Exogenous Metabolic System Research Articles

Sediment Mutagenicity and Liver Oxidative Stress in Fish Under Tannery Influence

The mutagenicity of interstitial water and organic extracts from the sediments in the Cadeia and Feitoria Rivers, RS, Brazil, were evaluated by Salmonella microsuspension bioassay using TA97a, TA98, TA100 and TA102 strains, in the absence and presence of S9 mix. At the contaminated site, the mutagenic responses for interstitial water, suggested the presence of frameshift and base pair substitution mutagens, including substances that generate oxidative stress. Organic extracts presented direct or indicative mutagenesis to the TA97a, TA98 and TA100 strains. In general, an exogenous metabolic systems decreased the mutagenicity of the samples. The livers of Gymnogeophagus gymnogenys, family Cichlidae, collected in this impacted area, compared to a non-polluted site, were analyzed for oxidative stress parameters. Compared to the controls, there was a significant increase in the activity of superoxide dismutase (SOD), levels of substances reactive to thiobarbituric acid (TBARS), and in the chemiluminescence of hepatic cells in fish collected at the polluted area. The concentration of cytochromes P450 and b5 decreased drastically in the fish at the polluted site, while the catalase activity did not change. It was possible to correlate the biological changes in the fish with the presence of mutagenic compounds in interstitial water in this area.

Mutagenicity of sediment and biomarkers of oxidative stress in fish from aquatic environments under the influence of tanneries

The mutagenicity of interstitial water and organic extracts from the sediments in the Cadeia and Feitoria Rivers, RS, Brazil, were evaluated by Salmonella microsuspension bioassay using TA97a, TA98, TA100 and TA102 strains, in the absence and presence of S9 mix. At the contaminated site, the mutagenic responses for interstitial water, suggested the presence of frameshift and base pair substitution mutagens, including oxidative substances. Organic extracts presented direct or indicative mutagenesis to the TA97a, TA98 and TA100 strains. In general, an exogenous metabolic systems decreased the mutagenicity of the samples. High concentrations of total chromium found in the sediment and interstitial water as well as total mercury in the sediment of the contaminated site, when compared to the control area, may help explain the mutagenic results. The livers of Gymnogeophagus gymnogenys collected in this impacted area, compared to a non-polluted site, were analyzed for oxidative stress parameters. Compared to the controls, there was a significant increase in the activity of superoxide dismutase (SOD) at levels of substances reactive to thiobarbituric acid (TBARS), and in the chemiluminescence of hepatic cells in fish in the polluted area. The concentration of cytochromes P450 and b5 decreased drastically in the fish at the polluted site, while the catalase activity did not change. It was possible to correlate the biological changes in the fish with the presence of mutagenic compounds in sediment and interstitial water in this area.

Verification of the BALB/c 3T3 cell transformation assay after improvement by using an ITES-medium

We carried out the first-step verification study on our ITES-medium-improved BALB/c 3T3 cell transformation assay. In order to estimate its potential use as a short-term screening method for putative carcinogens, 31 chemicals were tested in the improved transformation assay. The test chemicals consisted of 18 carcinogens and 13 noncarcinogens. The present improved transformation assay did not use an exogenous metabolizing system. Data analysis was carried out on 34 chemicals, including assay data for three chemicals reported previously by the authors. As a result, the improved transformation assay showed a concordance of 73.5% with a rodent bioassay, a sensitivity for carcinogens of 71.4%, and a specificity for detection of noncarcinogens of 76.9%. The improved transformation assay detected all of the genotoxic carcinogens, and five of 11 nongenotoxic carcinogens as positive. It can be expected that the improved transformation assay will be able to detect not only genotoxic carcinogens with high probability but also approximately 50% of nongenotoxic carcinogens within about 3 weeks. Hence, these preliminary findings suggest that our improved transformation assay will be a reliable and useful short-term test procedure of screening for potential carcinogens, and it encourages us to conduct further experiments on many carcinogens and noncarcinogens.

