Role of valence state and solubility of chromium compounds on induction of cytotoxicity, mutagenesis, and anchorage independence in diploid human fibroblasts.

We previously showed that carcinogenic nickel, arsenic, and chromium(VI) compounds induced anchorage independence (AI) in diploid human fibroblastic cells (HFC) derived from foreskins (K. A. Biedermann and J. R. Landolph, Cancer Res., 47: 3815-3823, 1987). To elucidate the role of the valence state of chromium and solubility of chromium compounds in inducing AI, we studied the ability of soluble and insoluble hexavalent [chromium(VI)] and trivalent [chromium(III)] chromium compounds to induce mutation and AI in HFC. Chromium(VI) compounds (PbCrO4, CaCrO4, Na2CrO4, and CrO3) were 1000-fold more cytotoxic to HFC (average 50% lethal dose 0.5 microM) than chromium(III) compounds (CrCl3, Cr2O3, Cr2S3; average 50% lethal dose 500 microM). However, equal concentrations (0.1-10.0 microM) of soluble or insoluble chromium compounds in either +6 or +3 valence states induced similar increases in frequencies of AI in HFC (100-200/10(5]. Chromium(VI)- and chromium(III)-induced AI was a stable phenotype. All soluble chromium(VI) and insoluble chromium(III) compounds studied induced mutation to 6-thioguanine resistance at cytotoxic concentrations in HFC. Insoluble PbCr(VI)O4 and a soluble form of Cr(III)Cl3 were inactive in this assay. Mutation induction by chromium(III) compounds only occurred at cytotoxic concentrations (100-1000 microM) 1000-fold greater than those concentrations of chromium(VI) compounds (0.25-1 microM) which were cytotoxic, mutagenic, and induced AI. Soluble hexavalent Na2(51)CrO4 was taken up facilely by cells at concentrations that induced cytotoxicity, mutation, and AI. At concentrations of 0.25-1.0 microM, which induced AI but were not cytotoxic or mutagenic, or concentrations of 1-1000 microM, which were cytotoxic and mutagenic, soluble trivalent 51CrCl3 was not taken up by cells. An insoluble form of CrCl3 was not taken up intracellularly but did avidly associate with cells over the concentration range 1 to 100 microM which induced AI, cytotoxicity, and mutagenicity. Therefore, both chromium(VI) and chromium(III) compounds induced genotoxic effects in human fibroblasts. Cellular uptake, cytotoxicity, mutagenicity, and AI induced by soluble chromium(VI) compounds all occurred at the low concentrations of 0.2 to 1.0 microM; hence mutagenicity and induction of AI may be coupled for soluble chromium(VI) compounds but not for insoluble PbCrO4, which induced AI but was not mutagenic. Cytotoxicity and mutagenicity of insoluble chromium(III) occurred at concentrations of 10-100 microM, but induction of AI occurred at concentrations of 0.1-10 microM, indicating that inductions of mutagenicity and AI were not coupled for chromium(III) compounds.(ABSTRACT TRUNCATED AT 400 WORDS)

Mutagenicity of 5-azacytidine and related nucleosides in C3H/10T 1/2 clone 8 and V79 cells.

