The mutagenicity of urine fractions from patients administered antineoplastic therapy

A concern among hospital personnel is their exposure to mutagenic drugs and in the incidental exposures that could occur in caring for the patients. In a recent published study the mutagenicity of urine from patients administered antineoplastic drugs was determined and techniques were developed to chemically inactivate the mutagenicity. A question still remained as to what components of the excreted urine were mutagenic. Urine samples from patients receiving mutagenic drugs were fractionated by high pressure liquid chromatography (HPLC) to then assay by the Ames test the collected and concentrated fractions to determine what were the mutagenic compounds in the urine. Urine samples from patients on single agent cancer treatment with cisplatin, cyclophosphamide, doxorubicin and mitomycin C were assayed. In general, all urine samples containing the cytotoxic agents studied were mutagenic because of the presence of the parent compound, except cyclophosphamide which requires activation and therefore an active metabolite was the major mutagenic constituent in the urine sample. This data indicates that the mutagenicity of urine from patients receiving these antineoplastic agents is the result of the parent compound or a single major metabolite.     

Amplification of doxorubicin mutagenicity by cupric ion.

There is a presumption that copper and anthracycline drugs will interact with DNA to produce genotoxicity. This is of concern because serum copper levels are increased in certain neoplastic diseases. To test the interaction, it was determined if the metal ion could alter the mutagenesis of doxorubicin and related drugs in the Salmonella microsome test. In the standard form of the test, doxorubicin was strongly mutagenic against frameshift-sensitive strain TA98. When cupric acetate was added with doxorubicin it amplified the mutagenesis of the antineoplastic with an increase of approximately 19% in peak mutagenic values. This apparent "chemoactivation" was evaluated by additional applications of the Salmonella test. Preincubation of cupric acetate, drug, and bacteria (+/- rat liver S9 fraction) also resulted in a copper-amplifying effect. In the preincubation method copper produced a drug concentration-related increase of more than 700% in the mutagenicity of doxorubicin. This large an increase occurred without S9 in the test. The effects observed in TA98 were not seen with TA102, a strain sensitive to oxidation mechanisms. Copper amplification in the mutagenicity of a positive control, aflatoxin B1, was also observed with TA98 but these effects were not seen with the chelator, EDTA, the antifolate antineoplastic drug, methotrexate, or a test negative amino acid, methionine. Results point to a direct frameshift mechanism to explain the increase in mutagenicity with copper. Amplification of mutagenicity found in this study provides initial experimental support that anthracycline-metal ion-DNA associations might contribute to genotoxicity as has been inferred in the literature.     

The Salmonella mutagenicity of water and sediments from the Porsuk River in Turkey

In this study, water and sediments from the Porsuk River were investigated for their potential mutagenicity in Salmonella typhimurium TA 98 and TA 100 strains by performing Ames test (plate incorporation assay) without metabolic activation. Different columns of XAD 4 and XAD 16 were used to fractionate the water samples. Positive results in XAD 4 extracts of water samples were obtained for TA 98 in two of these stations. Extracts of the sediment samples were assayed in five different doses of concentrations and mutagenic results were obtained for both of the two strains in different sampling sites. Total metal concentrations in the stream sediment samples were determined by atomic absorption spectrophotometry (AAS) in order to explain some of these mutagenic results. The presence of mutagens causing frameshift and base-pair substitution mutations in water and sediments of the Porsuk River was suggested by the results and this mutagenicity was discussed.     

Effect of pyrolysis temperature on the mutagenicity of tobacco smoke condensate.

