Genotoxicity of apomorphine and various catecholamines in the Salmonella mutagenicity test (Ames test) and in tests for primary DNA damage using DNA repair-deficient B. subtilis strains (rec assay)

Apomorphine, N-nor-N-propyl-apomorphine, dopamine, L-DOPA, 6-hydroxydopamine and adrenaline were evaluated for genotoxicity using the Ames test and DNA repair-deficient and DNA repair-proficient Bacillus subtilis strains (rec assay, H17/M45; HLL3g/HJ-15). In the absence of an S9 liver homogenate, apomorphine induced frame-shift mutations in Salmonella typhimurium, mainly in strain TA1537; no indication of DNA-damaging effects in B. subtilis was observed. N-Nor-N-propyl-apomorphine was tested using strain TA1537 only and found to be mutagenic. Dopamine, L-DOPA, 6-hydroxydopamine and adrenaline were non-mutagenic when tested without S9, whereas they were all more toxic for DNA repair-deficient than for DNA repair-proficient B. subtilis strains, indicating a DNA-damaging potential. In a second set of experiments the mode of action of apomorphine and the relevance of the positive Ames test data were investigated. Glutathione in physiological concentrations reduced the mutagenic effect of apomorphine in a dose-dependent way, both in the presence and the absence of S9. S9 also reduced the mutagenicity of apomorphine. By comparing the effects of a complete S9 mix with those of a preparation without glucose-6-phosphate and NADP, it became clear that S9 also had an activating effect, overshadowed under standard conditions by its deactivating activity. Apomorphine was not mutagenic under anaerobic conditions. Superoxide dismutase and catalase reduced the mutagenic effect of apomorphine. All test conditions which reduced the mutagenic effect also inhibited the dark discoloration of the tester plates, indicating a retardation of apomorphine oxidation. It can, therefore, be concluded that oxidation of apomorphine leads to mutagenic products which induce frame-shift mutations in Salmonella typhimurium. This oxidation was prevented both by glutathione in concentrations well below physiological levels and/or by catalase and superoxide dismutase. Under these conditions, apomorphine was non-mutagenic in therapeutic concentrations as well as at higher dose levels. The possibility of genotoxic side effects occurring in patients treated with apomorphine as an emetic drug is therefore considered to be very unlikely.     

Genotoxicity of silver nanoparticles evaluated using the Ames test and in vitro micronucleus assay

Silver nanoparticles (AgNPs) have antimicrobial properties, which have contributed to their widespread use in consumer products. A current issue regarding nanomaterials is the extent to which existing genotoxicity assays are useful for evaluating the risks associated with their use. In this study, the genotoxicity of 5 nm AgNPs was assessed using two standard genotoxicity assays, the Salmonella reverse mutation assay (Ames test) and the in vitro micronucleus assay. Using the preincubation version of the Ames assay, Salmonella strains TA102, TA100, TA1537, TA98, and TA1535 were treated with 0.15-76.8 μg/plate of the AgNPs. Toxicity limited the doses that could be assayed to 2.4-38.4 μg/plate; no increases in mutant frequency over the vehicle control were found for the concentrations that could be assayed. Human lymphoblastoid TK6 cells were treated with 10-30 μg/ml AgNPs, and additional cells were treated with water and 0.73 gy X-rays as vehicle and positive controls. Micronucleus frequency was increased by the AgNP treatment in a dose-dependent manner. At a concentration of 30 μg/ml (with 45.4% relative population doubling), AgNPs induced a significant, 3.17-fold increase with a net increase of 1.60% in micronucleus frequency over the vehicle control, a weak positive response by our criteria. These results demonstrate that the 5 nm AgNP are genotoxic in TK6 cells. Also, the data suggest that the in vitro micronucleus assay may be more appropriate than the Ames test for evaluating the genotoxicity of the AgNPs.     

