Genotoxic activity of m-nitrobenzaldehyde

m-Nitrobenzaldehyde (MNB) was evaluated for mutagenic activity using the Ames microbial mutagenicity test and for its ability to induce DNA single-strand breaks in rat hepatocytes as measured by alkaline elution. MNB was tested in S. typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100, both with and without pretreatment with liver microsomes (S9) isolated from rats pretreated with Aroclor 1254. MNB produced 2-fold or greater increases in revertants in TA1538, both with and without S9, and in TA100 with S9 only. A 2-fold increase in revertants was seen in TA98, but only at the highest dose tested which did not produce inhibition of background growth. MNB caused a greater than 3-fold increase in elution slope, with DNA alkaline elution assay, but only at highly cytotoxic doses and, therefore, is not considered genotoxic in this system. It is concluded that MNB possesses weak genotoxic activity.     

A comparative analysis of mutagenic and SOS-induced activity in three classes of chemical compounds

Mutagenic and SOS-inducing potential of 23 derivatives of fluorenone, phenanthrenequinone and biphenyl have been studied in tester strains of Salmonella typhimurium and in Escherichia coli strain PQ 37. 14 of these compounds revert the mutation hisD3052 (much less than -1 much greater than type), but none of them induce mutations in the strain TA 1535. Maximal mutagenic activity has been shown in strain TA 1538 for amide of 2,7-dinitrofluorenone-4-carbonic acid (580 revertants per nmol), 2,7-dinitrophenanthrenequinone (308 revertants per nmol), 2,4,7-trinitrophenanthrenequinone (306 revertants per nmol) and 2',4,4'-trinitrobiphenyl-2-carbonic acid (251 revertants per nmol). In plasmid-containing strain TA 98 the mutagenic potential of the compounds tested is lower than in the TA 1538 strain. It has been suggested that mutagenic activity of these compounds can be attributed to their acceptor properties, namely, the ability to form charge transfer complexes with DNA. SOS-inducing activity has been shown for 5 compounds, also positive in mutation induction. Mutagenic and SOS-inducing activities positively correlate in fluorenone derivatives. Among phenanthrenequinone derivatives, compounds with high mutagenic activity only can induce SOS response. None of the biphenyls tested induce SOS functions. The compounds giving the positive result in the SOS-chromotest have rigid co-planar structure.     

Mutagenicity of urine from rats after 1-nitropyrene and 2-nitrofluorene administration using new sensitive Salmonella typhimurium strains YG1012 and YG1024

1-Nitropyrene (1-NP) and 2-nitrofluorene (2-NF), two of the most abundant nitro-substituted polycyclic aromatic hydrocarbons (nitro-PAH) present in combustion products such as diesel engine exhaust, were administered intraperitoneally to rats at a dose of 5 mg per animal. Urine samples, 1-NP and 2-NF were tested in the Ames assay using the newly developed Salmonella typhimurium strains YG1012 and YG1024 (overproducing O-acetyltransferase) and their parent strains TA1538 and TA98. In urine, collected over 3 periods of 24 h after administration, most of the mutagens appeared during the first 24 h. The mutagenicity was found to be a factor 2-30 higher in the YG strains when compared to the TA strains. Addition of S9 mix and rat liver cytosol both with and without beta-glucuronidase increased the mutagenicity of urine samples from 1-NP-treated rats. Addition of beta-glucuronidase revealed that a considerable part of the mutagenic metabolites of 1-NP and 2-NF were excreted as glucuronide conjugates. The increase in mutagenicity of urine samples from 2-NF-treated rats after the addition of rat liver cytosol referred to N,O-acyl transfer as a step in activating 2-NF to strong mutagens. The high sensitivity of the YG tester strains indicated that these strains might be used to explore environments where people are exposed to nitro-PAH, such as work places with diesel emission sources.     