Tobacco smoke aerosols with fewer mutagens in the particulate fraction may present reduced risk to the smoker. The objective of this study was to test the hypothesis that the temperature at which tobacco is pyrolyzed or combusted can affect the mutagenicity of the particulate fraction of the smoke aerosol. Tobacco smoke aerosol was generated under precisely controlled temperature conditions from 250 to 550 degrees C by heating compressed tobacco tablets in air. The tobacco aerosols generated had a cigarette smoke-like appearance and aroma. The tobacco smoke aerosol was passed through a Cambridge filter pad to collect the particulate fraction, termed the smoke condensate. Although condensates of tobacco smoke and whole cigarette mainstream smoke share many of the same chemical components, there are physical and chemical differences between the two complex mixtures. The condensates from smoke aerosols prepared at different temperatures were assayed in the Ames Salmonella microsome test with metabolic activation by rat liver S9 using tester strains TA98 and TA100. Tobacco smoke condensates were not detectably mutagenic in strain TA98 when the tobacco smoke aerosol was generated at temperatures below 400 degrees C. Above 400 degrees C, condensates were mutagenic in strain TA98. Similarly, condensates prepared from tobacco smoke aerosols generated at temperatures below 475 degrees C were not detectably mutagenic in strain TA100. In contrast, tobacco tablets heated to temperatures of 475 degrees C or greater generated smoke aerosol that was detectably mutagenic as measured in TA100. Therefore, heating and pyrolyzing tobacco at temperatures below those found in tobacco burning cigarettes reduces the mutagenicity of the smoke condensate. Highly mutagenic heterocyclic amines derived from the pyrolysis of tobacco leaf protein may be important contributors to the high temperature production of tobacco smoke Ames Salmonella mutagens. The relevance of these findings regarding cancer risk in humans is difficult to assess because of the lack of a direct correlation between mutagenicity in the Ames Salmonella test and carcinogenicity.     

N,N-diethylphenylacetamide, an insect repellent: absence of mutagenic response in the in vitro Ames test and in vivo mouse micronucleus test

N,N-diethylphenylacetamide (DEPA), a promising new insect repellent, was tested for mutagenicity in the in vitro Ames Salmonella/microsome mutagenicity test and the in vivo mouse micronucleus test. For the Ames test, DEPA was assayed both in the presence and absence of Aroclor 1254-induced rat-liver S-9 mix (5 and 20% S-9 fraction), using five tester strains of Salmonella typhimurium--TA97a, TA98, TA100, TA102 and TA104. For the micronucleus test, mice were exposed to DEPA through ip injection for 2 and 5 days in separate experiments, and bone marrow and peripheral blood were sampled 6 and 48 hr after the final injection, respectively. DEPA did not induce a mutagenic response in the Ames test, and mouse bone marrow and peripheral blood micronucleus tests. DEPA was not considered cytotoxic, as a depression of the percentage PCE was not observed at any dose in the range of 1 to 100 mg/kg body weight with either treatment protocol of the micronucleus test.     

Study of mutagenicities of phthalic acid and terephthalic acid using in vitro and in vivo genotoxicity tests

Genotoxicities of phthalic acid (PA) and terephthalic acid (TPA) were examined using three mutagenicity tests: Ames, chromosome aberration (CA), and micronucleus (MN). In the Ames test, these two agents did not produce any mutagenic responses in the absence or presence of S9 mix on the Salmonella typhimurium strains TA98, TA100, TA102, TA1535, or TA1537. The CA test also showed that PA and TPA exerted no significant cytogenetic effect on Chinese hamster ovary (CHO) cells. In the mouse MN test, no significant alteration in occurrence of micronucleated polychromatic erythrocytes was observed in ICR male mice ip administered any of these agents at doses of 0, 20, 100, 500, 2500 or 12,500 microM/kg. These results indicate that PA and TPA produced no mutagenic effects using these in vitro and in vivo mutagenic test systems.     

Mutagenic activity of the feces of rats following oral administration of tartrazine

After oral administration of the azo dye tartrazine, bile and feces of treated rats were investigated for mutagenicity using the Ames test withSalmonella typhimurium strains TA 98 and TA 100 with and without metabolic activation. In the presence of S 9-mix fecal extracts developed a weak but reproducible dose-related response in strain TA 100. In bile no metabolites exerting mutagenic activity were found.     

