system composed of natural' "* ' rTonspeoficrhafriM ^uch^us^kin keratin and pH, of the natural mucociliary-barriers-as well as the normal bacterial flora that act lo prevent bacteria-from penetrating,. A third nonspecific mech anism begins operating when that first barrier has been breached. Macrophagei<mnd:polymdrphbnucIcar leucocytes, -together with sj>tcific:h u m o r a l ^ i s p l u b l e l y m p h o k l h e s , act in concert v` to sequester, ^degrade',*an d :neutralize"the material recognized as foreign, r-. --* If these natural and nonspecific barriers have been overwhelmed, *hc second and more specific defense mechanism comes into play. It involves the activation of lymphocytes that are preprogrammed lo combat, infection. T hese lymphocytes are the main element of this s^tcra and compose`the basis of the humoral, and cell medi ated imm un cVdcferise'' mecKanisms.' They are indistinguishable morphological!y,':*but; because of differences in the programming equcnces, therlymphocytes Have different capabilities.; Toti[x>tcnt.(or.-pluripotem):stem cells derived from the bone niarrow.are.distributcd lo the peripheral circulation and home in' on certain -tissue forcducation and processing. Lymphocyte clones ;C I 80 Environmental Toxicology arc processed by the thymus gland and are programmed with cer tain specific surface antigens and cellular memory for use in re sponse to fungi, parasites, intracellular vjral infections. tissue grafts and cancer cells. These are the^T-lvmphocvtcs^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 calle<^B-ceilsI)They respond to bacterial infection by ^transformation into a p lasma cell that secretes antibodies. The anTibodies are located in the peripheral blood, circulating about until they'tnpet an antigen and then begin the process of neutrali zation an (gorges tion. Each lymphocyte (T or BJ has received specific information during processing and is a clone with a response specificity. The specificity is due lfchemical 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 five for many year* and can. recirculate from 'lym ph node to lymph and to blood.*1 Another lymphocyte population that does not possess either immunoglubulin (B) or T-cell surface markers is present and they arc called null cells. They are considered to be involved in target cell killing termed antibody dependent cell mediated cytotoxicity (AD CC).* 5 Genetic Regulation Immune responses are controlled genetically by a series of linkedjgeaM known in mice as the major histocompatibility complcx^M H Cf-and in humains as th^HLA^compIex.- A similar series of genes has been identified in eacHmammalian species examin ed.^E lsj^henm enT was discovered when guinea pigs inocu lated with an antigenic complex of Dinitrophenyl conjugated poly 1-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 e rtvdcr U u rrd 10 contull on* of ih* currenily tvailibW le*U on mrnunoloy more dcuilc-d itifurmalion. specific r relations Furlhc amino ac responde . grouping other haii but strair respondet were invo respond. Further was linkc< control of rccognilL^. mouse complex i regions ha tissue tran and the "T The H-2k ` surface cnt\ reactions a: identifies : recognize a tion of an i altered as lymphocyte proliferate V individuals with idenii thymidine v response to be affected b and determi membranes fiedL c ccri re* jait.v siblc* )' the cr int 1y ni\LT), i and n bv The iboui tralioivcd ponsc led in -gh years zr im - rf are st cell CC).* ies of coniserics cxaminocud poly uction wcvei; tat the o lo jy lor ; I i ; i I lTnmunolox.ic(jUtgy and Chtmicat Carcinogtntsii 81 specific response is due to the carrier, poly 1-Iysinc, and its specific relationship to the I'LL gene, which has the capacity to respond-0 Further studies were carried out using variations of polymers of amino acids, ant(scpcratg)genes have been delineated. Strain #2 responded to a copolymer of glutamic acid and alanine (random grouping of AA's) but no response was seen in strain #13. On the other hand, strain #13 responded well to glutamic acid and tyrosine, but strain #2 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 M HC locus. Since that development, genetic control of immune responses and of an^gju^evoking^allograft recognition have been localized to thC H -2 gene complex of mouse chromosome #17, and th ejiu m an leukocyte antigeriJH L A )'' complex in the human on chromosom^Tft^-In the mouse, two 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 immun.responses. The H -2k and H r2d regions have been shown to code for the cell surface antigens that serve as targets for. cell mediated immune reactions as,,well as the antibody response. This mechanism, 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 o r altered.as in viral infection or neoplastic disease. The .m ixed 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.w ill be no-reaction,; and -no.thymidinc 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 arc expressions of genetic activity,-those surface membranes may have unusual conformations that could be identi- fied. 82 Erunronrricntei Toxicology n ImmuncosurYcIUncc Oell Surface Rccogixition System Within the immune defense system there exists a mechanism that has* the capacity to recognize the foreign nature of intrusions into a host. Bacterial infections occur, and the antibody response causes neutralization. Foreign tissue graft (allograft) causes the cell mediated response to reject'that tissue. Particulate material is sequestered, and phagocytosis causes the degradation of such mate- rial till it becomes an innocuous residual body or is discharged from the host. In each case, the immune system recognizes jhat this material is not identified as self, and sets about to remove the, mvasion^The immune system has developed and matured in each host,.whereby it has learned to recognize the tissue it was associated with during its process* of maturation and development. It can distinguish:betwcen self and nonself. ** - " - .The ^lym phocytes."