Mutagenic and genotoxic evaluation of bisphenol F diglycidyl ether (BFDGE) in prokaryotic and eukaryotic systems

The epoxy resin bisphenol F diglycidyl ether (BFDGE), was examined for its mutagenicity in prokaryotic assays ( Salmonella typhimurium His − and Escherichia coli Trp − tests) and its genotoxicity in eukaryotic systems (sister chromatid exchange (SCE) and micronucleus tests in human lymphocytes), in the presence or absence of an exogenous metabolizing system (S9 from rat liver). In the prokaryotic tests, the concentrations of BFDGE ranged between 100 and 5000 μg per plate, and in the eukaryotic assays from 12.5 to 62.5 μg/ml. The compound is able to induce mutagenic effects in bacterial strains TA100, TA1535, WP2 uvrA and IC3327, as revealed by the increase observed in the number of induced revertants. With respect to the genotoxicity assays, BFDGE induces an increase in the frequency of sister chromatid exchanges and micronuclei in human peripheral blood lymphocytes.

Mutagenicity of nitrosamines in methyltransferase-deficient strains of Salmonella typhimurium coexpressing human cytochrome P450 2E1 and reductase

Although dialkylnitrosamines are environmentally significant carcinogens, the use of short-term bioassays to assess the mutagenic potential of these compounds is problematic. The Ames test, a mutagenicity assay based on the reversion of Salmonella typhimurium histidine auxotrophs, is the most widely used bioassay in genetic toxicology, but the traditional Ames tester strains are largely insensitive to dialkylnitrosamine mutagenicity. We have constructed two mutagenicity tester strains that co-express full-length human cytochrome P450 2E1 and P450 reductase in S. typhimurium lacking ogt and ada methyltransferases (YG7104ER, ogt- and YG7108ER, ogt-, ada-). These new strains are susceptible to dialkylnitrosamine mutagenicity in the absence of an exogenous metabolic activating system (S9 fraction). Mutagenicity is dependent upon the coexpression of P450 2E1 with P450 reductase and is similar to or greater than that obtained with the parental strains in the presence of S9 fraction from ethanol-induced rat liver. These strains were also sensitive to nitrosamines with longer alkyl side chains including diethylnitrosamine, dipropylnitrosamine and dibutylnitrosamine. Mutagenicity decreased with alkyl chain length, consistent with the stringency of the ada-encoded enzyme for methyl and ethyl DNA adducts. These new strains may prove useful in the evaluation of nitrosamine contamination of food and environmental samples.

Genotoxicity of the coating lacquer on food cans, bisphenol A diglycidyl ether (BADGE), its hydrolysis products and a chlorohydrin of BADGE

The epoxy resin bisphenol A diglycidyl ether (BADGE), its hydrolysis products and a chlorohydrin of BADGE (BADGE·2HCl), were examined for their genotoxicity in the micronucleus test (MNT) with human peripheral blood lymphocytes in vitro, in presence and in absence of an exogenous metabolizing system S9 rat liver. The treatment was done using different compound concentrations up to cytotoxic doses. The concentrations tested ranged between 12.5 to 62.5 μg/ml of BADGE, 12.5 to 62.5 μg/ml of first BADGE hydrolysis product (BADGE·H 2O), 25.0 to 100.0 μg/ml of second BADGE hydrolysis product (BADGE·2H 2O) and 6.25 to 50.0 μg/ml of BADGE·2HCl. These compounds are able to induce both cytotoxic and genotoxic effects, as revealed by the increases observed in cytokinesis block proliferation index (CBPI) and in micronuclei (MN) frequencies, respectively.

Incorporation of S-9 activation into an ER-α transactivation assay ☆

We evaluated the feasibility of incorporating an exogenous metabolic activating system into an estrogen receptor-α transactivation assay. 17β-estradiol (E2), and the proestrogenic pesticide methoxychlor (MXC) were evaluated for activity in the presence and absence of Aroclor-1254 induced rat liver S-9 fractions. Both E2 and MXC responded consistently in the assay with average EC 50 values of 9.6 × 10 −11 M and 1.2 × 10 −5 M, respectively. In the presence of a 0.1% S-9 fraction, the EC 50 for E2 was increased to 1.4 × 10 −9 M and that for MXC decreased to 4.9 × 10 −7 M, with both compounds demonstrating increased secondary metabolite formation as evidenced by HPLC analysis. Consistent with these data, metabolites of E2 and MXC exhibited decreased and increased potencies, respectively, in the assay system relative to the parent molecules. S-9 was compatible with the MCF-7 reporter assay and has the potential to enhance detection of proestrogenic materials.