The anti-mutagenic and antioxidant effects of bile pigments in the Ames Salmonella test

The aim of this study was to explore the potential pro- and anti-mutagenic effects of endogenous bile pigments unconjugated bilirubin (BR), biliverdin (BV) and a synthetic, water soluble conjugate, bilirubin ditaurate (BRT) in the Ames Salmonella test. The bile pigments were tested over a wide concentration range (0.01-2 micromol/plate) in the presence of three bacterial strains (TA98, TA100, TA102). A variety of mutagens including benzo[alpha]pyrene (B[alpha]P), 2,4,7 trinitrofluorenone (TNFone), 2-aminofluorene (2-AF), sodium azide (NaN(3)) and tertiary-butyl hydroperoxide (t-BuOOH), were used to promote the formation of mutant revertants. Tests were conducted with (B[alpha]P, 2-AF, t-BuOOH) and without (TNFone, NaN(3), t-BuOOH) metabolic activation incorporating the addition of the microsomal liver preparation, S9. The bile pigments alone did not induce mutagenicity in any of the strains tested (p>0.05). Anti-mutagenic effects of the bile pigments were observed in the presence of all mutagens except for NaN(3) and the anti-mutagenic effects appeared independent of the strain tested. For TNFone induced genotoxicity, the order of effectiveness was BR> or =BRT>BV. However, the order was BV> or =BRT> or =BR for 2-AF. Antioxidant testing in the TA102 strain revealed bile pigments could effectively inhibit the genotoxic effect of t-BuOOH induced oxidative stress. The apparent antioxidant and anti-mutagenic behaviour of bile pigments further suggests their presence in biological systems is of possible physiological importance.     

Genotoxicity assessment of low-level doses of gamma radiation with the SOS chromotest and the Ames test

This is the first study to present data on the genotoxicity of low γ-irradiation doses for E. coli and S. typhimurium cells obtained using the SOS chromotest and the Ames test. The most pronounced effect was recorded in the first 24 h of γ-irradiation. After 72 h in the Ames test and after 96 h in the SOS chromotest, a significant effect of γ-irradiation on bacterial cells was detected. The absence of genotoxicity at the later stages can be explained by the adaptation of bacterial cells to the conditions of exposure. The findings allow the bacterial test system to be used for studying the effects of low doses at the early stages of exposure to radiation.     

Absence of mutagenic activity of ticlopidine in the Ames Salmonella/microsome test

Ticlopidine hydrochloride, 5-(o-chlorobenzyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine hydrochloride, a platelet aggregation inhibitor, was tested for mutagenic activity in the Ames Salmonella/mammalian microsome test. Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 were employed. Two of these strains (TA1535 and TA100) are sensitive to base-pair substitution mutagens, and the remaining 3 are sensitive to frame-shift mutagens. There was no evidence that ticlopidine hydrochloride had any mutagenic activity either in the presence or absence of a liver microsomal supplement.     

Genotoxicity of six pesticides by Salmonella mutagenicity test and SOS chromotest

Two in vitro tests (Ames test and SOS chromotest), one for bacterial mutagenicity and one for primary DNA damage, were assayed to determine the genotoxic activity of 6 pesticides (atrazine, captafol, captan, chlorpyrifosmethyl, molinate and tetrachlorvinphos). Assays were carried out both in the absence and presence of S9 fractions of liver homogenate from rat (Sprague–Dawley) pretreated with Aroclor 1254. Captan and captafol were genotoxic on both the Ames test and the SOS chromotest. Comparisons with mutagenesis data in Salmonella indicated that the SOS assay detected as genotoxic the pesticides that were mutagenic on the Salmonella test. Non-genotoxic effects were not detected in vitro either in the Salmonella/microsome assay nor in the SOS chromotest when bacterial tester strains were exposed to atrazine, molinate, chlorpyrifosmethyl and tetrachlorvinphos in the absence or presence of S9 mix.     

Mutagenicity tests of diflubenzuron in the micronucleus test in mice,the L5178Y mouse lymphoma forward mutation assay,and the Ames Salmonella reverse mutation test

Diflubenzuron, one of a new class of pesticides believed to act via inhibition of chitin synthesis in the developing insect cuticle, was tested for possible mutagenic activity using the micronucleus test in mice, the L5178Y mouse lymphoma forward mutation test at the thymidine kinase locus, and the Ames Salmonella/microsome reverse mutation test. No mutagenic effect was found.     