-responsible for cell mediated immunity, are part of the internal policing system that is supposed to recog nize aberrant cells and'remove them from the system. It is able to recognize these cells by surface determinants that reflect the aber rant-nature, of-the <cell-and would not be found on a normal cell.41 It is this change in surface antigen markers that separatesOhe-self from nonself recognition scheme. This concept was developed for experimental studies in which "nude" fathvmic) mice lack the ability to reject tissue grafts because of the defect tn the processing of lymphocytes into T-cells.44Thus, the animal cannot respond to the foreign' nature of a transplanted tumour celfand p rovides an acceptable medium for growth of various tumour cells, whereas, if a tum our is transplanted into a normal mouse, there is a rejection of th e 'lu m o u r tissue. It is now generally accepted that most transplanted'tum ours have tum our specific antigens that are the basis for the induction of the immune rejection reaction. They are called tum our specific transplantation antigens (TSTA's). : This-concept :of tum our specific-antigens has had a profound impact ori-tumour immunology. Mice that were inoculated with polyoma virus were later capable of rejecting a polyoma tumour that was grafted onto a synegenic mouse (genetically identical). This was interpreted as an example of the immune system's rec- \ / m m unoloxirnlogy and Chemical Carcinogenesis 63 mechanism f intrusions dy response } causes the e material is >(such mate* s discharged' ' ;ognizes that a remove the lured in each vas associated >ment. It can d immunity, to recog-; xt is able to :flect the abernorma] cell.45 sarates the self. developed for mice lack the the processing mot respond to .nd provides an ;ells, whereas, if :re is a rejection -pled that most ens that are the action. They are nSTA's). had a profound inoculated with polyoma tum our *,t tically identical), une system's rcc- ognition that the tumour tissue was indeed foreign and that prcimmuni/aiion was directed at these surface markers. It was also 1lypothesiVed that the immune system was rapablc of limiting tumour development. A logical extension of that concept held that if there is a condition! that exists in the animal that modifies o T alterT t^im m u n e system's capabilities to respond to a nonself entity (ini(nunemodulation), then the variant cell or foreign entity can take hofc 'develop and mature into perhaps a disease statc.*^"45" 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 directly occurs in immunization with microbial cells or products or indirectly as in passive transfer of serum, cells, or cell products among histocompatible donors and recip ien ts.^ There are also nonspecific or general responses that can occur as j with Bacillus calmette guerin (BCG), Corynebacterium pervum, levam- ; C.V?* if*?;. ^isole. and pokeweed, These cells o r products are referred to as j (^mitogenic since they are n o h sp ^fic.an d have.the ability to stimu- j late B and T cellsi as wcU as*m acrophag^rorlhe-com plem ent-4- - system. ' - Immunosuppression, on th e:othcr hancL .is the reduction of available immune elements directly or indirectly.' Lymphoid drain age, 'ey to x ic d ru g sT a ^ ^ direct, as is thy mectomy, which will reduce T-cell populations and response. Irra diation is treneral suppression. of4hffJmmune response. Bursectomy in fowl will lead to loss of B-cell response. Indirectly, one can also modify the response by increasing o r 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 chemotherapeulics. In the clinical sense, the goal is to suppress o r 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* cxtrcm cly^pid-grow th'rate-and, additionally, may have an abnormal'complcmenl of nucleic acids. Alkylating 84 Environmental Toxicology agents as well as blocking agents are used to interfere with metabolism of such cells. Because some neoplastic cells m etabolic accelerated rates, they are more likely to pick up higher dos^s nf ajitimetabolic 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 chemothera py.44 -------------------------------- ^Immune Deficiency and Malignancy { One of the most significant advances in cancer information is the finding that immunodefident states are associated with an increased inddence of malignancy. Kersey11has shown that patients who have naturally occurring states of jmmunodefidenev such as WIskott-Aldrich-diseasc, Ataxia telangiectasia, or agammaglobu linemia have an,unusually high inddence of malignant disease. Numerous experimental bioassays are died in his review, show ing that immune-suppression, fadlitates transplants of malignant cells, increases a.norm ally4"*.inddence of viral or chemically j induced cancers -and accelerates growth of mtastass. < 'Immunosuppressive therapy has been used for some twenty oddyears and has had a signmeant impact