Document NGGDM19m0EggLxQNEx6OZbD1b

J Bio ^ iocs on tlie - f.r; ifon'nenlu! :_;,i nj MeetI- ik. p 25. Else- .(,, I.nuida. K_ TipOtlSC Of S3Ch>r^rpla^ias in :-S^hydroxy- 65.1005. . Hill P-. Weisuirition and its 32. 237. y76f. Occupatio- Vol.271. !y induced dis* Si Weisburgr, ulral sterols on n skin of mice. of saccharin. In Report So. 3. '.irker. p. 153. i Mechanisms of lamine carcinoCarcinogenic and Is. Edited by S. 53. Monograph Institute. Beth- A 1 Chemical car?nce o f / 'oisons, issen & M. O. rk. Metabolism of rrehensice TreaBecker, p. 241. er tumour pro- . & van de Poll. Value o f 5/iorr- i-afutirifl. Elsensterdam. >31). Systematic point approach. . Tobacco and . Afed. 300. 894. f j r.nmH ill, Yul t`J. pp. 5ft7 lo J7& J^SI primed in Grcii Bntjin R p t'b m f Q j\fq ^ OOIS-fti- mi nyifc' - i'i'O ni n PiTpjrrhin Previ Ll . GENETIC AND NONGENETIC EVENTS IN NEOPLASIA W. T. Stott, R. H. Reitz, A. M. Schumann and P. G. IVatanabe Toxicology Research Laboratory, Dow Chemical USA, Midland, M / 48040, USA * (Received 2 February 9X1) i / ; Summary--It has become increasingly evident that all chemical carcinogens do not act via the same mechanism of tumorigenicity. Based upon the extent of a chemical's interaction with DNA, a general classification scheme of various mutational and nonmutational theories oT chemical carcinogenesis is presented. Compounds that directly interact with DNA are classified as genotoxic whereas those that do not interact directly with DNA are classified as epigenetic carcinogens. Under each general heading, several mutational and nonmutational mechanisms of carcinogenesis are believed to be possible. Data are also presented to support the existence of one such mechanism, an epigenetic-mutational theory of chemical carcinogenesis based upon recurrent cytotoxicity. In this case, increased regenerative DNA synthesis in response to tissue injury is believed to result in an enhancement of the normal spontaneous mutation rate, conceivably leading to a cellular transformation. The carcinogenic risk posed by such epigenetic carcinogens appears to differ greatly from that posed by genotoxic carcinogens. Thus, con sideration oi data concerning the possible mechanism of carcinogenicity of a chemical, along with pharmacokinetic data, will allow a better understanding of bioassay results and a more accurate assess ment of carcinogenic risk. Introduction Frequently in the routine rodent carcinogenesis bioassay of a chemical high levels of a test compound are administered for prolonged periods of time and, at the termination of the study, the numbers of tumours formed are counted. If a statistically significant excess of a particular tumour is observed the compound is classified as an animal carcinogen and an extrapola tion of risk-is made to humans. Yet in most cases, without knowledge of the fate of the chemical in several species (i.e. pharmacokinetics) as well as of the resultant macromolecular events to help understand ing of the bioassay results, there is little chance of a meaningful assessment of human carcinogenic risk. It is the integration of these three data bases (bioassay, pharmacokinetics and macromolecular events) to more realistically assess the potential carcinogenic risk of a given chemical that has been a continuing endeavour in our laboratory over the past several years. Recently, we have been involved in trying to understand the different mechanisms of tumour for mation and their implications for risk, assessment, and, more specifically, the role of cytotoxicity in the carcinogenic process. This latter area of research will be the focus of this paper. Classification of proposed mechanisms of chemical car cinogenesis Since the first experimental documentation of chemically induced carcinogenesis in animals by Yamagiwa & Ichikawa (1918), numerous theories have been proposed to explain the mechanisms of chemical carcinogenesis. Apart from the various dif ferentiation, protovirus and viral infection theories of carcinogenesis (Temin, 1974). most theories regarding the mechanisrnls) of chemical carcinogenesis may be generally classified ns `genetic' or `epigenetic' (nonge- netic) in nature. As discussed below, the distinction between these two general classes of mechanisms is of great significance in carcinogenic risk assessment. As used here, chemicals displaying a genetic mechanism of carcinogenesis are defined as those involved in di rect interaction with cellular DNA, In most cases this interaction is considered to result in a mutational event; however there may also be cases of nonmutational genetic mechanisms of carcinogenesis (c.g. Hol liday, 1979). Chemicals displaying an epigenetic mech anism of tumorigenesis are defined as those that do not directly interact with cellular DNA, and yet their action may indirectly