Diphenamid metabolism in plants.

Diphenamid, a herbicide, is metabolized to N-methyl 2,2-diphenylacetamide and 2,2-diphenylacetamide by the common soil fungi Trichoderma viride and Aspergillus candidus within 48 hours. The two metabolites are more toxic than diphenamid to both tomato and barnyard-grass seedlings under sterile conditions. This finding indicates that the phytotoxic moiety is not diphenamid but one of its metabolites-probably the N-methyl derivative.

Enhancement of oncogenesis in C3H/10T1/2 mouse embryo cell cultures by saccharin.

Impure and pure samples of saccharin (2 milligrams per milliliter) did not produce oncogenic transformation of C3H/10T1/2, clone 8, mouse embryo fibroblasts. However, after treatment of the cells with a nontransforming initiating dose (0.1 microgram per milliliter) of 3-methylcholanthrene, continuous treatment with either sample of saccharin (100 micrograms per milliliter) led to significant transformation. It is concluded that in this system saccharin is a cocarginogen, probably functioning as a promoting agent that is 1000-fold less active than the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate.

Inhibition of induced differentiation of C3H/10T 1/2 clone 8 mouse embryo cells by tumor promoters.

C3H/10T 1/2 cells were induced to differentiate into muscle cells by treatment with 5-azacytidine, and the effects of tumor promoters, nonpromoters, and inhibitors of tumor promotion on this induced differentiation were investigated. Cell morphology was dramatically changed within 30 min after treatment with phorbol ester-related tumor promoters and mezereine but not with other tumor promoters. There was a good correlation between the tumor-promoting activity of the compounds and their inhibitory action on differentiation except in case of phenobarbital; this promoter of liver carcinogenesis did not inhibit differentiation. Inhibitors of tumor promotion, dexamethasone, fluocinolone acetonide, retinoic acid, and antipain, also inhibited the 5-azacytidine-induced differentiation. When the cells were treated after induction of differentiation with 12-O-tetradecanoylphorbol-13-acetate, a potent tumor promoter, and simultaneously with one of those inhibitors of tumor promotion, the inhibitory action of 12-O-tetradecanoylphorbol-13-acetate was not affected. From these facts, it is evident that using a single phenomenon produced by one class of tumor promoter as a criterion for screening environmental tumor promoters is not justifiable.

The effect of modifiers of microsomal enzymes on chemical oncogenesis in cultures of C3H mouse cell lines.

Two cell lines, both derived from the C3H mouse and each having different responses (oncogenic and cytotoxic) to polycyclic aromatic hydrocarbon oncogens, were studied with respect to their drug-metabolizing enzymes. The 10T1/2CL8 cells (a C3H mouse embryo fibroblastic cell line) were much more effective in converting 3-methylcholanthrene (3-MC) to 3-MC water-soluble metabolites, 3-MC phenols, and 3-MC-bound cellular macromolecules than were CVP3SC6 cells (a new line of C3H mouse adult ventral prostate fibroblasts). Basal aryl hydrocarbon hydroxylase activity was higher in 10T1/2CL8 cells than in CVP3SC6 cells, while the reverse was found for epoxide hydrase activity (using 3-methylcholanthrene-11, 12-oxide as substrate. 3-MC or benz(a)anthracene induced epoxide hydrase activity in both cell lines to about the same extent. 3-MC did not induce aryl hydrocarbon hydroxylase activity in CVP3SC6 cells. Aryl hydrocarbon hydroxylase activity was markedly induced in both cell lines by benz(a)anthracene and was slightly induced in 10T1/2CL8 cells by 3-MC. In a chemical oncogenesis cell culture system, transformation of 10T1/2CL8 cells mediated by 3-MC could be increased two- to threefold by treating the cell cultures with: either benz(a)anthracene, styrene oxide, cyclohexene oxide, or 1,2,3,4-tetrahydrona=phthalene-1,2-oxide; or with cyclohexene or 1,2-dihydrona-phthalene, alkene precursors of cyclohexene oxide and 1,2,3,4-tetrahydronaphthalene-1,2-oxide, respectively. When 10T1/2CL8 cells were treated with a combination of benz(a)anthracene and cyclohexene, 3-MC-mediated transformation was increased 7.8-fold. CVP3SC6 cells that were not transformed by 3-MC or other hydrocarbon oncogens were transformed by a combined treatment with benz(a)anthracene, 1,2-dihydronaphthalene, and 3-MC.

