Complete Carcinogen Research Articles

The role of nongenotoxic mechanisms in arylamine carcinogenesis.

The growth of preneoplastic nodules during the feeding of a carcinogenic 2-acetylaminofluorene (2-AAF) regimen is preceded by several alterations in the physiologic homeostasis. Many of these alterations can be considered adaptive responses to the drug exposure. One property of AAF could be identified that clearly distinguishes this complete rat liver carcinogen from at least two other, incomplete rat liver carcinogens. Highly specific redox cycling in mitochondria was demonstrated in vitro, and this observation could well contribute an explanation of the morphologic and histochemical observations in vivo. It is emphasized that nongenotoxic effects may play an important role in the generation of tumors by genotoxic carcinogens.

The co-mitogenic effects of various estrogens for TGF-α-induced DNA synthesis in cultured female rat hepatocytes

The synthetic estrogens ethinyl estradiol (EE) and mestranol (M) are weak complete hepatic carcinogens and potent tumor promoters. In vivo, EE and M cause a rapid but transient increase in liver growth. However, studies in cultured female rat hepatocytes indicate that EE is not a strong complete hepatic mitogen but rather enhances epidermal growth factor (EGF)-induced DNA synthesis and is thus classified as a co-mitogen (Yager, J.D., Zurlo, J. and Ni, N. (1991) Proc. Soc. Exptl. Biol. Med., 198, 667–674). The endogenous estrogen 17β-estradiol (E 2) also exhibits co-mitogenic activity, enhancing the fraction of hepatocytes undergoing DNA synthesis induced by both EGF and transforming growth factor alpha (TGF-α) (Ni, N. and Yager, J.D. (1994) Hepatology, 19, 183–192). The objectives of the study reported here were: (1) to determine whether the co-mitogenic effects of EE and E 2 extend to other synthetic estrogens including mestranol and diethylstilbestrol, and to α-zearalanol, a natural product with estrogenic activity; (2) to compare the co-mitogenic effects of endogenous estrogens including E 2, estrone, estriol and the catechol metabolites 2- and 4-hydroxy-estradiol; and (3) to determine whether the conditioned medium from E 2-treated hepatocytes has co-mitogenic activity. Female rat hepatocytes in primary culture were exposed to the various estrogens ± TGF-α and DNA synthesis was determined by measuring [ 3H]thymidine incorporation into extracted DNA. The results show that the co-mitogenic effects previously observed with EE and E 2 also extend to all of these estrogens and to the E 2 catechol metabolites. Although the co-mitogenic potency of these estrogens does not correlate with their reported affinities to the estrogen receptor, their estrogenicity appears necessary since the non-estrogenic metabolite 2-methoxyestradiol lacks co-mitogenic activity. In addition, enhancement of TGF-α-induced DNA synthesis by conditioned medium from E 2-treated cells supports the notion that a metabolite mediates its co-mitogenic effect.

Polycyclic Aromatic Hydrocarbons (PAH) Induce Oxidative Stress and Oxidative DNA Modification - Characteristics of Tumor Promotion

Abstract Formation of PAH-DNA base adducts is an accepted tumor-initiating event. However, it is not yet known what constitutes tumor promotional processes engendered by complete carcinogens. It appears that induction of oxidative stress and oxidative DNA damage optimally correlates with the biological effects of tumor promoters. We showed that PAH-treated rat liver microsomes produce superoxide (O2·-) and hydrogen peroxide (H2O2) that cause oxidation of bases in DNA. Recently we found that 7,12-dimethylbenz[a]anthracene (DMBA) induces in vivo some of the same responses as those mediated by phorbol ester tumor promoters. These include inflammatory processes, H2O2 production and oxidation of bases in the epidermal DNA of topically-treated SENCAR mice. Those responses occurred in long (5 wk)- and short (4 day)-term DMBA treatments. Based on these findings, we propose that PAH-induced inflammation, generation of oxidants and oxidation of bases in DNA represent tumor promotional processes of complete carcinog...