Mutagenicity of nitro derivatives induced by exposure of aromatic compounds to nitrogen dioxide

Mutagenic nitro derivatives were readily induced when 6 kinds of chemicals were exposed to 10 ppm of nitrogen dioxide (NO2). Single nitro derivatives were formed from pyrene, phenanthrene, fluorene or chrysene. Carbazole and fluoranthene each produced 2 derivatives substituted with nitro groups at different positions. The formation of nitro derivatives was enhanced by exposure of pyrene to NO2 containing nitric acid (HNO3, less than 100-fold enhancement) or sulphur dioxide (SO2, less than 15-fold enhancement). After 24 h of exposure the yields of the nitro derivative were 0.02% with 1 ppm of NO2 in air and 2.85% with NO2 (1 ppm) containing traces of HNO3. The nitro derivatives from all but phenanthrene and carbazole were chemically identified by means of gas chromatography (GC) and mass spectrometry (MS), and the mutagenicity of the 4 kinds of authentic nitro derivatives was tested by using Salmonella strains TA98 and TA1538 with or without the S9 fraction from rat liver treated with Aroclor 1254. The nitro derivative induced from pyrene was determined to be 1-nitropyrene; that of chrysene was 6-nitrochrysene; that of fluorene was 2-nitrofluorene; and those of fluoranthene were 3-nitrofluoranthene, and 8-nitrofluoranthene. Tested with strain TA98 in the absence of the S9 fraction, the first 4 of these derivatives yielded, respectively, 3050, 269, 433 and 13 400 revertants per nmole. Thus, each nitro derivative formed was potentially a direct-acting frameshift-type mutagen. Each compound exposed to NO2 showed a decreased mutagenic activity when tested in the presence of S9 mix. A possible explanation comes from experiments in which 1-nitropyrene was incubated with the S9 mix at 37 degree C for 10 min, and 1-aminopyrene was formed. The mutagenic activity of 1-aminopyrene was appreciable, but only about one-tenth of that of 1-nitropyrene in the Ames test.     

The unusual effect of pKM101 on the mutagenicity of acetaldehyde oxime in Salmonella typhimurium

Acetaldehyde oxime was found to induce more revertants in Salmonella typhimurium strain TA1535 than in TA100 in the absence of S9 metabolic activation. TA100 was originally constructed from TA1535 by the addition of the plasmid pKM101, carrying mucAB which generally enhances sensitivity to the mutagenic effects of chemicals. The role of pKM101 in lowering the sensitivity to acetaldehyde oxime was explored by: (1) increasing the incubation time of the selective agar plates from 2 to 3 days; (2) using a new strain, isogenic to TA100, constructed by introducing pKM101 into the TA1535 isolate used in these experiments; (3) by testing a strain constructed by inserting into TA1535 a plasmid carrying mucAB but otherwise unrelated to pKM101. Each of these alterations increased the number of revertants per plate in the presence of acetaldehyde oxime, indicating that the apparent nonmutagenicity of this chemical in TA100 is due to multiple factors.     

A sensitive method for the detection of mutagenic nitroarenes: construction of nitroreductase-overproducing derivatives of Salmonella typhimurium strains TA98 and TA100

'Classical nitroreductase' is an enzyme involved in the intracellular metabolic activation of mutagenic nitroarenes. The nitroreductase gene of Salmonella typhimurium TA1538 was cloned into pBR322 and the plasmids harboring the gene were introduced into TA98 and TA100. The resulting strains (YG1021 and YG1026) had more than 50 times higher nitrofurazone-reductase activity than TA1538 containing pBR322, and were extremely sensitive to the mutagenic action of 2-nitrofluorene, 1-nitropyrene and 2-nitronaphthalene. These results indicate that the new strains permit the efficient detection of mutagenic nitroarenes.     

In vitro synthesis of 16 alpha-hydroxyestrone by female rat liver microsomes: its possible role in the etiology of breast cancer

Liver homogenates from female rat strains (Sprague-Dawley, Wistar and Fisher) were incubated in a NADPH regenerating medium in the presence of labelled and unlabelled estrone. Liver microsomes isolated from male rats and female mice were used as positive controls. Using HPLC and paper chromatography, under the experimental conditions used it was found that liver homogenates from female rats were able to convert estrone to various metabolites such as 16 alpha-hydroxyestrone. In a mutagenicity assay (Ames test), with 16 alpha-hydroxyesterone as test substance, two strains (TA98 and TA1538) of the five strains tested showed a 2-3-fold increase in the number of his+ revertants relative to the control values. Estrone did not cause any mutagens in the test used. It is concluded that female rats are able to synthesize 16 alpha-hydroxyestron in vitro. Whether this compound is risk factor for breast cancer remains unclear.     