Neoantigens on chemically transformed cloned C3H mouse embryo cells.

Using an in vitro cytotoxicity test for cell-mediated immunity and a membrane immunofluorescence test, the appearance of new antigens was detected on cloned C3H mouse embryo cells undergoing malignant transformation in vitro following treatment with 3-methylcholanthrene or 7,12-dimethylbenz[a]anthracene. These antigens were recognized by specifically immunized syngeneic mice and were individually unique for each of eight chemically transformed cell lines tested, all of which were derived from the same control parent clone. Very few cross-reactions were seen between lymphoid cells or antibody from mice immunized against a given cell line and target cells of other cell lines. New antigens could not be detected on two spontaneously transformed lines. Lymphoid cells from multiparous pregnant or embryo-immunized cmice were used to search for fetal antigens on control and transformed cells. Fetal antigens were detected on seven of the chemically transformed cell lines and one spontaneous transformant, but not on nontransformed control cells. It is concluded that individually specific new antigens are characteristic of chemically transformed cells, but the expression of fetal antigens may be a more common feature of transformed cells in general.

Effects of praziquantel, a new antischistosomal drug, on the mutation and transformation of mammalian cells.

Praziquantel (PQ), a tetrahydroquinoline derivative, is a new and clinically effective antischistosomal drug, which has been shown to lack or to possess very weak mutagenic activity. However, in bacteria, this compound can act as a weak comutagen that increases the mutagenicity of several chemical mutagens and carcinogens. We have found that PQ can act as a very weak comutagen in animal cells. At 10 to 50 micrograms/ml, PQ increased the mutagenicity of N-methyl-N'-nitro-N-nitrosoguanidine and 2-methoxy-6-chloro-9-[3-(2-chloroethyl)amino-propylamino]acridine dihydrochloride about 2-fold in Chinese hamster V-79 cells. In C3H/10T 1/2 mouse embryo cells, PQ exhibited only negligible comutagenic activity. PQ did not oncogenically transform C3H/10T 1/2 cells but had a pronounced effect on 3-methylcholanthrene-induced transformation of these cells. When PQ was coadministered with or added after 3-methylcholanthrene treatment, the number of type III foci produced was about 5-fold lower than in cultures treated with 3-methylcholanthrene alone. Therefore, PQ can inhibit type III focus formation in C3H/10T 1/2 cells.

Biochemical characterization of fluoropyrimidine-resistant murine leukemic cell lines.

Twenty clones stably resistant to 5-fluorouracil, 5-fluoro-2'-deoxyuridine, or 5-fluorouridine have been isolated from L1210 or P388 murine leukemia cells by a one-step mutation and selection procedure. The activities of enzymes of the pyrimidine salvage pathway relevant to the activation of these drugs have been determined in order to elucidate the mechanisms of resistance in these cells. Cell line resistant to 5-fluorouracil have 7 to 50% of the pyrimidine phosphoribosyltransferase activity found in the wild-type cells, with 5-fluorouracil, uracil, or orotate as substrate. Cells selected for resistance to 5-fluoro-2'-deoxyuridine have no detectable thymidine kinase activity. 5-Fluorouridine-resistant cells have 3 to 25% of the uridine kinase activity measured in the wild-type cell lines. No significant changes were observed in the activities of thymidylate synthetase, nucleoside phosphorylases, or 5-fluorouridylate kinase in any of the resistant cell lines. These findings have relevance to the treatment of human cancer, since pyrimidine phosphoribosyltransferase, thymidine kinase, or uridine kinase could be assayed in tumor biopsies in order to predict whether the fluoropyrimidines would be effective in individual patients.