in producing secondary and tertiary-cancers in patients who were under such treatment. Penn46 has Oeen m aintaining 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 S4.percent had evidence of transmitted cancers. Cessa tion of immunosuppressive therapy 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 homografis, 32 of 364 patients developed cancer, an inei- labozo at >C5 Of cells calcr ough nical on is h an .ients ch as obu- nant ally enty dary lent. who five rom nt to -one :ssa; rcims. .'3 Of ncj- t fm m unoloxicofogy end Chemical Carcinogenesis 65 dence of 5.7 percem .V he Denver Transplant Tumor Registry has dau on dm d* novo cancers that have occurred in 378 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. Azofhm pn'n^ anrj.lymphocyte globulin (ALG), Actinomvein, cvclophosphnmidp- Radiaiion, splenectomy, thymectomy and thoracic duct fistula procedures were also in use. In conjunctio n ^ itb ALG treatment, 6-mercaptopurine, methotrexate, and azaserine were also used. patients receiving irradiation, spleneclorny, thymectomy, or thoracic duct drainage treatment accounted for 217 de novo cancers, while the p a tie n ts ' on pharmarnlngic therapy accounted for'the remaining 164 cancers. T he development of malignancy could not be related to the use of any one agent, but appeared to be 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 times more common. The lymphoma, patients were slightly younger than the other cancer patients (S6.5 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......................... i V*. v r. . . 2 Hodgkins Discase............................................. -- 1 Histiocytic Reticulosis (?).............................................................. 1 1 ri i ri i r i 66 Emi'ronrncnta/ Toxicology Non Transplant Patients Treated with Immunosuppressives (Antiinflammatory therapy) Data has been collected that indicates that of seventy paiinm who have been under antiinflammatory therapy, seventy-two ran . cers developed during treatment with various agents. Disease No. Cases T herapy N o. No. Cases C ancer Tvpe C asa Psoriasis 24 Methotrexate Aminopterin Other 23 Lymphoma A 3 Leukemia 1 8 Skin 5 Misc. 16 Renal Disease IS Azathioprine 7 Cyclophosphamide 7 Other 10 Skin Lymphoma Misc. 5 2 6 Rheumatoid Arthritis *" ` 10 Cyclophosphamide 8 Lymphoma Other . 8 Leukemia Lymphoma : Misc. 5 3 2 4 Systemic Lupus Erythematosus 7 ' Cyclophosphamide Azotheoprine Other 3 6 6 Kaposi's Sarcoma 1 Other Xnflamatory Diseases 16 Agents Used 31 (as above, alone or as combined) Cancers 16 (Lymphomas, Leu- kemias, Hodgkins. and cancer of skin. bladder, colon. bronchus.) Neoplastic Diseased Patients W ithout 'Transplants 'In nontransplant patients with' neoplasms receiving imtnunosuppressive cancer therapy, Penn46 and Kcrscv81 showed that sec ond and even third tumours have arisen during treatment wiili chemotherapcutics. Of 185 patients with tumours treated H'>th either Mclphalan, cyclophosphamide, busulfun, 6-mcrcap|f'purinc, Iv' Immunoioxicology and Chemical Caranugcncsii * 67 t 1 i chlornaphazine, chlorambucil, ihiotepa, methotrexate, and pred nisone* alone or in combination. 194 new malignancies developed. patients -two can- Various leukemias accounted for cightv-iwo ot the 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 JVo. < Cases 4 2 5 16 t 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 ( 5 data and identify a significant association between the develop- ' 2 merit of malignancy of lymphoid elements and the use of chemi- j 6 cals that have the capability of suppressing elements in the j 5 3 2 4 ' immune/host.defense system. (Chlornaphazine is the one agent j capable of directly causing cancer in man since ilimetabolhes to --* beianaphihylamine. It has caused bladder cancer in aniline dye workers.) . ` ______ - . -. Leukemias and Chemical Immune Modulation 1 16 , Leuof skin. lit sccwith with jrinc. Of the four major types of cytologieal 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. T he lymphatic type has th e`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.** * An epidemiological study of solvent exposure and leukemia was conducted among rubber workers by McMichacl-et al.4*indicating that an association between leukemia and jobs that involved using solvents existed. Prior to tins time. Kessler and Lillienfeld {1969) ) as well as Viglfani and Saita4* had prepared papers indicating they thought that the current evidence su p p o ricd b enzcno, phenyl* buta?onc, and chloramphenicol as being luckcinogcns: however, they had found little epidemiological evidence to support this  `/ S c 65 Eninronmcntat Toxicology posture. McMichacl cl al.a 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 pro- 1 portional 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 the^vmpKatid>leulcelTrrrrstands out P as associated with solvent exposunTTobs. 