result in a mutational event. Thus, the terms genetic and epigenetic in chemical carcinogenesis, as used here, refer to the likelihood of direct chemical-DNA interaction, and in either case may or may not involve a mutational event. The most common interpretation of a genetic mechanism of tumorigenesis is embodied in the soma tic mutation theory of carcinogenesis first proposed by Boveri (1929). Basically, this theory dictates that the direct interaction of a chemical with DNA (e.g. by alkylation, intercalation) can result in a somatic cell mutation (via point or deletion mutation, gene dupli cation or gross chromosomal disruptions) which may or may not ultimately lead to a transformed or neo plastic cell. As reviewed by Hanawalt. Friedberg & Fox (1978) mitigating factors in this progression are the DNA-repair enzymes which remove the chemi cally induced `lesion' before (excision repair) or after (post-replication repair) DNA replication (i.e. before the lesion is fixed as a mutation). It also appears that errors in this repair, possibly by an inducible errorprone DNA-repair enzyme system analogous to SOS repair observed in bacteria, may be responsible for the mutation rather than the initial chemicallyinduced mispairing of bases (Kon.do. 1976 &. 1977: San Stich, 1975; Sarasin & Benoit, 19S0; Witkin. 1976). 567 003721 I i J -r r 568 W. T. Stott, R. H. Reitz. A. M. Schumann and P. G. Watanabf. Comings' (1973) "general theory of carcinogenesis' provides a possible scenario of events leading to a state of anaplasia after a mutation occurs. In this case the significant events would be a double mutation of one or more regulatory genes (normally present in all cells), in turn derepressing corresponding structural genes capable of coding for cellular transforming factors. In the case of chemical carcinogenesis follow ing an apparent two-step progression towards neo plasia ("two-step theory of carcinogenesis'), this dere pression of genes following a mutational event (in itiation) may be promoted by epigenetic stimuli, chemical or physical (Boutwell, 1974; Trosko & Chang, 1978). Substantial evidence has been accumulated to link mutagenic events with the neoplastic transformation of cells. As reviewed by Trosko & Chang (1978), find ings regarding the clonal nature of tumours, the mutagenicity of many carcinogens, the correlation of high cellular mutation frequency with increased cancer rates among humans suffering from deficien cies in DNA-repair enzymes, the correlation of in vitro DNA damage with cell transformation frequen cies and neoplasia, the involvement of mutation in the initiation phase of some hydrocarbon-induced carci nogenesis. the effects of age on mutagenesis and the incidence of various hereditary tumours, are all sup.portive of a mutagenic origin of cancer. However, several findings cannot be reconciled with a muta genic basis of carcinogenesis. These have included ob servations regarding the fact that all carcinogens are not mutagens (Jensen, 1974; Lippman, 1975; Segaloff; 1975), the discrepancy between gene mutations and cell transformation rates in short- i\ long-lived ani mals (Huberman. Mager & Sachs. 1976; Peto, 1977), the induction of tumours by plastics- or metal-film implantation and hormone imbalance (Berenblum, 1978). the ability of teratoma cells to produce appar ently normal cells upon transplantation into normal mosaic mouse blastocytes (Mintz & Ulmensee, 1975; Papaioannou. McBurney, Gardner & Evans. 1975) and the ability of Lucke frog adenocarcinoma cells to reproduce normal cells upon nuclear transplantation into anucleate eggs indicating a totipotency of the tumour-cell genes (McKinnell, Deggins & Labat, 1969). Thus, there is also substantia! evidence for the existence of one or more epigenetic nonmutational mechanism of carcinogenesis. Several theories have attempted to reconcile these different mechanisms of carcinogenesis either by pro posing an alternative to mutagenesis or by accommo dating both genetic mutational and epigenetic non mutational mechanisms. A recent example of the former situation is the nonmut3genic theory proposed by Holliday (1979). This theory proposes that gene regulation, as directed by specific DNA base methylation and recognition by several DNA methylases, may be altered during repair of DNA-chemical alkylation sites. The resultant loss of specific methylated recog nition sites for DNA methylases results in loss of gene regulatory control and ultimately in a transformed cell. Since the direct interaction of the chemical with the DNA is required to deregulate genes in this nonmutayenic theory, it is still consistent with a genetic mechanism of tumorigenidty as defined above. An example of the latter "coexistence' type of theory is the "integrative theory' of carcinogenesis of Trosko & Chang (1978). These authors propose'that neoplasms may arise from a mutagenic event alone (regulatory locus) if the genes affected are in a transcribable state, from a mutagenic event if there is a coupled nonmuta tional alteration of the mutated genes (promotion), or from the abnormal derepression of g^nes at critical developmental stages that prevent normal gene regu lation. Based upon the apparent realization that some chemicals may cause tumours via several different mechanisms of action (i.e. there is no single unifying mechanism), it has been possible to propose a classifi cation scheme for chemical carcinogens even though mechanistic details remain to be elucidated. Weisburger & Williams (1980) have thus proposed that carcinogenic chemicals be categorized under the gen eral division of genotoxic chemicals (direct-acting or primary carcinogens, procarcinogens or secondary carcinogens, inorganic carcinogens) and epigenetic carcinogens (solid-state carcinogens, hormones, immunosupressive agents, co-carcinogens, promoters). A diagrammatic representation of the various theories of carcinogenesis is presented in Fig. 1. Concept of the cytotoxic mechanism of tumour formation An epigenetic mechanism of tumorigenesis that is not frequently considered in terms of tumour produc tion is that of recurrent cytotoxicity. The simplest example of this mechanism is the production of sar comas following subcutaneous injection of inert sol utions such as saline or glucose. It is envisaged that repeated subcutaneous injection of these nonreaciive materials results in inflammation, necrosis and cellu lar division which may increase the error rate in nor mal DNA replication or DNA repair, resulting in cri tical-site mutation and ultimately in a transformed or neoplastic cell. In general terms, this theory represents a modification of Virchow's `irritation theory' in which hyperplasia was believed to be the driving force behind carcinogenesis (Berenblum, 1944). It is likely that some chemicals may illustrate both genetic and epigenetic effects. In such an instance it will be impor tant to determine which mechanism is predominantly responsible for the formation of tumours. With the concept of testing maximum doses in ani mals in order to enhance the probability of detecting a carcinogenic response, one of the primary issues is the relevance of such testing in terms of the real risk for humans of a chemical shown to be carcinogenic under such test conditions. Frequently in such studies tumours are observed at dose levels that also cause recurrent cytotoxic responses, and a logical question would be whether an analogy to sarcoma production following subcutaneous injection may be made. In understanding this relationship, it-is enlightening to explore the molecular basis for increasing mutation as a consequence of increased DNA synthesis and cell division. DNA docs not exist in vivo ns a pristine molecule even in the absence of a measurable genotoxic chal lenge. So-called spontaneous mutations appear to occur at a rale dependent upon exposure to unavoid able exogenous (e.g. cosmic radiation) and endogen- Ahemi-WUtfivniLiWkmauMit i*4 * ,, S' 3I C2.b! *^aL-OPG- nn" u3 m*o*r3 3. :P?. "o=Sc.' n2> ?2T..K1-/1. ts!o3. 2uV. ia2a\ , "Q> / S?' iaasi -gQ3.m3S' wfn?> >?s *2f^.ifE3=e*5u-?=' ^*iia?'i 8uaS, #Cg =rt;, O_ *- J --< /i ? # GENETIC D irect In te ra ctio n with gen etic m aterial (DNA) EPIGENETIC No d ire c t In te ra ctio n with DNA MUTATIONAL EVENT e .g . Mutation in c r it ic a l s it e on DNA (somatic mutation theory) NONMUTATIONAL EVENT* e .g . Loss o f DNA regu latio n markers' due to rep air of alkylation sites MUTATIONAL EVENT e .g . Repeated c y to to x ic ity leading to enhanced number o f errors In normal r e p lic a tio n and c r i t i c a l NONMUTATIONAL EVENT e .g . Induction o f preexisting oncogenic factors by alteration of sp ecific cellu lar metabolic functions S o lid -state carcinogenesis Hormone-Induced carcin o gen esis Genetic and nonger.etic events in neoplasia TRANSFORMED CELL Fig. I. General classification of some of the proposed mechanisms of carcinogenesis, excluding the differentiation, oncogenic, viral infection and protovirus theories. The role or DNA repair in mutagenesis is not included. 'Believed to be a readily reversible alteration (Holliday, 1979). 570 W. T. Stott, R. H. Reitz, A. M. Schumann and P. G. Watanabe ous factors, DNA-repair competency (e.g. relative to age) and the basic thermo-stability of the DNA mol ecule itself. With regard to the latter point it has been estimated that thermal decomposition may account for between 2000 and 10,000 depurinations, several hundred dspyrimidinations and several base deamina tions of DNA molecules in a mammalian cell per gen eration (Lindahl Sc. Karlstrom, 1973; Lindahl & Nyberg. 