To determine whether 5-azacytidine (5-AzaCR)-induced transformation and/or differentiation of C3H/10T 1/2 clone B (10T 1/2) cells might have a mutational basis, we studied whether 5-AzaCR and structurally related nucleoside analogs could mutate 10T 1/2 and Chinese hamster V79 cells. In an assay for mutation to ouabain resistance in 10T 1/2 cells, which detects base substitution mutations but not frameshift mutations, 5-AzaCR and 6-azacytidine were not significantly mutagenic. 5-Aza-2'-deoxycytidine, 5-fluoro-2'-deoxycytidine, 5,6-dihydro-5-azacytidine, 5-fluoro-2'-deoxyuridine (FUdR), 5-bromo-2'-deoxyuridine (BUdR), and 1-beta-D-arabinofuranosylcytosine (ara-C) were only weakly mutagenic. In an assay for mutation to ouabain resistance in V79 cells, which also detects base substitution mutations but not frameshift mutations, 5-AzaCR, 5-aza-2'-deoxycytidine, FUdR, and ara-C were not detectably mutagenic, and BUdR was moderately mutagenic at highly cytotoxic concentrations. In an assay for mutation to 8-azaguanine resistance in V79 cells, which detects base substitution and frameshift mutations, 5-fluoro-2'-deoxycytidine and ara-C were weakly mutagenic, BUdR was moderately mutagenic at very cytotoxic concentrations, and 5-AzaCR, 5-aza-2'-deoxycytidine, FUdR, 6-azacytidine, and 5,6-dihydro-5-azacytidine were not significantly mutagenic. Therefore, 5-AzaCR and related cytosine analogs can be considered as negligibly mutagenic. This study does not provide support for a mutational basis for 5-AzaCR-induced differentiation in 10T 1/2 cells. Further, there was no correlation between the mutagenicity of the nucleosides 5-AzaCR, ara-C, BUdR, and FUdR studied here and their previously reported abilities to transform 10T 1/2 cells. The mutagenicities of 5-AzaCR and FUdR were so low that the biological significance of these effects is uncertain. Hence, it is not clear to what extent, if any, mutation contributes to the transformation caused by these two compounds, and other possible mechanisms of transformation should also be investigated.

Induction of anchorage independence in human diploid foreskin fibroblasts by carcinogenic metal salts.

We studied whether arsenic, nickel, and chromium compounds that are human carcinogens could induce transformation of cultured primary human diploid foreskin cells (HFC). All nickel compounds tested, PbCrO4, K2Cr2O7, CrO3, Na2HAsO4, NaAsO2, and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) caused significant (p = 0.001) dose-dependent inductions of anchorage-independent colonies in HFC. KH2AsO4, CaCl2, MnCl2, and Hg(CH3CO2)2 did not induce anchorage independence. Optimal expression times for induction of anchorage independence in HFC were observed as early as 11 days following treatment with MNNG, Ni3S2, Ni(C2H3O2), or NiSO4. Cell strains derived from anchorage-independent colonies showed 33 to 429-fold higher plating efficiencies in soft agar than parental populations, and the anchorage-independent phenotype was stable for eight passages, at which time cells senesced. Anchorage-independent cell strains derived from metal salt-treated cells were not resistant to the cytotoxicity of metal salts, indicating metal salts induced rather than selected for anchorage independence. Nine of 10 cell strains derived from metal compound- or MNNG-induced anchorage-independent colonies displayed the same or lower saturation densities than untreated human fibroblasts. None of these cell strains escaped senescence or showed definitive morphological transformation. MNNG (1 micrograms/ml) induced anchorage independence and mutation to ouabain resistance and 6-thioguanine resistance in HFC, but concentrations of Ni2S3 that induced anchorage independence did not induce mutation at either locus in HFC. These results demonstrate that carcinogenic metal salts induce stable anchorage independence early in human diploid foreskin fibroblasts, and this anchorage independence is independent of other in vitro markers of fibroblast transformation, such as focus formation or immortality. Metal salt induction of anchorage independence can now be used as an assay to study mechanisms of genotoxicity exerted by carcinogenic metal compounds in human cells.

Quantitative studies of the toxicity of benzo(a)pyrene to a mouse liver epithelial cell strain in culture.

The toxic effects of the carcinogen benzo(a)pyrene (BaP) were studied in a well-characterized epithelial cell strain NMuLi, derived from the livers of weanling Namru mice. These cells were extremely susceptible to the toxicity, 99% dying after a 6-day exposure to BaP, 5mug/ml. The toxic effects began between 11 and 24 hr postapplication of BaP to the cells and increased exponentially with the time of treatment. The toxicity was concentration dependent in cells treated for a specific time period. The survival curves were exponential and extrapolated to a survival fraction of 1.0. The toxic effects of BaP to logarithmically growing NMuLi were inhibited 40% by 7,8 benzoflavone, and the inhibition was concentration dependent. The 7,8-benzoflavone also inhibited aryl hydrocarbon hydroxylase (AHH) from NMuLi cell homogenates and microsomes by 99%. The concentration dependence for AHH inhibition by 7,8-benzoflavone paralleled its inhibition of cellular toxicity. The toxicity of BaP to these cells increased exponentially with the number of population doublings. Hence, the toxicity was 130 times greater in exponentially growing cells than in confluent cells. Levels of AHH, the enzyme that metabolizes BaP to its cytotoxic derivatives, were only 2.4 times higher in exponentially growing than in confluent cells, suggesting that cell division was responsible for the large differential toxicity. In addition, a toxic BaP metabolite was preferentially toxic to log-phase cells. The results indicate that the metabolism of BaP by AHH to produce cytotoxic metabolites, which may cause lesions that are expressed upon cell division, is responsible for the cytotoxicity of BaP to NMuLi.

