Effects of glutathione transferase activity on benzo[a]pyrene 7,8-dihydrodiol metabolism and mutagenesis studied in a mammalian cell co-cultivation assay

This study deals with the role of glutathione transferase (GST)-mediatedconjugation of (+)-7ß,8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene(BPDE) in two mammalian cell lines, human mammary carcinomacells (MCF-7) and rat hepatoma cells (H4IIE), in relation tothen-capacity to metabolize (–)-trans-7,8-dihydroxy-7,8-dihydro-benzo[a]pyrene[(–)-BP-7,8-diol] to products that induce mutations inco-cultivated V79 cells. Both MCF-7 and H4IIE cells metabolized(–)-BP-7,8-diol to BPDE, but mutations in co-cultivatedV79 cells were only detected with MCF-7 cells. However, depletionof glutathione (GSH) in H4HE cells increased the mutagenidtyof (– )-BP-7,8-diol to a similar level to that found withMCF-7 cells. Measurements of GST activity using GSH and post-microsomalsupernatants from H4IIE, V79 and MCF-7 cells indicated a substantialdifference in conjugation capacity. Although preparations fromall three cell-lines showed GST activity with l-chloro-2,4-dlnitro-benzeneas the substrate, GST activity towards BPDE could only be detectedin supernatants from H4HE cells. This is consistent with thepresence of GST 7-7 an isoenzyme highly efficient hi catalysingBPDE-GSH conjugation. The difference in GSH-conjugation activitytowards BPDE was confirmed using intact H4IIE and MCF-7 cellsin culture. These results indicate that GSH-conjugation playsa pivotal role in mutagenesis induced by polycyclk aromatichydrocarbons (PAH). Accordingly, a deficiency in GSH-conjugationcapacity may be regarded as one important factor in defininga target cell population with an increased risk for tumour initiationfollowing exposure to PAH.     

Effects of tamoxifen and protein kinase C inhibitors on hyperthermic cell killing

The effects of the anti-estrogen, tamoxifen, and the protein kinase C inhibitor, 1-(5-isoquinolinylsulfonyl)2-methylpiperazine (H7), on hyperthermic cytotoxicity were studied. Three cell lines were used, a human colon cancer cell line (HT-29), a human mammary carcinoma cell line (MCF-7), and Chinese hamster V79 lung fibroblasts. With all three cell lines, tamoxifen at concentrations greater than 7.5 microM during heating or with a 3-hr exposure prior to heating significantly sensitized cells to heat. When cells were preincubated with 10-20 microM tamoxifen for 1-2 hr at 37 degrees C prior to heat treatment, washed free of extracellular tamoxifen, heated to generate thermoresistance, and examined 18 hr later for thermoresistance, tamoxifen treated HT-29 and MCF-7 cells were significantly more heat sensitive than thermotolerant controls not previously exposed to tamoxifen. In contrast, the degree of induced thermoresistance of V79 cells was unchanged after tamoxifen treatment. H7, but not its structural analogue and low affinity protein kinase C inhibitor, HA1004, also sensitized cells to heat. Neither H7 nor HA1004 had any apparent effect on the degree of heat-induced thermoresistance in the three cell lines tested.     

Expression of stably transfected murine glutathione S-transferase A3-3 protects against nucleic acid alkylation and cytotoxicity by aflatoxin B1 in hamster V79 cells expressing rat cytochrome P450-2B1.