SOS induction of selected naturally occurring substances in Escherichia coli (SOS chromotest)

Naturally occurring substances were tested for genotoxicity using a modified laboratory protocol of the Escherichia coli PQ37 genotoxicity assay (SOS chromotest) in the presence and in the absence of an exogenous metabolizing system from rat liver S9-mix. Aristolochic acid I, II, the plant extract aristolochic acid and psoralene were genotoxic; cycasine, emodine, monocrotaline and retrorsine were classified as marginal genotoxic in the SOS chromotest in the absence of S9-mix. In the presence of an exogenous metabolizing system from rat liver S9-mix aristolochic acid I, the plant extract, β-asarone, cycasin, monocrotaline, psoralen and retrorsine showed genotoxic effects; aristolochic acid II marginal genotoxic effects. Arecoline, benzyl acetate, coumarin, isatidine dihydrate, reserpine, safrole, sanguinarine chloride, senecionine, senkirkine, tannin and thiourea revealed no genotoxicity in the SOS chromotest either in the presence or in the absence of an exogenous metabolizing system from rat liver S9-mix. For 17 of 20 compounds, the results obtained in the SOS chromotest could be compared to those obtained in the Ames test. It was found that 12 (70.6%) of these compounds give similar responses in both tests (6 positive and 6 negative responses). The present investigation and those reported earlier, the SOS chromotest, using E. coli PQ37, was able to detect correctly most of the Salmonella mutagens and non-mutagens.

Effects of β-Carotene, Retinal, Riboflavin, α-Tocopherol and Vitamins C and K 1 on Sister-chromatid Exchanges Induced by 3-Amino-1-methyl-5 H-pyrido[4,3- b]indole (Trp-P-2) and Cyclophosphamide in Human Lymphocyte Cultures

The vitamins and related compounds cited in the title were investigated for their abilities to modulate sister-chromatid exchanges (SCEs) induced by Trp-P-2 or cyclophosphamide (CP) in human peripheral lymphocyte cultures in the presence of an exogenous metabolizing system from rat liver. When inducer and test substances were given simultaneously, β-carotene, retinal and α-tocopherol caused a dose-dependent decrease of SCE frequencies induced by Trp-P-2 and CP. Vitamin K 1, however, brought about an identical effect with Trp-P-2 only, while with CP an initial decrease of SCEs was followed by a statistically significant re-increase at higher concentrations. Vitamin C was ineffective against Trp-P-2, but caused an overall increase of SCEs induced by CP. When blood cultures were preincubated with vitamins before the addition of CP or Trp-P-2, basically identical effects were observed with β-carotene, retinal, α-tocopherol, vitamin K 1 and vitamin C. Riboflavin decreased SCEs induced by Trp-P-2 in all treatment schedules, although statistically confirmed minima were observed in the dose–response curves, except in post-treatment experiments. On the other hand, riboflavin only reduced SCEs induced by CP when it was preincubated with lymphocytes. When vitamins were applied in a post-treatment schedule after removal of Trp-P-2 or CP, again, basically identical results against both genotoxins were observed with β-carotene, retinal and α-tocopherol with vitamin K 1, however, only with respect to Trp-P-2, and with vitamin C only with respect to CP. In the post-treatment schedule, vitamin K 1 caused a decrease of SCE frequencies induced by CP, and vitamin C a decrease of SCEs induced by Trp-P-2.

Lack of Mutagenicity of Polycyclic Musk Fragrances in Salmonella typhimurium

Synthetic musks are widely used as fragrances and therefore found in various environmental samples. Residues were identified in river and waste water, animal and human tissues and breast milk. In the present study, six artificial polycyclic musk fragrances—Galaxolide, Tonalide, Celestolide, Phantolide, Cashmeran and Traseolide—were tested for mutagenicity using the Salmonella/mammalian-microsome assay with Salmonella typhimurium strains TA97, TA98, TA100 and TA102 in the presence (+S-9) and absence (−S-9) of an exogenous metabolizing system. All compounds tested exhibited no mutagenicity in the Salmonella assay. These results could be rated as one indicator of the biological inactivity of this group of chemicals with respect to genotoxicity.