Transformation of C3H/10T1/2 mouse embryo cells to focus formation and anchorage independence by insoluble lead chromate but not soluble calcium chromate: relationship to mutagenesis and internalization of lead chromate particles.

The genotoxicity of soluble and insoluble hexavalent chromium compounds was studied in mammalian cell assays which detect base substitution, deletion, addition, and frameshift mutations [6-thioguanine resistance in Chinese hamster ovary cells], primarily base substitution mutations [ouabain resistance in Chinese hamster ovary and C3H/10T1/2 Cl 8 mouse embryo fibroblasts (10T1/2)] and morphological transformation [focus formation] in 10T1/2 cells. Soluble hexavalent CaCrO4, administered in either acute (5-h) or subacute (24-h) dosing regimens, induced dose-dependent cytotoxicity and mutation to 6-thioguanine resistance in Chinese hamster ovary cells but no mutation to ouabain resistance or focus formation in transformation assays, although the acute treatment induced a high frequency of conversion of 10T1/2 cells to adipocytes. Cell lines established from cloned adipocytic cells were not morphologically transformed and did not grow in soft agarose. PbCrO4 did not induce mutation to either 6-thioguanine or ouabain resistance but did induce a reproducible dose-dependent, low frequency of focus formation in 10T1/2 cells. Cell lines established from PbCrO4-induced foci stably formed foci when coseeded with 10T1/2 cells, had 3-5-fold increased saturation densities relative to nontransformed 10T1/2 cells, and formed colonies in soft agarose, indicating their likelihood to be neoplastic. Long term exposure of 10T1/2 cells to either CaCrO4 or PbCl2, even at 85% cytotoxic concentrations, or pretreatment of cells with either CaCrO4 or PbCl2 followed by treatment with the alternate compound, did not induce morphological transformation. Treatment of cells with insoluble hexavalent PbCrO4 resulted in progressive and extensive vacuolization of cells in contact with the particles. Progressive cytoplasmic engulfment of PbCrO4 particles was observed using scanning electron microscopy, although PbCrO4 particles were not observed inside vacuoles. These results indicate that the soluble clastogens K2Cr2O7 and CaCrO4 were probably mutagenic by a non-base substitution mechanism but could not transform 10T1/2 cells. In contrast, PbCrO4 was not detectably mutagenic but induced transformation, which could not be explained solely by acute or chronic exposure to dissolution products of either lead or chromate alone. Since PbCrO4 particles were found to be intracytoplasmic in extensively vacuolated cells, we suggest that the unique physiochemical properties of PbCrO4 particles, leading to their internalization and the resultant associated cellular stress response, may be related to the transformation induced by this compound.

Study of the ability of phenacetin, acetaminophen, and aspirin to induce cytotoxicity, mutation, and morphological transformation in C3H/10T1/2 clone 8 mouse embryo cells.