Antimutagenic activity of soybeans fermented with basidiomycetes in Ames/Salmonella test

Soybeans fermented with either Phellinus igniarius or Agrocybe cylindracea inhibited the mutagenicity of the directly-acting mutagens: 4-nitro-o-phenylenediamine on Salmonella typhimurium strain TA 98 and NaN₃ on S. typhimurium strain TA 100; and indirectly-acting mutagens, 2-aminofluorene using strain TA 98 and benzo[a]pyrene using strain TA 100, in the presence of a supernatant solution from mammalian hepatic microsomes.     

Mutagenicity of five food additives in Ames/Salmonella/microsome test

The mutagenic activity of five food additives (K₂S₂O₅: potassium metabisulphite, KMB; K₂SO₄: potassium sulphate, KS; Na₂SO₃: sodium sulphite, SS; KNO₃: potassium nitrate, KN; NaNO₃: sodium nitrate, SN) were investigated using histidin auxotrophs TA98 and TA100 strains ofSalmonella typhimurium in the presence or absence of S9 mix. The test substance were investigated for their mutagenic effects at non toxic concentrations of 0.83, 1.66, 3.33 and 5.00 mg/plate with and without S9 mix. All the test substances were not mutagenic on TA98 and TA100 strains ofSalmonella typhimurium in the presence or absence of S9 mix except KS and SN. KS and SN showed a weak mutagenic effect on TA100 strain in the absence of S9 mix.     

Absence of mutagenic activity of fodder proteins in the Ames Salmonella/microsome test

The mutagenicities of fodder proteins (Pekilo, L-lysine and Orsan) were tested towards Salmonella typhimurium in the plate-incorporation assay in the presence or absence of metabolic activation with a rat-liver S9 preparation. Filtrates and 2-, 5- and 10-fold-concentrated filtrates of saline- or ethanol-soluble fodder proteins were tested. No mutagenic activity was observed.     

Mutagenicity tests of diflubenzuron in the micronucleus test in mice, the L5178Y mouse lymphoma forward mutation assay, and the Ames Salmonella reverse mutation test.

Diflubenzuron, one of a new class of pesticides believed to act via inhibition of chitin synthesis in the developing insect cuticle, was tested for possible mutagenic activity using the micronucleus test in mice, the L5178Y mouse lymphoma forward mutation test at the thymidine kinase locus, and the Ames Salmonella/microsome reverse mutation test. No mutagenic effect was found.     

Absence of mutagenic activity of acidity regulators in the Ames Salmonella/microsome test

The activities of various concentrations of 4 acidity regulators (anhydrous citric acid, phosphoric acid, malic acid and lactic acid) used in food industries in Iraq was assayed using the Salmonella/microsome mutagenicity assay. None of the samples was mutagenic in the absence or in the presence of S9 to any of the tester strains of Salmonella typhimurium.     

Genotoxicity of 1,3-dichloro-2-propanol in the SOS chromotest and in the Ames test. Elucidation of the genotoxic mechanism

1,3-Dichloro-2-propanol (1,3-DCP-OH, glycerol dichlorohydrin) is of great importance in many industrial processes and has been detected in foodstuffs, in particular in soup spices and instant soups. It has been shown to be carcinogenic, genotoxic and mutagenic. Its genotoxic mechanisms are, however, not yet entirely understood. We have investigated whether alcohol dehydrogenase (ADH) catalysed activation to the highly mutagenic and carcinogenic 1,3-dichloroacetone or formation of epichlorohydrin or other genotoxic compounds play a role for mutagenicity and genotoxicity. In our studies, no indications of ADH catalysed formation of 1,3-dichloropropane could be found, although we could demonstrate a clear activation by ADH in the case of 2-chloropropenol. Formation of allyl chloride could also be excluded. We found, however, clear evidence that epichlorohydrin formed chemically in the buffer and medium used in the test is responsible for genotoxicity. No indication was found that enzymatic formation of epichlorohydrin plays a role. Additional mutagenicity and genotoxicity studies with epichlorohydrin also confirmed the hypothesis that genotoxic effects of 1,3-DCP-OH depend on the chemical formation of epichlorohydrin.     