Comparative mutagenicity studies of azo dyes and their reduction products in Salmonella typhimurium

The arabinose-resistant and Ames assay systems of Salmonella typhimurium were used to evaluate the mutagenic potential of azo dyes and their aromatic amine reduction products. Azo dyes, namely direct black 38, direct blue 15, and direct red 2, were mutagenic in the arabinose-resistant and Ames assays with both hamster and rat liver S9 activation. Both assays gave relatively higher mutagenic responses with hamster S9. Reduction products of these dyes, namely benzidine, o-dianisidine, and o-tolidine, were mutagenic in the Ames assay. Benzidine was weakly mutagenic and o-dianisidine and o-tolidine were nonmutagenic in the arabinose-resistant assay. These results indicate that both arabinose-resistant tester SV50 and Ames tester TA98 were sensitive in detecting mutagenicity of azo dyes. The use of the standard plate protocol with Ames tester TA98 is more efficient than the modified azo dye protocol in detecting mutagenicity of aromatic amine reduction products. Additional modifications in either the standard plate or modified azo dye protocols may improve detection of mutagenicity of these compounds in the arabinose-resistant assay system.     

Biological and chemical studies on overheated brewed coffee

Vapour formed from overheated decaffeinated coffee was condensed and tested for mutagenicity using the Ames assay in Salmonella typhimurium strains TA98 and TA100. Vapour produced at 73 and 100 degrees C exhibited no mutagenicity. The basic fraction of vapour produced at 350 degrees C showed weak mutagenicity towards strains TA98 with metabolic activation. The chemical analysis of this fraction identified pyridines and pyrazines as the major constituents. None of the compounds identified in this fraction has been reported as mutagenic when tested in the Ames assay.     

Mutagenicity of Mouriri pusa Gardner and Mouriri elliptica Martius

Mouriri pusa Gardner and Mouriri elliptica Martius are fruit-bearing plants of the Melastomataceae family, popularly known in Brazil as puçá-preto or jaboticaba-do-cerrado, and they are used in folk medicine for the treatment of gastric ulcers. In this study, we employ the Ames test to assess the mutagenicity of compounds obtained from the leaves of these species. The methanol extract of the M. pusa was mutagenic to the Salmonella typhimurium strains TA98, TA97a and TA100, with or without metabolic activation. The methanol extract of M. elliptica induced mutagenic activity in TA98 when metabolized with S9 fraction and TA97a with and without S9, but with lower mutagenicity index (MI) and potencies values than those for M. pusa. Enriched fractions of flavonoids and tannins of M. pusa were also evaluated and they demonstrated positive mutagenicity. The highest values of MI and potency were obtained with the flavonoid fraction, which contains large amounts of quercetin, quercetin glycosides and myricetin. These compounds are probably related to the mutagenicity observed in the Ames test. The dichloromethane extract was not mutagenic in any of the test conditions employed.     

The mutagenic properties of N-nitrosopeptides in the Ames test

N-(N-Acetyl-L-prolyl)-N-nitrosoglycine (APNG) and N-(N-acetylvalyl)-N-nitrosoglycine (AVNG) are shown to exert mutagenic activity in the Salmonella/mammalian microsome mutagenicity (Ames) test. Positive responses are apparent for base-pair substitution mutation-detecting strains (TA1535, TA100 and TA102) both with and without the addition of S9-mix. It is concluded that both APNG and AVNG are direct-acting mutagens.     

Mutagenicity of thionitrites in the Ames test. The biological activity of thiyl free radicals

Thiols, such as glutathione and cysteine, are mutagenic in the Ames test, using Salmonella typhimurium strain TA 100 and rat kidney S-9 preparation [Glatt et al. Science 220, (1983)]. Formation of thiyl free radicals, RS., has been implicated in this effect. We have prepared thionitrite (nitrosylmercaptan) derivatives of glutathione and other thiols. These unstable derivatives decompose by homolysis, yielding RS. radicals. Glutathione thionitrite is mutagenic to strains TA 100 and TA 102, in the absence of activation by mammalian S-9 preparations. We suggest that this mutagenicity is evidence for the role of thiyl free radicals as biological reactive intermediates. Since alkyl nitrites readily convert thiols to thionitrites, our findings have implications for the toxicology of nitrosating drugs, such as amyl nitrite.     