1972 & 1974). These thermal lesions can result in base transitions (e.g. deamination of 5mcthylcytosine to form thymine) which conceivably may be `fixed* (irreparable) by DNA replication or repair processes resulting in a functional mutation. Indeed, errors in the process of DNA replication itself may occur as a result of base mispairing. DNA poly merase base selection errors and mismatch repair errors (see review by Hartman, 1980). Based upon es timates of the mutation rates per human gene per sexual generation, it has been suggested that 10% of all human gametes contain a new mutation of their own plus several inherited mutated genes as well (Drake, 1978). Thus the mere fact that DNA synthesis and cellular division is enhanced following cytotoxic responses will result in a decreased time for repair of `naturally' occurring DNA lesions before replication, an increased number of replication errors, and an enhanced somatic mutation rate. Additionally, since errors in DNA may occur throughout the cell cycle or during DNA synthesis, the DNA-repair process can be thought of as a con tinuous process. However, as noted, some forms of DNA repair are error-free while others may be errorprone. Therefore the fidelity of DNA repair can affect the somatic mutation rate during cell division. A recent report by Shank & Barrows (1980) on studies of the noncarbonatious chemical hydrazine has shown that cytotoxic doses of hydrazine cause abnor mal methylation of nucleic acid bases, in the liver. Furthermore, evidence was presented which suggests that at hepatotoxic doses other chemicals (e.g. carbon tetrachloride) produce a similar alteration in the DNA as a result of cytotoxicity. Whether this effect is due to an alteration in the DNA polymerase or in the fidelity of DNA repair following DNA replication is not known, but it is an example of how a cytotoxic agent at high dose levels can cause abnormal nucleic acid bases to occur in cellular DNA. DNA synthesis and cell division can also enhance the susceptibility to mutation by exogenous and en dogenous agents. For example, Berman, Tong .& Wil liams (1978) have reported an increased mutation fre quency in actively dividing adult rat-liver epithelial cells exposed to the mutagenic chemical methyl methanesulphonate compared with that in quiescent (nondividing) cells. In this study the number of azaguanine resistant colonies per 10 colony-forming units was used as a measure of mutation frequency. The muta tion frequency in the actively dividing cultures wasincreased five-fold when compared with the quiescent culture, indicating that dividing cells were more sus ceptible to mutation. Finally, DNA synthesis and cell division can also enhance mutation frequency by altering the amount of DNA repair prior to DNA replication. An example of this is given by the work of Maher, Dorney, Mendrala, Konze-Thomas & McCormick (1979) using normal human fibroblasts and fibroblasts lacking normal DNA excision repair. Cell surviyal and muta tion rate in celts exposed to ultraviolet (UV) radiation was observed to be related to DNA-repair compe tency. Survival of normal cells was higher and muta tion rate lower per UV dose than for deficient cells. Normal cells were also able to survive a usually lethal and mutagenic UV dose by being held in confluence (nondividing) for a period of time before assessment of survival and mutation rate, presumably because this allowed a greater time for DNA repair to occur before replication. In addition to a molecular basis for enhancing mutagenic events as a result of increased DNA syn thesis, there is an experimental basis for the role of DNA synthesis in carcinogenesis. Tumours often de velop in chronically inflamed or scarred tissue; colon cancer is frequently observed in patients with chronic colitis; skin cancer occurs in burn scars; liver tumours are associated with chronic cirrhosis of the liver (Berenblum, 1944; Chan, 1975; Laroye. 1974). Repeated tissue damage with a physical agent (dry ice) and resultant regeneration has induced tumours in mice (Berenblum, 1929). Physical trauma such as partial hepatectomy has also been shown to enhance the tumorigenic effect of thioacelamide, N-nitrosodimethylamine (NDMA) and N-nilrosodielhylamine (Craddock, 1978; Date. Gotoskar & Bhide, 1976). Dimethylbenzanthracene-induced skin tumours pro moted by the classic phorbol esters have been ob served to be inhibited by anti-inflammatory steroids which inhibit inflammation, DNA synthesis and cell proliferation (Slaga, Fisher, Viaje. Berry, Bracken, LeClerc & Miller, 1978; Weeks, Slaga, Hennings, Gleason & Bracken, 1979). Finally, as noted above, tumours can be induced at the site of subcutaneous injection of nonreactive chemicals, such as glucose, saline or distilled water (Grasso & Golberg, 1966). Having