Aflatoxin B1 (AFB1) is activated to AFB1-8,9-oxide (AFBO), a potent mutagenic and carcinogenic metabolite of AFB1. In the mouse, AFBO has been shown to be most efficiently detoxified by a specific isozyme of alpha-class glutathione S-transferase (GST), mGSTA3-3 (mGST-Yc). A hamster V79 cell line (V79MZr2B1, originally designated V79/SD1) previously transfected with the rat cytochrome P450-2B1 was stably transfected with an mGSTA3-3 expression vector, to study the chemopreventive role of GST in protecting against cytotoxicity or genotoxicity of AFBO. Immunoblotting demonstrated strong expression of an alpha-class GST in the mGSTA3-3 transfected cell line, whereas no detectable alpha-class GST protein was observed in the control (empty vector-transfected) cells. Previous studies with the V79MZr2B1 cell line indicated that it can activate AFB1 to a mutagenic metabolite via a transfected rat P450-2B1 stably expressed in the cells. We examined the ability of the expressed mGSTA3-3 to protect against AFB1-induced cytotoxicity or [3H]-covalent adduct formation in cellular nucleic acids. Exposure of empty vector-transfected control cells and mGSTA3-3 expressing cells to up to 600 nM [3H]-AFB1 indicated that a 70-80% reduction in DNA and RNA adducts was afforded by the expression of mGSTA3-3 in the transfected cells. Clonogenic survival assays showed that the mGSTA3-3 cell line was 4.6-fold resistant to AFB1 cytotoxicity as compared with the empty vector-transfected control SD1 cells, with IC50 values of 69 and 15 microM, respectively. The results of these studies demonstrate that mGSTA3-3 confers substantial protection against nucleic acid covalent modification and cytotoxicity by AFB1 in this transgenic cell model system.     

Species specificity in the metabolism of N-nitrosobis(2-oxopropyl)amine and N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine to mutagens by isolated rat and hamster hepatocytes

The metabolic activation of the carcinogens N-nitrosobis(2-oxopropyl)amine (BOP) and N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP) by Fischer rat and Syrian hamster hepatocytes was investigated in order to determine the existence of species differences in the induction of cell mutation. The conversion of BOP and HPOP into forms mutagenic to V79 cells was studied by using the hepatocyte-mediated mutagenicity assay. Mutations at the hypoxanthine:guanine phosphoribosyltransferase locus and the Na-K-ATPase locus were scored by the induction of 6-thioguanine resistance (TGr) or ouabain resistance (Ouar), respectively. Hepatocytes of both species were capable of converting BOP and HPOP to mutagens for V79 cells in a dose-dependent manner. Metabolism of BOP by rat hepatocytes resulted in higher mutation frequencies than that by hamster hepatocytes. At a BOP concentration of 240 microM, rat hepatocyte metabolism yielded 90.7 TGr mutants and 19.5 Ouar mutants per 10(5) V79 cells. At the same concentration, hamster hepatocyte metabolism of BOP yielded 54.1 TGr mutants and 13.0 Ouar mutants per 10(5) V79 cells. These results did not correlate with the known carcinogenic potency of BOP in the hamster as compared to the rat. Hamster hepatocytes carried out the catabolism of BOP to CO2 at faster rates than rat hepatocytes; therefore, the species difference in mutagenic activation was not due to a defect in BOP uptake or metabolism by hamster hepatocytes. In contrast, metabolism of HPOP by hamster hepatocytes resulted in significantly higher mutation frequencies than that by rat hepatocytes. At an HPOP concentration of 240 microM, hamster hepatocyte metabolism yielded 83.5 TGr mutants per 10(5) V79 cells; rat hepatocyte metabolism yielded only 19.8 TGr mutants per 10(5) V79 cells. This species difference in mutagenic activation correlated well with the known potency of HPOP as a carcinogen for the hamster as compared to the rat. Since hamster pancreatic cells and subcellular fractions are known to have very limited capacity to perform the metabolic activation of HPOP, the results of this study imply that liver metabolism plays an important role in the conversion of HPOP to an agent(s) which subsequently affects the hamster pancreas. The mutagenic potency of BOP versus HPOP was compared after metabolism by hepatocytes from both species. Following their metabolism by hamster hepatocytes, the two compounds were nearly equivalent in mutagenic potency. After metabolism by rat hepatocytes, BOP was significantly more potent mutagen than HPOP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of dietary selenium on biotransformation and excretion of mutagenic metabolites of N-nitrosodimethylamine and 1,1-dimethylhydrazine in the liver perfusion/cell culture system