Mechanism of Fumonisin Toxicity and Carcinogenesis

What are the molecular events that fumonisin-induced porcine pulmonary edema syndrome and equine leucoencephalomalacia have in common? Do these animal diseases relate mechanistically to fumonisin toxicity in laboratory rats? There is considerable data indicating that disruption of sphingolipid metabolism plays an important early role in all of these diseases. In vitro studies have revealed that fumonisins and structurally related Alternaria alternata f. sp. lycopersici-toxin (AAL-toxin) are potent inhibitors of the enzyme sphinganine (sphingosine) N-acyl transferase (ceramide synthase). Soon after cultured cells or animals are exposed to fumonisins there is a dramatic increase in the free sphingoid base, sphinganine, in tissues, serum and/or urine. Also, free sphingosine concentration increases, complex sphingolipid concentration decreases, and sphingoid base degradation products and other lipid products also increase. It is hypothesized that disruption of sphingolipid metabolism is an early molecular event in the onset and progression of cell injury and the diseases associated with consumption of fumonisins. However, the exact mechanisms responsible for the diseases will not be easily revealed since the role of sphingolipids in cellular regulation is very complex and not yet fully understood. While fumonisin B1 is non-genotoxic it is a complete carcinogen in rat liver. Recent studies indicate that fumonisins inhibit hepatocyte proliferation in rat liver. It has been hypothesized that hepatotoxicity and effects on hepatocyte proliferation are critical determinants for fumonisin B1 cancer initiation and promotion. Alternatively, recent studies have found that fumonisin B1 has mitogenic activity in cultured fibroblasts. It is conceivable that the mitogenic, cytostatic and cytotoxic potential of fumonisin may all contribute to the animal diseases including liver cancer in rats.

The effect of intraperitoneal N-methyl- N-nitrosourea on hamster palatal gingiva and intermolar mucosa

Fifty 4- to 6-week-old male random-bred golden hamsters were injected intraperitoneally with a weight-related dose (12.5 mg/kg body weight) of N-methyl- N-nitrosourea (NMU) three times a week for 4 weeks. Groups of seven animals were killed 10, 16 and 22 weeks after the first injection. The palatal gingiva from six animals and the intermolar mucosa from 21 animals was examined. Seven male age-matched untreated control animals were killed at each period. Although all NMU-treated hamsters showed dysplastic and neoplastic changes similar to those in human oral squamous-cell carcinoma, other changes such as acantholytic dyskeratosis, invading cysts, duct-like structures and basaloid islands and cords were not. The extent and severity of the changes increased with time so that by 22 weeks there was extensive involvement of the palatal bone and marrow spaces, the molar periodontal ligament and the greater palatine neurovascular bundle by neoplastic epithelium. The invading epithelium was derived from the junctional, crevicular and palatal gingival and intermolar epithelium. The latent period for the crevicular and junctional epithelia was shorter than that for the palatal gingival and intermolar epithelium. The invasive changes from the latter epithelium were often preceded by exophytic changes such as epithelial projections, papillae and papillomas. Such changes were infrequent for the gingival, crevicular and junctional epithelia. The study shows that intraperitoneal NMU acts as a complete carcinogen on the palatal gingival and intermolar epithelium in hamsters.

Mechanism of Fumonisin Toxicity and Carcinogenesis

What are the molecular events that fumonisin-induced porcine pulmonary edema syndrome and equine leucoencephalomalacia have in common? Do these animal diseases relate mechanistically to fumonisin toxicity in laboratory rats? There is considerable data indicating that disruption of sphingolipid metabolism plays an important early role in all of these diseases. In vitro studies have revealed that fumonisins and structurally related Alternaria alternata f. sp. lycopersici-toxin (AAL-toxin) are potent inhibitors of the enzyme sphinganine (sphingosine) N-acyl transferase (ceramide synthase). Soon after cultured cells or animals are exposed to fumonisins there is a dramatic increase in the free sphingoid base, sphinganine, in tissues, serum and/or urine. Also, free sphingosine concentration increases, complex sphingolipid concentration decreases, and sphingoid base degradation products and other lipid products also increase. It is hypothesized that disruption of sphingolipid metabolism is an early molecular event in the onset and progression of cell injury and the diseases associated with consumption of fumonisins. However, the exact mechanisms responsible for the diseases will not be easily revealed since the role of sphingolipids in cellular regulation is very complex and not yet fully understood. While fumonisin B1 is non-genotoxic it is a complete carcinogen in rat liver. Recent studies indicate that fumonisins inhibit hepatocyte proliferation in rat liver. It has been hypothesized that hepatotoxicity and effects on hepatocyte proliferation are critical determinants for fumonisin B1 cancer initiation and promotion. Alternatively, recent studies have found that fumonisin B1 has mitogenic activity in cultured fibroblasts. It is conceivable that the mitogenic, cytostatic and cytotoxic potential of fumonisin may all contribute to the animal diseases including liver cancer in rats.

