Evaluation of the mutagenic potential of endosulfan using the Salmonella/mammalian microsome assay

The mutagenic potential of endosulfan, a polychlorinated insecticide, was assessed using the highly sensitive Salmonella tester strains TA97(a), TA98, TA100 and TA102. It exhibited a toxic effect at dose levels of 50 micrograms/plate and higher. Plate incorporation studies did not show mutagenic response with any of the tester strains used. A modification of the assay using a preincubation procedure showed mutagenic activity with and without metabolic activation with TA97(a) only. Increased toxicity was observed after addition of phenobarbital-induced S9 mix.     

Single-strand nicks induce homologous recombination with less toxicity than double-strand breaks using an AAV vector template

Gene targeting by homologous recombination (HR) can be induced by double-strand breaks (DSBs), however these breaks can be toxic and potentially mutagenic. We investigated the I-AniI homing endonuclease engineered to produce only nicks, and found that nicks induce HR with both plasmid and adeno-associated virus (AAV) vector templates. The rates of nick-induced HR were lower than with DSBs (24-fold lower for plasmid transfection and 4- to 6-fold lower for AAV vector infection), but they still represented a significant increase over background (240- and 30-fold, respectively). We observed severe toxicity with the I-AniI ‘cleavase’, but no evidence of toxicity with the I-AniI ‘nickase.’ Additionally, the frequency of nickase-induced mutations at the I-AniI site was at least 150-fold lower than that induced by the cleavase. These results, and the observation that the surrounding sequence context of a target site affects nick-induced HR but not DSB-induced HR, strongly argue that nicks induce HR through a different mechanism than DSBs, allowing for gene correction without the toxicity and mutagenic activity of DSBs.     

Measurements of Mutagenic and Antimutagenic Activities of Bile Acids by Rec-assay

To study the carcinogenic activity of bile acids, we examined the mutagenic activity of bile acids by Rec-assay using B. subtilis H17 and M45 strains. Cholic, chenodeoxycholic, lithocholic, and glycolithocholic acids exerted much weaker mutagenicity than mitomicin C (MMC), and deoxycholic and glycodeoxycholic acids showed toxicity toward the bacteria. Most of the conjugated bile acids (glycocholic, taurocholic, and taurodexycholic acids) and their amino acid components (glycine and taurine) were neither toxic nor mutagenic. No bile acids enhanced the mutagenicity of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), but glycine enhanced both toxicity and mutagenicity of MNNG in a dose-dependent manner. On the other hand, taurine decreased the mutagenicity of MNNG, and most of the bile acids decreased the mutagenicity of MMC. Furthermore, taurocholic acids decreased toxicity and/or mutagenicity of other bile acids. These results suggested that the mutagenic and comutagenic activities of bile acids can be disregarded, but they are antimutagenic in some situations.     

Toxic and mutagenic effects of formaldehyde in Salmonella typhimurium

Toxic and mutagenic activities of formaldehyde were studied in Salmonella typhimurium strain TM677, using forward mutation to 8-azaguanine (8-AG) resistance both in the absence and in the presence of Aroclor-induced rat-liver postmitochondrial supernatant (PMS). The results showed that formaldehyde was toxic and mutagenic to the bacteria in both systems, but toxicity and mutagenicity were reduced in the presence of PMS. The minimum concentration required to induce toxicity and mutagenicity was 0.17 mM in the absence of PMS and 0.33 mM in the presence of PMS.     

Simultaneous measurement of toxicity and mutagenic activity

The assay of mutagenic activity of toxic drugs is difficult to perform and analyze, because one needs to know the kinetics of both effects in order to draw reliable conclusions. This is the case with niflumic acid (NA), which reduced the viability of S. typhimurium TA1535 100 times in the Ames test, but the background microcolonies show no difference from controls and the number of revertants was not altered by the drug. A test which measures the kinetics of growth of viable bacteria and mutants in liquid medium has been developed and applied to NA. No mutagenic activity was detected and elimination of the toxicity from the medium is suggested.     