The mutagenicity of N-nitrosodimethylamine (NDMA) and 1,1-dimethylhydrazine (UDMH) has been studied in an isolated liver perfusion/cell culture system. The liver donors, male Wistar rats, were either selenium (Se)-deficient or had a physiologically adequate Se status (Se-supplemented). Mutagenicity was measured in perfusate and bile with Chinese hamster V79 cells as the genetic target. Se deficiency increased the mutagenic effect of NDMA in the perfusate, whereas no mutagenicity was detected in the bile of either Se-deficient or Se-supplemented livers. No significant increase in the mutagenicity of UDMH was seen in the perfusate with Se deficiency, but the bile became mutagenic. Se deficiency thus increased the mutagenicity of both NDMA and UDMH: with NDMA, the effect was observed in the perfusate, and with UDMH, in the bile.     

Microsome-mediated mutagenesis in V79 Chinese hamster cells by various nitrosamines.

A microsome-mediated mutagenesis system has been established with the V79 Chinese hamster cell line. The cells, grown in monolayer, were treated with various nitrosamines in the presence of a postmitochondrial fraction ((S15) from rat liver and a reduced nicotinamide adenine dinucleotide-generating system for 1 hr, washed and incubated for 2 to 3 hr in fresh culture medium, and then plated for toxicity and mutagenicity assays. Mutation was determined by resistance to 20mug 8-azaguanine per ml. In this assay system, the S15 fraction and cofactors by themselves were not toxic to the cells; dose-related mutagenicity and cytotoxicity were induced by N-nitrosodimethylamine (DMN) only in the presence of the S15 fraction and cofactors. Pretreatment of rats with phenobarbitone led to an approximately 2-fold increase in the mutation rate over that with tissues from untreated rats with concentrations of DMN from 10 to 50 mM, while aminoacetonitrile pretreatment reduced the mutagenic effect. Methylcholanthrene pretreatment resulted in an increase in the mutation frequency with a higher concentration of DMN (50mM). Various other nitrosamines were also assayed in the presence or absence of the S15 fraction from phenobarbitone-pretreated rats and a reduced nicotinamide adenine dinucleotide phosphate-generating system. With the exception of N-nitrosomethylphenylamine, the carcinogenic nitrosamines (DMN, N-nitrosodiethylamine, N-nitrosodi-n-propylamine, N-nitrosodi-n-butylamine, N-nitrosodi-n-pentylamine, N-nitrosomethyl-n-propylamine, N-nitrosomorpholine, N-nitrosopyrrolidine, N-nitroso-N'-methylpiperazine, and N-nitrosomethylphenylamine) were mutagenic to the V79 Chinese hamster cells in the presence of the S15 fraction and cofactors. Neither N-nitrosodiphenylamine nor N-nitrosomethyl-tert-butylamine had a mutagenic effect. These findings show that chemical carcinogens can be tested for mutagenicity in cultured mammalian cells in the presence of a metabolic activation system. The results are discussed in relation to the carcinogenicity and mutagenicity of these compounds in other test systems.     

Cell-mediated cytotoxicity against human bladder cancer

Chemically non-reactive carcinogens, such as polycyclic hydrocarbons, have to be metabolized by cellular enzymes in order to exert their biological effects including mutagenicity. Chinese hamster V79 cells can be efficiently mutated from 8-azaguanine susceptibility to resistance by N-methyl-N-nitro-N-nitrosoguanidine. But these cells do not metabolize polycyclic hydrocarbons and were therefore not mutated by these compounds. A system of cell-mediated mutagenesis with carcinogenic hydrocarbons has been developed, by co-cultivating V79 cells with lethally irradiated rodent cells that can metabolize the carcinogens. The number of mutations was dependent on the number of metabolizing cells and the carcinogens were not mutagenic when V79 cells were co-cultivated with non-metabolizing cells. Inhibition of the hydrocarbon metabolizing enzymes by 7,8-benzoflavone, inhibited mutagenicity. Cell-mediated mutagenicity was obtained with the carcinogenic hydrocarbons 7,12-dimethylbenz (a)anthracene, benzo(a)pyrene and 3-methylcholanthrene and there was no mutagenicity with the non-carcinogenic hydrocarbon benz(a)anthracene. The degree of mutagenicity was related to the degree of carcinogenicity and the method detected mutagenicity with 0.1μg/ml. It is suggested that cell-mediated mutagenesis with human cells should provide a useful system to test for environmental chemicals hazardous to humans that have to be metabolically activated.     

