In vitro genotoxicity studies using complex hydrophobic mixtures: efficient delivery of a petroleum sample to cultured C3H/10T1/2 cells via lipid vesicle incorporation.

Petroleum fractions are a diverse group of extremely hydrophobic mixtures, some of which display strong carcinogenicity in animal skin painting experiments. Interpretation of in vitro genotoxicity experiments with these samples is complicated by inefficient delivery of these hydrophobic substances inside target cells. We therefore developed methods to assess and improve the efficiency of delivering a petroleum sample (Matrix, A.P.I. 81-17) to cultured C3H/10T1/2 cells for genotoxicity studies via lipid vesicle incorporation. Three radiolabeled compounds (14C-benzo(a)pyrene, 14C-decane, and 14C-naphthalene) of widely differing volatilities, broadly representative of the spectrum of compounds in petroleum samples, were separately added to Matrix. Lipid vesicles containing Matrix and radiolabeled compounds were prepared by the classical methods for preparing neutral, positive, and negatively charged multilamellar and unilamellar liposomes. Of these, the classical methods for preparing neutral unilamellar liposomes were the most successful for delivering radiolabeled compounds in Matrix to cells. Vesicles optimal for the delivery of tracers in Matrix were prepared with DSPC:cholesterol:lyso-PC (8.8:0.8:0.4, molar ratio) in a Matrix to lipid ratio of 31:69 (w/w). This new method of delivery resulted in proportional, dose-dependent, and reproducible uptake of all tracers. Further, cells treated with this preparation took up 2.5-fold more 14C-decane, 1.5-fold more 14C-BaP, and 18-fold more 14C-naphthalene added to Matrix than did cells treated with Matrix emulsified in tissue culture medium. In contrast, tracers were not taken up in a proportional or reproducible manner when emulsions were used, and in fact, uptake of 14C-naphthalene was consistently very small. Two petroleum fractions, C(2)029188 and C(3)029194, were 4- and 6-fold more cytotoxic, respectively, when delivered to C3H/10T1/2 cells by lipid vesicles than emulsions. The carcinogenic petroleum fraction C(5)0292202 induces type II transformed foci in C3H/10T1/2 cells when cells were treated with C(5)0292202 incorporated into lipid vesicles. The methods for lipid vesicle incorporation described here are effective in delivering hydrophobic petroleum fractions to cells and provide an alternative to the current inefficient and artifactual methods of emulsification currently used. With further validation and standardization, lipid vesicle incorporation of petroleum fractions and treatment of cells with these vesicles should be useful for studying the genotoxicity of complex hydrophobic mixtures in cell culture systems.

Assay and time course of 5-fluorouracil incorporation into RNA of L1210/0 ascites cells in vivo.

A method for determination of levels of incorporation of nonradiolabeled 5-fluorouracil (FUra) into RNA (F-RNA) in tissue samples is shown to be applicable to tissues in vivo. BDF1 mice bearing L1210 ascites cells were injected intraperitoneally with [14C]FUra, 100 mg/kg. The time course of F-RNA levels in L1210 cells was determined by following the radiolabeled drug, and by NaB3H4 labeling of isolated and derivatized nucleoside. RNA ribonucleotides were obtained by KOH hydrolysis of perchloric acid precipitates of cell sonicates. FUMP nucleotides were separated from remaining nucleotides by DEAE-cellulose chromatography. FUMP fractions were treated with alkaline phosphatase, and FUrd was separated from non-FUrd nucleoside contaminants by additional DEAE-cellulose chromatography. FUrd was quantitated by periodate oxidation of ribose and NaB3H4 reduction of the resulting nucleoside dialdehydes. Isolation of tritiated FUrd-trialcohol from remaining tissue contaminants and background radioactivity was done by silica gel thin layer chromatography. Comparison of results obtained by isolation of [14C]FUrd with results of NaB3H4 labeling of the same samples showed parallel results with comparable biological standard deviations, although the tritium method consistently gave slightly lower values. The peak level of F-RNA at 3 hr was 1 base substitution per 174 normal nucleotides. The level of F-RNA after 3 hr declined slowly, so that at 96 hr there still remained 1 FUra base per 597 normal nucleotides. Serial determinations of RNA content showed marked decreases, on the basis of either DNA or protein level, that continued up to 96 hr after FUra administration. These biochemical effects are among the most prolonged reported for FUra, suggesting the possibility that F-RNA represents a storage compartment for release of toxic metabolites and emphasize the need for additional study of RNA effects at long time points. Our method for assay of F-RNA appears to be suitable for study of biopsy specimens of tumors and normal tissues following nonradiolabeled-FUra administration.

Oncogenic transformation of C3H/10T1/2 Cl 8 mouse embryo fibroblasts by inhibitors of nucleotide metabolism.