Comutagenesis of sodium arsenite with ultraviolet radiation in Chinese hamster V79 cells

Summary Solar ultraviolet radiation has been associated with the induction of skin cancer. Recent studies have indicated that near-ultraviolet, especially UVB, is mutagenic. Exposure to trivalent inorganic arsenic compounds has also been associated with increased skin cancer prevalence. Trivalent arsenic compounds are not mutagenicper se, but are comutagenic with a number of cancer agents. Here, we test the hypothesis that arsenite enhances skin cancer via its comutagenic action with solar ultraviolet radiation. Irradiation of Chinese hamster V79 cells with UVA (360 nm), UVB (310 nm) and UVC (254 nm) caused a fluence-dependent increase in mutations at thehprt locus. On an energy basis, UVC was the most mutagenic and UVA the least. However, when expressed as a function of toxicity, UVB was more mutagenic than UVC. Nontoxic concentrations of arsenite increased the toxicity of UVA, UVB and UVC. Arsenite acted as a comutagen at the three wavelengths; however, higher concentrations of arsenite were required to produce a significant (P < 0.05) comutagenic response with UVB. The increased mutagenicity of UVB and UVA by arsenite may play a role in arsenite-related skin cancers.     

Microbioluminescent study of the general toxicity and mutagenicity of pollutants

Bacterial bioluminescence was applied to detection of general toxicity (MIT test) and genotoxicity (SOS-lux test) of some chemicals, seawater, and fresh water. The SOS-induced luminescence of E. coli WP2s (cda::luxCDABE) cells was higher than in E. coli C 600 (cda::luxCDABE) at 37 degrees C and pH 6.5. The mutagenic effect of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), mitomycin C, and hydrogen peroxide determined from the induction of E. coli WP2s cell luminescence was detected at lower concentrations than in the assessment of reversion frequencies. General toxicity was demonstrated by using luminescence inhibition for hydrogen peroxide, Zn2+, and Cd2+ at low concentrations. Regions of the Krasnodar Krai where sea and fresh waters exerted toxic action on luminescence were determined by the microbioluminescent method.     

Mutagenic and recombinogenic consequences of DNA-repair inhibition during treatment with 1,3-bis(2-chloroethyl)-1-nitrosourea in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae has been used as a model system to explore whether the clinical combination of the antitumour agent BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) with DNA-repair inhibitors would affect the drug's mutagenic or recombinogenic potential. Preliminary experiments suggested that mitotic crossing-over and other mutagenic events are controlled in a separate fashion. BCNU was more toxic in yeast derivatives with specific defects in any of the three recognised major DNA repair pathways than in the DNA-repair-proficient parent strain. However, in a diploid homozygous for rad18, BCNU showed enhanced mutagenic and recombinogenic potential. Both of these effects were reduced in a comparable rad3 strain, and mitotic crossing-over but not other types of mutagenic event eliminated in the rad52 derivative. Experiments were performed in the presence of three DNA-repair inhibitors which are currently in clinical use and which might be available for combination chemotherapy. Hydroxyurea and amsacrine themselves caused mitotic crossing-over and other events, and did not reduce mutagenic or recombinogenic potential of the BCNU. Hydroxyurea actually decreased toxicity of the BCNU. Caffeine, however, showed some effect in enhancing toxicity and decreasing both mutagenic and recombinogenic potential of the drug. Development of more specific repair inhibitors related to amsacrine or to caffeine, using these repair-deficient strains as model systems, might lead to an enhanced clinical potential of this bisalkylating drug and related compounds.     

Drug residue formation from ronidazole, a 5-nitroimidazole. VI. Lack of mutagenic activity of reduced metabolites and derivatives of ronidazole

The potential toxicity of ronidazole residues present in the tissues of food-producing animals was assessed using the Ames mutagenicity test. Since ronidazole is activated by reduction, reduced derivatives of ronidazole and metabolites formed by enzymatic reduction of ronidazole were tested for mutagenicity. When tested at levels several orders of magnitude higher than that at which ronidazole was mutagenic, 5-amino-4-S-cysteinyl-1,2- dimethylimidazole , a product of the dithionite reduction of ronidazole in the presence of cysteine, the 5-N-acetylamino derivative of ronidazole and 5-amino-1,2- dimethylimidazole all lacked mutagenic activity in Ames strain TA100. The metabolites of ronidazole formed by the incubation of ronidazole with microsomes under anaerobic conditions were also not mutagenic. These data demonstrate that although ronidazole is a potent mutagen, residues from it which may be present in the tissues of food-producing animals lack any mutagenic activity.     