Nitric oxide enhancement of melphalan-induced cytotoxicity.

The effects of the diatomic radical, nitric oxide (NO), on melphalan-induced cytotoxicity in Chinese hamster V79 and human MCF-7 breast cancer cells were studied using clonogenic assays. NO delivered by the NO-releasing agent (C2H5)2N[N(O)NO]- Na+ (DEA/NO; 1 mM) resulted in enhancement of melphalan-mediated toxicity in Chinese hamster V79 lung fibroblasts and human breast cancer (MCF-7) cells by 3.6- and 4.3-fold, respectively, at the IC50 level. Nitrite/nitrate and diethylamine, the ultimate end products of DEA/NO decomposition, had little effect on melphalan cytotoxicity, which suggests that NO was responsible for the sensitization. Whereas maximal sensitization of melphalan cytotoxicity by DEA/NO was observed for simultaneous exposure of DEA/NO and melphalan, cells pretreated with DEA/NO were sensitized to melphalan for several hours after NO exposure. Reversing the order of treatment also resulted in a time-dependent enhancement in melphalan cytotoxicity. To explore possible mechanisms of NO enhancement of melphalan cytotoxicity, the effects of DEA/NO on three factors that might influence melphalan toxicity were examined, namely NO-mediated cell cycle perturbations, intracellular glutathione (GSH) levels and melphalan uptake. NO pretreatment resulted in a delayed entry into S phase and a G2/M block for both V79 and MCF-7 cells; however, cell cycle redistribution for V79 cells occurred after the cells returned to a level of cell survival, consistent with treatment with melphalan alone. After 15 min exposure of V79 cells to DEA/NO (1 mM), GSH levels were reduced to 40% of control values; however, GSH levels recovered fully after 1 h and were elevated 2 h after DEA/NO incubation. In contrast, DEA/NO (1 mM) incubation did not reduce GSH levels significantly in MCF-7 cells (approximately 10%). Melphalan uptake was increased by 33% after DEA/NO exposure in V79 cells. From these results enhancement of melphalan cytotoxicity mediated by NO appears to be complex and may involve several pathways, including possibly alteration of the repair of melphalan-induced lesions. Our observations may give insights for improving tumour kill with melphalan using either exogenous or possibly endogenous sources of NO.     

Expression of human glutathione S-transferase P1 confers resistance to benzo[a]pyrene or benzo[a]pyrene-7,8-dihydrodiol mutagenesis, macromolecular alkylation and formation of stable N2-Gua-BPDE adducts in stably transfected V79MZ cells co-expressing hCYP1A1

Transgenic cell lines were constructed to study dynamic competition between activation versus detoxification of benzo[a]pyrene (B[a]P) and its metabolites. Transfected V79MZ cells expressing human cytochrome P4501A1 (hCYP1A1) alone, or expressing hCYP1A1 in combination with human glutathione S-transferase P1 (hGSTP1), were used to determine how effectively GST protects against macromolecular damage or mutagenicity of B[a]P or its enantiomeric dihydrodiol metabolites (+)-benzo[a]pyrene-7,8-dihydrodiol [(+)B[a]P-7,8-diol] and (-)-benzo[a]pyrene-7,8-dihydrodiol [(-)-B[a]P-7,8-diol]. Mutagenicity of B[a]P at the hprt locus was dose- and time-dependent in cells that expressed hCYP1A1. Mutagenicity was reduced in cells further modified to co-express hGSTP1. Dose-response and time-course studies indicated that mutagenicity was reduced up to 3-fold by hGSTP1 expression, compared with cells expressing hCYP1A1 alone. Mutagenicity induced by the B[a]P 7,8-dihydrodiols was also dose-dependent, and was reduced 2- to 5-fold by hGSTP1. Expression of hGSTP1 reduced B[a]P adducts in total cellular macromolecules by 3.8-fold, which correlated with the reduction in B[a]P mutagenicity and with reduction in the formation of the proximate metabolite B[a]P 7,8-dihydrodiols from B[a]P. However, measurement of total B[a]P metabolites bound to DNA isolated from cells incubated with [3H]-B[a]P revealed only 12, 33 and 24% reduction at 12, 24 and 48 h, respectively, by GSTP1 expression. Nevertheless, (32)P-post-labeling analysis demonstrated nearly total prevention of the known B[a]P-DNA adduct, N2-guanine-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), in cells co-expressing hGSTP1. This adduct, thought to be the most mutagenic of the stable B[a]P adducts, accounts for 15% or less of the total DNA adducts observed. These results indicate that the reduction in hCYP1A1-mediated B[a]P mutagenesis by hGSTP1 is probably largely due to prevention of the N2-guanine-BPDE adduct. However, the significant fraction (30-40%) of this mutagenesis and the majority of the total DNA binding that are not prevented together suggest formation by hCYP1A1 of a subset of mutagenic metabolites of B[a]P that are not effectively detoxified by hGSTP1.     