Morphological or oncogenic transformation of mouse embryo, C3H/10T1/2 Cl 8 fibroblasts was induced by methotrexate, 5-fluorouracil and 5-fluorodeoxyuridine. It is known that these compounds cause inhibition of thymidylate synthetase and, hence, depletion of deoxythymidine triphosphate (dTTP) and an increased ratio of deoxycytidine triphosphate (dCTP) to dTTP in the deoxyribonucleotide pools that are used for DNA synthesis in mammalian cells. This ratio is, in effect, increased by treating mammalian cells with arabinosyl cytosine and 5-azacytidine, which are converted into analogs of dCTP in mammalian cells and also induce oncogenic transformation of C3H/10T1/2 cells. By contrast, trifluorothymidine, 5-bromodeoxyuridine and 5-iododeoxyuridine, which are analogs of thymidine that in effect reduce the dCTP:dTTP ratio, did not induce oncogenic transformation. Moreover, thymidine was selectively lethal to tumorigenic C3H/10T1/2 cells and inhibited oncogenic transformation in cells treated with 5-fluorodeoxyuridine. These observations suggest that treatments that effectively increase the dCTP:dTTP ratio in mammalian cells facilitate oncogenic transformation of C3H/10T1/2 cells, whereas treatments that have the effect of decreasing this ratio inhibit transformation. However, dCyd did not induce oncogenic transformation of C3H/10T1/2 cells, although it has been shown to increase the dCTP:dTTP ratio in mammalian cells. Thus, increasing this ratio may not be sufficient to cause the transformation.

S-9 metabolic activation enhances aflatoxin-mediated transformation of C3H/10T1/2 cells.

The use of a rat liver S-9 metabolic activation system to enhance aflatoxin B1-mediated transformation of C3H/10T1/2 cells was studied. Under conditions of metabolic activation, the cytotoxicity of Aflatoxin B1 (AFB) was increased approximately 10-fold over that observed in the absence of activation. Similarly, activation increased the transformation of these cells treated with 1 microgram/ml AFB1 greater than 10-fold when cells were treated in the absence of activation with 1 microgram/ml AFB1 for 3 hr, and four-to-fivefold over the transformation observed when cells were treated for 48 hr with 1 microgram/ml AFB1. This same activation procedure also induced the transformation of these cells treated with 10 and 20 micrograms/ml cyclophosphamide in the absence of activation. The use of S-9 metabolic activation greatly increased the sensitivity of AFB1-mediated transformation of C3H/10T1/2 cells, and should expand the range of chemical carcinogens, requiring metabolic activation, in particular mycotoxins related to AFB1, that can be effectively detected and studied in the C3H/10T1/2 cell transformation assay.

Morphological correlates of transformation in cultured C3H/10T1/2 mouse embryo cells.

In efforts to determine common and consistent morphological parameters of transformation in C3H/10T1/2 cells, 15 cell lines transformed by different carcinogens were examined with the scanning electron microscope (SEM) and compared to non-transformed cells. Cell lines were studied at different passages, at different cell densities, and after growth as anchorage dependent or independent cultures. The transformed cell lines could be distinguished in the SEM from non-transformed cultures by the expression of one or more of the following morphological characteristics: formation of mini- or macro-foci (random piling of cells on top of each other), pleiomorphism in cell size and shape, and cell surface complexity. The extent to which these characteristics were expressed varied widely in the different transformed cell lines. Light microscopic scoring for different types of foci also revealed broad variability among the different transformed lines. At the SEM level, cell lines could not be characterized as transformed on an individual cell basis. However, all transformed cell lines could be definitively characterized as transformed on a population basis due to the presence of mini-foci. The various transformed cell lines were classified semi-quantitatively into categories based on the extent of expression of the different morphological characteristics. There was broad correspondence between the morphological classification and the relative plating efficiencies of the cell lines in soft agarose.

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.

Defective binding of 3-methylcholanthrene to the Ah receptor within C3H/1OT1/2 clone 8 mouse fibroblasts in culture.

C3H/1OT1/2 clone 8 mouse fibroblasts (C3H/1OT1/2 cells) exhibit induction of aryl hydrocarbon hydroxylase (cytochrome P1-450) when exposed in culture to benzo(a)pyrene, benz(a)anthracene or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), but do not display the induction response when treated with 3-methylcholanthrene (MCA), the classical inducer of cytochrome P1-450. Induction of cytochrome P1-450 is regulated by the Ah receptor which initially binds inducing chemicals in the cytoplasm, after which the inducer x receptor complex translocates into the nucleus. Cytosolic and nuclear forms of the Ah receptor can be detected in C3H/1OT1/2 cells using [3H]TCDD as the radioligand in culture, but specific Ah receptor binding is not detectable within C3H/1OT1/2 cells incubated with [3H]MCA. In contrast, in Hepa-1c1 cells, which exhibit cytochrome P1-450 induction when treated with MCA, cytosolic and nuclear Ah receptor can be detected by incubation of the cells either with [3H]MCA or with [3H]TCDD. Nonradioactive MCA is able to compete with [3H]TCDD for Ah receptor sites in C3H/1OT1/2 cells, but the relative potency of MCA as a competitor is lower within C3H/1OT1/2 cells than in C3H/1OT1/2 cytosol during extracellular incubation. Specific binding of [3H]MCA to Ah receptor can be detected by incubation of [3H]MCA with C3H/1OT1/2 cytosol outside the cell. The selective loss of response to MCA as a cytochrome P1-450 inducer (while retaining response to other inducers) appears to be due to defective interaction of MCA with the Ah receptor within the intracellular environment. The specific molecular alteration which makes the MCA x receptor complex ineffective within C3H/1OT1/2 cells is unknown. Some fibroblast lines other than C3H/1OT1/2 also selectively fail to respond to MCA; thus, this variation in Ah receptor function may not be due to a mutational change in the Ah regulatory gene which codes for the Ah receptor.