explored the molecular as well as experi mental basis for the effects of cytotoxic responses on the carcinogenic process, the obvious question arises about the impact of cell division and increased DNA synthesis on genotoxic chemical carcinogens. For genotoxic carcinogens it is likely that cytotoxicity will cause an increased incidence and/or a decreased latency in the production of tumours. A good example of this is the induction of liver tumours by NDMA (Terracini, Magee & Barnes, 1967). At low doses of NDMA that preclude cytotoxic effects it the liver, tumours can still be induced. At higher levels where both cytotoxic as well as genotoxic effects act in concert, liver tumours are observed at an increased frequency as well as at a decreased latency. For chemical carcinogens displaying a cytotoxic mechan ism with little to no genotoxic activity it is likely that a prolonged, recurrent cytotoxic response throughout a large portion of the animal's life will be required for the induction of tumours. The implications of these carcinogenic mechanisms are as follows. (1) For genotoxic carcinogens, the defence mechanisms such as DNA repair, detoxifica tion and excretion of the reactive species will be criti cal for modifying the carcinogenic response. Risk esti mates need to consider the dose-related kinetics of the lesions produced and the extent and persistence of those genetic lesions. (2) For epigenetic carcinogens 1 s ! i s5 >* causi igeni duce voul injur It resen to ep activi vated with well : respo genet the n genet rcacti poter pensi qualii these outco majoi result they i also i such with strain lar tu cal e: tance result Exper Tht has b entiati cytotc pararr geneti alkyla induct ecule. total I tion, d critica bases, potent selecte of D^ treat m those selecte lating low-mi weight benzof appror effects, mcasu: out. T; ethxlv!' The chlorot Genetic and nongcnelic events in neoplasia 571 causing tumours via a cytotoxic mechanism of lumorigenictiy. tumours will be induced at doses that pro duce recurrent cytotoxicity and no carcinogenic risk would be predicted at doses that preclude any tissue injury. It is expected that chemical carcinogens will rep resent a spectrum of activity ranging from genotoxic to epigenetic. Those chemicals having strictly genetic activity are often those that either are, or can be acti vated to, strongly electrophilic species. Chemicals with intermediate activity may have both a genetic as well as an epigenetic component to their tumorigenic response and some chemicals that show little to no genetic activity may produce tumours strictly through the mechanism of recurrent cytotoxicity or other epi genetic mechanisms. This is not to say that chemical reactivity is always directly relatable to carcinogenic potency but only that such compounds show a pro pensity to react with macromolecules. Obviously, the qualitative character or reaction site specificity of these reactions will have a profound impact upon the outcome of the DNA-chemical interaction. Indeed, a majority of interactions would not be expected to result in any biologically significant event because they involve noncritical-site alkylation or repair. It is also important not to rule out endogenous factors such as genetic predisposition working in concert with epigenetic mechanisms since all species and strains are susceptible to the development of particu lar tumours that are not related to exogenous chemi cal exposure. Tnis concept is of paramount impor tance in assessing the relevance of extrapolating test results in sensitive animal species to humans. Experimental approaches: some examples The experimental approach taken in our laboratory has been to develop some objective criteria to differ entiate between the genetic effect and the epigenetic, cytotoxic mechanism of tumour production. The two parameters that were selected as representative of a genetic effect were (1}*DNA damage^ measured by alkylation of DNA in vivo and (2) DNA repair, induced presumably by damage to the DNA mol ecule. While there are inherent limitations to using total DNA alkylation as an index of genetic interac tion, due to the overall lack of information on specific critical sites of reaction on all of the nucleic acid bases, it was one of the only means of assessing the potential for in vivo genetic interaction. NDMA was selected as a genetic-acting carcinogen and the extent of DNA alkylation and of DNA repair following treatment with the test chemicals were compared with those after NDMA treatment. NDMA was also selected because it represented an electrophilic methy lating agent and the chemicals we were testing were low-molecular-weight molecules. If high-molecularweight heteroaromatic molecules are being tested, bcnzo[u]pyrcne or the naphthylamines may be 3Ppropriate. For