1His leukemia tends to be : the m^loSlasie^or the stem cell type. McMichael had indicated that tfrei^ujcemogenic effects may have been the result of concom itant exposure to other solvents that were used in the rubber industry. However, Infante el al.50 studied a population of workers who were occupationally exposed to onlv 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. T he observations are in agreement with Vigliani and Saita,* whereby a specific type of leukemia is associated with the expo sure to benzene. T he myelogenous or monocytic-leukemia has been shown to correlate very, well with the exposure and confirms, the suspicion that benzene is a powerful bone_marTow 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, the alteration of stem cell function is apparent.515LM Occupationally^exposed persons as well as laboratory animal stud ies have shown chromosomal abnormalities, and correlates well with the known clinical picture of benzene toxicity.505-56Goldsicjn XifLJt ,I jj h* I*- (k- u*l or In ilii chi cyt di: 1 ch: cyt *1 8PI fig vne. clci im am! Mer. fun V that to I cull M.I cml to b tru occu I mm A< OJ W who a~i w( c Im m unolozicology and Chemical Carcinogcntjif 89 has reported-that in mice chronically exposed to 100 ppm of benzene, leukopenia, due primarily to lymphocytopenia, was clearly demonstrated. These manifestations of pancytopenia may repre- sent a destruction of the stem cells, failure of the cells to mature, or prevention of 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 Chloromycetin, vinblastine, mitomycin, puromycin, colchicine and rytochala$in B. Many of these latter compounds have been used in , clinical treatment of malignancy. Fomi et al.52 carried out cytogenic studies and have confirmed chromosomal aberrations occurring in peripheral blood Ivmpho- cytes, due to benzene exposure. " 17 one examines the ontogeny of lymphocytes, it is readily apparent that bone marrow stem cells develop into two different lines of cells. T he one line produces hemopoietic precursors, and the second line produces the lymphoid cells. Logically, interfer ence at, th e `-stem*..cell-level is bound to affect both the blood dements^xjvwell ks ;:lhe.-imraune-^system;elements...Laboratory investigations have observed pancytopenias such as monocytic, and myelogenous.-leukemias due to benzene' exposure also show stem cells that have produced abnormal mature erythrocytes, which / further supports'stem cell nuclear function interference., *-"Wohnanw has indicatedthat. ample.evidence is present to show that .chromosomal aberrations can be induced through exposure lo benzene, (japs*and junction 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 phytohcmaglutinin (PHA) exposed to benzene for seventy-two hours revealed both numerical and I structural '-alterations. Aneuploidy and chromosome breakage occurred seven and eight times more frequently in treated cells. lrnmun&chanicats and Cancer 3 'Advances in pharmacological therapy has produced problems ** well as remissions in.the treatment ofSliseasc''stalcs.'Patients 1 iJwho h*avc-been-treated with antihypertensive, antiarrvihmic agents T " ** well as am ubcrcular or anticonvulsivc agents have shown lo be ii h 1 ( I I 1 i I i 1 t i i1 J I ! it( [ f c / 90 E nvironm ental Toxicology susceptible to a syndrome related to the immune disease ralln;! systemic lupus erythematosus fSLEl.112*44-^ In SLE, the patient develops antibodies stimulated by (he patient's own cellular material, especially thcjyuleatelements. It affects various tissues, and has a fatal form. One of theynajor findings in diagnosis has been the presence of n LE cell ^polymorphonuclear leucocyte-- PMNJ that contains pKagocytjzed~nuclear material. This coll is formed as a consequence of reaction between an antibody present 7 that can bind cellular nucleoproteins. Presumably the mechanism involves the lymphocytic nuclei that reacts with the antibody. Lymphocytes become saturated on their active sites, and PMN cells come to engulf the swollen lymphocytes. Digesting away (phagocytosis) the remainder of the lvmphocyte, the PMN-pak^ ages the nucleoprotein materiaP^gfraVesidual body :called LE. '} t\.; Although this disease isusually virallv fffdiieed. therapeutic agents-'- such as diphenylhvdantoinTisbmazid.jiydralazine. and procainamide have induced the syndrome that generally disappears upon with 9 drawal of the drug. These mechanisms that induce .the SLE syn drome are not yet welLiinderstood;'however,-chemical* influence on the immune systenrhas produced* antibodies that include-anti- DNA, antinucleoprotein, antihistones,, antinucleolar RNA, and antibodies to fibrous or particulate nucleoproteins. T he 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 biological dysfunction. * Zarrabt et al.37 studied four groups of humans under treatment for some two and one-half years, with chlorpromazine and various other antipsychotic drugs. The*main observation was the preva lence of immunologic and thromboplastic coagulativc disorders. In patients on long-term chlorpromazine,- the authors found that patients had increased levels of serum IgM. -405 5,5 (in chlorpro mazine treated patients), whereas controls had 157 23. and nor* mal is considered to range from 60 to 230: patients who were given combined therapy, chlorpromazine plus another drug, for the same length of time showed levels of scrum IgM at 493 75. Other c Irnmvnotoxicology end Chetnical Cercinogtneiis 91 sniipsychotics used were thioridizinc, lrifluoperazine, thiothixene, perphenazine, haloperidol. lithium, and amilryplilene. 