Use of the analgesic compounds acetylsalicylic acid (aspirin), phenacetin, and acetaminophen has been correlated with increased risk of renal cancer in humans. Hence, we studied these compounds for ability to induce cytotoxicity, mutation to ouabain resistance, and morphological transformation in cultured C3H/10T1/2 clone 8 (10T1/2) mouse embryo cells. All three compounds were cytotoxic from 0.5-mg/ml to 2-mg/ml concentrations as evidenced by decreased plating efficiency. None of the compounds induced detectable base substitution mutations to ouabain resistance even at cytotoxic concentrations. Aspirin did not induce morphological transformation. Both phenacetin and acetaminophen induced low but concentration-dependent numbers of atypical, weak type II morphologically transformed foci; at equimolar concentrations, phenacetin was 1.1- to 3.0-fold more active in inducing these foci. Neither phenacetin nor acetaminophen was cotransforming with 3-methylcholanthrene, and neither compound promoted cell transformation when added to 3-methylcholanthrene-initiated 10T1/2 cells. The focus-inducing potency of both compounds was increased by addition of an Arochlor-induced hamster liver S9 fraction as an exogenous metabolizing system. However, seven putative metabolites of phenacetin and acetaminophen that were tested--N-hydroxyphenacetin, p-phenetidine, p-aminophenol, p-nitrosophenol, benzoquinone, acetamide, and N-acetyl-p-benzoquinoneimine--were inactive in transformation assays at the concentrations reducing plating efficiency of treated cells to 50% of the plating efficiency of nontreated (control) cells. Several acetaminophen- and phenacetin-induced foci were cloned, expanded into cell lines, and characterized. These cell lines stably formed type II foci when maintained at confluence for 2 to 4 wk in reconstruction experiments with nontransformed 10T1/2 cells; however, they did not exhibit significantly increased saturation density compared to 10T1/2 cells, and they did not grow in soft agarose. These results suggest that metabolic intermediates of high concentrations of phenacetin and acetaminophen induce a low frequency of nonneoplastic morphological transformation of 10T1/2 mouse embryo cells.

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.

Enhanced expression of c-myc and decreased expression of c-fos protooncogenes in chemically and radiation-transformed C3H/10T1/2 Cl 8 mouse embryo cell lines.

c-abl, c-fos, c-Ha-ras, c-myc, and c-mos were expressed whereas c-sis, c-fms, c-rel, c-src, and c-myb expression was not detectable in C3H/10T1/2 Cl 8 (10T1/2) cells and in eight chemically and radiation-transformed 10T1/2 cell lines. The expression of c-abl, c-fos, c-Ha-ras, and c-myc was growth-related in nontransformed 10T1/2 cells. c-abl and c-fos expression increased at confluence by 5- and 9-fold, respectively, compared to that in log phase cells. c-Ha-ras and c-myc transcripts were most abundant in log phase cells and decreased by 70 and 50%, respectively, in confluent cells. There were no significant growth-related changes in the expression of c-Ha-ras, c-myc, or c-abl in methylcholanthrene-transformed Cl 15 cells. The c-fos transcript was not detected in Cl 15 cell cultures. c-abl, c-fos, c-ras, and c-myc were expressed in whole C3H mouse embryo tissue, mouse liver, and 10T1/2 cells. Sizes of these protooncogene transcripts in 10T1/2 cells were the same as those in whole embryo tissue, except that 10T1/2 cells did not express the 8.2-kilobase abl transcript. At subconfluence, equivalent low levels of c-mos expression were observed in nontransformed and in the eight transformed 10T1/2 cell lines. The level of c-abl expression was similar in the nontransformed and in the eight transformed cell lines, but there was a new 8.2-kilobase transcript in the transformed MCA Cl 15 cell line. c-fos was expressed in 10T1/2 cells but was not detectable or greatly reduced in eight transformed cell lines. c-Ha-ras was expressed to a similar extent in eight transformed cell lines and in nontransformed 10T1/2 cells. In the UVC-4 transformed cell line, extra 3.3-kilobase Ha-ras and 7.5-kilobase Ki-ras transcripts were observed. c-myc was expressed at 4- to 7-fold higher levels in six transformed cell lines compared to 10T1/2 cells. There were no major rearrangements in or amplification of the c-myc gene in three transformed cells overexpressing this gene 5-fold. These studies show that enhanced expression of c-myc and decreased expression of c-fos correlate with the chemically and radiation transformed states of 10T1/2 cells. Changes in c-fos and c-myc oncogene expression may be casually linked to late stages of neoplastic transformation in these chemically and radiation transformed 10T1/2 cell lines.

Oncogenic transformation and mutation of C3H/10T 1/2 clone 8 mouse embryo fibroblasts by alkylating agents.