The mutagenicity of dibenz[a,j]acridine, some metabolites and other derivatives in bacteria and mammalian cells

Dibenz[a,j]acridine (DBAJAC) was studied because of its close structural relationship with a number of important carcinogenic polycyclic and azaaromatic hydrocarbons. It was of particular relevance to examine the mutagenicity of known or proposed 'bay-region' metabolites, which may be proximate or ultimate carcinogenic derivatives of DBAJAC. Trans-1,2-, 3,4- and 5,6-dihydrodiols, the 4- and 6-phenols, the 5,6-oxide and N-oxide derivatives, and anti- and syn-3,4-diol 1,2-epoxides of DBAJAC were examined for their mutagenicity in Salmonella typhimurium TA98 and TA100 and in V79 Chinese hamster lung cells. Of all the compounds studied which require metabolic activation, the 3,4-dihydrodiol was the most active in both TA100 and in V79 cells. The activity of the 3,4-dihydrodiol enantiomers was also tested in strain TA100 where no difference was observed from that of the racemic mixture. In V79 cells only the 3R,4R-dihydrodiol was active, the activity being about three times that of the racemic material. Salmonella strains TA98 and TA100 also differed in their sensitivity towards DBAJAC dihydrodiols, the 1,2-isomer being of greatest activity in TA98. The most mutagenic compounds in both mammalian and bacterial cells were the 'bay-region' diol epoxides of DBAJAC which did not require metabolic activation by S9 mix. The anti-DBAJAC 3,4-diol 1,2-epoxide was more mutagenic than the syn form in V79, TA98 and TA100 cells. Overall these results suggest that the in vivo biological activity of DBAJAC metabolites is likely to reflect previous findings with other similar polycyclic aromatic hydrocarbons.     

Cell-mediated mutagenesis and tumor-initiating activity of the ubiquitous polycyclic hydrocarbon, cyclopenta[c,d]pyrene

The ubiquitous polycyclic aromatic hydrocarbon (PAH), cyclopenta[c,d]pyrene(CPP), was tested to determine its mutagenicity for 6-thioguanineand ouabain resistance in Chinese hamster V79 cells and itstumor-initiating activity in the skin of the tumor susceptibleSencar mice. The potent carcinogen/mutagen, benzo[a]pyrene (BP),was included for comparison. Inasmuch as V79 cells do not metabolizePAHs, mutagenesis was tested both in the presence and in theabsence of X-irradiated golden hamster embryo fibroblasts capableof metabolizing PAH. Neither CPP nor BP showed mutagenicityfor V79 cells in the absence of the embryo cells. In the presenceof these cells (in the cell-mediated assay) both PAHs elicited,in a dose dependent manner, a cytotoxic and mutagenic responsein V79 cells. CPP was however less active than BP in inducingboth of these responses. At the optimal expression time andat the dose range of 0.1–1 µg/ml, CPP induced 2–86-thioguanine resistant mutants per 10⁵ colony forming cellscompared to 9–50 mutants induced by BP. Similarly, thesedoses of CPP induced 1–9 ouabain resistant mutants per10⁶ colony forming cells compared to 7–75 mutants inducedby BP. CPP was also active in initiating skin tumors in a dosedependent manner in the Sencar mice yielding tumors in 60% ofthe mice at 200 µg, the highest dose tested. BP was moreefficient in tumor initiation and yielded a similar responsewith 10 µg. These results indicate that CPP and BP elicit,in the cell mediated assay, a mutagenic response similar tothe activity of these PAH in the skin of Sencar mice.     