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, tjjrnifieanl 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 antibody lest 163%1. both had nucleoprotein antibodies (58%), and~ hath-grSupsTiaci antibodies to n^ n w J 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 f immunological analyses was the finding that the percentage nf Tlvmphocytes were below normal in thirteen out of forty-one patients i treated with Chlorpromaxine and twenty out of forty-two patients ^ under single or combined treatment developed splenomegaly. Recently, Doctor I. Fidler*1* at the Detrich Maryland Cancer Research Center,-working'with 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 tn viVro;-.however/ a lymphokine released by an, activated lymphocyte, referred to as"'macrophage activating factor (MAFJ 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 (lipopolysaccharide--LPS), endotoxins, pyran copolymers, double stranded RNA, or during chronic infection with obligate bacteria. The in vitro studies were slopped and. in certain cases, the procedure modified because the PEMs were found to have lost the cytotoxic capability. A careful examination of the occurrence of lost activity seemed to correlate.with a. m inor change in the drinking water used for the mouse colonies. Water .fed to the mice had a chlorine level of about twelve to sixteen pans-.per million (ppm). This high Ievel.,is necessary to *TMucc the'rafc*:of e a rly death syndrome in the colonies due to Pxudomonas infection 'since the mice had been lelhally irradiated. 92 E nvironm ental Toxicology Due to an unusually high incidence of such early death syn dromes, the chlorine level was raised to 25 to 30 ppm, and the 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 wo<-l later the level of PEMs in the mice receiving hypephltM;inau-d water had decreased to 13 at 2 x 106 per mouse. Controls'were yielding 25 3 x 106 per mouse. On conseculive^victX PEM yield increased from the control mice, but mice on hyperchlorinated water had reduced PEMs or remained lower than controls. Mice that were receiving tap water yielded macrophages that when stimulated were tumorcidal in rirro 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 levels of cytotoxic ability for the first two weeks, and, by the endoT 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 PEMs. Lower levels (10-15 ppmj may also exert such influence although over a longer period of time. "" * \ Supporting these effects was a report by Fdler 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 about a dnaturation of the hemoglobin by direct oxidation ami also by inhibition of the direct oxidative pathway (Hexose mono phosphate shunt) of red blood cells (RBCs). T he damage to RBCs was found to be cumulative over several periods of dialysis. s Although the mechanism for this depression of macrophage tumoricidal activity is unknown, Fidler suggests several possibili ties. T he vacuoles of the macrophage system are probably involved in the cytotoxic-mechanism. This has been shown by Hibbs ami 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. -7 & <7 7 -Je'-r ,-hv syn. the the veek ated were 5EM isied Vlice -'hen sr to JF, as ered M of ->n p roiy of encc I that jlyiic verse ught i and 10110<BCs hage ibilijlved * and f the at ion yclro- Im m unotoxicology and Chemical Ccrcinogcncru 93 Macrophages that have been activated by Ivmphokincs have enhanced bactericidal activity, and mctubolically 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 HMPS pathway5* (that is the major path for glucose oxidation in the RliC's), there is a possibility that chlorine and/or its compounds mav sufficiently inhibit glucose oxidation to the point whereby macrophages that have reduced tumouricidal capac ity will allow aberrant cells to continue growing and localizing! 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 thtlTmacrbphage activity is compromised. Since if carries a major role-in host defense against neoplastic disease, the posstbifity exists thatTa host who is exposecTto such compounds may in fact become immunocompromised. Silica, carageenan, and trypan blue are substances that also have the ability, to suppress macro phages. in lab. studies'have been rep o rted io decrease host'resist ance against transplantable tumours. Dandliker et al.wrecently 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 LD50 dissolved in 1 ml of com oil. Arochlor 1260, Dinoseb, Paralhion, pentachloronitrobenzene, piperonyl buloxidc, mixed pyrethrins, and resmithrin were administered iniragastrically.-The animals were examined for an end point of - redness and swelling" (inflammatory response) and change. in> temperature of the. foot pads, as well as histological exam. aficr/an^niigcnicchaUcngc. Serum antibody titer, binding affinity, nnd^hetcrogcneity^wcrc determined by fluorescent polari- ' TTicSanimals^werc^firsl.given an injection of fluorescein labeled . ovalbumin, allowed-only water ad lib for twenty-four hours, and then given a bolus of-Tood with the pesticide by iutrjgastric feed ing tube twentv-four hourT'aftcr thc~ immunization. The most c 94 E nvironm ental Toxicology striking feature reported by the authors after immunanalysis inch* cated marked humoral and cellular immunosuppression to single doses of Dinoscb and Parathion, and a marked stimulation of (he cellular response by resmethrin. 