Metabolic activation of benzo(a)pyrene by transformable and nontransformable C3H mouse fibroblasts in culture.

Incubation of 2.5 microM benzo(a)pyrene (BaP) with C3H/10T 1/2 or CVP3SC6 (CVP) mouse fibroblasts for 48 hr resulted in the metabolism of 36 to 42% of the BaP to organic soluble derivatives, which cochromatographed with 7,8-trans-dihydroxy-7,8-dihydrobenzo(a)pyrene, 9,10-trans-dihydroxy-9,10-dihydrobenzo(a)pyrene, 3-hydroxybenzo(a)pyrene, and 9-hydroxybenzo(a)pyrene, or to water-soluble derivatives. The formation of both organic and water-soluble metabolites during the 48-hr period increased proportionally with time, except in the case of BaP phenols, which increased initially but then remained the same or decreased. The distribution of organic soluble metabolites in the extracellular culture medium consisted primarily of BaP diols and was significantly different from that found inside the cells. The intracellular profile of organic soluble metabolites produced by both cell lines consisted predominantly of BaP phenolic derivatives and was qualitatively similar to the spectrum of metabolites produced by the incubation of BaP with C3H/10T 1/2 or CVP cell microsomes. The nature of the BaP water-soluble derivatives produced by the C3H/10T 1/2 and CVP cell lines was investigated by hydrolysis of culture medium with beta-glucuronidase and arylsulfatase. Although sulfation was not a major conjugation pathway for BaP in these cells, glucuronidation of BaP phenols was found to account for 30% of the total water-soluble derivatives. The similarity in the kinetics and qualitative nature of the metabolism of BaP by C3H/10T 1/2 and CVP cells indicates that both cell lines are equally capable of biosynthesizing the proximal carcinogen, 7,8-trans-dihydroxy-7,8-dihydrobenzo(a)pyrene. Analysis of the water-soluble metabolites produced by these cells suggests further that the nonresponsiveness of the CVP cells to BaP-induced transformation cannot be accounted for on the basis of an increased detoxication of 7,8-trans-dihydroxy-7,8-dihydrobenzo(a)pyrene.

Isolation of fluoropyrimidine-resistant murine leukemic cell lines by one-step mutation and selection.

The effectiveness of the clinically useful fluoropyrimidines in the treatment of human cancer is often limited by the development of resistance to the drugs by the tumor. In order to systematically study the mechanisms of resistance to 5-fluorouracil and its nucleoside derivatives, several cell lines resistant to these drugs have been derived from murine leukemia cells by a one-step mutation and selection procedure. Logarithmically growing suspension cultures of L1210 and P388 cells were treated with ethyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, or ICR-191 at concentrations which result in 20 to 30% cell survival. After a 10-day expression time, mutagenized cells were plated into soft-agarose medium that contained 10(-5) M 5-fluorouracil, 10(-5) M 5-fluoro-2'-deoxyuridine, or 10(-6) M 5-fluorouridine. Twenty stable clones were isolated and found to be 5- to 28-fold resistant to growth inhibition by 5-fluorouracil, 4,000- to 25,000-fold resistant to 5-fluoro-2'-deoxyuridine, or 8- to 220-fold resistant to 5-fluorouridine. The clones retain their drug-resistant phenotype after repeated passaging in the absence of selection. Since the biochemical changes responsible for resistance to one drug can render the cells collaterally sensitive to other drugs, the growth-inhibitory effects of antimetabolites that inhibit other steps in pyrimidine metabolism were examined in the wild-type cells and in the fluoropyrimidine-resistant sublines. Although cross-resistance to 5-azacytidine was found in L1210 cells selected for resistance to 5-fluorouridine, none of the cell lines tested demonstrated collateral sensitivity to methotrexate, 1-beta-D-arabinofuranosylcytosine, 5-azacytidine, or N-(phosphonacetyl)-L-aspartate.