Epidermal enzyme-mediated mutagenicity of the skin carcinogen, 2-aminoanthracene

Using four Salmonella typhimurium tester strains (TA1537, TA1538, TA98 and TA100) and the promutagen 2-aminoanthracene, an epidermal S9-mediated mutagenicity assay was developed. Using an activation mixture derived from whole skin of the rat, mutagenicity was observed in tester strain TA98 whereas an activation mixture derived from the dermis resulted in mutagenicity in tester strains TA1538, TA98 and TA100. Activation mixtures from both the epidermis and the liver produced a positive response in all of the tester strains studied. Activation mixtures from liver were shown to have the highest specific activity followed in decreasing order of potency by epidermis, dermis and whole skin. These results indicate that the skin, a target tissue directly exposed to environmental chemicals, is capable of converting 2-aminoanthracene to mutagenic moieties. Since the skin of the rat is known to be susceptible to tumor induction by 2-aminoanthracene our findings re-emphasize that membrane-bound enzymes can influence toxic responses including mutagenicity to xenobiotics in cutaneous tissue.     

Mutagenicity of azo dyes in the Salmonella/microsome assay using in vitro and in vivo activation

The mutagenicity of 6 azo dyes, including direct black 38 (DB38), direct black 19 (DB19), direct brown 95 (DB95), solvent yellow 3 (SY3), trypan blue (TPB), and food black 2 (FB2), was examined in the Salmonella/microsome assay. The effect of chemical azo reduction (dithionite) and in vivo metabolism on the mutagenicity of the dyes was also studied. In vivo azo-dye metabolites were isolated from the urine of rats intubated with dyes by XAD-2 column chromatography. Urinary metabolites from all the treated animals, except animals treated with FB2, induced frame-shift mutations in strains TA1538 and TA98 in the presence of liver S9 activation. The control urine did not increase the incidence of revertants in strains TA1538 and TA98. Thus, XAD-2 chromatography can be used to isolate genotoxic metabolites from the urine of animals intubated with azo dyes.     

Sensitive method for the detection of mutagenic nitroarenes and aromatic amines: new derivatives of Salmonella typhimurium tester strains possessing elevated O-acetyltransferase levels

Acetyl-CoA: N-hydroxyarylamine O-acetyltransferase is an enzyme involved in the intracellular metabolic activation of arylhydroxylamines derived from mutagenic nitroarenes and aromatic amines. The acetyltransferase gene of Salmonella typhimurium TA1538 was cloned into pBR322 and the plasmids harboring the gene were introduced into TA98 and TA100. The resulting strains (YG1024 and YG1029) had about 100 times higher 2-hydroxyamino-6-methyldipyrido[1,2-a:3',2'-d]-imidazole (N-hydroxy-Glu-P-1) O-acetyltransferase activity than TA1538 containing pBR322, and were extremely sensitive to the mutagenic actions of 2-nitrofluorene, 1-nitropyrene, 1,8-dinitropyrene, 2-amino-6-methyldipyrido[1,2-a:3',2-d)-imidazole (Glu-P-1), 2-aminofluorene and 2-aminoanthracene. These results indicate that the new strains permit the efficient detection of the mutagenicity of environmental nitroarenes and aromatic amines.     

Bacterial reversion assay and micronucleus test carried out on hydrogenated glucose syrups 'Malti-Towa' (powder) and maltitol crystal

Two preparations of maltitol (4-O-alpha-D-glucopyranosyl-D-sorbitol), hydrogenated glucose syrups and maltitol crystal, were examined for genotoxic potential by a battery of short-term tests. In the bacterial reversion assay, maltitol induced no detectable revertants in any of the tester strains, Salmonella typhimurium TA98, TA100, TA1535, TA1537, TA1538, or Escherichia coli WP2/pKM101 at doses of 0.5-50 mg per plate with and without rat liver S9 mix. In the micronucleus test, no significant increase in the frequency of micronucleated erythrocytes was observed in bone marrow of mice after administration of the two preparations at 3.75-30 g per kg by gastric intubation.     

