Comparative genotoxic effects of IQ and MeIQ in Salmonella typhimurium and cultured mammalian cells

The food mutagens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) were studied for their genotoxic potential using hepatocytes isolated from untreated and Aroclor 1254 (PCB) pretreated rats as an activation system. Monolayers of hepatocytes co-incubated with Salmonella typhimurium TA98 activated IQ and MeIQ to bacterial mutagens, with MeIQ being about twice as potent as IQ. The mutagenic activities of IQ and MeIQ were increased by using hepatocytes from PCB-pretreated rats. IQ and MeIQ also caused primary DNA damage in the hepatocytes as determined by increases in the rate of alkaline elution of DNA, as well as increases in DNA-repair synthesis. Furthermore, exposure of V79 cells co-cultured with PCB-pretreated hepatocytes to IQ and MeIQ showed evidence of increased sister-chromatid exchanges and a low and variable increase in the number of 6-thioguanine-resistant mutants. The genotoxic potency of IQ and MeIQ in mammalian cells was low or virtually absent compared to their extreme potency in bacteria. This could be due to a lower capacity of mammalian cells to further metabolize the so-called directly acting bacterial mutagens, formed by a cytochrome P-450 dependent N-hydroxylation, to their ultimate reactive forms.     

Structure-activity relationships of epoxides: induction of sister-chromatid exchanges in Chinese hamster V79 cells

Analysis of SCE frequencies in Chinese hamster V79 cells was used to investigate structure-activity relationships of epoxides in mammalian cells. For this purpose the SCE-inducing potency of 58 epoxides was determined. Of these, 16 failed to induce SCE in V79 cells. According to the substitution of the oxirane ring the results show general agreement with results obtained in the Ames test. Mono-substituted epoxides had the highest genotoxic potency compared to di- and tri-substituted epoxides. In detail, there are differences in genotoxic potency between bacteria and mammalian cells which can be explained by differences in the cellular uptake of the compounds and by detoxification reactions.     

Structure-activity relationships of epoxides: induction of sister-chromatid exchanges in V79 cells by enantiomeric epoxides.

Analysis of SCE frequencies in Chinese hamster V79 cells was used to investigate the influence of the stereoisomeric forms of epoxides in mammalian genotoxicity tests. The SCE-inducing potency of 12 pairs of (R)- and (S)-enantiomeric epoxides which differed in the degree of substitution of the oxirane ring was determined. Of these, 2 pairs of epoxides failed to induce SCE. Different SCE-inducing potencies between the (R)- and (S)-enantiomers were shown for 5 epoxides. This study demonstrates that stereoselectivity might play an important role in genotoxicity testing of chemicals with asymmetric C atoms.     

In vivo cytogenetic effects of the cooked-food-related mutagens Trp-P-2 and IQ in mouse bone marrow

Sister-chromatid exchange and chromosomal aberrations were measured in vivo in mouse bone marrow following intraperitoneal injection of the cooked food mutagens, Trp-P-2 and IQ. Trp-P-2 produced a significant positive dose response for both endpoints while IQ produced only a weak but significant sister-chromatid exchange response. The relative potency of these two chemicals is similar to that seen in mammalian cells in vitro but opposite to their potency in Salmonella.     

Cytotoxic and genotoxic effects of energetic compounds on bacterial and mammalian cells in vitro.