evidence of epigenetic, cytotoxic ejects. DNA synthesis in the target tissue was measured and classic histopathology was also carried out. The chemicals that were tested were perchlorocln>!ene. chloroform and 1.4-dioxane, hi C resu*ls the carcinogen bioassay on perc orocthylene conducted by the National Cancer Institute and of studies conducted in our laboratory are summarized as follows. Mice exposed by gavage to approximately 500 or 1000 mg/kg/day throughout their lifetime showed an increase in liver tumours (hepatocellular carcinomas), but rats exposed to equally high doses did not show any tumorigenic re sponse (National Cancer Institute, 1977). J n inhala tion studies, exposure to 300 or 600 ppm perchloroethylene daily for 1yr (subsequent observation for 18 months) did not prove to be tumorigenic in rats (Rampy, Quast, Leong & Gehring, 1978). It is impor tant to emphasize that the B6C3Ft mice in which liver tumours were enhanced by perchloroethylene treatment have an average control incidence of hepatocellular carcinoma of 10-15% (some nearly 50%). Therefore, this species and strain has a marked genetic predisposition for liver neoplasms. . Studies designed to characterize differences in the pharmacokinetics of perchloroethylene in rats and mice showed the following (Table 1; Schumann, Quast & Watanabe, 1930; Watanabe, Reitz, Schu mann, McKenna, Quast & Gehring, 1980). When exposed to a 10 ppm perchloroethylene atmosphere for 6 hr mice metabolized and activated nine times more perchloroethylene than did rats. This resulted in a seven-fold greater macromolecular binding in the liver of mice than rats. Importantly, binding to hepa tic DNA in mice at a tumorigenic dose level of 500 mg/kg was not observed. The detection limit for the perchloroethylene-DNA binding studies in mice was 10 alkylations per 106 nucleotides. When parameters of cytotoxicity were assessed (Table I) the liver weights were increased in treated mice but not in rats. DNA synthesis caused by tissue injury was increased by 82% in the mice but was not significantly increased in rats, and finally histopathological alterations in the liver were evident in mice but not in rats. Another possible indication of direct genetic inter action is that of gross chromosomal damage and of in vitro mutagenesis, if a possible nonmutational gene deregulation mechanism of carcinogenesis (e.g. Holli day's theory) is ruled out. When mutagenicity data for perchloroethylene were examined (Table 2), it wasnoted that negative results in Salmonella have been reported from three laboratories and there has been one report of a positive finding in the Ames-type strain of Salmonella typhimurium. Further, in a cyto genetics study no chromosomal abnormalities attribu table to perchloroethylene exposure were observed. The overall conclusion is that perchloroethylene has little to no genetic activity. In summary of the perchloroethylene data, DNA alkylation was not delectable at a detection limit of ten alkylations per 106 nucleotides; DNA-rcpair assays were not conducted because they are generally less sensitive than the alkylation studies. Mutagenicity data is predominantly negative in bacterial systems as well as negative in an animal cytogenetics study. Cytotoxic responses indicated by increased DNA syn thesis, two-fold over that of control, and histopatho-. logical examination were evident in the mouse at tumorigenic dose levels. In contrast, a classic genotoxic carcinogenic agent (e.g. NDMA, ethylnitrosourea) would alkylate DNA at a le\d of hundreds to thousands per 10h nucleotides; DNA repair would 572 W. T. Stott. R. H. Reitz. A. M. Schumann and P. G. Watanabe Tabic 1. Perchloroethyiene metabolism, genotoxicity and cy/oio.xiciry data / (Schumann, Quasi & Watanabe, 9SO; Watanabe, Reitz, Schumann, McKenna A Cehring, 980) Perchloroethylene treatment Parameter Response in t Rats Mice Inhalation exposure to 10 ppm for 6 hr Single oral dose of 500 mg/kg body weight Twelve oral doses of 500 mg/kg body weight/day Total metabolized (pmol-equiv/kg body weight) Total macromolecular binding (pmol-equiv/g hepatic protein) Hepatic DNA binding Liver weight;body weight (% increase) Hepatic DNA synthesis (% increase)* Hepatic histopathological changest 10-5 002 . 