In vitro mutagenicity and cell-transformation screening of N-(2,3-epoxy-propyl)-phthalimide.

N-(2,3-Epoxy-propyl)-phthalimide (EPP) was tested for genetic activity in the Salmonella/microsome mutagenicity test. Concentration-dependent mutagenicity was demonstrated in S. typhimurium strains TA1535, TA1537 and TA100 with and without rat S9. It was inactive in strain TA1538, and active without rat S9 in TA98 at the high dose. EPP induced 6-thioguanine-resistant mutants of Chinese hamster ovary cells in the absence of an exogenous activating system. EPP produced dose-dependent enhancement of SA7 virus transformation of primary hamster-embryo cells, and transformed secondary hamster-embryo cells in a non-dose-related fashion. At a dose of 5 g/kg p.o. or i.m., EPP was inactive in the host-mediated assay using C57Bl/6XC3H mice and S. typhimurium strain TA1535. Murine testicular DNA synthesis was not inhibited by oral administration of EPP at 1000 mg/kg.     

Optimum associations of tester strains for maximum detection of mutagenic compounds in the Ames test

The Ames test is now widely used as a short-term test for the detection of mutagens. Different strains are available with various genetic characteristics, and in the past decade various authors have recommended different associations of strains to give maximum detection potential. However, few studies have been done to compare the sensitivity of individual strains towards a wide range of compounds in a single study. In order to define the best association of strains for screening or regulatory purpose, we have tested 103 direct mutagens (reference genotoxins or in-house compounds) on 7 strains of Salmonella typhimurium: TA1535, TA1537, TA1538, TA97, TA98, TA100 and TA102. 126 different associations of strains have been studied in terms of sensitivity and percentage overlap. Optimum associations of 2, 3, 4 or 5 strains included strains both with and without plasmid pKM101. However, the specificity of detection is greatly diminished by the presence of plasmid pKM101 in the strain, as shown by the high degree of overlap in associations constituted entirely of strains containing the plasmid. The association of strains TA1538 and TA100 detected 86% of the chemicals tested and is therefore recommended for large-scale screening. A rate of detection of 100% was obtained when 6 strains were used. The best associations of 4 and 5 strains, which detected 97 and 99% chemicals respectively, all contained strains TA1537, TA1538 and TA102. Finally, the associations of 4 strains (TA1537, TA1538, TA100, TA102) or 5 strains (TA1535, TA1537, TA1538, TA97, TA102) seemed well adapted to the optimum detection of mutagenic compounds.     

Glycosphingolipid patterns of the gastrointestinal tract and feces of germ-free and conventional rats

Acid and non-acid glycosphingolipids of stomach, small and large intestine, and stimulated feces of germ-free and conventional rats of the same stain have been isolated and characterized. The glycosphingolipid patterns of the intestinal organs were chemically and immunologically very similar between the two groups of rats and relatively unaffected by the presence of an intestinal microbial flora. The major exception was the presence of hematoside with N-glycoloylneuraminic acid (NeuGc) (NeuGc alpha 2----3Gal beta 1----4Glc beta 1----1Cer) in the stomach of conventional rats not found in the stomach of germ-free animals. Glycosphingolipids of stimulated feces of germ-free animals were derived from epithelial cells mainly of the small intestine and showed no signs of degradation. Glycosphingolipids of feces of conventional rats completely retained the pattern of blood group A-, B-, and H-active glycolipids as found in sterile feces but contained less of hematoside and more of lactosylceramide. This effect was probably due to degradation by bacteria, as demonstrated in vitro with the production of lactosylceramide after treatment of the isolated acid glycolipids of sterile feces with neuraminidase from Clostridium perfringens. The amount of total non-acid glycosphingolipids per dry weight was similar for stomach, was 50% higher for small intestine, and 300% higher for large intestine of germ-free animals compared to conventional animals. Due to the presence of large amounts of mucins the dry sterile feces contained 12% less non-acid glycolipids than conventional feces. However, calculated per rat per day the germ-free animal excreted more of non-acid glycosphingolipids (1.8 and 1.2 mg, respectively).     