Testing of SOS induction of artificial polycyclic musk fragrances in E. coli PQ37 (SOS chromotest)

Synthetic fragrances are widespread in the environment. Residues were found in animals, human tissues and breast milk. Therefore, six artificial polycyclic musk fragrances—Galaxolide, Tonalide, Celestolide, Phantolide, Cashmeran and Traseolide—were tested for SOS induction using the Escherichia coli PQ37 genotoxicity assay (SOS chromotest) in the presence (+S9) and absence (−S9) of an exogenous metabolizing system. All compounds tested exhibited no SOS inducing potency with the SOS chromotest. These results could be rated as one indicator of the biological inactivity of this group of compounds with respect to genotoxicity.

Chemopreventive properties of chlorophylls towards aflatoxin B 1: a review of the antimutagenicity and anticarcinogenicity data in rainbow trout

The anticarcinogenic activity of chlorophyllin (CHL), a water-soluble derivative of chlorophyll, was first reported in rainbow trout. This review describes certain experiments which set the stage for long-term tumor bioassays, in trout and other species, using CHL and various food-borne carcinogens. Initial work with trout and rat liver enzymes in the Salmonella assay showed that CHL was a potent antimutagen towards heterocyclic amines, polycyclic aromatic hydrocarbons, aflatoxins and other classes of mutagen. Antimutagenic activity was further demonstrated using the corresponding direct-acting mutagens in the absence of an exogenous metabolizing system. Mutagen-inhibitor interaction (molecular complex formation) was identified in spectrophotometry studies, suggesting that CHL acts as an `interceptor molecule'. In vivo, CHL reduced hepatic AFB1-DNA adducts and hepatocarcinogenesis when the inhibitor and carcinogen were co-administered in the diet. Finally, co-injection of inhibitor and AFB1 into trout embryos established that CHL was more effective than chlorophyll a in reducing AFB1-DNA adducts 2 weeks after injection, and liver tumors after 1 year.

In vitro genotoxicity of polycyclic musk fragrances in the micronucleus test

The synthetic polycyclic musk fragrance compounds galaxolide (1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-(g)-2-benzopyrane), tonalide (7-acetyl-1,1,3,4,4,6-hexamerthyltetraline), celestolide (4-acetyl-1,1-dimethyl-6- tert-butylindane), phantolide (6-acetyl-1,1,2,3,3,5-hexamethylindane), cashmeran (6,7-dihydro-1,1,2,3,3-pentamethyl-4-(5H) indanone) and traseolide (5-acetyl-1,1,2,6-tetramethyl-3-isopropylindane) were examined for their genotoxicity in the micronucleus test (MNT) with human lymphocytes in vitro in the presence and absence of an exogenous metabolizing system containing rat liver S9 and the metabolically competent human hepatoma cell line Hep G2. Compound concentrations were employed up to cytotoxic doses. Galaxolide, tonalide, celestolide, phantolide, cashmeran and traseolide revealed no genotoxicity in the micronucleus test with human lymphocytes and with the human hepatoma cell line Hep G2.

Cytotoxic and cell transforming activities of the fungicide methyl thiophanate on BALB/c 3T3 cells in vitro

Cytotoxic and cell-transforming activities of methyl thiophanate, a systemic fungicide capable of entering plant cells and thus controlling fungal diseases that have already started, were studied in an in vitro medium-term (6–8 weeks) experimental model utilizing BALB/c 3T3 cells. Cells were exposed to the chemical, dissolved in dimethyl sulfoxide, in the absence or presence of an exogenous metabolizing system derived from rat livers supplemented with cofactors (S9 mix). In the absence of metabolic activation, methyl thiophanate exerted cytotoxic activity, evidenced through the formation of cell colonies, at low doses (>10 μg/ml). However, the cytotoxic activity was greatly reduced by the S9 mix-induced metabolic activation of the chemical. Without bioactivation, cell-transforming potential, evidenced through the induction of transformation foci, was observable only at the highest (weakly toxic) dose employed (25 μg/ml). On the contrary, in the presence of metabolic activation, the cell-transforming activity was detectable at all tested doses (i.e. from 20 to 200 μg/ml) and it was particularly evident in a level-II transformation amplification test when the cells were allowed to perform active proliferative activity. These results, providing further information on the activity of methyl thiophanate in multistep carcinogenesis as possible genotoxic and/or co-carcinogenic agent, may contribute to better evaluate the oncogenic risk to man.