5 -- 89-5 0-147 ND 25 + ND =* Not delected ' Present as a percentage increase in DNA synthesis in treated compared with control animals as measured by [6-3H] thymidine uptake. tA treatment-related response was observed (+). or no treatment-related response was observed ( --). illustrate a dose-response relationship; in vitro muta genicity would be clearly evident. In contrast to these genotoxic effects, there would be little histopathological alteration or increased DNA synthesis in the target organs at low, though tumorigenic, dosages, thus providing evidence of a lack of a significant role oi an epigenetic, cytotoxic mechanism. Our overall conclusion is that the liver tumours observed in mice following lifetime administration of high doses of perchloroethylene were a result of the recurrent hepatotoxic effect of perchloroethylene which caused an enhancement of the normal background liver tumour incidence in a strain of mice with genetic predisposition towards the development of such tumours. Most importantly, hepatotoxic effects in ani mals and humans would be observed only at dose levels that are well above levels encountered in the occupational environment. Thus the relevance of the data that has been obtained for mice which suggests a carcinogenic risk to humans exposed at occupational or environmental levels of perchloroethylene is highly questionable. The next example is that of chloroform (Reitz. Quasi, Stott, Watanabe & Gehring. 19S0). The carci nogenicity data for male mice given chloroform are summarized in Table 3. This represents a compilation of more than one study, but these data clearly show that following administration of chloroform at high doses male mice are susceptible to the development of liver and kidney tumours. It is noteworthy that at 17 mg/kg/day no significant incidence of tumours at any site was observed. At high doses chloroform also induces tumours in the rat, but for the sake of space we shall limit discussion here to mouse data. At an oral dose level of 240 mg chloroform/kg body weight, mouse liver and kidney DNA was alkylated at a rate of three and one alkylations per 106 nucleotides respectively (Table 4). These values represent the upper bounds of chloroform alkylation since no actual DNA adducts have been identified. In contrast. Table 2. In vitro mutagenicity data and cytogenetics data on perchloroethylene Bioassay system Results* Reference Escherichia coli Salmonella strains Cytogenetics - + * -- h-- Greim, Bonse. Radwan, Reichert & Henschler (1975) National Institute for Occupational Safety and Health (1977). Cern.t & Kypcnovd (1977) Bartsch, Malavetlle, Barbin & Planche'(1979) National Toxicology Program (1980) Cerna & Kypenov (1977) Positive (+ ) and negative ( --) responses are indicated. **r*%V\^'^*** * ' .vv MW i> */.-M -V, Genetic and nongcnetic events in neoplasia 573 Table 3. Data from lumorigenicity assays o f chloroform given orally to mate mice Chloroform dose 277 mg/kg/day, 5 days/wk for 78 wk 60 mg/kg/day, 6 days/wk for 80 wk 60 mg/kg/day. 6 days/wk for 80 wk 17 mg/kg/day, 6 days/wk for 80 wk Strain B6C3F, C57BL ' ICI ICI Percentage of excess tumours (site) 92 (liver) Reference t National Cancer Institute (1976) ^ ND Roe, Palmer, Worden & Van Abb (1979) 2 3 (kidney) Roe et at. (1979) ND Roe cl at. (1979) ND = Not detected ene is highly j form (Reitz, j; |. The caret- ;; oroform are * compilation clearly show -y arm at high V elopment of 1 rthy that at -- j tumours at 4-j roform also - j jke of space ^jj ata. rm. kg body - *j alkylated at -jt nucleotides - *| present the - * i since no - 1 In contrast, -\ a dose of 10 mg NDMA/kg results in approximately 540 alkylations per 106 nucleotides in the liver. Simi larly. a tumorigenic dose of 240 mg chloroform/kg was not observed to induce hydroxyurea-resistant DNA repair (Table 4) while NDMA showed a doserelated increase in DNA repair following doses of 3, 10 and 20 mg; kg body weight. Cytotoxicity, as evidenced by increased DNA syn thesis in both the liver and kidney of chloroformtreated mice, showed an increasing dose-response re lationship at 60 and 240 mg chioroform/kg body weight, but it was normal at a nontumorigenic dose of I5mg/kg (Table 4). Concomitant with the increase in DNA synthesis, histopathological alterations were ob served at the higher dose levels. On the other hand, NDMA, at 3mg.ke body weight, showed no propen sity towards increasing DNA synthesis or histopatho logical alterations as evidence of cytotoxicity. , In summary cf the chloroform data, DNA alkytation in the liver following a tumorigenic dose level of chloroform was a maximum of three alkylations per 106 nucleotides. DNA repair was not detectable. A review of the literature has also shown that chloro form is not mutagenic in a mammalian cell and several bacteria! mutagenesis assay systems (Kirkland, Smith & Van Abb, 1981: Reitz et aL, 1980). Evidence of chloroform's cytotoxicity has come from studies using mice dosed with a tumorigenic level of 240 mg/kg body weight. DNA synthesis was observed to be increased 14-fold in the liver and 25-fold in the kidneys of these animals, and this was associated with histopathological observations of toxicity. It appears that the genetic potential of chloroform is nil to very little, while the component of recurrent cytotoxicity is very significant. Thus, as with perchloroethylene, it appears that chloroform causes tumours in mice via a cytotoxic mechanism rather than by a purely genetic mechanism. The final example is that of 1,4-dioxane. When chronicaliy administered to rats at a concentration of 0*75 to 1-8% in their drinking-water, 1,4-dioxane has been observed to cause an excess incidence of nasal and hepatocellular carcinomas (Argus, Arcos & Hoch-Ligeti, 1965; Hoch-Ligeti, Argus & Arcos. 