The migration of Langerhans' cells into and out of lymph nodes draining normal, carcinogen and antigen-treated sheep skin.

Epidermal Langerhans' cell (LC) migration to the regional lymph node and beyond into central lymph was examined in sheep following topical application of the complete chemical carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) or the contact sensitizing antigen 2,4,6-trinitrochlorobenzene (TNCB). This was facilitated by cannulating previously constructed pseudoafferent lymphatic vessels draining the skin treated with these agents or alternatively, the efferent lymphatic vessel of the regional lymph node. Application of DMBA resulted in a biphasic increase in LC migration. There was an initial increase in LC migration at 25 h with the maximum response (3.6 x 10(7) LC/h) occurring approximately 5 days after DMBA treatment. In contrast, the contact sensitizing antigen TNCB caused enhanced LC migration within minutes of the application of antigen (3.3 x 10(6) LC/h) and peak migration at 8-12 h. Examination of efferent lymph cells from the regional lymph node after DMBA treatment showed uncharacteristically large numbers of LC traversing the lymph node. These LC migration patterns suggest different mechanisms may trigger the migration of LC from skin after the application of DMBA to those associated with the normal processes of antigen presentation.

Risk Assessment of Carcinogenic Chemicals in the Netherlands

The Health Council of The Netherlands advises the Dutch government on scientific issues concerning health and environmental protection. In 1978 a committee of the Council established mechanistic criteria for quantitative risk assessment of carcinogenic substances. For this purpose it adopted the multistage model of carcinogenesis. Based on this model two categories of carcinogens were distinguished. The first category includes complete carcinogens and tumor initiators: these agents induce irreversible modification of DNA and act by a stochastic mechanism. The second category includes promotors and other substances acting as cocarcinogens; these agents act by nonstochastic mechanisms. Stochastic action implies that there is no threshold for the contribution to the carcinogenic effect, whereas nonstochastic action implies a threshold dose. Recently, reevaluation of the method for carcinogenic risk assessment was considered due because of remarkable scientific progress since 1978, such as the identification of cellular oncogenes and tumor suppressor genes, and the partial clarification of mechanisms at the cellular and/or molecular level by which tumor promotors stimulate tumor development. The present paper discusses the Dutch method for carcinogenic risk assessment in the light of the recent scientific developments. It concludes that this method for risk assessment is still a valid and appropriate one.

Effect of 4-acetylaminofluorene and other tumour promoters on hepatocellular growth and binucleation.

The complete liver carcinogen 2-acetylaminofluorene (2-AAF) promoted the outgrowth of large neoplastic liver nodules and hepatocellular carcinomas in diethylnitrosamine-treated rats. 2-AAF did not alter the overall proliferative activity of normal hepatocytes, but suppressed binucleation and induced, on a long-term basis, an increase in proliferative activity and in the fraction of diploid hepatocytes relative to control animals. The analogue 4-acetylaminofluorene (4-AAF) was much less effective than 2-AAF as a promoter of large nodules and carcinomas, but promoted the outgrowth of medium-sized nodules (1 < 2.5 mm). While 2-AAF specifically stimulated the growth of cells in enzyme-altered foci, the cells responding to 4-AAF were more randomly distributed throughout the liver tissue. In contrast to 2-AAF, 4-AAF strongly stimulated the growth (DNA synthesis) of normal hepatocytes, but like 2-AAF it suppressed binucleation and caused a long-term increase in the proliferative activity and in the fraction of diploid hepatocytes. Other liver tumour promoters (cyproterone acetate, alpha-hexachlorocyclohexane, methylclofenapate) likewise stimulated the growth and suppressed the binucleation of normal hepatocytes. All hepatocellular ploidy classes were affected virtually equally by mitogenic stimulation, but at low proliferation rates the mononuclear cells were more proliferative than the binuclear cells. Since this difference could be eliminated by increasing the mitogen dose, it would seem that mononuclear cells may be somewhat more sensitive towards mitogens than binuclear cells. In contrast to previously reported results [Styles et al. (1990) Carcinogenesis, 11, 1149-1152], methylclofenapate was not found to specifically stimulate binuclear hepatocytes. Our results indicate that liver tumour promoters in general tend to induce a non-binucleating, non-polyploidizing hepatocellular growth pattern, similar to that observed during liver regeneration. 4-AAF is confirmed to be, at best, a very weak promoter of liver carcinogenesis, but appears to be an effective promoter of benign tumours.