Effect of 12-O-tetradecanoylphorbol-13-acetate on the morphology and growth of C3H/10T1/2 mouse embryo cells.

The effects of the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on the morphology and growth properties of C3H/10T1/2 clone 8 cells were examined. The morphology of these cells was changed within 30 min following treatment with 0.1 microgram of TPA per ml; they became smaller and refractile with long beady processes. Such changes were observed in both logarithmic and confluent cultures and lasted about 72 hr. Subsequent treatments were much less effective in inducing these changes. Scanning electron microscopy showed cell retraction and rounding as the most significant immediate effects of TPA treatment; many cells remained partially rounded 48 hr afterwards. Long-term surface modifications ascribable to TPA treatment were not detected. TPA had only minor effects on the growth of cultured C3H/10T1/2 cells in the presence of 10% fetal calf serum. Slight increases in plating efficiencies and saturation densities were generally observed in the presence of TPA but not with the related non-tumor-promoting compound phorbol. The cells grew slowly in 1% fetal calf serum and demonstrated serum batch-dependent alterations in their growth properties when exposed to TPA. Under conditions that produce doubling times of 70 hr or greater, TPA, but not phorbol, reduced the doubling time to about 50 hr. Saturation densities were also increased by TPA in 1% fetal calf serum. The effects of TPA on the growth of an oncogenically transformed variant of C3H/10T1/2 were quite different. While minimal effects of TPA were observed quite different. While minimal effects of TPA were observed when transformed cells were treated in the presence of 10% total calf serum, TPA treatment in 1% fetal calf serum significantly inhibited cell growth.

Specificity of pyrimidine nucleoside phosphorylases and the phosphorolysis of 5-fluoro-2'-deoxyuridine.

Isoelectric focusing and studies with 1-(2'-deoxy-beta-D-glucopyranosyl)thymine (GPT), a specific inhibitor of uridine phosphorylase activity, were used to determine the substrate specificities of mammalian pyrimidine nucleoside phosphorylases and their cleavage of 5-fluoro-2'-deoxyuridine (FdUrd). Isoelectric focusing profiles for the cytosol fractions from Ehrlich ascites cells and from Novikoff hepatoma cells each consisted essentially of one peak of nucleoside phosphorylase activity [isoelectric points (pl) 5.4 and 5.8, respectively] that cleaved both uridine and thymidine (dThd), as well as FdUrd. By contrast, cytosol fractions from HeLa (S3) cells, mouse liver, and normal human leukocytes each exhibited a major peak of activity (pl 4.6, 6.5, and 4.9, respectively) that cleaved only dThd and FdUrd, while mouse liver exhibited a second peak (pl 5.2) that cleaved primarily uridine. To distinguish clearly between (a) uridine phosphorylases that cleave primarily uridine and that are inhibited by GPT and (b)dThd phosphorylases that cleave only deoxynucleosides and that are not inhibited by GPT, we propose the term "uridine-deoxyuridine phosphorylases" to define those pyrimidine nucleoside phosphorylases that cleave both uridine and dThd and that are inhibited by GPT. On the basis of this definition and studies with GPT in nonfocused cytosol preparations, we conclude that FdUrd is cleaved to 5-fluorouracil by uridine-deoxyuridine phosphorylase activity in Ehrlich ascites cells and in Novikoff hepatoma cells, and by dThd phosphorylases in mouse liver, in normal human leukocytes, and in HeLa (S3) cells.

Mutation of Chinese Hamster V79 cells and transformation and mutation of mouse fibroblast C3H/10T1/2 clone 8 cells by aflatoxin B1 and four other furocoumarins isolated from two Nigerian medicinal plants.