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 pcsticide^waa-given orally at th e,IH35<H ^ h e ^ .l-I^D*^:7ivc--~ days before, two days before, o r two days after immunization-with 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 immunization exhibited no significant reduction in the antibody plaque forming cell num bers. T he author indicates that a lack of information prevents a conclusion as to the efficacy'of these compounds on modulating! the immune system, SinceThe methodology only uses one test lor the .erythrocyte receptor, little can be concluded. :This is:why m ultiple parameter assays are necessary. (^Paith and L u s te r^ have performed extensive investigations on the p re- and postnatal effects of Tetrachlorodibenzodioxin (TCDD) on the immune system and have found that TCDD appears a relatively excellent im m unosuppressive in the FischcrAVlsUr rat strains. T he Fischer strain is reputed to be less of a responder than its Fischer/Wistar cousin. However, dosing of nursing females has shown that the TCDELhas the capability of causing immunosup pression in liuerrnpr^i. T he effects have lasted as long as 270 days, from three doses to the m other at days zero; seven, and fourteen, applied at five micrograms p er kilogram (ug/kg) body weight. At days eighteen and thirty-five, both female and m a le - litiermatcs showed depressed body weight as well as depressed thymic:wcightsThese depressed values'were evident at day 128 posldqsing. The weight of the spleens were also found to be affected. Effecu^oi TCDD__exposurc on the homing patterns of lymphocytes were also studied in these same rat strains. Splenic cells taken from the * lite I celts TCL horn ft ct that otter * 1 imm n abili mon* t*X|>C susc< hype imm La plum Fidlc was 1 t-drp Kuril mixtt p-tcr comj- Oe phrn two a rates, to su spire UALl three C [mmunoloxcology and Chintii Carcinogenesis 95 lhe TCDD exposed rats were injected mo noncxposed rats, and the thymus was found to significantly increase the uptake of 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. The authors proposed that a change jn cellular metabolism occurred altering the cell membrane, or that insertion of the TCDD into the membrane caused surface altrations, and this change in the cell modified its normal hom ing patterns. Various investigators have shown such alterations in immujie_function due to TCDD exposure.45,1*.6* (^Th)gpen^ has shown that subdinicahloses of T CDD had the ability to affect host responseTwhcn subsequent exposure to Sal monella 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"hypcrsensivity-response. (Suppression^)! the T-cell dependent "immune functions appears to occur as an isolated response.) ^ ^ P h c n o l ' *'" ' - ''Tjava^t" al.64 also had occasion io come upon the effects of . ph^iioTon immune function, th ro u g h 'a case^of serendipity, as did. Fdleri Phenol was being used to'*disinfect wejeages.of mice, and 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-phenylphonol (5.0%), o-benzyl-p-phenol (4.5%), and p-leri-amylphenol (1.0%). OPP, OBP. and FTA, respectively. Such comjxmnds are used in biocides throughout the world. (uehm e47has studied the metabolism of one compound, o-phenylphehol (OPP) in the cat and dog and has found that although the two animals metabolize OPP according to two different routes and Rtles^ excessive tissue levels are found in the spleen. Lavia44.decided to study this phenomena on immune function knowing that the spleen plays a major role in the immune response. Groups of BALB/c female mice were dosed witli the phnol derivatives (all three derivatives were used, but each group of mice received only I 96 Environmental Toxicolaty a single compound) at levels of 0.46 milligrams per kilogram (mg/kgj of^OPP, 0.41 inc/kg QBP. and 0.09 mg/kg PTA in ihcir drinking water. 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. 1he response after lour weeks showed depression to be occurring in 77 percent ofth e dosed mice, comparison of OFF with'a mrXTtn^"Cf"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 analyzed for their capacity to phagocytize yeast cells. This capacity was significantly reduced as compared to controls. Measurement of T and B lymphocyte numbers in rnnrrol and f)PP_ exposed . mice showed no significant differences. The net effect was that the macrophages appear to have sus- tained some defect that reduced their ability to present antigen foF phagocytosis or may have just resulted in a reduced num ber of circulating macrophages that could affect the cooperative cell-cell activation upon 'B rlymphocytes. Archer* has also studied the suppression of the immune system using gallic acid, a phenolic derivative/ anci 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^that the immune system is in fact a very sensitive system that-maybe an"ideal vehicle lor indicating potential . toxicity to humans. Although ultrastructural morphology or DNA damage studies have been used prior to this time for indications of toxicity, it appears that immune cell biochemistry Tfray*be more sensitive to such low level closes that are prevalenLin the envi-^ j ; : . * 1 ; ; ronmc Extc tin con Sc non tion of ihc-spltionshi of thyn pronoi periph reverse exposu tin chic showee. n-dod lri-n-ob . atrophy data dc ^ cstablis ; infectio is also s conclue B-Iymp modifie rabbits million primary depress acetate 1 cells to lion, sir ! associt stimult live anti lo rats jwciglus c iIo f ram in their nuniicd Cj. Four FC) was ^ u re , 45 response percent f phenol ne. indiimmune nance in also anacapacity urement exposed ave susfi^en for . er of cell-cell died the phenolic ^11s that' system -se data ssion has xposure, posures. late this is not donc by a very. potcntial or DNA ations of% bc more:hc envi-' | fm m unolxicotogy and Chemical Carvinogcncstf 97 I, r o n m e n t a n d w o u id co m p l c m c n t- s u ch-bioas.saY _5.