Comparative evaluation of the alkaline comet assay with the micronucleus test for genotoxicity monitoring using aquatic organisms

A comparative analysis between the in vivo comet assay and the in vivo micronucleus test (MNT) was carried out in three aquatic organisms suitable for genotoxicity monitoring, carp (Cyprinus carpio), rainbow trout (Oncorhynchus mykiss), and clam (Spisula sachalinensis), using a direct-acting mutagen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and an indirect mutagen, benzo[a]pyrene (B[a]P). By optimizing the conditions for cell isolation, gill and liver (or digestive glands) were selected as test tissues of the comet assay for MNNG and B[a]P. The MNT employed the erythrocytes (or hemocytes), the most universal cell type for the assay. The analysis of DNA strand breaks using the comet assay and the micronucleus frequencies using the MNT revealed dose- and time-dependent increases between animals exposed to several concentrations of mutagens. But the statistical significance (P<0.05) obtained was higher by the comet assay than by the MNT. When the time profiles of genotoxic signals resulting from B[a]P exposure to carp were plotted representatively, clear distinctions between all concentrations were made in the comet assay, but not in the MNT. The correlation index defined in this study also showed a higher correlation between concentration and signal in the comet assay than in the MNT. It is suggested that the standardization of the comet assay is necessary for its methodological evaluation and use as a genotoxicity biomarker. We conclude that the comet assay has an excellent suitability for aquatic genotoxicity monitoring because of its high and reliable sensitivity.     

Benzo(a)pyrene-induced morphologic and developmental abnormalities in rainbow trout

To determine if the widespread environmental mutagen, benzo(a)pyrene (BaP), was embryotoxic or teratogenic in the rainbow trout (Salmo gairdneri R.), newly fertilized eggs were reared on sand experimentally coated with 1 to 500 ppm BaP. The system produced relatively constant aqueous BaP concentrations ranging from 0.08 to 2.99 ppb, levels comparable with those found in polluted rivers. Rainbow trout alevins reared in 2.40 ppb aqueous BaP for 36 days accumulated an average of 12.34 ppm BaP, and autoradiographic examination revealed accumulation of¹⁴C-benzo(a)pyrene primarily in the yolk sac and in developing neural and ocular tissues. Although no differences in either survival or hatching success were found between control and BaP-treated eggs, exposure to BaP did alter the length of the hatching process. Morphological abnormalities were significantly increased in BaP-treated alevins compared to controls at aqueous exposures of 0.21, 2.40, and 2.99 ppb. Insufficient yolk sacs, lack of body pigment, kyphosis, and abnormalities or absence of the eyes were among the anomalies present in alevins exposed to BaP.     

Interaction of stilbene compounds with human and rainbow trout estrogen receptors

Compounds with stilbene structures are widely used as pharmaceuticals and personal care products (PPCPs) and are present in plants. A suite of stilbene-related compounds, including PPCPs and plant-derived compounds were tested in vitro for interactions with the human and rainbow trout estrogen receptors and in vivo with rainbow trout using vitellogenin levels as a biomarker. Among the compounds with antagonistic activity, the common structural similarity was (in addition to the stilbene backbone) the presence of 4-hydroxy substitution. Stilbene-related compounds found to act as inhibitors at the estrogen receptor included the plant-derived compound resveratrol and two formulations of fluorescent whitening agents used in detergents, 4,4'-bis(2-sulfostyryl)biphenyl and diaminostilbene-1. In the yeast estrogenicity screening assay, the concentrations which caused a 50% inhibition in estrogenic response (IC50s) with the human estrogen receptor ranged from 2.56 x 10(-6) to 2.56 x 10(-6) M. In the rainbow trout estrogen receptor assay, the IC50s ranged from 7.75 x 10(-8) to 1.11 x 10(-5) M. However, in the in vivo rainbow trout vitellogenin assay, tamoxifen was the only stilbene of the compounds tested to have a significant effect as an inhibitor of estrogenicity.     