The mutagenicity and toxicity of energetic compounds such as 2,4, 6-trinitrotoluene (TNT), 1,3,5-trinitrobenzene (TNB), hexahydro-1,3, 5-trinitro-1,3,5-triazine (RDX) and octahydro-1,3,5,7-tetranitro-1,3, 5,7-tetrazocine (HMX), and of amino/nitro derivatives of toluene were investigated in vitro. Mutagenicity was evaluated with the Salmonella fluctuation test (FT) and the V79 Chinese hamster lung cell mutagenicity assay. Cytotoxicity was evaluated using V79 and TK6 human lymphoblastic cells. For the TK6 and V79 assays, TNB and 2, 4,6-triaminotoluene were more toxic than TNT, whereas RDX and HMX were without effect at their maximal aqueous solubility limits. The primary TNT metabolites (2-amino-4,6-dinitrotoluene, 4-amino-2, 6-dinitrotoluene, 2,4-diamino-6-nitrotoluene and 2, 6-diamino-4-nitrotoluene) were generally less cytotoxic than the parent compound. The FT results indicated that TNB, TNT and all the tested primary TNT metabolites were mutagenic. Except for the cases of 4-amino-2,6-dinitrotoluene and 2,4-diamino-6-nitrotoluene in the TA98 strain, addition of rat liver S9 resulted in either no effect, or decreased activity. None of the tested compounds were mutagenic for the V79 mammalian cells with or without S9 metabolic activation. Thus, the FT assay was more sensitive to the genotoxic effects of energetic compounds than was the V79 test, suggesting that the FT might be a better screening tool for the presence of these explosives. The lack of mutagenicity of pure substances for V79 cells under the conditions used in this study does not preclude that genotoxicity could actually exist in other mammalian cells. In view of earlier reports and this study, mutagenicity testing of environmental samples should be considered as part of the hazard assessment of sites contaminated by TNT and related products.     

Mutagenic activity of 4 active-principle forms of pharmaceutical drugs. Comparative study in the Salmonella typhimurium microsome test, and the HGPRT and Na+/K+ ATPase systems in cultured mammalian cells

The nitroimidazole derivatives used in human therapy have shown a strong mutagenic activity in bacterial tests using Ames strains of Salmonella typhimurium. Our study compared the response of 4 products of this family on bacterial target cells as well as on mammalian target cells (Chinese hamster V79 cells). The strong positive response on TA100 was greatly reduced on the nitroreductase-deficient strain TA100 Frl. Furthermore, no mutagenic activity was found in V79 mammalian cells that we examined for ouabain and 6-thioguanine resistance.     

Comparative genotoxic effects of the cooked-food-related mutagens Trp-P-2 and IQ in bacteria and cultured mammalian cells

As part of a major study to evaluate the mutagenicity of chemicals produced during the cooking of foods, we examined the responses of bacteria and cultured Chinese hamster cells to the compounds Trp-P-2 (3-amino-1-methyl-5H-pyrido[4,3-b]indole) and IQ (2-amino-3-methylimidazo[4,5-f]quinoline), constituents identified in cooked beef and fish. In the Ames/Salmonella tester strain TA1538, both compounds were confirmed to be extremely potent mutagens that were active at levels below 1 ng/plate in the presence of hamster-liver S9 microsomal fraction. 50-fold higher doses of both compounds were required for mutagenicity in the uvr+ tester strain TA1978. Trp-P-2 also behaved as a strong mutagen in CHO cells using the standard exogenous activation with hamster-liver S9 fraction. At concentrations below 1 microgram/ml it produced dose-dependent increases in cell killing, mutations at the hprt and aprt loci, sister-chromatid exchanges, and chromosomal aberrations. An excision-repair-deficient strain was about 2-fold more sensitive than the normal CHO cells with respect to these genotoxic effects of Trp-P-2. IQ had unexpectedly weak activity for all genetic endpoints in the CHO cells, and it produced clear-cut responses only in the repair-deficient cells and only above a concentration of 10 micrograms/ml. The toxicity that was observed with IQ was not affected by the repair capacity of the cells and was not associated with chromosomal aberrations, indicating that damage to cellular structures other than nuclear DNA was likely the predominant pathway for cell killing. Because the culture conditions normally used for CHO cell exposure were shown to be competent in producing bacterial mutagenicity with IQ, it was concluded that the active metabolite of IQ was present in the medium but was somehow ineffective in reaching the DNA of CHO cells and/or reacting with it. These results suggest that the relative mutagenic potency of compounds in Salmonella may bear no direct relationship to relative mutagenicity in CHO cells, emphasizing precaution in attempting to extrapolate microbial data to mammalian somatic cells. This study illustrates the use and merits of a multi-endpoint assay for genetic damage in CHO cells, the utility of using CHO cells that are defective in excision repair of DNA, and the importance of comparative testing between bacterial and mammalian systems.     