Comitogenic effects of estrogens on DNA synthesis induced by various growth factors in cultured female rat hepatocytes

Ethinyl estradiol is a weak complete carcinogen and potent tumor promoter. In vivo, ethinyl estradiol causes a rapid but transient increase in liver growth, whereas in cultured female hepatocytes it enhances DNA synthesis induced by epidermal growth factor and is thus classified as a comitogen. The objectives of this study were to determine: (a) whether estradiol also has comitogenic activity; (b) whether the comitogenic effects of estrogen extend to complete hepatic mitogens other than epidermal growth factor and (c) whether inhibition of hepatocyte DNA synthesis by transforming growth factor-beta can be blocked by estradiol. Female rat hepatocytes in primary culture were exposed to estradiol with and without various growth factors, and we determined DNA synthesis by measuring [3H]thymidine incorporation into extracted DNA or by determining the nuclear labeling index by means of autoradiography. The results show that estradiol, like ethinyl estradiol, has comitogenic activity for epidermal growth factor, although it is somewhat less potent. Four complete hepatic mitogens showed different abilities to stimulate DNA synthesis, with hepatocyte growth factor > transforming growth factor-alpha > epidermal growth factor > acidic fibroblast growth factor. Estradiol enhanced DNA synthesis occurring in response to all four of these complete hepatic mitogens. This finding suggests that the mechanism of estrogen comitogenesis may involve effects at a point where the different signal-transduction pathways leading from the growth factors converge. The level of estrogen-mediated enhancement of DNA synthesis was similar for all four mitogens, ranging from 1.5 to 2.2-fold, depending on the experiment. Furthermore, determination of the nuclear labeling index showed that estrogen enhancement of DNA synthesis was associated with an increase in the percentage of hepatocytes that responded to the growth factors. Finally, estradiol did not specifically block the growth-inhibitory effects of transforming growth factor-beta.

Tumors in rat kidney generated by initiation with trans-4-acetylaminostilbene and several promoting treatments.

trans-4-Acetylaminostilbene (AAS) is a complete carcinogen in rats and produces quite selectively tumors in Zymbal's glands. On the basis of DNA adduct formation, it has been proposed that this model arylamine initiates neoplastic transformation of cells in many tissues, particularly liver and kidney, which, in the classical sense are considered to be non-target tissues for this chemical. In the present study an initiating treatment with AAS was followed by unilateral nephrectomy and the application of two nephrotoxic substances, gentamycin or beta-cyclodextrin which, among other activities, stimulate cell proliferation specifically in kidney. The initiating dose of AAS, given alone, gave rise to Zymbal's gland and mammary tumors in female Wistar rats within 88 weeks but not to liver or kidney tumors. When the initiation treatment was followed by unilateral nephrectomy, alone or in combination with gentamycin, or by beta-cyclodextrin, four tumors in two out of ten animals, eight tumors in three/ten, and seven tumors in three/ten, respectively, were observed in the kidney. The administered dose of gentamycin was not sufficient to induce tumors on its own. The results support the view that the genotoxic effects of AAS produce promotable lesions in rat kidney. None of the animals that had been treated with AAS, with or without other treatments, developed tumors or the predominant types of preneoplastic lesions in the liver within 88 weeks; this supports the notion that liver, like kidney, is not a target for complete carcinogenesis for this chemical.