Mutagenicity to mammalian cells in culture by (+) and (-) trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrenes and the hydrolysis and reduction products of two stereoisomeric benzo(a)pyrene 7,8-diol-9,10-epoxides

The mutagenicity for mammalian cells of benzo(a)pyrene (BP) and 9 of its derivatives was tested by resistance to ouabain in Chinese hamster V78 cells. The derivatives included the (-) and (+) enantiomers of trans-7,8-diol; the racemic (+/-)trans-7,8-diol; two triols, (7/8,9)-triol and (7,9/8)-triol; and four tetrols, (7,10/8,9)-tetrol, (7/8,9,10)-tetrol, (7,9/8,10-triol and (7,9,10/8)-tetrol. Since V78 cells do not metabolize polycyclic hydrocarbons, mutagenesis was tested both in the presence and in the absence of Golden hamster cells capable of metabolizing polycyclic hydrocarbons. Neither BP nor any of its 9 tested derivatives showed mutagenicity for V78 cells in the absence of normal Golden hamster cells. However, in the presence of these cells, BP and the optically active and racemic trans-7,8-diols exhibited a mutagenic response that was dose-dependent. All other derivatives were inactive. The most active mutagenic hydrocarbon was (-) trans-7,8-diol, and activity decreased in the order (+/-)trans-7,8-diol, (+) trans-7,8-diol and BP.     

Antineoplastic drug sensitivity of human MCF-7 breast cancer cells stably transfected with a human alpha class glutathione S-transferase gene

Studies have suggested that the alpha class glutathione S-transferase (GST) may protect cells from the chemotherapeutic drugs chlorambucil and melphalan. In order to further define the function of human alpha class GST, a complementary DNA which encodes it was ligated into an expression vector under the direction of the human metallothionein-IIA promoter and stably transfected into human MCF-7 breast cancer cells in conjunction with the G418-selectable plasmid pSV2neo. Clonal cell lines were identified which expressed increased levels of GST enzyme activity (2.2- to 5.6-fold). The transfected cell lines also had increased peroxidase activity using cumene hydroperoxide as the substrate (1.9- to 3.8-fold) which is consistent with the intrinsic peroxidase activity of alpha class GSTs. Southern blot analysis indicated that genomic DNA from these cells contained a fragment indistinguishable from the transfected alpha class GST complementary DNA (850 base pairs); Northern blot analysis of total cellular RNA indicated that these cells contained appropriately sized alpha class GST RNA (980 nucleotides); and Western blot analysis indicated that, while MCF-7 cells contained no detectable alpha class GST protein, the transfected cells contained markedly elevated levels of alpha class GST but no detectable mu or pi class GST. These alpha class GST transfected cells had increased resistance to ethacrynic acid (2.1- to 3.0-fold). However, the transfected cells failed to show any increased resistance measured at the drug dosage which inhibited 50% of the colony formation to the chemotherapeutic drugs chlorambucil, melphalan, Adriamycin, or cisplatin under conditions of either continuous or 1-h drug exposure. Neither was there any change in sensitivity to the cytotoxins benzo(a)pyrene, benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide (anti), or 1-chloro-2,4-dinitrobenzene. These studies indicate that expression of this human alpha class GST by itself in MCF-7 human breast cancer cells does not confer resistance to the chemotherapeutic drugs tested under the conditions used in these studies.     

What role do glutathione S-transferases play in the cellular response to ionizing radiation?