Dose finding in the Ames Salmonella assay

Threshold dose/concentration values, such as the lowest effective dose, minimum effective dose or the lowest effective concentration (LED, MED or LEC, respectively) are in use as an alternative to the mutagen potency measures based on the 'rate' measurements (e.g., the slope of the initial part of the dose-response curve). In this respect, several statistical procedures for the corresponding so-called 'dose finding' were proposed during the last decades. However, most of them disregard the discrete nature of responses such as the plate colony count in the Ames Salmonella assay. When the plate counts agree with the Poisson assumption, two procedures considered here seem to be appropriate for the dose finding. One is based on the stepwise collapsing of the homogeneous control and dose counts; another consists of constructing the confidence limits for the mutation induction factor (MIF). When the dose and control counts are non-overlapping, the simple 'visual' non-parametric estimation of LED is possible. Applicability and validity of the methods is demonstrated with the two data sets on the mutagenicity of the beta-carboline alkaloid, harmine, and one of the oxidation products of apomorphine.     

Genotoxicity investigation of ELF-magnetic fields in Salmonella typhimurium with the sensitive SOS-based VITOTOX test

We performed a genotoxicity investigation of extremely low-frequency (ELF) magnetic fields (MFs, 50 Hz, 100 and 500 μT, 1 and 2 h exposure) alone and in combination with known chemical mutagens using the VITOTOX test. This test is a very sensitive reporter assay of Salmonella typhimurium bacteria based on the SOS response. Our study showed that ELF-MFs do not induce SOS-based mutagenicity in S. typhimurium bacteria and do not show any synergetic effect when combined with chemical mutagens.     

A comparison of alternative measures of mutagenic potency in the Salmonella (Ames) test

Both the spontaneous and the induced mutation rates in Salmonella tester strains vary among different laboratories, and also within the same laboratory over time. If there is an association between spontaneous and induced mutagenesis, a measure of mutagenic potency that incorporates the background may be more consistent than the simple measure of the induced slope. We have used the statistical procedures recently described by Bernstein et al. (1982), and a large data-base of Salmonella test results to examine the association between spontaneous and induced mutation and to compare several alternative measures of mutagenic potency. A correlation analysis indicated an association between spontaneous and induced mutation for TA98, TA1537 and TA1535; TA1538 was close to being significant. This was observed over a wide range of chemicals. In addition, for TA98, for which we observed the strongest association, we obtained a rough estimate of the relationship between slope and intercept by using least squares to fit K and p in the power curve beta = k alpha p. We then chose 3 simple potency measures: the slope, the ratio of slope to spontaneous background, and the ratio of slope to the square-root of spontaneous background. These corresponded to the range of p's estimated from the least-squares fit procedure. The reproducibility of these measures was compared and no significant differences were found. Though there were some differences in the relative potency ranking of chemicals using the different measures, they were highly correlated.     

An interlaboratory study of an EPA/Ames/Salmonella test protocol

7 laboratories participated in a collaborative study to evaluate an EPA standard protocol for the Ames test. The study utilized Salmonella typhimurium (strains TA98 and TA100) and 3 metabolic activation levels (0%, 2%, and 10% S9 in the S9 mix). 6 pure chemicals and 2 complex mixtures were tested as coded unknowns. Ability to obtain qualitative results in agreement with published data was less (% agreement) than that reported in an earlier study (% agreement) by de Serres and Ashby (1981) in which each laboratory used its own protocol. The conclusion from analysis of the quantitative data from this interlaboratory Ames study was that both intralaboratory and interlaboratory variations were substantial. Results for the same substance varied by an order of magnitude or more (CV of 115%) when the mutagenic response was measured as the slope of the dose response in revertants/microgram. Taking interlaboratory variation into account, one chemical must be more than an order of magnitude more mutagenic than another (ratio of slopes greater than 10) to have only an even chance of finding a statistically significant difference between the two chemicals at the 5% level. Such large variations must be taken into account when evaluating Ames/Salmonella data.     