Mutation by aflatoxin B1 (AFB1), imperatorin, marmesin, chalepin, and 8-methoxypsoralen (MOP), with and without black light (BL; long-wavelength ultraviolet light) activation, was determined at the hypoxanthine-guanine phosphoribosyltransferase locus (8-azaguanine resistance) in Chinese hamster V79 cells and at the ouabain locus in mouse C3H/1OT1/2 cells. Transformation by these furocoumarins under the same activation conditions was also investigated in C3H/1OT1/2 cells. In V79 cells, AFB1 induced a 4-fold maximum mutation frequency over controls under BL activation at a concentration of 5 micrograms/ml; marmesin induced a 2-fold increased mutation frequency at 1.5 micrograms/ml; MOP induced a 19-fold increase at 10 micrograms/ml; chalepin induced a 3-fold increase at 5 micrograms/ml; and imperatorin induced a 20-fold increase at 10 micrograms/ml. Essentially no mutation was observed at the ouabain-resistant (Ouar) locus in C3H/1OT1/2 cells with any of these compounds. In the transformation assays, type II and type III foci were observed at a 1-microgram/ml addition of AFB1 with or without BL activation; while with MOP and imperatorin, these types of foci were observed only with BL activation. Marmesin, although relatively more cytotoxic than the other furocoumarins studied, with a 50% lethal dose of less than 0.5 micrograms/ml, was not as mutagenic or potentially carcinogenic as were AFB1, imperatorin, or MOP with BL activation. These furocoumarins are considered to be involved in the etiology of the high incidence of skin cancer in Nigeria. Our experiments reinforce that concept and suggest that exposure to these furocoumarins may constitute a real carcinogenic hazard.

Two-stage chemical oncogenesis in cultures of C3H/10T1/2 cells.

The initiation and promotion stages of chemical oncogenesis have been demonstrated in cultured C3H/10T1/2 mouse embryo cells. Treatment of the cells with a subeffective concentration of 3-methylcholanthrene, benzo(a)pyrene, or 7,12-dimethylbenz(a)anthracene, followed 4 days later by a nontransforming amount of tetradecanoylphorbol acetate (TPA), phorbol didecanoate, or 4-alpha-phorbol didecanoate, produced transformation. Phorbol was ineffective. TPA did not select for transformed cells. When TPA treatment preceded 3-methylcholanthrene, no enhancement of transformation was observed. When TPA was added immediately after hydrocarbon treatment, there was a significant inhibition of transformation. TPA did not exert promoting activity when the hydrocarbons were in high enough concentrations to produce appreciable transformation. The promoting action of TPA cannot be attributed only to a stimulation of cell division.

Influence of benzoflavone on aflatoxin B1-induced cytotoxicity, mutation, and transformation of C3H/10T1/2 cells.

Aflatoxin B1 (AFLB1), a metabolite of the fungus Aspergillus flavus, is hepatotoxic and hepatocarcinogenic in several animal species and is thought to play an etiological role in human liver cancer. C3H/10T1/2 clone 8 mouse embryo fibroblasts are killed, mutated, and morphologically transformed byAFLB1. 7,8-Benzoflavone, a known inhibitor of aryl hydrocarbon hydroxylase, inhibits this enzymatic activity in C3H/10T1/2 cells. Furthermore, benzoflavone inhibits the binding of AFLB1, to the DNA of C3H/10T1/2 cells. Benzoflavone also inhibits AFLB1-induced cytotoxicity and mutation of C3H/10T1/2 cells, as well as inhibiting the activation of AFLB1 into mutagenic metabolites capable of reverting the Ames Salmonella tester strain TA98. Interestingly, benzoflavone had no effect on the oncogenic transformation of these cells by AFLB1. Therefore, benzoflavone inhibits the DNA binding, cytotoxic, and mutagenic effects of AFLB1 but does not reduce the morphological transformation of C3H/10T1/2 cells by this mycotoxin.