Mutagenicity of plant flavonols in the Salmonella/mammalian microsome test: activation of flavonol glycosides by mixed glycosidases from rat cecal bacteria and other sources.

Over 70 naturally occurring and synthetic flavonoids were screened for mutagenicity with 5 tester strains in the Salmonella/mammalian microsome assay: TA1535, TA100, TA1537, TA1538 and TA98. Frameshift mutagenicity was confined to the flavonols (flavon-3-ols) in strain TA98, TA1537 and TA100. The two most mutagenic falvonols, namely, quercetin (3,3',4',5,7-pentahydroxyflavone) and kaempferol (3,4',5,7-tetrahydroxyflavone), exhibiting 12 and 7 revertants/nmol in TA98 respectively, are also the most common flavonols occurring in plants. Other flavonols exhibited less activity (revertants/nmol): galangin (2.0), rhamnetin (0.45), kaempferide (0.24), fisetin (0.14), myricetin (0.12), robinetin (0.06) and morin (0.05). All of these flavonols apparently exhibited significant activation by Aroclor 1254 induced rat-liver microsome preparations (S9). However, subsequent study revealed that only those flavonols either lacking or possessing one B ring hydroxyl group had an absolute requirement for microsomal activation. Alternatively, quercetin with two B-ring OH groups is not activated by microsomal enzymes, but by soluble (S100) enzymes from liver which are apparently constitutive and not subject to the usual chemical induction. 3 flavonol glycosides, namely, quercetrin (quercetin-3-O-rhamnoside), rutin (quercetin-3-O-rutinoside) and robinin (kaempferol-3-O-galactosido-rhamnoside-7-O-rhamnoside), were found to be nonmutagenic. They could, however, be activated by a variety of mixed glycosidases incorporated in the usual pour plate procedure. The most effective enzyme mixtures were obtained from rat cecal bacteria and from the snail Helix pomatia.     

Bacterial mutagenicity of aceanthrylene: a novel cyclopenta-fused polycyclic aromatic hydrocarbon of low molecular weight

Aceanthrylene, a non-alternant cyclopenta-fused hydrocarbon, was shown to be weakly mutagenic without S9 and strongly mutagenic with S9 in the Ames Salmonella plate incorporation assay. The compound was most active in strain TA100 (35 revertants/nmole in the presence of 0.3 mg of S9 protein), and less active in strains TA98, TA1537 and TA1538 (20, 10 and 3.1 rev/nmole respectively, + S9). Strain TA1535 was unresponsive, suggesting that this compound induces frameshift mutations rather than base-pair substitutions. The mutagenic potency of aceanthrylene is consistent with predictions of its activity based on the relatively large delocalization energy (delta E deloc/beta = 0.931) of the carbonium ion which would result from oxirane ring opening of the 1,2-epoxide, a potential active metabolite.     

Failure of liver cirrhosis induction by thioacetamide in Nagase analbuminaemic rats

Repeated administration of thioacetamide (TA), either intraperitoneally or in drinking water, produced liver cirrhosis in normal Sprague-Dawley rats (SDR) with significant histological alterations similar to those observed in human cirrhosis. In the present study, we evaluated the ability of TA to induce liver cirrhosis in mutant Nagase analbuminaemic SDR. Thioacetamide was administered either intraperitoneally up to 4 months or in drinking water up to 6 months to normal and to Nagase analbuminaemic SDR. Nagase analbuminaemic rats (NAR) were also administered TA in drinking water up to 10 months. Liver cirrhosis development was determined by macroscopic and microscopic analysis. In contrast to normal SDR, no histological characteristics of cirrhosis could be observed in NAR submitted to a 4 or 6 months treatment with TA. Such failure to induce cirrhosis in Nagase rats was confirmed even after prolonged TA administration in drinking water for up to 10 months. In contrast, fibrosis and cholangiolar proliferation occurred in the 10-month TA-treated analbuminaemic rats, suggesting that the mechanisms involved in cirrhosis induction are different from those involved in fibrosis development and carcinogenesis. It is unlikely that the protective effect against TA-induced cirrhosis observed in analbuminaemic rats is related to the absence of albumin in this rat strain, since a co-administration of TA with albumin in analbuminaemic rats did not restore the potential for TA to induce cirrhosis in this rat strain. In conclusion, the fact that induction of cirrhosis by TA is prevented in the inherently hyperlipidaemic and hypercholesterolaemic analbuminaemic rats could be considered for potential application in the treatment of clinical cirrhosis.     