Peroxisome proliferators: Paradigms and prospects

Peroxisome proliferators are a structurally diverse group of chemicals. They include fibrate hypolipidaemic drugs, phthalate ester plasticisers, phenoxy acid herbicides, azole antifungal drugs, and perfluorinated fatty acids. This presentation will focus on the common pleotropic responses produced by these compounds including hepatomegaly (hyperplasia and hypertrophy), activation of cell cycle S-phase ploidy changes, cytochrome P4504A1 induction, morphometric/biochemical analysis of peroxisome proliferation and stimulation of growth factors, and oncogene activation. Consideration will also be given to the role of recently described Peroxisome Proliferator Activated Receptor in these diverse hepatic responses. Peroxisome proliferators are uniformly negative in a wide range of genotoxicity tests, but nevertheless are complete carcinogens, particularly in rodent liver. Mechanisms of nonmutagenic carcinogenesis will be discussed including the active oxygen hypothesis involving 8-hydroxydeoxyguanosine adducts and the possibility of peroxisome proliferators promoting preexisting lesions by clonal expansion, eventually resulting in frank tumorigenesis. Finally, a consideration of the risk assessment of peroxisome proliferation to humans will be discussed.

Factors affecting carcinogenic potential of mixtures

Historically, exposure to complex mixtures such as soot, coal tar, mineral oils, and cigarette smoke has been associated with increased cancer mortality. Benzo[a]pyrene (BaP) has been used to predict the carcinogenic potency of mixtures. Two complete carcinogenicity C3H/HEJ mouse skin bioassays were undertaken to determine the effect of low doses of BaP on the carcinogenic potential of mixtures. A toluene solution containing 0.1% each of five noncarcinogenic polycyclic aromatic hydrocarbons (PAHs), anthracene, chrysene, pyrene, fluoroanthene, and phenanthrene, produced tumors in 23% of the mice with a latent period of 73 weeks. With the addition of a 0.001% BaP to the above solution, 47% of the mice produced tumors with a latent period of 66 weeks. In the second study, coal tar in toluene, which was determined to contain 0.0006% BaP, produced tumors in 51% of mice with a latent period of 73 weeks. In both studies the BaP solutions by themselves did not produce tumors. In a third study, the 9-, 2-, and 3-methylbenz[a]-anthracene compounds were noncarcinogenic using toluene as the solvent. With the substitution of n-dodecane for toluene all three compounds produced significant numbers of tumors. The results indicate that (1) low dose levels of BaP can have an impact on the carcinogenic potential of mixtures, (2) the presence or absence of BaP is not always sufficient to account for the observed potency and the synergistic effects of other substances which might be present, and (3) that certain noncarcinogenic methylbenz[a]anthracenes can have their carcinogenic potential altered by a change in the solvent used.

Asbestos-induced lung disease.

This review attempts to deal with two major questions concerning asbestos-induced lung disease: How does inhaled asbestos cause cell proliferation and fibrosis? and Will there continue to be risk from exposure to asbestos in schools and public buildings? The first is a scientific question that has spawned many interesting new experiments over the past 10 years, and there appear to be two hypothetical schemes which could explain, at least in part, the fibroproliferative effects of asbestos fibers. One supports the view that toxic oxygen radicals generated on fiber surfaces and/or intracellularly are the central mediators of disease. The second hypothesis is not mutually exclusive of the first, but, in my opinion, may be integral to it, i.e., the cellular injury induced by oxygen radicals stimulates the elaboration of multiple varieties of growth factors and cytokines that mediate the pathogenesis of asbestosis. There is increasing evidence that molecules such as platelet-derived growth factor and transforming growth factor beta, both synthesized and secreted by activated lung macrophages, are responsible, respectively, for the increased interstitial cell populations and extracellular matrix proteins that are the hallmarks of asbestos-induced fibrosis. The challenge today is to establish which combinations of the many factors released actually are playing a role in disease pathogenesis. The issue of continued risk currently is more a question of policy and perception than science because a sufficient database has not yet been established to allow full knowledge of the circumstances under which asbestos in buildings constitutes an ongoing health hazard. The litigious nature of this question does not help its resolution. In as much as public policy statements and risk assessment are not within my purview, I have focused on the state-of-the-art of asbestos as a complete carcinogen. It appears to be generally nongenotoxic, but all asbestos fiber types can induce chromosomal mutations and aneuploidy, perhaps through their ability to disrupt normal chromosome segregation.

Effects of benzo[a]pyrene and (+-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene on mitosis in Chinese hamster V79 cells with stable expression of rat cytochrome P4501A1 or 1A2.