Two-hr treatments with N-methyl- and N-ethyl-N'-nitro-N-nitrosoguanidines and ethyl methanesulfonate induced ouabain-resistant mutants in C3H/10T 1/2 cells. The alkylnitronitroso-guanidines gave linear dose-response curves and were more potent mutagens than were ethyl methanesulfonate and methyl methanesulfonate. These differences in potency were largely due to differences in the half-lives of the alkylating agents in culture medium. Differences in mutation frequencies at equitoxic concentrations of the alkylating agents are considered to reflect differences in the chemical mechanisms of alkylation and mutagenesis by the compounds. However, the frequencies of mutations produced at equitoxic concentrations were not uniformly associated with the nucleophilic selectivities of the compounds as expressed by their published Swain-Scott substrate constants. Whether or not followed by repeated replating, the yield of oncogenically transformed foci of asynchronous cells after treatment with the alkylating agents was so low that we could not obtain dose-response curves, and the yield may not be significant. By contrast, in previous experiments with N-methyl-N'-nitro-N-nitrosoguanidines and polycyclic hydrocarbons in Syrian hamster embryo fibroblasts and with ultraviolet light and polycyclic hydrocarbons in C3H/10T 1/2 cells, transformation occurred to an equal or greater extent than mutation measured in the same cells.

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.

Increased expression of the glucose-regulated gene encoding the Mr 78,000 glucose-regulated protein in chemically and radiation-transformed C3H 10T1/2 mouse embryo cells.

Expression of the gene coding for the Mr 78,000 glucose-regulated protein (GRP78) was examined in nontransformed and chemically and radiation-transformed C3H 10T1/2 Cl 8 mouse embryo cells. When cells were grown in complete medium with 10% fetal bovine serum, GRP78 mRNA was increased 4- to 9-fold in 3-methylcholanthrene (MCA; Clones 15 and 16)-, bleomycin (Bleo 1)-, and ultraviolet light (UV-C3)-transformed cell lines compared to nontransformed 10T1/2 clone 8 cells (Cl 8) at similar cell number and growth phase. Increased steady-state levels of GRP78 protein in MCA Cl 15 compared to Cl 8 cells were confirmed by 2-dimensional gel electrophoresis. Under these conditions transformed MCA Cl 15 exhibited increased GRP78 RNA within 24 h after addition of fresh glucose-containing medium, whereas nontransformed Cl 8 cells did not increase expression of this gene even after 5 days of culture in conditioned medium. Incubation of Cl 8 and MCA Cl 15 in glucose-free medium for 16 h caused a 3- and 15-fold induction of GRP78 RNA, respectively. In addition, chemically transformed cells were highly sensitive to glucose deprivation and responded by rounding up and detaching from the substratum. Cl 8 cells exhibited no such sensitivity to glucose deprivation. These results extend earlier reports on virally transformed cells to include chemically and radiation-transformed cells and expand earlier reports to include mRNA expression and 2-dimensional gel electrophoresis of GRP78 protein.

Enhanced expression and state of the c-myc oncogene in chemically and X-ray-transformed C3H/10T1/2 Cl 8 mouse embryo fibroblasts.

We examined expression of the c-myc oncogene in nontransformed, in three chemically transformed, and in two X-ray-transformed C3H/10T1/2 Cl 8 cell lines. In nontransformed C3H/10T1/2 cells, c-myc was expressed when cells were logarithmically growing, and expression decreased as cells reached confluence. In a methylcholanthrene-transformed cell line, MCA Si 24, c-myc expression was similar to that observed in nontransformed cells, while in two chemically transformed cell lines, Bleo Cl 2 and DMBA Cl 2, and in two radiation-transformed cell lines, F17 and F29, steady-state levels of the c-myc transcript were 5-7-fold greater than observed in nontransformed C3H/10T1/2 cells. All cell lines, both transformed and nontransformed, produced a 2.3-kilobase c-myc transcript. There was no detectable amplification or rearrangement of c-myc DNA sequences in any of the cell lines examined as determined by DNA dot blot and restriction endonuclease-Southern blotting analyses. In addition, the c-myc gene in nontransformed and transformed cell lines showed similar methylation patterns as determined by HpaII/MpsI digestion analysis, except that F19 and F29 cells lost a 0.95-kilobase HpaII band, suggesting extra region-specific methylation in these two cell lines compared to C3H/10T1/2 cells. Therefore, increased c-myc expression in the four transformed lines did not generally correlate with changes in DNA methylation in the vicinity of the c-myc gene. Our results suggest that expression of the c-myc gene is growth related and that elevated steady-state levels of c-myc RNA in certain chemically and X-ray transformed C3H/10T1/2 cell lines, such as Bleo Cl 2, DMBA Cl 2, F17, and F29, are correlated with and may participate in conversion to or maintenance of cells in the transformed state.