>_ ; Extensive investigations have been made of the effects of organo ! tin compounds on the immune system and it has been shown by 1 Soincn and PenninksMthat Di-n-octyl-tin chloride causes a dcplc . non of lymphocytes in the thymus and thymus dependent areas in ; the spleen and lymph nodes. There was a dose dependent rela- . tionship showing a decrease in the number as well as the viability : of thymocytes. Spleen cell numbers and viability were slightly less pronounced, and no-eiiect..was_ound on bone m arrow and/or peripheral lymphocytes and/or monocytes. Thymic atrophy : reversed upon discontinuation of the exposure. The results of exposure to Di-n-buiyl tin chloride w as identical to the Di-n-ootyl tin chloride. Di-n-ethyl-, and Di-n-propyl tin chloride compounds j showed less pronounced effects. In contrast, Di-n-melhyl-, Di| n-dodecyl-, and Di-n-octadecvl tin chlorides as well as monooctvl-, i tri-n-octyl-, and letraoctyltinchlorides 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 tfce major effect 'isJan increased susceptibility to infection by gram negative bacteria'that con tain endotoxin. L e a d ', "Is also shown to decrease *the resista' nce to"vir-al dis' ease, , * one can \) .conclude.that the humoral response is somehow affected-causing / B-lymphocyte defects in antibody generation'/'or perhaps in-they modification of circulating antibody molecule itself; Roller,70using rabbits dosed with lead acetate, 2200 mg/Iiter (equal parts per million) in water showed that after ten weeks of exposure, both the primary and secondary response to pseudorabics virus had been depressed. Roller and Kovadc71 showed that mice dosed with lead acetate had a significantly increased number of IgM plague forming cells to SRBC. T his further adds to the complexity of the situa tion, since the effect may be occurring in the lymphoid organfs) associated with development of B-cells. Additional celfs^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 posma tally by'Faith el al.7? showed the thymus weights to be suppressed along with'decreased responsiveness to ___ ___ 77 7*^ t I I I ! I j I ttI ! i ( 98 Eninronmental 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 ppm 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 iacet ol the immune function has been altered. T he 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 j indicator, covert toxicity may in fact be occurring72"74 even at low j levels. Hoffman and Niyogi74 have 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 i^ interfere with the DNA at a small lymphocyte blast stage perhaps.a maior clonal species of lymphocyte coutdiye~pet~nr.tTrenilv impaired! ~ Additional studies by Vos et al.73 using hexachlorobenzene on rats has shown the immune system to be stimulated, which con- * trasts other data presented. Sharma76 has exposed mice to vinyl i chloride and found the immune system lymphocytes to be pro- \ foundly stimulated. M iller77 and Kagan and M iller71 also have observed im m uno stimulation in patients who have asbestosis. 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 lgA, and a variety of autoamibodics. T he reason' forTEis activity is not clear; however, asbestos studies done on the lung suggest that macrophages trying to digest^the mineral .fibers are damaged, spilling out lysosomal enzymes, which may bring on autoimmune pathnhn. iaiiM 'j n the puli impair*.*' that mat th ere is hut who " Mecxu-- llivlll. tl* of (he it Am titcitncr.' availab tells ol mecha i taut in tells. It tal chcr iiulivid The creas with ct cancers im im ir <( u r n * U**t*ettc m a g,* inuiiui AlftMna i- r . WM- njUT'fm L II /m inunoloxicoiogy and Chemical Carcinogenesis 99 pathology. Drath el al.Mhave established lhat smoke from tobacco causes morphological biochemical and functional alterations in ihc pulm onary 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, Jm t whose total impact is yet to be discovered. ------j Because of a known role~in the immunosurveillance mecha- _ nism, the possibility of modulation by environmental chemicals : of the immune cells certainly seems an exciting speculation^) Summary The influence of environmental chemicals upon the public health is of considerable importance. Determining how such chem icals may..cause adverse effects upon -humans is a continuing problem that-perplexes tall phases of 'scientific-inquiry.- Genetic chronic, toxicity- and dose responses are defined from animal bioassays and microbial DN'A studies for human use, and consti tute a m ajor source of controversy'among predictive toxicolfigists^ A major- step .forward:, in resolving such problems ^could.,,be-* attained with the use of human lymphoid cells,*which-are readily * available from the peripheral circulating blood. T he lymphoid j cells of the immune system are an integral part of the defense j mechanism known as immune surveillance and are very im por- j tant in the recognition and rerrtoyal of abberant o r malignant J cells. It is this relationship lhat inav^be disturbed bv environ men- ' lal 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 witK 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 discas is also found in individuals with ^ n c lic immunodeficiencies at a higher frequency than in immune normal hosts. So it has become apparent that.malignant response '' in a given host may in fad be very seriously dependent upon an immune system that has been somehow compromised. 