Dimethylglycine and chemically related amines tested for mutagenicity under potential nitrosation conditions

Dimethylglycine (DMG) and the chemically related amino acids glycine, sarcosine (monomethylglycine) and betaine (trimethylglycine) were tested in Salmonella typhimurium strain TA100 after treatment with sodium nitrite under acidic conditions using a modified Ames Salmonella/microsome assay as reported by Colman et al. (1980). The increase in the number of revertants observed both with and without metabolic activation was also induced in the control mixtures without adding the amines. From the subsequent testing of the individual components of the mixtures, we concluded that non-consumed nitrite was responsible for the mutagenic responses observed in the different reaction mixtures, and not the amines themselves. There were no consistent indications of mutagenic activity of the DMG test mixture as compared to the control mixture which exhibited both consistent mutagenic activity and a toxic effect which was not increased by the addition of DMG. In fact, DMG seemed to decrease the toxicity of the control reaction solution to the Salmonella which was clearly observed at the higher doses. DMG cannot be considered mutagenic under the test conditions employed. The same can be said of the other amino acids as well.     

Genotoxic evaluation of the organophosphorous pesticide temephos

The chemical compound temephos (0,0,0',0'-tetrametyl-0,0'-thiodi-p-phenylene phosphorothioate) is an organophosphorous pesticide that has been used in Brazil since 1967 in control campaigns against the mosquito Aedes aegypti, the vector of dengue and yellow fever. We used single cell gel electrophoresis (SCGE), SOS/umu and Ames/Salmonella assays to test the toxicity and mutagenicity of temephos. Temephos was genotoxic in the SCGE assay, inducing severe DNA lesions (type IV lesions) at doses above 1.34 micro M. It was mutagenic, but not toxic, in the SOS/umu assay to Escherichia coli strain PQ37, but not to PQ35, at concentrations above 1.33 micro M, particularly when the S9 mixture was not used in the assay. Temephos was not mutagenic in the Ames assay with S. typhimurium strains TA97, TA98, TA100 and TA102, both with and without metabolic activation. However, temephos at concentrations above 3.33 micro M was mutagenic to TA98NR, YG7104 and YG7108, both with and without metabolic activation. In conclusion, temephos was genotoxic and mutagenic in all the three tests used, and in two of them at concentrations similar to those routinely used to combat Aedes aegypti.     

Relationships between structure, toxicity and genetic effects in Salmonella typhimurium and Saccharomyces cerevisiae for substituted aniline mustards

A series of 4-substituted aniline mustards of widely varying reactivities have been evaluated for their mutagenic effects in Salmonella typhimurium strains of varying uvrB gene and plasmid status, and for their ability to cause mitotic crossing-over in Saccharomyces cerevisiae. The 4-methyl aniline mustard N,N-bis(2-chloroethyl)-4-methylaniline and its corresponding half-mustard N-(2-chloroethyl)-4-methylaniline showed widely different effects in the various bacterial strains, with the half-mustard being much less toxic than the full mustard in the uvrB- strain TA100. However, in the uvrB+ strain TA1978+, possessing an intact excision repair system, both compounds were equally toxic and the full mustard was the more mutagenic. Both compounds were equally effective in promoting mitotic crossing-over in yeast. For a series of 4-substituted full mustards, the toxicity in S. typhimurium strain TA100 correlated with substituent electronic parameters in the same way as does mammalian cell toxicity, supporting the view that the primary mode of toxicity is via DNA cross-linking, even for unreactive analogues. However, there were no obvious correlations between substituent physiochemical properties and mutagenic potential in bacteria, suggesting that mutagenic events are subject to a variety of influences other than the reactivity of the mustard group. In contrast, the most chemically reactive compounds were the most toxic and most recombinogenic in yeast.     

Evaluation of extracts from Coccoloba mollis using the Salmonella/microsome system and in vivo tests

The common everyday use of medicinal plants is an ancient, and still very widespread practice, whereby the need for studies on their possible toxicity and mutagenic properties. The species Coccoloba mollis has been much used in phytotherapy, mainly in cases involving loss of memory and stress. In order to investigate its genotoxic and mutagenic potential, ethanolic extracts from the leaves and roots underwent Salmonella/microsome assaying (TA98 and TA100 strains, with and without exogenous metabolism - S9), besides comet and micronucleus tests in vivo.There was no significant increase in the number of revertants/plate of Salmonella strains in any of the analyzed root-extract concentrations, although the extract itself was extremely toxic to the Salmonella TA98 strain in the tests carried out with S9 (doses varying from 0.005 to 0.5 μg/plate). On the other hand, the leaf-extract induced mutations in the TA98 strain in the absence of S9 in the highest concentration evaluated, although at very low mutagenic potency (0.004 rev/ μg). Furthermore, there was no statistically significant increase in the number of comets and micronuclei, in treatments involving Swiss mice. It was obvious that extracts of Coccoloba mollis, under the described experimental conditions, are not mutagenic.     