Mutagenicity testing of protein-containing and biological samples using the Ames/Salmonella plate incorporation test and the fluctuation test.

Mutagenicity testing of biological samples and proteins is complicated by the presence of histidine and histidine-related growth factors which may produce a false positive result in the Ames/Salmonella plate incorporation test. A bioassay method, utilizing an automated dispenser-photometer and Salmonella typhimurium strain TA1535 as the indicator bacteria, was used to estimate the presence of histidine-related growth factors in three enzyme solutions submitted for mutagenicity testing. One of the solutions was clearly positive in the Ames/Salmonella test and also contained the highest amount of L-histidine-HCl-equivalents. The two other solutions, with low or undetectable amounts of L-histidine-HCl-equivalents, gave equivocal and negative results, respectively, in the Ames/Salmonella test. Studies were also performed with strains TA98, TA100 and TA1535 to determine the amount of added L-histidine-HCl that would result in a 'positive' result in the Ames/Salmonella test. Because the minimum amount of L-histidine-HCl required to double the number of revertant colonies was 150 nmol/plate, and the maximum amount of L-histidine-HCl-equivalents supplied by the enzyme preparations was 40 nmol/plate at the highest tested dose, the mutagenicity test results of the enzyme solutions cannot be explained solely by histidine or related compounds. Smokers' and non-smokers' urines, concentrated with liquid extraction (CHCl3) and adsorbent (XAD-2 and XAD-2/Sep-Pak C18) techniques, were studied to reveal differences in efficiencies to extract histidine and histidine-related compounds in the urines. Amounts of 'histidine' in concentrates of urine were measured using the bioassay method and a chemical method employing derivatization with fluorescamine. The fluorescamine method also efficiently detected 3-methyl-L-histidine, a product of muscle metabolism excreted in urine, which was found to be unable to support auxotrophic growth in TA1535, leading to exaggerated estimations of the auxotrophic growth enhancing properties of urine extracts. The urine extracts, and pure L-histidine-HCl, were tested using a two-step fluctuation test to estimate auxotrophic growth factor effects in this type of test. Because of a strong dilution effect when adding the histidine-free selection medium, the fluctuation test employed in this study was not found to be particularly sensitive to growth factors. The results of this study indicate that use of a bioassay, employing the same indicator bacteria as the mutagenicity test themselves, is a reliable way to measure histidine-related growth factors in biological samples.(ABSTRACT TRUNCATED AT 400 WORDS)     

An international collaborative study of ''genetic drift'' in Salmonella typhimurium strains used in the Ames test

The efficiency of Weigle reactivation of ultraviolet light-irradiated single and double-stranded phi X174 DNA by wild-type and excision repair-defective E. coli hosts was determined. After limited exposure to ultraviolet light, the efficiency of Weigle reactivation by an ultraviolet light-irradiated wild-type host was greater for double-stranded phi X174 DNA than for its single-stranded counterpart. However, the efficiency of inducible recovery of the double-stranded DNA molecule decreased as its exposure to ultraviolet light increased until it became constant at a value 1.5 times less than that for single-stranded form of phi X174 DNA. The efficiency of Weigle reactivation of the single-stranded DNA molecule by the same host, however, was independent of the dose to the DNA, as were the efficiencies of reactivation for both forms of phi X174 DNA by ultraviolet light-irradiated excision repair-deficient hosts. In excision repair-defective hosts the efficiency of Weigle reactivation of double-stranded phi X174 DNA was also 1.5 times less than that for the single-stranded molecule. These results suggest that the Weigle reactivation of double-stranded phi X174 DNA is mediated in part by an excision repair process, and that this component of Weigle reactivation eventually can be saturated by ultraviolet light-induced DNA damage leaving other repair processes, such as trans-damage synthesis, responsible for the remaining inducible reactivation.