The effect of bioactivation of benzo[a]pyrene (B[a]P) and (+-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-diol) on spindle disturbances and toxicity has been investigated in V79 Chinese hamster cells genetically engineered to express cytochrome P4501A1 (CYP1A1) and cytochrome P4501A2 (CYP1A2). B[a]P induces spindle disturbances in native V79 Chinese hamster cells. This effect was enhanced by the expression of CYP1A1 but not CYP1A2. The increased effect seen in the CYP1A1-expressing cell line could be brought back to the level seen in the native cell line by alpha-naphthoflavone in a dose-dependent manner. This strongly suggests that a CYP1A1-dependent metabolite, conceivably (+-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BPDE) accounts for the increased spindle disturbing effect. B[a]P-7,8-diol induced spindle disturbances at remarkably low concentrations, 10(-8) M, irrespective of expression of the two CYPs. Our data suggest that B[a]P-7,8-diol is the most potent spindle-disturbing metabolite, whereas BPDE is the most important metabolite concerning mutagenesis. The concentrations inducing spindle disturbances correspond to those that are positive in mutation assays. We hypothesize that B[a]P is a complete carcinogen because of its ability to induce both aneuploidy and mutations after metabolic conversion of low non-cytotoxic concentrations.

Factors Affecting Carcinogenic Potential of Mixtures

Historically, exposure to complex mixtures such as soot, coal tar, mineral oils, and cigarette smoke has been associated with increased cancer mortality. Benzo[a]pyrene (BaP) has been used to predict the carcinogenic potency of mixtures. Two complete carcinogenicity C3H/HEJ mouse skin bioassays were undertaken to determine the effect of low doses of BaP on the carcinogenic potential of mixtures. A toluene solution containing 0.1% each of five noncarcinogenic polycyclic aromatic hydrocarbons (PAHs), anthracene, chrysene, pyrene, fluoranthene, and phenanthrene, produced tumors in 23% of the mice with a latent period of 73 weeks. With the addition of a 0.001% BaP to the above solution, 47% of the mice produced tumors with a latent period of 66 weeks. In the second study, coal tar in toluene, which was determined to contain 0.0006% BaP, produced tumors in 51% of mice with a latent period of 73 weeks. In both studies the BaP solutions by themselves did not produce tumors. In a third study, the 9-, 2-, and 3-methylbenz[a]-anthracene compounds were noncarcinogenic using toluene as the solvent. With the substitution of n-dodecane for toluene all three compounds produced significant numbers of rumors. The results indicate that (1) low dose levels of BaP can have an impact on the carcinogenic potential of mixtures, (2) the presence or absence of BaP is not always sufficient to account for the observed potency and the synergistic effects of other substances which might be present, and (3) that certain noncarcinogenic methylbenz[a]anthracenes can have their carcinogenic potential altered by a change in the solvent used.

Chemically induced skin carcinogenesis in a transgenic mouse line (TG.AC) carrying a v-Ha-ras gene.

A transgenic mouse line (TG.AC) created in the FVB/N strain, carries a v-Ha-ras gene fused to a zeta-globin promoter gene. These trangenic mice have the properties of genetically initiated skin and have been shown to be sensitive to 12-O-tetradecanoylphorbol-13-acetate (TPA), a well-described promoter of skin papillomas in the two-stage mouse skin tumorigenesis model. It was of interest to determine whether the TG.AC mouse strain was also responsive to other known promoters. Groups of heterozygous or homozygous TG.AC mice were treated topically, 2x/week, for up to 20 weeks with benzoyl peroxide (BPO), 2-butanol peroxide (2-BUP), phenol (PH), acetic acid (AA), TPA and acetone (ACN), the vehicle control. Skin papillomas were induced in all groups treated with TPA, BPO and 2-BUP. Papillomas were observed in some treatment groups as early as 3 weeks. The relative activity of the promoters was TPA > 2-BUP > BPO > PH = AA = ACN. No papillomas were observed in any of the uninitiated FVB/N mice treated in a similar manner and which served as treatment control groups. Studies to determine the sensitivity of TG.AC mice to TPA, indicated that a total dose of 25-30 micrograms of TPA administered in 3 or 10 applications, was sufficient to induce an average incidence of 11-15 papillomas per mouse. The papilloma incidence continued to increase and was maintained up to 15 weeks after TPA treatment was terminated. The short latency period and high incidence of papilloma induction indicate that TG.AC mice have a high sensitivity to known skin promoters. The TG.AC line should prove to be a sensitive model for identifying putative tumor promoters or complete carcinogens.