Stimulation of mutations suppressing the loss of replication control by small alcohols

Transient exposure of lysogenic Escherichia coli cells to small alcohols stimulated the frequency of mutations suppressing the lethal loss of replication control from a prophage fragment of bacteriophage lambda. The stimulation in mutation frequency paralleled the effect of mutagenic agents, and in this sense the alcohols behaved as mutagens. 10-min treatments above distinct threshold concentrations at 23%, 18%, 10% and 4% (v/v) were required in order for methanol, ethanol, isopropanol and propanol to evoke mutagenic effects. The selected mutant cells were, in general, equally or more sensitive to ethanol than the starting cells. The mutagenicity of methanol and ethanol was detected only with E. coli strains with lambda fragments that included the site-specific and general recombination genes found within the phage int-kil gene interval; whereas, stimulation of the frequency of phenotypically identical mutations by nitrosoguanidine or ionizing radiation did not require that the lambda fragment encode these genes. Treatments of lysogenic cells with mutagenic concentrations of ethanol did not trigger prophage induction and were concluded not to induce a cellular SOS response nor to denature the prophage repressor, or to disrupt repressor-operator binding. The toxicity of ethanol was pH-dependent. Cellular sensitivity to ethanol toxicity was unaffected by the integrated lambda fragment(s) or by an intact lambda prophage; but, it was increased by deletions of the E. coli chromosome extending rightward from bio into uvrB, and rightward from chlA.     

Frameshift mutagenesis by nitracrine analogues in wild-type, uvrB, polA and recA strains of Salmonella typhimurium, with and without plasmid pKM101

The mutagenic potential of 9-[(3-dimethylaminopropyl)amino]-acridine and its 1-, 2-, 3- and 4-nitro derivatives was studied in several strains of Salmonella typhimurium carrying the frameshift marker hisC3076. The strains all carried deep rough (rfa) mutations, and were either wild-type with respect to DNA repair capacity or carried recA, uvrB, polA1 or polA3 (amber) mutations. Derivatives with and without plasmid pKM101 were also studied. The des-nitro compound resembled 9 aminoacridine and other simple intercalating compounds. Both toxicity and mutagenesis were apparently unaffected by the uvrB and recA mutations or by the presence of plasmid pKM101. However, mutagenicity was reduced by the polA1 mutation, and virtually eliminated by the polA3 mutation. The drug was substantially more toxic in the latter, slightly more toxic in the former, of these polA- strains. Plasmid pKM101 enhanced mutagenesis and protected from toxicity in both polA1- and polA3- strains, although it did not restore either of these parameters to the level in the wild-type strain. The 2-nitro compound was generally similar to the des-nitro compound, except that it was considerably more toxic and apparently non-mutagenic in the recA-bearing strain. By contrast, mutagenicity of the 3- and 4-nitro compounds was enhanced by the uvrB mutation and by the presence of the plasmid. These compounds were highly toxic but non-mutagenic in the recA- strain, and showed some increased toxicity in polA1- and polA3- strains. The 1-nitro compound has been previously found to cross-link DNA. Unlike well-characterised cross-linkers such as mitomycin C it was highly mutagenic in the uvrB- strain, and this mutagenesis was enhanced by plasmid pKM101, but eliminated by the recA mutation. At high doses, where the drug was completely toxic towards uvrB- or recA-carrying strains, it became mutagenic in the DNA-repair-proficient strains. This 'high-dose' mutagenesis was enhanced by plasmid pKM101, but reduced by the polA1 mutation and almost eliminated by the polA3 mutation. Although there are several possible interpretations of these data, they are compatible with the suggestion that the lesion induced by high doses (but not by low doses) of nitracrine is a cross-link, but that this is not the major mutagenic lesion.     

Toxicity, mutation frequency and mutation spectrum induced by dacarbazine in CHO cells expressing different levels of O(6)-methylguanine-DNA methyltransferase

The toxicity and mutagenicity (including the mutation spectrum induced) of dacarbazine, a methylating cytostatic drug, was examined in CHO cells expressing different levels of the repair enzyme O(6)-methylguanine-DNA methyltransferase (MGMT). Expression of low or high levels of a transfected human MGMT gene under the control of the metallothionein promoter protected the cells against dacarbazine-induced toxicity and mutagenesis. In the absence of MGMT expression, the mutation spectrum in the HPRT locus was dominated by GC-->AT transitions (mostly found at 5'Pu-G sequences), while there were also a few AT-->GC transitions. Expression MGMT was associated with a substantial decrease of GC-->AT mutations, suggesting that these mutations arose primarily via O(6)-methylguanine. These data illustrate the important role of the latter lesion in the drug's mutagenic and cytotoxic activity.     