The glutathione S-transferases (GST's) are cytosolic dimeric proteins that are composed of three family members, alpha, pi, and mu, and a fourth microsomal member. These four family members are primarily involved in cellular detoxification of xenobiotics and hydroperoxides. Recently, a strong correlation has been found between the overexpression of GST's and resistance to chemotherapeutic drugs. In comparison to chemotherapy, little is known about the role of GST's in the cellular response to ionizing radiation. To determine which GST's may be involved in this response, we have identified Chinese hamster ovary cell lines that possess different levels of alpha and pi GST isozyme activity. The survival of these cell lines to ionizing radiation was similar to that of wild-type Chinese hamster ovary-KI cells from which they were derived. Although differences in GST levels did not affect ionizing radiation sensitivity per se, we found that ionizing radiation decreased the amount of cytosolic pi GST without affecting alpha GST levels. Taken together, these data suggest that GST's are involved in the cellular response against oxidative stress generated by ionizing radiation.     

Mutagenicity of oxidative DNA damage in Chinese hamster V79 cells

We have investigated the mutagenicity of oxidative DNA damage induced in V79 Chinese hamster lung fibroblast, and measured 8- hydroxydeoxyguanosine (8OHdG) levels as an indicator of this damage. A hydroxyl radical generator, N,N'-bis(2-hydroxyperoxy-2-methoxyethyl)- 1,4,5,8-naphthalene-tetra -carboxylic-diimide (NP-III), induced 8OHdG in V79 upon irradiation with 366 nm ultraviolet light (UV) for 15 min. 8OHdG was determined by HPLC with electrochemical detection after anaerobic sample processing. The 8OHdG level in the cells treated without NP-III was 0.49 per 10(5) dG, whereas levels in the cells treated with 5, 10 or 20 microM NP-III and UV irradiation were 1.84, 4.06 or 6.95 per 10(5) dG, respectively. The 8OHdG induced by 20 microM NP-III with UV irradiation decreased rapidly, and the half-life of the induced 8OHdG was approximately 6 h. NP-III with UV irradiation also induced DNA strand breaks in all cells uniformly, as determined by single cell gel assay. Mutant frequencies at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in V79 were determined as the number of 6-thioguanine-resistant cells per 10(6) cells. Mutant frequency of the cells without NP-III was 8.0, and frequencies of the cells treated with 5, 10 or 20 microM NP-III and UV irradiation were 14.9, 20.6 or 24.7 respectively. Treatment with 20 microM NP-III and UV irradiation decreased the cell number, determined 3 days after the treatment, to 20.8%. These findings indicate that acutely induced oxidative DNA damage including mutagenic 8OHdG is only weakly mutagenic in V79.      

Effect of norharman on DNA strand breaks and mutation of Chinese hamster V79 cells by chemical carcinogens

The effect of norharman, which shows a comutagenic activityin the Salmonella mutation assay, was examined on the actionof mutagens towards Chinese hamster V79 cells. Norharman reducedthe DNA strand breaks by N-methyl-N'-nitro-N-nitrosoguanidine(MNNG), but enhanced the 4-hydroxy-aminoquinolnie 1-oxide (4HAQO)-inducedDNA strand breaks. Norharman also reduced the cytotoxicity andthe mutagenicity of MNNG but enhanced the cytotoxicity of 4HAQOto V79 cells. In the Salmonella mutation assay, norharman showedno effect on the mutagenic activity of MNNG but reduced themutagenic activity of 4HAQO. The effect of norharman on theaction of mutagens to V79 cells appeared dependent on mutagensand did not correlate with the effect observed in Salmonellamutation assay.     