Initiation of enzyme-altered foci by the synthetic steroid cyproterone acetate in rat liver foci bioassay.

Cyproterone acetate (CPA) is a synthetic steroid which is used in oral contraceptive and anti-androgen formulations. It has previously been classified as a tumor promoter in rat liver. Recent studies have shown that CPA induces DNA repair synthesis in isolated hepatocytes, and this implies that CPA is genotoxic. We studied the initiating activity of CPA in vivo by means of a rat liver foci bioassay, using weanling female Sprague-Dawley rats. The results show that CPA induces adenosine-triphosphatase-deficient and gamma-glutamyltranspeptidase-positive putative preneoplastic foci in a dose-dependent manner. This indicates that CPA has not only promoting but also initiating activity and may therefore act as a complete carcinogen in rat liver.

Vinyl carbamate epoxide, a major strong electrophilic, mutagenic and carcinogenic metabolite of vinyl carbamate and ethyl carbamate (urethane).

Vinyl carbamate epoxide (VCO) was found to possess strong electrophilic, mutagenic and carcinogenic activities. It reacted with water at 37 degrees C and pH 7.4 (phosphate buffer) to form glycolaldehyde and several related reducing compounds; none of these products were mutagenic for Salmonella typhimurium TA1535. Under these conditions VCO had a half-life (determined chemically and mutagenically) of approximately 10.5 min. This half-life was progressively lowered by increasing concentrations of chloride ion (liver, serum and isotonic levels). This ion reacted with VCO to form chloroacetaldehyde. VCO also reacted with other nucleophiles such as glutathione, DNA and its constituent guanine and adenine bases. The purine adducts formed by VCO in DNA in vitro and in vivo were released by weak acid treatment and consisted of 7-(2'-oxoethyl)guanine and N2,3-ethenoguanine as major products with 1,N6-ethenoadenine as a minor product. VCO was a strong direct mutagen in Salmonella typhimurium TA1535 and TA100 but was only weakly active in the TA98 mutant. VCO was a stronger initiator of carcinogenesis in the skin of CD-1 mice and in the liver of infant male B6C3F1 mice than its metabolic precursors vinyl carbamate (VC) and ethyl carbamate (EC). Unlike VC and EC, VCO was a strong complete carcinogen in the skin of CD-1 mice and induced papillomas and carcinomas following repetitive administration of sub-ulcerogenic doses. VCO also exhibited some carcinogenic activity in the lungs of mice and in the s.c. and mammary tissue of female Sprague-Dawley rats. These data and those from other recent studies support the conclusion that VCO is a major strong electrophilic, mutagenic and carcinogenic metabolite of EC and VC in the mouse.

Cytoskeleton modifications induced by phenobarbital, 2-acetylaminofluorene and 4-acetylaminofluorene in normal and initiated/selected hepatocytes: relation with the "resistant" phenotype.

Initiated/selected (ISH) and normal (NH) rat hepatocytes were used to study cytoskeleton modifications induced by three liver acting chemicals: 2-AAF, a liver complete carcinogen; PB, a liver tumor promoter; and 4-AAF, a non-carcinogen analogue of 2-AAF. Cytoskeleton alterations were visualized by disappearance of F-actin fibers and tubulin depolymerization. The three drugs induced actin fragmentation in normal hepatocytes; a net loss of actin protein was observed with PB. They also induced varied tubulin depolymerization. The principal difference between chemicals is that 2-AAF led to non-reversible effects, in comparison with PB and 4-AAF which induced reversible damages on cytoskeleton. By contrast to normal hepatocytes, the cytoskeleton of ISH obtained from rats subjected to the "resistant" hepatocyte protocol was much less susceptible to the effect of the three chemicals. Moreover, we observed a lack of LDH release in the culture medium and a very rapid inducibility of GST activity after exposure of ISH to drugs. The moderate effect of the three chemicals on actin and tubulin in ISH could thus be explained by the "resistant" metabolic profile of these cells.