Activation of N-acetoxy- and N-hydroxy-2-acetylaminofluorene to mutagenic and cytotoxic metabolites by V79 Chinese hamster cells

N-Acetoxy-2-acetylaminofluorene (AAAF) and N-hydroxy-2-acetylaminofluorene (OH-AAF) are mutagenic to V79 cells, causing the induction of 6-thioguanine-resistant clones, and are cytotoxic. The presence of the deacetylase inhibitor, paraoxon, drastically reduces both the mutagenic and cytotoxic effects. This strongly suggests that deacetylated metabolites are the major active species. Furthermore, when Salmonella typhimurium TA98 is used as target organism, addition of homogenate of V79 cells strongly potentiates the mutagenicity of OH-AAF. To our knowledge, this is the first report demonstrating a significant biological effect due to the metabolism of a mutagen by V79 cells.     

Studies by means of the SCE assay in V79-E Chinese hamster cells on the mode of action of tri-substituted nitrosoureas

The genotoxic activity of 3,3-diethyl-1-methyl-1-nitrosourea ( DEMNU ), 1,3-dimethyl-3-phenyl-1-nitrosourea ( DMPNU ) and 1-chloroethyl-3-methyl-3-phenyl-1-nitrosourea ( CEMPNU ) was studied in the SCE assay in V79-E cells in vitro. These compounds are very stable in aqueous solutions, but are directly acting genotoxins . The SCE rates increase linearly with the length of the incubation period. This direct activity is presumably due to an intracellular catalytic decomposition. Whereas the SCE-inducing effect of DMPNU and CEMPNU is not influenced by addition of S9 mix, that of DEMNU is strongly potentiated by rat and Syrian hamster S9 mix. This DEMNU activation is an NADPH-dependent enzymatic reaction and is inducible by phenobarbital. The absence of a direct mutagenic effect of DEMNU in the Ames test, as reported by other authors, is probably caused by a striking insensitivity to tri-substituted nitrosoureas of the Salmonella assay. This assumption was substantiated by long-term application of very low DMPNU doses to V79-E. Long-term simultaneous treatment with DMPNU and bromodeoxyuridine (BUdR) significantly diminished the rate of SCE induction.     

Differential mutagenic activity of IQ (2-amino-3-methylimidazo[4,5-f]quinoline) in Salmonella typhimurium strains in vitro and in vivo, in Drosophila, and in mice

IQ, a heterocyclic aromatic amine which is formed during the frying of meat, was prepared by chemical synthesis. Its genotoxic potential was studied in bacteria, Drosophila and in mice. A mutagenic effect of IQ (frameshift induction) was detected in Salmonella typhimurium in experiments without metabolic activation; this effect was several orders of magnitude lower than that observed in the presence of an activation system. Ames tests with liver-homogenate S9 fraction from PCB-induced mice and rats confirmed the high mutagenic potency of IQ metabolites (Kasai et al., 1980a). Comparative studies on diagnostic Salmonella strains revealed that the high frameshift-inducing activity is independent of the plasmid pkM101; it is, however, greatly reduced by an intact excision-repair system for DNA lesions. The mutagenic activity of the metabolite(s) formed in vitro by S9 mix has a half-life of ca. 14 min. In the fruit fly, Drosophila melanogaster, IQ induced when used at sublethal concentrations, X-chromosomal recessive lethal mutations in male germ cells in a dose-dependent manner. In mice, tests were performed to detect somatic mutations: chromosomal anomalies (micronuclei) in bone marrow, and gene mutations (affecting coat pigmentation) in mice exposed to IQ in utero. No genotoxic effects were observed in these assays. However, the formation of mutagenic metabolites in the liver of IQ-treated mice was unequivocally demonstrated in host-mediated assays using Salmonella as mutagen probes in mice. The data demonstrate genotoxic activity of IQ in prokaryotic and eukaryotic organisms. The possible reasons for the different response of mammalian systems in vivo and the Salmonella system are discussed.     