1970; Kociba, McCollister, Park, Torkelson & Gehring, 1974). Ingestion oflower doses (0-1%) in drinkingwater (Kociba et at. 1974) and inhalation of 1,4-diox ane (Torkelson, Leong, Kociba, Richter & Gehring, Table 4. Data on chloroform and hi-nilrosotlimethylamine (NDMA) genotnxicity amt cyto toxicity in mate CD-J mice (Reitz, Quast, Stott, Watanube & Gehring. i 9H) Treatment Hepatic DNA ----------------------------Binding (alkylations/ 106 nucleotides) Repair* DNA synlhesist in Liver Kidney Histopathological changes in Liver Kidney Chloroform 240 mg/kg 60mg/kg 15 mg/kg NDMA: 20 mg/kg 10 mg'kg 3 mg/kg 3 -- -- -- 540 -- ND X 14 X25 -- X2*2 X82 -- ND ND 7*37 -- 304 -- 1*60 ND -- -- -- + -- -- -- -- - + + -- -- -- -- ND = Not detected ` Ratio of treated control values. Values > 10 indicate an increase in DNA repair, !I : :nti t Presented as multiples of control values as measured by [6-AMjlhymidine incorporation. iA treatment-related response was observed ( + ) or no treatment-related effect was ob served ( - ) , Chloroform was administered as a single oral dose; NDMA vus given as a single ip injection. *!* J.. 574 W. T. Stott. R. H. Reitz. A. M. Schumann and P. G. Watanabe 1974) have failed to produce excess tumours. At a tumorigenic dose level (1015 mg/kg/day or 1% in water), 1,4-dioxane was also observed to cause pro nounced hepatic degenerative and regenerative changes (Kociba et ai. 1974). No genotoxicity has been observed in SpragueDawley rats dosed with a single oral carcinogenic dose level of 1,4-dioxane (1000 mg/kg); no increased DNA repair or hepatic DNA alkylation was detected. In vitro mutagenicity assays carried out following the methods of Ames, Durston. Yamasaki & Lee (1975) and using S. typhimurium strains TA93, 100, 1535, 153? and 1538 also gave negative results. No DNA repair was detected in an in vitro primary rat hpato cyte RNA repair assay following the method of Wil liams (1977). Yet, as shown in Table 5, repeated administration of a carcinogenic dose level of 1,4-di oxane (1000 mg/kg body weight/day) was observed to be cytotoxic to rat hepatic tissue as evidenced by a 1-5-fold increase in hepatic DNA synthesis and abnor mal histopatholoey relative to the controls. No cyto toxic changes were observed in rats dosed with a nontumorigenic level of 1,4-dioxane (10 mg/kg body weight/day). The lack of genotoxicity and the observed cytotoxi city of 1,4-dioxane at tumorigenic dose levels indi cates that as with perchloroethylene and chloroform in the mouse, 1,4-dioxane may cause tumours in the rat ria an epigenetic-cytotoxic mechanism of action. However, the pronounced hepatocellular hypertro phic response observed in rats exposed to a high dose level of 1,4-dioxane (Table 5) and the observation that 1,4-dioxane induces hepatic drug-metabolizing enzyme systems in the rat (this laboratory) also sug gest another epigenetic mechanism of tumorigenic action characteristic of metabolic inducing agents such as phnobarbital. It has been suggested that this mechanism of action may be a result of degranulation of rough endoplasmic reticulum resulting in alter ations in protein synthesis and gene expression or by a hyperplasia-induced enhancement of the expression of pre-existing oncogenic factors (Tennekes, 1979; Wright, Akintowa & Wooder, 1979). Thus from the available data it appears that repeated exposure to high doses of 1,4-dioxane may cause tumours in rats via one or more epigenetic (mutational or nonmutational) mechanisms of action. Conclusion Obviously, much work remains to be done to fill in the gaps in our understanding of the mechanisms of chemical carcinogenesis. Future work to define criti cal sites on macromolecules (DNA, histones, nonhis tone proteins) that may be important in the process of gene expression and cellular transformation will be of particular importance. However our understanding of the mechanisms of chemical carcinogenesis has reached a point whereby this knowledge may be used to generally classify carcinogens and in human risk assessment, even though the more definitive mechan istic details remain to be elucidated. The studies cited above, and others, have shown that all carcinogens do not act by the same mechanism. Some, which have genetic mechanisms of carcinogenicity, pose greater risks than others, which act by epigenetic mechan isms. 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