A comparative study, with 40 chemicals, of the efficiency of the Salmonella assay and the SOS chromotest (kit procedure)

A comparison was made, for 40 compounds belonging to different chemical classes, of the mutagenicity as measured by the Salmonella assay and of the SOS-inducing potency as measured by the SOS chromotest kit procedure. It was found that most (78%) of the chemicals described as mutagens/carcinogens (14 compounds) were detected with a simplified Ames test procedure, using 3 strains (TA 97, TA 98, TA 100) and 3 concentrations of the tested material. The SOS chromotest, carried out following the recommendations of the commercially available kits, revealed that only 4 Ames test-positive compounds were mutagenic towards E. coli strain PQ 37.     

2-(N-nitroso-N-methylamino)propiophenone, a direct acting bacterial mutagen found in nitrosated Ephedra altissima tea

A new N-nitroso compound identified in a nitrosated tea extract made from the plant Ephedra altissima and shown to be formed under in vivo conditions was identified as 2-(N-nitroso-N-methylamino)propiophenone (NMAP). N-Nitrosoephedrine (NEP), another N-nitroso compound detected in nitrosated Ephedra altissima tea and NMAP are shown to exert mutagenic activity in the Salmonella/mammalian microsome mutagenicity (Ames) test. Base-pair substitution mutation-detecting strains (TA100 and TA1535) showed both compounds to be weak direct-acting mutagens without the addition of S9-mix. The identification, synthesis and mutagenicity of NMAP are discussed.     

Effects of using liver fractions from different mammals, including man, on results of mutagenicity assays in Salmonella typhimurium

Twenty chemicals, including 16 aromatic amines, were studied in the Salmonella/mammalian-microsome mutagenicity test using the bacterial strains TA100 and TA98 to compare the activation potential of liver preparations from several mammalian species. The hepatic post-mitochondrial supernatants (S-9 fractions) of rat, mouse, hamster, dog, monkey and man were used for metabolic activation. Striking quantitative and even qualitative differences were apparent in the capacity of the different preparations to activate the compounds to mutagens. All compounds that gave positive results in the Ames test when activated with a liver preparation from Aroclor-pretreated rats were also identified as mutagens when tested in the presence of S-9 from one or more other species. Four substituted anilines, however, were converted to mutagenic metabolites only in the presence of a post-mitochondrial fraction of hamster liver. Three human carcinogens, 2-aminoanthracene, benzidine and cyclophosphamide were detected as mutagens under various experimental conditions, including metabolic activation by human or monkey liver S-9. There were no qualitative differences in the mutagenic responses obtained in assays with human and monkey liver S-9.     

Experimental models for the biological detection of N-nitroso compounds formed from amines and nitrite

The secondary and tertiary amines morpholine, aminopyrine and cimetidine as well as their nitroso products were examined for mutagenicity with the Ames Salmonella typhimurium microsome test (strains TA1535 and TA100) and the host-mediated assay. The formation of mutagenic nitroso compounds from morpholine and aminopyrine in the presence of nitrite could be demonstrated in artificial gastric juice and was confirmed in the stomach of mice in vivo. In contrast, the positive response of the chemical nitrosation in vitro with cimetidine did not match with the mammalian host-mediated assay results. To enhance sensitivity the role of modifiers of nitrosation, such as ascorbic acid and thiocyanate as well as the influence of biotransformation were studied.     

Absence of genotoxicity of potato alkaloids alpha-chaconine, alpha-solanine and solanidine in the Ames Salmonella and adult and foetal erythrocyte micronucleus assays.

To assess whether reported toxicities of potato-derived glycoalkaloids could be the result of interactions with cellular DNA, the genotoxic effects of alpha-solanine, alpha-chaconine and solanidine were studied, using the Ames test (Salmonella strains TA98 and TA100), the mouse peripheral blood micronucleus test and the mouse transplacental micronucleus test. The Ames test for mutagenicity with alpha-solanine was weakly positive in TA100 with S-9 activation (29 revertants per millimole per plate). However, pooled data from duplicate tests gave a negative effect. Pooled data from two experiments with alpha-chaconine gave a weak positive response in TA98 without microsomes (17 revertants per millimole per plate). The micronucleus tests for clastogenicity using male mouse and foetal blood were negative. The absence of mutagenicity and clastogenicity suggests lack of damage to intracellular DNA for potato alkaloid toxicity.     