Mutagenicity of glyceryl trinitrate (nitroglycerin) in Salmonella typhimurium

The recent finding that the clinical nitrovasodilator, glyceryl trinitrate (GTN), is mutagenic in Salmonella typhimurium strain TA1535 has been examined in closer detail, with emphasis on its mechanism of action. GTN increased the number of His⁺ revertants to a maximum of 4 times over background at a GTN dose of 5 μmol/plate. Hamster liver S9 depressed the toxicity of high GTN doses and increased the maximum number of revertants to 5 times over background at 10 μmol/plate. GTN did not cause significant reversion in any of the six other S. typhimurium strains tested (TA1975, TA102, TA1538, TA100, TA100NR, YG1026), although signs of toxicity were observed. Therefore, the mutagenicity of GTN was manifest only in the repair-deficient (uvrB and lacking in pKM101) strain which is responsive to single base changes. Oligonucleotide probe hybridization of TA1535 revertants showed that virtually all of the GTN-induced mutants contained C → T transitions in either the first or second base of the hisG46 (CCC) target codon, with a preference for the latter. A similar mutational spectrum was seen previously with a complex of spermine and nitric oxide (NO) which releases nitric oxide. This suggests that NO, which can be derived from GTN via metabolic reduction, may be responsible for GTN's mutagenic action. The known NO scavenger oxymyoglobin did not substantially alter the dose response of GTN, indicating that extracellular NO was not mediating reversion. The data are consistent with the hypothesis that intracellular nitric oxide is responsible for the observed mutations.     

Mutagenicity evaluation of the two antimalarial agents chloroquine and mefloquine, using a bacterial fluctuation test.

The two antimalarial agents chloroquine and mefloquine have been tested for mutagenicity in Salmonella typhimurium strains TA1535, TA1537 and TA1538. Chloroquine was found to revert strain TA1537 at concentrations of 100 and 250 micrograms/ml, most likely due to intercalation. No mutagenicity was found with mefloquine at concentrations up to 2.5 micrograms/ml, neither without nor with metabolic activation by Ca2+-precipitated rat liver microsomes. Higher concentrations of mefloquine and chloroquine inactivated the bacteria.     

A comparative study of mutagenic and SOS-inducing activity of biphenyls, phenanthrenequinones and fluorenones

A total of 23 chemicals--biphenyls, phenanthrenequinones and fluorenones--were tested for mutagenicity towards Salmonella typhimurium strains TA1538, TA1535 and TA98. SOS-inducing activity of the same chemicals was studied in terms of the SOS-inducing potency in Escherichia coli PQ37, using an automated instrument controlled by a dedicated computer program for the SOS Chromotest. Of the 23 chemicals studied 14 induced His+ revertants in S. typhimurium TA1538 hisD305 (-1 frameshift); none induced His+ reversions in TA1535 (base-pair substitution). The mutagenicity of the chemicals in S. typhimurium TA98 (pKM 101) was lower than in TA1538. There was a close correlation between mutagenicity and SOS-inducing activity of fluorenones and phenanthrenequinones. None of the biphenyls tested induced SOS response and this property does not depend upon the mutagenic activity of the chemicals. SOS Chromotest is particularly valid in detecting chemicals which give rise to base-pair substitutions through SOS induction. If positive results are obtained, the Salmonella assay may be omitted. However, this test cannot replace the Ames test especially for the primary screening of mutagenicity of chemicals with unknown structure.