Nitro musk compounds genotoxic activity

Five nitro musk compounds are widely used as fragrance ingredients in perfumes, lotions and detergents; as food additives in cigarettes and fish baits, and in such technical products as herbicide formulations and explosives. Several studies identified nitro musk compounds in aquatic environment samples, human milk and fat samples as highly lipophilic and persistent bioaccumulating environmental pollutants. To examine the compounds for genotoxic activity, musk xylene (1-tert.-butyl-3, 5-dimethyl-2, 4, 6-trinitrobenzene), musk ketone (4-tert.-butyl-3, 5-dinitro-2, 6-dimethylacetophenone), musk ambrette (l-tert.-butyl-4-methyl-6-methoxy-3, 5-dinitrobenzene), musk moskene (l, 1, 3, 3, 5-pemamethyl-4, 6-di-nitroindane) and musk tibetene (1-tert.-butyl-3, 4, 5-trimethyl-2, 6-dinitrobenzene) were tested for SOS inducing potency in the SOS chromotest with E. coli PQ37 and for sister-chromatid exchange inducing activities in human lymphocytes in vitro both in the presence and absence of an exogenous metabolizing system from rat liver S9-Mix. Nitro musks revealed no genotoxicity either in the SOS chromotest with E. coli PQ37 or in the sister-chromatid exchange test with human lymphocytes.

Genotoxicity of selected pesticides in the mouse bone-marrow micronucleus test and in the sister-chromatid exchange test with human lymphocytes in vitro

Selected pesticides, (aldicarb, 1,3-dichloropropene, methidathion, parathion, triadimefon, vinclozolin) were tested for their clastogenic and aneugenic activities in the mouse bone-marrow micronucleus (MN) test in vivo and for their sister-chromatid exchange-inducing activities in human lymphocytes in vitro in the presence and absence of an exogenous metabolizing system from rat-liver S9. 1,3-Dichloropropene significantly increased the frequencies of micronucleated polychromatic erythrocytes (PCE) in bone-marrow cells of female mice from 3.3 MN/1000 PCE to 15.3 MN/1000 PCE (187 mg per kg body weight). 1,3-Dichloropropene (100 μM) induced 16.0 SCE/metaphase after 24 h of incubation as compared with the basal rate of 11.2 SCE/metaphase (− S9) and of 15.4 SCE/metaphase as compared with 10.5 SCE/metaphase of the control (+ S9). These values were statistically significantly different from each other. The other pesticides tested did neither increase the rate of micronuclei significantly in polychromatic erythrocytes in male nor in female animals. Aldicarb and methidathion induced a significant increase in SCEs in human lymphocytes in vitro only without the metabolic activating system: aldicarb, 5 μm, 24 h incubation: 15.5 SCE/metaphase; control: 12.6 SCE/metaphase; methidathion, 100 μM, 24 h incubation: 15.8 SCE/metaphase, control: 11.1 SCE/metaphase. Parathion, triadimefon and vinclozolin did not have any SCE-inducing effects.

Malachite Green: A Toxicological Review

Malachite green, an N-methylated diaminotriphenylmethane dye, is used primarily as a therapeutic agent in aquaculture. In solution, the dye exists as a mixture of the cation (chromatic malachite green) and its carbinol base, with the ratio depending on the pH of the solution; the dye also can undergo chemical and metabolic reduction to a leuco derivative. Analysis offish tissue after exposure to malachite green indicates the presence of both chromatic and leuco forms, with the latter having a much longer tissue half-life. Malachite green intercalates with DNA, with a preference for A:T-rich regions, and the leuco derivative bears a structural resemblance to carcinogenic aromatic amines that can form covalent DNA adducts. Malachite green is mutagenic in Salmonella typhimurium TA98 in the presence of an exogenous metabolizing system. In mammalian cells, it shows marked cytotoxicity and the ability to induce cell transformation and lipid peroxidation. Results from carcinogenicity bioassays with malachite green have been equivocal; however, it appears to act as a tumor promoter, perhaps because of its ability to induce the formation of reactive oxygen species. These characteristics, plus its close structural similarity to carcinogenic triphenylmethane dyes (e.g., gentian violet) suggest that additional data are required to determine if human exposure to malachite green results in adverse health effects.