Hepatocyte-mediated SCE induction by indirect mutagens: importance of hepatocyte density and cell-to-cell contact

The SCE-inducing effects of the indirectly acting mutagens cyclophosphamide (CP), dimethylnitrosamine (DMN) and aflatoxin B1 (AFB1) were analysed in hepatocyte (hpc)/mammalian cell coculture systems with regard to the importance of the hpc density. V79 cells and human lymphocytes served as target cells. For all 3 compounds steadily increasing genetic effects were observed when the hpc density was increased from 3.2 X 10(4) up to 3.2 X 10(6) viable hpc per culture (25-cm2 flask), i.e. the more hpc available for metabolisation, the more genetic effects induced. The frequency distributions of the CP-induced SCE values were clearly different from those obtained with DMN, especially when high hpc densities were used: distribution patterns obtained for the mutagen with stable metabolites (CP) are characterized by the presence of distinct maxima and the absence of cells with SCE control values, whereas distribution patterns for the mutagen with very short-lived metabolites (DMN) can be described by the absence of maxima and the presence of cells with SCE control values. The frequency distributions of the AFB1-induced SCE values were more similar to the CP type than to the DMN type. From these results it is deduced that close contact between metabolising and target cells is necessary for the detection of the genotoxic effect of DMN. For CP and AFB1 a direct contact seems not to be essential, i.e. reactive intermediates may also be transported via the culture medium to the target cells.     

Cold atmospheric plasma jet effects on V79-4 cells

The effects of cold plasmas are due to charged particles, reactive oxygen species (ROS), reactive nitrogen species (RNS), UV photons, and intense electric field. In order to obtain a more efficient action on mammalian cells (useful for cancer therapy), we used in our studies chemically activated cold plasma (He and O2 gas mixture). V79-4 cells were exposed to plasma jet for different time periods (30, 60, 90, 120 and 150s), using different combinations of helium and oxygen inputs (He:2.5l/min + 02:12.5ml/min; He:2.51/min + O2:25ml/min; He:2.51/min + O2:37.5 ml/min). Using MTT test we demonstrated that plasma jet induced cell viability decrease in all cases. The effect of chemically activated cold plasma--apoptosis or necrosis--depends on gas mixture and treatment period. Taking into account that ROS density in cell microenvironment is related to O2 percent in the gas mixture and treatment period, we can presume that cell death is due to ROS produced in plasma jet.     

Genotoxicity of naturally occurring hydroxyanthraquinones

A variety of structurally related hydroxyanthraquinones (HA) were investigated in a test battery for the evaluation of mutagenicity and cell-transforming activity. The tests were: (1) the Salmonella typhimurium mutagenicity assay, (2) the V79-HGPRT mutagenicity assay, (3) the DNA-repair induction assay in primary rat hepatocytes and (4) the in vitro transformation of C3H/M2 mouse fibroblasts. In Salmonella, most of the tested compounds were mutagenic in strain TA1537, but only a few were active in other strains. Among these were HA with a hydroxymethyl group, such as lucidin and aloe-emodin. In V79 cells, only HA with 2 hydroxy groups in the 1,3 positions (1,3-DHA, purpurin, emodin) or with a hydroxymethyl sidechain (lucidin and aloe-emodin) were mutagenic. The compounds found to be active in V79 cells were also active in the DNA-repair assay and in the C3H/M2 transformation assay. Thus, it appears that the genotoxicity of HA is dependent on certain structural requirements.     

Lack of activity of the bacterial mutagen emodin in HGPRT and SCE assay with V79 Chinese hamster cells

Genotoxicity of emodin was studied in the Salmonella/microsome assay, the sister-chromatid exchange (SCE) assay and the hypoxanthine-guanine-phosphoribosyltransferase (HGPRT) forward mutation assay with V79 Chinese hamster cells. In the Salmonella/microsome assay, emodin was found to be positive in TA97, TA100 and TA1537 in the presence of liver homogenate. In TA1537 a weak direct mutagenicity was also observed. In both mammalian test systems, no genotoxicity was found either with or without metabolic activation.     