DT-diaphorase activity and the cytotoxicity of quinones in C3H/10T1/2 mouse embryo cells.

A permanent mouse fibroblast cell line derived from C3H mouse embryos, C3H/10T1/2 C18, was used to study the cytotoxicity of some model quinones under conditions in which DT-diaphorase (EC 1.6.99.2) activity was induced or inhibited. Sudan III [1-[[4-(phenylazo)phenyl]azo]-2-naphthalenol] and 3-methylcholanthrene (MCA), but not butylated hydroxyanisole (BHA), induced DT-diaphorase in a concentration-dependent manner. Induction of DT-diaphorase activity was dependent upon new RNA and protein synthesis, as shown by experiments employing actinomycin D and cycloheximide respectively. Induction of DT-diaphorase by Sudan III or MCA was associated with protection against the cytotoxicity of quinones as measured by a colony survival assay. When control and induced cells were also exposed to dicoumarol, a specific and potent inhibitor of DT-diaphorase, the cytotoxicity of the quinones in both control and induced cells was enhanced markedly. The results support the hypothesis that DT-diaphorase competes with one-electron quinone-reducing enzymes (such as cytochrome P-450 reductase) which generate auto-oxidizable semiquinones and forms more stable hydroquinones as an initial step in the detoxification of quinones in 10T1/2 cells.

Molecular mechanisms of transformation of C3H/10T1/2 C1 8 mouse embryo cells and diploid human fibroblasts by carcinogenic metal compounds.

Carcinogenic arsenic, nickel, and chromium compounds induced morphological and neoplastic transformation but no mutation to ouabain resistance in 10T1/2 mouse embryo cells; lead chromate also did not induce mutation to ouabain or 6-thioguanine resistance in Chinese hamster ovary cells. The mechanism of metal-induced morphological transformation was likely not due to the specific base substitution mutations measured in ouabain resistance mutation assays, and for lead chromate, likely not due to this type of base substitution mutation or to frameshift mutations. Preliminary data indicate increases in steady-state levels of c-myc RNA in arsenic-, nickel-, and chromium-transformed cell lines. We also showed that carcinogenic nickel, chromium, and arsenic compounds and N-methyl-N-nitro-N-nitrosoguanidine (MNNG) induced stable anchorage independence (Al) in diploid human fibroblasts (DHF) but no focus formation or immortality. Nickel subsulfide and lead chromate induced Al but not mutation to 6-thioguanine resistance. The mechanism of induction of Al by metal salts in DHF was likely not by the type of base substitution or frameshift mutations measured in these assays. MNNG induced Al, mutation to 6-thioguanine resistance, and mutation to ouabain resistance, and might induce Al by base substitution or frameshift mutations. Dexamethasone, aspirin, and salicylic acid inhibited nickel subsulfide, MNNG, and 12-O-tetrade-canoylphorbol-13-acetate (TPA)-induced Al in DHF, suggesting that arachidonic acid metabolism and oxygen radical generation play a role in induction of Al. We propose that nickel compounds stimulate arachidonic acid metabolism, consequent oxygen radical generation, and oxygen radical attack upon DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphological and neoplastic transformation of C3H/10T1/2 Cl 8 mouse embryo cells by insoluble carcinogenic nickel compounds.