DNA-DAMAGE CAUSED BY ANTIBIOTIC DRUGS - QUINOLONES

In the present investigation, the genotoxic potencies (SOSIP) of 10 antibiotic quinolones (topoisomerase LT inhibitors) were tested in the sfiA::lacZ fusion containing strain Escherichia coli PQ37 using a modified procedure of the SOS chromotest. A number of quinolones exhibited extremely high DNA damaging effects in the absence of an exogenous metabolizing system. The highest SOS inducing potencies (SOSIP) exhibited sparfloxacin with 2,400 Delta IF/nmole, ciprofloxacin (SOSIP = 184 Delta IF/nmole) and norfloxacin (SOSIP=120 Delta IF/nmole), whereas pipemic acid (SOSIP=4.6 Delta IF/nmole), cinoxacin (SOSIP=0.5 Delta IF/nmole) and nalidixic acid (SOSIP=0.5 Delta IF/nmole) showed only weak genotoxicity. The possibility of mutagenic effects caused by quinolones in eukaryotic cells is discussed.

Evaluation of the Dysmorphogenic Effects of Procarbazine, Benzylazoxyprocarbazine, and Methylazoxyprocarbazine in Whole Embryo Culture

Serum from procarbazine (PCZ)-treated rats is dysmorphogenic to rat embryos cultured in vitro, but PCZ is not effective when added directly to culture medium, even with an exogenous metabolizing system. Methylazoxyprocarbazine (MPCZ) is a metabolite which we have identified by HPLC in the dysmorphogenic serum of PCZ-treated rats. PCZ, MPCZ, and benzyl-azoxyprocarbazine (BPCZ, an isomer of MPCZ) were tested in rat whole embryo culture to determine their effects on embryo development. The parent compound, PCZ, produced no effect on embryo growth or development at concentrations up to 200 μg/ml. MPCZ proved to be the most potent of the agents tested. There was significant embryo lethality at concentrations of ≥10 μg/ml while 25 μg/ml had significantly reduced embryonic developmental score (DEVS), and 35 μg/ml reduced DEVS, head length, and somite number. There was 89% embryo lethality at the 50 μg/ml exposure level. At concentrations >5 μg/ml, there were significant increases in anomalies, primarily, failure of neural tube closure, erratic neural seam, and microcephaly. In contrast, BPCZ produced embryo lethality and reductions in DEVS only at 100 μg/ml. These data suggest that MPCZ, which has been identified in PCZ-treated rat serum, may be the proximate dysmorphogenic metabolite of PCZ.

The anticlastogenicity of β-carotene evaluated on human hepatoma cells

The efficiency of β-carotene as a modulatory agent against clastogenicity induced by a cyclophosphamide (CPA), an indirect-acting mutagen, and mitomycin C (MMC), a direct-acting mutagen, was evaluated in human hepatoma cells (Hep G2) using three different treatment regimes. Six doses of β-carotene, 0.25, 0.5, 1.0, 2.0, 4.0 and 6.0 μM, were tested as pre-treatment, simultaneous treatment and pre- + simultaneous treatment. Since these cells are able to activate mutagens without any exogenous metabolizing system (S9 mix), some problems related to the use of S9 mix were eliminated. The data obtained show a statistically significant decrease in the frequency of micronuclei (MN) induced by CPA when the cells were treated with β-carotene, for all treatments, and no effect of this provitamin on the clastogenicity of MMC was found. These results reinforce the anticlastogenicity of β-carotene showing and its action is independent of the treatment regime used. On the ther hand, the fact that β-carotene had a protective action only on CPA-induced MN suggests an effect on activation of the promutagen and emphasizes the important utility of cell lines capable of metabolizing chemical mutagens, in such basic studies.