Enhancement of X-ray transformation by 12-O-tetradecanoyl-phorbol-13-acetate in a cloned line of C3H mouse embryo cells.

A phorbol ester promoting agent, 12-O-tetradecanoyl-phorbol-13-acetate, enhances X-ray transformation in vitro in a two-stage fashion similar to that shown previously for ultraviolet radiation and chemical carcinogens. In studies with a mouse embryo-derived cell line (C3H/1OT 1/2 clone 8), there were clear interactive effects between X-radiation and 12-O-tetradecanoylphorbol-13-acetate. These were particularly marked when a minimally transforming X-ray dose (50 or 100 rads) was followed by 12-O-tetradecanoylphorbol-13-acetate treatment beginning either immediately after the radiation exposure or 48 to 96 hr later.

Comparison of adriamycin- and ouabain-induced cytotoxicity and inhibition of 86rubidium transport in wild-type and ouabain-resistant C3H/10T1/2 mouse fibroblasts.

Ouabain (OUA) inhibited 86Rb uptake (50% inhibitory concentration = 0.8 X 10(-4) M) over concentration ranges close to those at which it caused a reversible cytotoxicity (50% lethal dose = 2.5 X 10(-4) M) in growing wild-type C3H/10T1/2 cells. On the other hand, Adriamycin (ADM) inhibited 86Rb uptake (50% inhibitory concentration = 2 X 10(-3) M) but at concentrations 10(4)-fold higher than those causing irreversible cytotoxicity in growing wild-type cells (50% lethal dose = 3 X 10(-8) M). While OUA inhibited 86Rb uptake more in wild-type cells than in a OUA-resistant mutant, ADM inhibited 86Rb uptake to the same extent in confluent wild-type and OUA-resistant cells. Further, three OUA-resistant mutants were not cross-resistant to ADM- or daunomycin (DM)-induced cytotoxicity during log phase or to ADM-induced cytotoxicity at confluence. In addition, ADM, DM, or 5-iminodaunomycin did not displace the cardiac glycosides digoxin or digitoxin from their respective antibody complexes. The order of potency of anthracycline derivatives in inhibiting 86Rb uptake in confluent wild-type cells was the same as their order of inhibiting the growth of wild-type cells and in detaching confluent wild-type cells (DM > ADM > 5-iminodaunomycin) but did not correlate with their cardiotoxic potentials (ADM > DM > 5-iminodaunomycin). Therefore, in this model system, ADM cytotoxicity is mediated differently from OUA cytotoxicity. Further, we find no biological evidence consistent withADM binding to the OUA site on the cell surface (Na+-K+) adenosine triphosphatase and therefore no evidence in this model system that ADM cardiotoxicity could be a digitalis-type toxicity per se.

The isolation and characterization of polycyclic hydrocarbon-binding proteins from mouse liver and skin cytosols.

The major protein to which metabolites of methylcholanthrene are covalently bound has been purified from C3H mouse liver cytosol. Its properties are identical to the mouse skin h-protein, which may be primary arget of carcinogenic hydrocarbon metabolites during transformation to caner. It has a molecular wight of 44,000, consists of 2 subunits o- M.W. 20,000, has an isoelectric point (pI) of 8.05 to 8.6, and a sedimentation coefficient of 3.6 S. These physical properties are rather similar to those of ligandin, a hepatic protein that binds carcinogen metabolites, steroid anionic metabolites, bilirubin, and exogenous organic anions, but not to those of the rat liver azo dye carcinogen binding 'slow h-2-5S' protein. The h-protein and ligandin consistently give different pl values. Two minor basic proteins (molecular weights around 44,000 each), to whcih methylcholanthrene metabolites are convalently bound, have been separated from the h-protein by carboxymethyl-cellulose chromatography. Prelininary results indicate that these 2 minor proteins are related to ligandin. A protein to which methylcholanthrene is noncovalently bound was also identified in the acidic fraction of the mouse liver and skin sytosols and has been partially purified and characterized. It has a molecular weight of 60,000, a pl of 5.0, and a sedimentation coefficient of 4.5S.