Mutagenicity and cytotoxicity of N-methyl-2-pyrrolidinone and 4-(methylamino)butanoic acid in the Salmonella/microsome assay

The industrial solvent N-methyl-2-pyrrolidinone (NMP) and its hydrolysis product, 4-(methylamino)butanoic acid (N-MeGABA), were examined for mutagenicity and cytotoxicity in the Ames Salmonella/microsome assay. In order to detect a broad range of possible mutagenic endpoints, the following strains were used in the assay: base-pair substitution strains TA100, TA102 and TA104; frameshift strains TA97 and TA98; and repair proficient strains TA2638, UTH8413 and UTH8414. In the standard plate incorporation assay, six log-linear doses of each compound were tested; doses ranged from 0.01 to 1000 mumol/plate for NMP, and 0.01 to 316 mumol/plate for N-MeGABA. Neither compound was detectably mutagenic when tested in the presence and absence of metabolic activation by Aroclor-induced rat liver S9. NMP did show significant responses with strains TA102 and TA104 that were less than two-fold over background, but no clear dose-response relationships were evident. A preincubation modification of the assay was also performed, using strains TA98 and TA104. Mutagenic activity was not observed for NMP, while N-MeGABA showed significant responses with TA104 but dose-related mutagenicity was not established. Preincubation testing revealed both NMP and N-MeGABA to be cytotoxic to the test population of Salmonella at the highest treatment doses.     

Mutagenicity and anti-mutagenicity of Acanthopanax divaricatus var. albeofructus

The beneficial effects of Acanthopanax divaricatus var. albeofructus (ADA) extracts have been assessed by mutagenic and anti-mutagenic activities by Ames test. Mutation of Salmonella typhimurium strains TA 98, TA 100, TA1535, TA1537, and Escherichia coli WP2 uvr A was assayed in duplicates by the procedure of Maron and Ames in the presence or absence of S9 mix. As a result, ADA extracts were not mutagenic for S. typhimurium strains TA 98, TA 100, TA1535, TA1537, and E. coli by the Ames assay. Anti-mutagenic activity was assayed by the Ames mutagenicity assay using histidine mutant of S. typhimurium strains TA 98 and TA 100, using the plate-incorporation method. 2-Aminoanthrancene (2-AA), 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2), and sodium azide (NaN(3)) were used as the mutagens. ADA extracts showed a strong anti-mutagenic activity against 2-AA-induced mutagenesis which requires liver-metabolizing enzymes, and the same extract exhibited inhibitory effects on AF-2 and NaN(3)-induced mutagenesis in the absence of liver-metabolizing enzymes. The data indicate that ADA extracts contain anti-mutagenic activities against typical mutagens. The anti-mutagenic property of ADA provides additional health supplemental value to the other claimed therapeutic properties of the plant.     

Monitoring sãto paulo state rivers in brazil for mutagenic activity using the ames test

Organic extracts of raw water from 11 water courses of São Paulo State, Brazil, were collected during one year bimonthly and tested for mutagenicity using the Ames test, with strains TA98 and TA100 of Salmonella typhimurium with and without metabolic activation. The samples were extracted with XAD₂ resin and eluted with methanol and methylene chloride. From the 75 samples analyzed, 14 showed positive responses and 8 were considered marginal, making up 29% of mutagenic samples. The percentage of mutagenic samples in October (spring) was 9%, increasing to 64% in February (summer), and decreased to 9% again in August (winter). Paraiba do Sul river showed the higher percentage of mutagenic samples (67%) and Capivari river the highest mutagenic sample (1787 and 3265 revertants per liter for TA98 without and with S9, respectively). The amplitude of the mutagenic response was 39–3265 revertants per liter for TA98 and 83–467 for TA100. The mutagenic samples showed direct and indirect mutagens, and TA98 detected the majority of responses, indicating prevalence of frameshift mutagens in these samples. © 1993 John Wiley & Sons, Inc.