Bovine bladder urothelial cell activation of carcinogens to metabolites mutagenic to Chinese hamster V79 cells and Salmonella typhimurium

The ability of bovine bladder urothelial cells to activate genotoxic chemicals to mutagens was examined by cocultivating bladder cells with Chinese hamster V79 cells or Salmonella typhimurium as mutable targets. Activation of test chemicals to mutagenic intermediates by urothelial cells was detected by induction of 6-thioguanine resistance in V79 cells or by induction of histidine revertants in Salmonella. In the bladder cell-mediated V79 cell mutagenesis system, a significant increase in mutation frequency was induced by exposure to 7,12-dimethylbenz(a)anthracene and dimethylnitrosamine. The aromatic amines 2-aminofluorene, 2-acetylaminofluorene, and 4-aminobiphenyl were weakly mutagenic to V79 cells with bladder cell activation, while no mutagenic activity was detected with 1-naphthylamine, 2-naphthylamine, or benzidine. Because the mutagenic activity of the aromatic amines was low with V79 cells as the target, a bladder cell-mediated S. typhimurium system was developed for these chemicals. The aromatic amines 2-aminofluorene, 2-acetylaminofluorene, 4-aminobiphenyl and 2-naphthylamine were mutagenic to S. typhimurium TA98 and TA100 in the presence of bladder cells but not in their absence. Benzidine was mutagenic to TA98 but not to TA100. The putative noncarcinogen 1-naphthylamine was not mutagenic in the system. In contrast to the V79 data, 7,12-dimethylbenz(a)anthracene and dimethylnitrosamine were not mutagenic with either bacterial strain. Mutagenic responses were related to both the number of bladder cells used for activation and the concentration of test chemical in the Salmonella assay. The data demonstrate that bovine bladder urothelial cells can activate carcinogens from three chemical classes to mutagens and indicate the different sensitivities of V79 cells and S. typhimurium to genotoxic agents.     

A quantitative comparison of the mutagenicity of carcinogenic polycyclic hydrocarbon derivatives in cultured mammalian cells.

The mutagenicity of a series of reactive polycyclic hydrocarbon derivatives has been studied using Chinese hamster (V79) cells in culture and, as a mutational marker, resistance to the purine analogue 8-azaguanine. The compounds were compared by relating mutation frequency to the dose applied (mutagenic effectiveness) to induced cytotoxicity (mutagenic efficiency) and to the extent of reaction of the hydrocarbon with DNA (absolute mutagenic efficiency). In each case anti-benzo(alpha)pyrene (BP)-7,8 dihydrodiol-9,10 oxide, the suspected ultimate carcinogenic form of benzo(alpha)pyrene, was by far the most potent of the compounds tested. Furthermore, the mutagenicity of the syn- and anti-BP-diolepoxide isomers correlated positively with their documenrences in the ability of each derivative to form a carbonium ion. Variations in mutagenic efficiency and absolute mutagenic efficiency were more difficult to explain. The latter findings are discussed in relation to the types of hydrocarbon-DNA product obtained with each compound and also to the possibility of a variable cellular response to more subtle differences in the chemistry of the hydrocarbon-DNA interaction.     

Influence of the alkyl substituent on mutagenicity and covalent DNA binding of bay region diol-epoxides of 7-methyl- and 7-ethylbenz(a)anthracene in Salmonella and V79 Chinese hamster cells

The anti-isomers of the bay region diol-epoxides of the strong carcinogen 7-methylbenz(a)anthracene and of the weak carcinogen 7-ethylbenz(a)anthracene were investigated for mutagenicity in Salmonella typhimurium (reversion of the his - strains TA98 and TA100 to prototrophy) and V79 Chinese hamster cells (acquisition of resistance to 6-thioguanine and ouabain; formation of micronuclei). In addition, in the V79 cells, the levels of the DNA adducts formed were determined by 32P-postlabeling analysis. In terms of mutations per nmol compound administered, the methyl derivative was four to 10 times more potent, depending on the genetic endpoint, than its ethyl congener. However, when the results were expressed as mutations per adduct, the difference between the two diol-epoxides was small. Therefore, a higher level of DNA modification appears to be the major reason for the stronger mutagenicity of the methyl derivative. However, both diol-epoxides had similar half-lives (about 9 min) in physiological buffer, as determined from the decline in mutagenic activity after preincubation of the test compound. These results suggest that the effect of the 7-alkyl group on the extent of reaction with DNA is more a result of steric factors than of a change in the intrinsic chemical reactivity of the diol-epoxides.