100 Environmental Toxicology In vitro and vivo studies using pesticides, melallo-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 \ imonocytes, which play a vital role-in-Jthe~B-cell/T<ell in tprara tions, are shown to be very severely disturbed by excessive amounts j . of chlorine or hemoglobin degradation products. So, direct or / indirect eiiecis 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. T he cell inav/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- i isis, 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 axe not understoodr~In fact, there are many who have asked questions that may^ilTthii^excuemenTover the im m unosurveillance theory. HoweverTtheV have"not been sufficicntly substantiated. What has been elucidated in this presenta- $ lion is that we cannot deny the influences external chemicals have j on the cells and products of tissue from the lymphoid-immune system. T he role of chemically induced malignancy in the immune suppressed patients receiving therapy or the excess occurrences of malignant disease in dmmunodcficient individuals is significant and cannot be ignored. The complexity of the immune system, details of mechanisms, in fact, whether cells or products may be inpuencing this system is in most cases not known. H o w e v e r, it can be stated that many of the substances mentioned here today and iiics hcsc and lood erac>unts :t or r (he ifluircct ay be lasm dear all .1crial well iman gical any the suf- latve lie nc of ant 'm. be . it today I Im m unotoxicology and Chemical Carcinogenesis 101 are in the environment and they do have immunomodulaling effects. How does this role of immunomodulation 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 nou^allows a^neoplastic response to occur? Immunotoxicologv is the new kid oh the b lo c k e d is able to ask some very difficult question?. Questions-for'wKich 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. Establishing a tissue culture proce dure with human lymphocytes and/or macrophages, even though in vitro, would allow* function and surface identification studies as well as biochemical investigations to procd and perhaps pro mote greater confidence when possibly identifying toxic responses to humans. Other human cells have been cultured,-such as fibrpblasti-- --__ ^n d 'flieH eL a cells, surely the same could be done for the immune i system cells. It would go far in impacting the program -of public -- ' health for which we are all responsible. References 1. Kraybill. H . F.: Carcinogenesis induced by trace contaminants in potable water. Bull /Vcir Yori Arad Medicine. W.T13. 1978. 2. Smith, J. Ji.: G overnm ent says cancer rate is increasing. Science, 209:998, 1980, f.'. / * 102 Environmental Toxicology .1. Doll. Sir Richard: Strategy for detection of cancer h aurcb lo man, Ao/li*. 263*89, 1977. 4. Scheidcrm an. M., and Brown. C- C.: Estimating cancer risks io a popula tion. Env Health Perspectives T2:\ 15. 1978. 5. Kali. David P.: Difficulties in extrapolating the results of toxicity studies in laboratory animals to m an. Environmental mearrA. 7:360. 1969. 6. Dolt. Sir Richard: Epidemiology of cancer: Current perspectives. Amt r Jount of Epidemiology, 4:396, 1976. 7. Bishop. Yvonne M.: Statistical methods for haxard and health. Env Health Perspective^ 30:149, 1977. 6. Lepkowski. Wil; Extrapolation of carcinogenesis data. Env Health Ptnpeetii^es 22:173. 1978. 9. Rail. David P.: Chemical carctnogcttesis and mutagenesis: Introduction to symposium III. Proceedings of the European Society of Toxicology. 1978. 10. Vos. J . G.: Im m une suppression as related to toxicology. CRC CriticalReview in Toxicology S:67, 1977. 11. Cantor. K. P.. and McCabe. L. J.: T h e epidemiologic approach to the evaluation of organics in d rin k in g water. US.EPA^ 2nd Conference on environm ental impact of chlorination. 1977. 12. R ubin. P hillip: Com m ent: Cancer epidemiology.//43M , 225:1557, 1973. 13. Epidemiology subcommittee of the safe drinking water committee: Epi dem iological studies o f cancer frequency and c e n t in organic constituents of drinking water. A review of recent published and unpublished literature. U.S.E.PA. by National Academy of Sciences NRC. Wash. D.C. 1978. 14. Ames. Bruce, Durston, Wm. E^ Yamasaki. E_. and Lee, Frank: Carcinogens are mutagens; a sim ple test system com bining liver homogenates for setivation and bacteria for detection. Proc N a tl Acad o f Sciences / 0.2281, 1973. 15. M cCann, Joyce. Chio, Edw., Yamasaki, Edith, and Ames, Bruce: Detection of carcinogens as m utagens in the &/m onr/Za/miaosome test: assay of 300 chemicals. Proc Het7 Acad o f Sciences 72:5135, 1975. 16. Fox. Jeffrey L.: Ames lest success paves way for short-term cancer testing. CAcm and Eng Hevx, p. 34, Dec. 12, 1977. 17. Bridges, Bryn A .: Short term screening tests for carcinogens. Hature, 257:195. May 20, 1*976. 18. Devorel, R.: Bacterial tests for potential carcinogens. Scientific American. 241 (2JAQ. 1979. 19. Smith, Aileen M.: Does the Ames test work? H evt Engineer, p. 25. Apr. 1977. 20. Cairns, T hom as: T he EDot study: Introduction, objectives and experimen tal design. Env Path and Tax, J :l. 1979. 21. National Academy ot Sciences-National Research Council, Principles lor evaluating environm ental chemicals. N.A.S. Wash. D.C. 1977. 22. Jones, T. C.: M am m alian and avian models of disease in man. Federation Proc, 28{1J:162, 1969. 23. Schein, P.. and A nderson, T.: T h e efficacy of animal studies in predicting clinical toxicity of cancer chem otherapeutic drugs. / CA"<i Phams S3.22S, 3 3 3 37 7* I