Mutagenicity experiments on L-cysteine and D-penicillamine using V79 cells as indicators and for metabolic activation

Previous studies have shown that cysteine and penicillamine induce gene mutations in Salmonella typhimurium, the effect being strongly potentiated in the presence of mammalian tissue preparations. It has now been demonstrated that homogenate of V79 Chinese hamster cells is an efficient activator of thiol amino acids as well. Nevertheless, L-cysteine and D-penicillamine did not induce gene mutations (acquisition of resistance towards 6-thioguanine) in V79 cells. This was true even in the presence of the most efficient activating system, kidney postmitochondrial fraction. The result suggests the existence of an effective protective system in mammalian cells against the natural amino acid L-cysteine and its therapeutically used derivative, D-penicillamine.     

Study of the mutagenic activity of 6 hepatotoxic pharmaceutical drugs in the Salmonella typhimurium microsome test, and the HGPRT and Na+/K+ ATPase system in cultured mammalian cells

Several pharmaceutical drugs show strong hepatotoxicity during therapeutic use. We have studied 6 of them: aminophenazone, clofibrate, nifuroxazide, oxamniquine, perhexiline maleate, tienilic acid. Their mutagenicity was assessed in the Ames test on 6 strains of Salmonella typhimurium, and in V79 Chinese hamster lung cells using a rat-hepatocyte-mediated metabolic activation system and the HGPRT and Na+/K+ ATPase assay. Nifuroxazide was positive in the Ames test in two Salmonella strains (TA100, and TA100 Fr1). In the hepatocyte-mediated mammalian V79 cell system, nifuroxazide, clofibrate and aminophenazone were negative; oxamniquine and tienilic acid were positive with and without metabolic activation in tests looking for ouabain and 6-thioguanine resistance. Perhexiline maleate was negative for the direct induction of 6-thioguanine resistance without metabolic activation, and positive after metabolisation mediated by primary rat's hepatocytes. These results suggest the need for some caution in the use of some pharmaceutical drugs because of hepatotoxicity and because 3 out of 6 drugs were shown to be slightly mutagenic in mammalian cells.     

Mutagenic effects of TPA-induced clastogenic factor in Chinese hamster cells

Previous work in our laboratory has shown that the clastogenic and SCE-inducing effects of 12-O-tetradecanoylphorbol 12-acetate (TPA) are mediated by secondary products formed by the cell in response to the tumor promoter. A low-molecular-weight clastogenic factor (CF) was isolated from the medium of TPA-treated human leukocytes and caused chromosome aberrations and sister-chromatid exchanges (SCE) in fresh cultures not exposed to TPA itself. In the present study, we show that Chinese hamster fibroblasts (V79 cells) also produce CF when exposed to TPA. CF from V79 cells induced SCE not only in hamster cells, but also in human lymphocytes. Vice versa, CF from human leukocyte cultures induced SCE in hamster cells. It also increased the frequency of 6-thioguanine-resistant mutants in this cell system. All cyto- and geno-toxic effects of TPA-induced CF were prevented if the cells were treated with superoxide dismutase before exposure. The lipophilic CF seems to be derived from arachidonic acid of cell membranes released as a consequence of oxidative damage and subsequently degraded to genotoxic aldehydes in an autoxidative process. CF is formed only under culture conditions with low antioxidant content in culture media and sera. This may explain the discordant results obtained by different laboratories with regard to the genotoxic effects of TPA.     

Radiosensitization of hypoxic cells by a nitrofuran; dose-modifying and shoulder effects.