Separation of glutathione S-transferase activities with epoxides from the mouse liver h-protein, a major polycyclic hydrocarbon-binding protein.

The C3H mouse liver h-protein is a cytoplasmic protein to which metabolites of carcinogenic polycyclic hydrocarbons bind covalently following i.p. injection. It has a number of physical properties similar to those of the glutathione S-transferases (EC 2.5.1.18). These properties include molecular weight (40,000), number of subunits (2), basic isoelectric point around 8.0, sedimentation coefficient (3.5S), and subcellular localization. In this communication, we have shown that glutathione S-transferase activities with 1,2-epoxy(3-p-nitrophenoxy)propane and benz[a]anthracene 5,6-oxide as substrates were separated from the h-protein on carboxymethylcellulose and isoelectrofocusing columns. The purification of the mouse h-protein as a [3H]-7,12-dimethylbenz[a]anthracene conjugate or as the free form is also described.

Effects of promoters on DNA synthesis in C3H/10T1/2 mouse fibroblasts.

The synthesis of DNA has been studied by autoradiography and by measurements of tritiated thymidine ([3H]TdR) incorporation in cultured C3H/10T1/2 mouse embryo fibroblasts. The cells were first treated with 3-methylcholanthrene as an initiator and then with promoters according to schedules that produce oncogenic transformation. The levels of 3-methylcholanthrene used did not affect the growth or [3H]TdR incorporation of the cells. Treatment during the log phase of growth with 12-O-tetradecanoyl-phorbol-13-acetate, phorbol didecanoate, or 4alpha-phorbol didecanoate produced a transient inhibition of [3H]TdR incorporation with the maximum at 12 hr after treatment. This resulted in a temporary delay of growth followed by recovery of the normal cell-doubling time. Phorbol did not produce these effects, suggesting that the inhibition of DNA synthesis is associated with the process of promotion. Although treatment of the cells with 12-O-tetradecanoyl-phorbol-13-acetate during stationary phase resulted in a 2- to 3-fold stimulation of [3H]TdR incorporation, multiple treatments spanning log and stationary phases were found to be necessary for promotion.

Induction of cytotoxicity, mutation, cytogenetic changes, and neoplastic transformation by benzo(a)pyrene and derivatives in C3H/10T1/2 clone 8 mouse fibroblasts.

Benzo(a)pyrene (BaP), a series of its metabolic derivatives, and benzo(e)pyrene, a very weakly carcinogenic isomer, were tested for their biological effects on transformable C3H/10T1/2 cells. These cells were used as targets in a series of assays designed to measure oncogenic transformation, mutation to ouabain resistance, cytotoxicity, and induction of cytogenetic changes, as evidenced by chromosomal aberrations and sister chromatid exchange. Of all the compounds tested, only the parent hydrocarbon, BaP, an (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene were found to be significantly active in producing transformation and cytogenetic alterations. BaP, (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene, and (+/-)-7 alpha, 8 beta-dihydroxy-9 beta, 10 beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, however, were all effective inducers of mutation in C3H/10T1/2 cells. (+/-)-7 alpha, 8 beta-Dihydroxy-9 beta, 10 beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene was the most potent agent in tests for cytotoxicity, Benzo(e)pyrene was inactive in all assays examined. Among the compounds tested, there was a correlation between the ability to induce cytogenetic changes and the ability to produce mutation and transformation. These results support the demonstrated role of (+/-)-trans-7,8-dihydroxy-7,8,-dihydrobenzo(a)pyrene as a proximal carcinogenic form of BaP and illustrate the utility of the C3H/10T1/2 cell system as an important tool for the detection of genotoxic damage by carcinogenic chemicals.