Isolation of the mutagenic and DNA adduct-inducing components from a commercial preparation of HC blue 1 using Salmonella (TA98) bioassay-directed HPLC fractionation.

In the present study we report the separation of the mutagenic impurities from the nitrophenylenediamine hair dye HC Blue 1. This was accomplished by bioassay-directed HPLC fractionation, using Salmonella strain TA98 and reverse phase HPLC analysis. The mutagenic fraction eluted between 80 and 90% methanol, whereas the HPLC fraction containing the parent compound HC Blue 1 eluted with 30% methanol and was non-mutagenic. 100% of the mutagenic activity applied to the column was recovered in fractions that did not possess the blue color of HC Blue 1. Also, HPLC-purified HC Blue 1 did not form DNA adducts (32P-postlabeling) in Salmonella strain TA98. On the other hand, commercial HC Blue 1 and the mutagenic fraction derived from commercial HC Blue 1 (HPLC-isolated) gave similar DNA-adduct profiles that consisted of 7 adducts. DNA adduction was examined concomitantly with mutagenicity and toxicity studies on the HC Blue 1 samples in TA98. The data indicated that, in Salmonella, both the mutagenicity and DNA adduction of commercial HC Blue 1 are due to impurities and not the parent compound.     

Evaluation of mutagenic activities of leachates in landfill sites by micronucleus test and comet assay using goldfish

To develop a simple system for monitoring the presence of mutagens/carcinogens in the leachates from landfill sites, we used a micronucleus test and a single cell gel electrophoresis (comet) assay originally developed for mice and rats on goldfish (Carassius auratus). The goldfish were exposed for 9 days to the leachate with chemical and biological treatment (treated leachate) or without treatment (raw leachate). The goldfish exposed to several samples died because of the high concentrations of NaCl or ammonium ion (NH4+). In the comet assay using peripheral erythrocytes, the raw leachates showed higher mutagenic activity than the treated leachates. In the micronucleus test, it was difficult to detect the micronuclei in peripheral erythrocytes. On the other hand, the frequency of micronuclei was high in gill cells of goldfish exposed to the raw leachates compared to the treated leachates. A combination of the two bioassays was shown to be useful to evaluate the mutagenic activity of the leachates. We also propose a new scoring method for determination of water quality by using acute toxicity and mutagenic activity.     

Evaluation of mutagenic and cytotoxic effects of sodium fluoride on mammalian cells influenced by an acid environment

The mutagenic activity of sodium fluoride at reduced pH was studied in the V79/HGPRT system. Statistical analysis of the results of mutagenicity testing suggests that, despite its high toxicity, sodium fluoride has no mutagenic effects at reduced pH on hamster V79 cells. Short-term treatment of cells with sodium fluoride at reduced pH inhibits growth activity of cells as well as synthesis of pulse-labeled nascent DNA and cumulative RNA synthesis and proteosynthesis. From the results of this study we suggest that an acid environment which supports formation of hydrogen fluoride increases toxic but not mutagenic potencies of sodium fluoride.     

Microsomal activation to mutagens of antischistosomal methyl thioxanthenones and initial tests on a possibly non-mutagenic analogue.

Five methylthioxanthenone and methylbenzothiopyranoindazole analogues, including lucanthone (Miracil D), are non-mutagenic for Salmonella typhimurium but are activated to mutagens by a rat liver microsome preparation. Hydroxymethyl analogues, including hycathone (Etrenol), are mutagenic in the absence of microsomes. It seems reasonable to assume that the hydroxymethyl derivatives are the more proximal mutagens and that Salmonella is unable to carry out the hydroxylation necessary for mutagen activation. During the pase 24 years, several million patients with schistosomiasis have been treated with lucanthone, and in recent years about 700 000 persons with hycanthone. The possible long-term deleterious effects of these agents for man even now remain to be determined. Our studies indicate that particular modifications in the structure of thioxanthenones drastically alter their mutagenicity. One apparently non-mutagenic thioxanthenone has been found. A number of the less mutagenic compounds also exhibit decreased acute toxicity in the mouse while retaining appreciable antischistosomal activity, suggesting that genetic and schistosomicidal activities may be dissociated from each other.