We studied induction of cytotoxicity and morphological transformation in C3H/10T1/2 Cl 8 (10T1/2) mouse embryo fibroblasts by soluble and insoluble carcinogenic nickel compounds. Soluble nickel sulfate and nickel chloride caused dose-dependent cytotoxicity in the concentration range from 0.5 microM to 100 microM after 48 hr treatments, but neither compound induced morphological transformation even at concentrations causing up to 94% cytotoxicity. Insoluble nickel subsulfide, nickel monosulfide, and nickel oxide caused dose-dependent cytotoxicity and a low, dose-dependent frequency of morphological transformation in the concentration ranges from 0.5 to 40 microM, 5 to 50 microM, and 50 to 400 microM, respectively, after 48 hr exposure of cells to these compounds. Foci were predominantly of type II morphology; type III foci were rare. The insoluble nickel compounds studied caused no induction of base substitution mutations to ouabain resistance in 10T1/2 cells over concentration ranges that induced morphological transformation. Nickel subsulfide and nickel monosulfide were taken into cells by phagocytosis, since particles were visible in intracytoplasmic vacuoles. Numerous nickel oxide particles were found associated with cells, but true phagocytic uptake was difficult to detect since no vacuoles were observed. We twice cloned type II and type III foci induced by insoluble nickel compounds, established independent cell lines, and characterized their phenotypes. Four of seven of these cell lines had three- to fourfold increased saturation densities compared to 10T1/2 cells, formed type II and type III foci in reconstruction assays, and grew in soft agarose. One cell line induced by nickel oxide formed tumors in nude mice. These data indicate that insoluble carcinogenic nickel compounds induced type II foci in 10T1/2 cells, some of which were tumorigenic, and that the 10T1/2 cell system is suitable for studying mechanisms of nickel compound-induced morphological transformation in mammalian cells.

Biochemical basis for the acquisition of resistance to benzo[a]pyrene in clones of mouse liver cells in culture.

In a Namru mouse liver epithelial cell strain designated NMuLi, aryl hydrocarbon hydroxylase (AHH) activity peaked at 12 h post-induction with 1 microgram/ml of benzo(a)pyrene (BaP) in both confluent and growing cells. Maximal levels of AHH activity were reached on day two post-plating. This induced activity was inhibited in vitro 78% by gassing the incubation mixture with carbon monoxide for 15 s, and inhibited 93% by addition of 40 microgram/ml of 7,8 benzoflavone(BF). Induced AHH levels were higher in epithelial clones that were sensitive to the toxicity of BaP than in resistant clones. The survival fraction of clones from NMuLi and of subclones derived from a sensitive clone of NMuLi after BaP treatment was a negative exponential function of the maximal induced AHH activity in the clones. One of the clones, NMuLi cl 8, was extremely susceptible to the toxic effects of BaP, the +/-(trans)-7alpha, 8beta-dihydroxy-7,8-dihydro-BaP(7,8-diol), and the (+/-)-7alpha, 8beta-dihydroxy-9beta, 10beta-epoxy-7,8,9,10-tetrahydro-BaP (diol-epoxide), known metabolites of BaP. The toxicity of BaP and the 7,8 diol to this clone was inhibited by BF, suggesting that these cells possessed an enzyme activity inhibitable by BF that could epoxidize BaP to the 7,8 oxide and then epoxidize the resultant 7,8 diol to the diol-epoxide. Another clone derived from NMuLi, clone 7, was relatively resistant to the toxic effects of BaP and the 7,8-diol, but still extremely susceptible to the toxic effects of the diol-epoxide. The slight toxicity to BaP in this clone was inhibited by BF, but the toxicity of the 7,8-diol to this clone was not inhibited by BF. A typical cytochrome P450 inhibitor, metyrapone, had no effect on the toxicity of BaP, the 7,8-diol, or the diol-epoxide to either clone 7 or clone 8. The results suggest that these liver cells possess two enzymes that play some role in polycyclic hydrocarbon-induced toxicity. Enzyme A, a BaP-inducible enzyme that is inhibitable by BF, efficiently metabolizes BaP to the 7,8-diol and the 7,8-diol to the diol-epoxide. It is responsible for most of the hydrocarbon toxicity. Enzyme B is not inhibitable by BF and metabolizes the 7,8-diol less efficiently to the diol-epoxide or efficiently to other, less toxic products.

Microcell-mediated transfer of carcinogen-induced ouabain resistance from C3H/10T1/2 Cl 8 mouse fibroblasts to human cells.