Cell transformation by chemical agents--a review and analysis of the literature. A report of the U.S. Environmental Protection Agency Gene-Tox Program.

The literature on cell transformation by chemical carcinogens has been critically reviewed. This subject is highly relevant to carcinogenesis in vivo, because the phenotypic changes that are collectively referred to as cell transformation usually involve the acquisition of tumorigenicity on inoculation into suitable rodent hosts. The systems chosen for review fall into 3 categories: cell strains (cells with a limited lifespan); cell lines (cells with an unlimited lifespan); and oncogenic viral-chemical interactions involving cells (Fischer rat embryo cells expressing an endogenous retrovirus, mouse embryo cells expressing the AKR leukemia virus, chemical enhancement of a simian adenovirus, SA7 transformation of Syrian hamster or rat embryo cells). Of the entire literature reviewed, 117 papers have been accepted for data abstraction by pre-defined criteria; these include 41 references to cell strains, 40 in cell lines, and 38 in viral-chemical interactions including cells. Because different systems have been reviewed, it would be meaningless to group all the compounds. The overall summary of the systems is as follows (many compounds have been tested in more than one system and, hence, are duplicated in these totals). (Chart: see text) In general, there is a reasonably good correlation between the results of the cell transformation systems and in vivo carcinogenesis. However, the many deficiencies of the EPA Merged Carcinogen List preclude definitive comparisons. Moreover, a number of 'false negatives' were obtained in systems that did not employ external metabolic activation. Further validation of all systems is required, but it seems very probable that several cell transformation systems will become valuable in assaying (with reasonable time and cost) the carcinogenic potential of environmental chemicals.

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.

Induction of ouabain-resistant mutants by chemical carcinogens in rat prostate epithelial cells.

We determined optimal conditions to quantitatively select ouabain-resistant (Ouar) mutants induced by chemical carcinogens in a rat prostate epithelial cell line (RPYK). These conditions included selection of Ouar mutants in 3 mM ouabain, an expression time of two days following a two-day treatment with carcinogens, and a reseeding density of 2 X 10(5) mutagenized cells per 100 mm dish to select mutants in ouabain. Ouar mutants induced by N-methyl-N'-nitro-N'-nitrosoguanidine (MNNG) remained stably Ouar when passaged in nonselective medium. Hemicyst formation, a characteristic of epithelial cells, was reversibly inhibited by ouabain in wild-type cells and was resistant to ouabain in Ouar cells. The direct-acting carcinogens MNNG and methylazoxymethanol-acetate (MAMA) and the environmentally widespread procarcinogens aflatoxin B1 and benzo(a)pyrene increased the frequency of Ouar mutants in RPYK cells. The procedures developed here now make it possible to detect some environmental carcinogens likely to cause prostate cancer by virtue of their ability to mutate cultured prostate epithelial RPYK cells. The sensitivity of the RPYK cell line to aflatoxin-induced cytotoxicity and mutagenesis also makes it a useful cell system in which to study enzymes governing the conversion of aflatoxin to genotoxic metabolites.

Therapeutic response of leukemic mice treated with fluorinated pyrimidines and inhibitors of deoxyuridylate synthesis.

The therapeutic efficacy of combinations of fluorinated pyrimidines and inhibitors of either ribonucleotide reductase or deoxycytidylate deaminase was evaluated for the treatment of the L1210 mouse leukemia in DBA/2 mice. Therapeutic synergisms were observed with optimal combinations of 5-fluor-2'-deoxyuridine and either hydroxyurea or guanazole. In addition, mice treated with guanazole combined with 5-fluorouracil survived longer than was observed with any dose of guanazole or with 5-fluorouracil alone. Tetrahydrodeoxyuridine, a potential prodrug of a transition-state analog of deoxycytidylate deaminase, did not have antitumor activity by itself nor did it improve the therapeutic response of leukemic mice to 5-fluoro-2'-deoxyuridine. These results are consistent with the hypothesis that deoxyuridylate accumulation was limited by inhibition of ribonucleotide reductase but not by administration of tetrahydrodeoxyuridine. It is suggested that combination chemotherapy with fluorinated pyrimidines and inhibitors of deoxyuridylate synthesis may improve the therapeutic response to these drugs.

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.

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.

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.

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.