The radiosensitizer nifurpipone dihydrochloride (5-nitro-2-furaldehyde N-methyl piperazino acetyl hydrazone dihydrochloride) sensitizes hypoxic V79 mammalian cells by at least two mechanisms. Sensitization is by a reduction of ? in addition to an increase in slope. Both these affects are absent under oxygenated conditions. When hypoxic V79 cells are irradiated in the presence of nifurpipone dihydrochloride combined with Ro-07-0582, sensitization greater than that due to air alone is observed; this effect is due to a reduction in ? and an increased slope. Again this effect is absent under oxygenated conditions. Rapid-mix studies using Serratia marcescens show that full senitization occurs with a pre-irradiation contact time of 4 msec; this contrasts with data for V79 cells where a pre-irradiation contact time of 40 msec is insufficient for any sensitization to occur. This sensitizer also exerts a differential toxic effect, being more toxic to hypoxic cells than to oxygenated ones. It is concluded from these results that nifurpipone dihydrochloride sensitizes by at least two mechanisms, one of which resembles that of the electron-affinic type.     

Niacin, poly(ADP-ribose) polymerase-1 and genomic stability

This study demonstrates that cupric 8-quinolinoxide (CuQ) has induced genetic toxicity in bacteria and mammalian cells through a mechanism of reactive oxygen species (ROS) generation. In the Ames test with rat liver S9, CuQ dose-dependently caused a point mutation in Salmonella typhimurium TA100. The effect of CuQ on DNA damage in HL60 and V79 cells identified in the comet assay is direct and enhanced by the addition of S9. Meanwhile, the tailing length of comet DNA is related to the increasing dosage of CuQ. The genotoxic effect of CuQ on either gene mutation in bacteria or DNA damage in culture cells can be generally blocked by several antioxidants, e.g. pyrrolidinedithiocarbamate, N-acetylcysteine, Vitamins C and E. Supportive of this observation, ROS generation induced by CuQ can be demonstrated both in vitro and in vivo by using the DCFH-DA fluoroprobe. The CuQ-induced intracellular ROS level is also dramatically inhibited by the above antioxidants. Above results imply that the CuQ-induced genotoxicity could be mediated by ROS generation. The nature of ferrous-dependent and S9-enhancing in CuQ-induced ROS generation hints a Fenton-like reaction or some specific enzymes activation could be involved in this process. Furthermore, a DNA damage- and oxidative stress-dependent protein, P53, could also been induced by CuQ treatments in a time-course and dose-dependent manners. Its expression level is recoverable by antioxidants too. In conclusion, our current study strongly suggests that CuQ induces gene mutation, global DNA damage, and P53 expression through a ROS-dependent mechanism.     

Genotoxicity of 1,3- and 1,6-dinitropyrene: induction of micronuclei in a panel of mammalian test cell lines

1,3-Dinitropyrene (1,3-DNP) and 1,6-dinitropyrene (1,6-DNP) were assessed for their potential to increase the frequencies of micronuclei in a panel of test cell lines consisting of H4IIEC3/G, 5L, 5L/r-1,3-DNP1, 208F, V79, V79/r-1,6-DNP1, HepG2 and BWI-J cells, which have been partially characterized for their expression of xenobiotic metabolising enzymes. The micronuclei were analyzed for the presence or absence of kinetochores indicating the occurrence of aneuploidy or chromosome breakage, respectively. 1,3-DNP caused a substantial increase in the frequency of micronuclei only in V79 cells. 1,6-DNP was strongly genotoxic in lines H4IIEC3/G⁻, 208F, V79 and, to a minor degree, in 5L/r-1,3-DNP1. It caused the formation of kinetochore-positive as well as kinetochore-negative micronuclei in V79 cells but only of kinetochore-negative micronuclei in H4IIEC3/G⁻ and 208F cells. 1,6-DNP-induced formation of micronuclei was paralleled by the appearance of multinucleated cells. Treatment of V79 cells with 1,3-DNP resulted in the same types of damage as treatment with 1,6-DNP, although considerably higher concentrations were required.The results show that 1,6-DNP can be highly genotoxic in mammalian cells, whereas, at least in the panel of test cell lines used, 1,3-DNP possesses only a low genotoxic activity. 1,3-DNP appears to be activated to genotoxic products in V79 cells by the same pathway(s) as 1,6-DNP.