We previously developed a quantitative assay for measuring the induction of ouabain-resistant (Ouar) variants in transformable C3H/20T1/2 Cl 8 mouse fibroblasts following treatment of the cells with chemical carcinogens. To further define the nature of the Ouar phenotype, we conducted microcell-mediated chromosome transfer studies using Ouar cell lines induced by chemical carcinogens in C3H/10T1/2 Cl 8 cells as donors and 8-azaguanine-resistant (Azgr) derivatives of the human cell lines, D98/AH2 and HT 1080, as recipients. Microcells prepared from one spontaneous and two carcinogen-induced Ouar mouse cell lines were able to transfer resistance to 0.01 and 1 mM Oua to ouabain-sensitive D98 and HT 1080 cells. The frequency of microcell hybrid formation ranged from 10(-6) to 10(-5). Karyotypic analysis of the microcell hybrids indicated that the Ouar phenotype of C3H/10T1/2 Cl 8 derivatives mapped to mouse chromosome 3, the chromosome to which the wild-type murine Oua-1 allele had previously been assigned. These studies show that both spontaneous and chemically induced high level Ouar phenotypes of C3H/10T1/2 Cl 8 mouse fibroblasts can be transferred via microcell-mediated chromosome transfer, and provide strong genetic evidence that chemically induced Ouar phenotypes of C3H/10T1/2 Cl 8 cells arise from mutations at Oua-1. In addition, this study sufficiently standardizes microcell-mediated chromosome transfer in the C3H/10T1/2 Cl 8 cell line so that this technique can be used to investigate the nature of other phenotypic changes in these cells, such as the chemically transformed phenotype.

Molecular and cellular mechanisms of transformation of C3H/10T1/2 Cl 8 and diploid human fibroblasts by unique carcinogenic, nonmutagenic metal compounds. A review.

Work from our laboratory showed that carcinogenic metal salts of arsenic, nickel, and chromium induced morphological transformation of cultured C3H/10T1/2 Cl 8 (10T1/2) mouse embryo cells, and that many of the transformants grow in soft agarose and form tumors in nude mice. Concentrations of arsenic, nickel, and chromium compounds that induced morphological transformation did not induce mutation to ouabain resistance in 10T1/2 cells. This indicated that the mechanism of metal induced morphological transformation was likely not caused by induction of base substitution mutations, and in the case of lead chromate, likely not caused by frameshift or deletion mutations. In addition, we showed that carcinogenic arsenic, nickel, and chromium compounds, and MNNG, induced anchorage independence in diploid human fibroblasts. Anchorage-independent cell strains derived from anchorage-independent colonies were stable but did not form foci and eventually senesced, therefore, arsenic and nickel compounds and lead chromate induced stable anchorage independence as an isolated phenotype. Nickel compounds and lead chromate induced anchorage independence but not mutation to ouabain resistance or to 6-thioguanine resistance. Hence, the mechanism of induction of anchorage independence by these metal salts in human fibroblasts was likely not via induction of base substitution, frameshift, or deletion mutations that would be measured in these mutation assays.(ABSTRACT TRUNCATED AT 250 WORDS)

Soluble vs insoluble hexavalent chromate. Relationship of mutation to in vitro transformation and particle uptake.

Soluble CaCrO4 and insoluble PbCrO4 were tested for induction of mutation to 6-thioguanine (base-substitution, deletion, addition, and frameshift mutations) or ouabain (base-substitution mutations) resistance in Chinese hamster ovary cells and morphological transformation in C3H/10(1/2) mouse embryo cells. CaCrO4 induced dose-dependent cytotoxicity and mutation to 6-thioguanine resistance, but did not induce mutation to ouabain resistance or morphological transformation. Highly cytotoxic amounts of CaCrO4 induced conversion of 10T1/2 cells to adipocytes, but cell lines derived from such cells were not transformed. PbCrO4 was not mutagenic in either mutation assay but induced a dose-dependent, low frequency of focus formation. Cell lines established from these foci had a 3-5-fold increased saturation density, grew in soft agarose, and were tumorigenic in nude mice. Chronic exposure to CaCrO4 or PbCl2 did not induce transformation, PbCl2 was inactive even at acutely cytotoxic concentrations, and sequential treatments with CaCrO4 and PbCl2 did not induce transformation. Light and scanning electron microscopy showed progressive cytoplasmic engulfment of PbCrO4 particles and extensive vacuolization of cells in contact with the particles. No particles were observed inside of vacuoles. We suggest that internalization of PbCrO4 and the associated cellular stress response may be related to PbCrO4-induced neoplastic transformation of 10T1/2 cells.