Comparison of the sensitivities of common in vitro and in vivo assays of estrogenic activity: application of chemical toxicity distributions

A number of contaminants in municipal effluent discharges are estrogen agonists to fish. Whereas several in vitro and in vivo techniques have been developed to assess the estrogenic activity of these compounds or ambient environmental samples, previous comparisons of the relative sensitivities of these approaches remain inconclusive. We employed a probabilistic hazard assessment approach using chemical toxicity distributions (CTDs) to perform a novel evaluation of relative sensitivities of six common in vitro and in vivo assays. We predicted that there was an 8.3% (human breast ademocarcinoma cell line, MCF-7, assay), 6.3% (yeast estrogen screen assay), or 1.9% (fish hepatocyte vitellogenin, VTG, assay) probability of detecting a compound in aquatic systems that will elicit an estrogenic response at concentrations at or below 0.1 microg/L, suggesting that the MCF-7 assay was the most sensitive in vitro assay evaluated in this study. The probabilities of eliciting the estrogenic response of VTG induction at a concentration less than 0.1 microg/L in rainbow trout, fathead minnow, and Japanese medaka were determined at 29.9, 26.2, and 18.8%, respectively. Thus, rainbow trout VTG induction was the most sensitive in vivo assay assessed. Subsequently, CTDs may provide a useful technique for hazard assessment of chemical classes for which exposure data are limited and for chemicals with common toxicological mechanisms and modes of action.     

Acacia salicina extracts protect against DNA damage and mutagenesis in bacteria and human lymphoblast cell K562 cultures

Three extracts were prepared from the leaves of Acacia salicina: aqueous, methanol, and ethyl acetate extracts. The antigenotoxic properties of these extracts were investigated by assessing the inhibition of mutagenicity of the indirect-acting mutagen benzo[a]pyrene using the Ames assay and the genotoxicity of the direct-acting mutagen, hydrogen peroxide, using the “Comet assay.” Aqueous, methanol, and ethyl acetate extracts at doses of 500, 50, and 500 μg per plate reduced benzo[a]pyrene mutagenicity by 95%, 82%, and 40%, respectively, in Salmonella typhimurium TA98 strain and by 91%, 66% and 63%, respectively, at the same doses with a TA97 assay system. Human lymphoblast cells K562 were pretreated with 50% inhibition concentration of each extracts and then treated by H₂O₂, for the Comet assay. The Comet assay results showed that ethyl acetate and methanol extracts decreased the DNA damage caused by H₂O₂ by, respectively, 34.8% and 31.3%. We envisaged also the study of the antioxidant effect of these extracts by the enzymatic xanthine/xanthine oxidase assay. Results indicated that methanol and ethyl acetate extracts were potent inhibitors of xanthine oxidase and superoxide anion scavengers. We conclude that these integrated approaches to antigenotoxicity and antioxidant assessment may be useful to help compare the beneficial effects associated with using A salicina as medicinal and dietary plant.     

Estimating the mass of mutagens in indeterminate mixtures

A method is shown for estimating the quantity (mass) of genotoxic compounds in complex mixtures without prior identification of components. This method uses fractiles of the probability distribution of responses from the assay of interest and dose-response of the mixture. The method depends upon the assumption of additivity, on average, in the interaction of mutagens and on lognormality of the distribution of mutagen molecular weights. Mass estimates are necessary for hazard characterization, risk estimation, and risk assessment. The method is illustrated using Ames assay results from a coke plant wastewater.     

Some effects of selenium deficiency on glutathione peroxidase (EC 1.11.1.9) activity and tissue pathology in rainbow trout (Salmo gairdneri)

1. Two duplicate groups of rainbow trout (Salmo gairdneri; mean weight 27 g) were given diets of differing selenium content (deficient 0.025 mg Se/kg; supplemented 1.022 mg Se/kg) for 30 weeks. 2. There were no significant differences between treatments in weight gain but packed cell volume, liver vitamin E and liver and plasma Se concentrations were all significantly lower in the Se-deficient trout. 3. Ataxia occurred in about 10% of the Se-deficient trout and histopathologies were evident in nerve cord (damage to axon sheath) and liver (loss of integrity in endoplasmic reticulum and mitochondria with appearance of increased vesiculation). 4. Glutathione peroxidase (EC 1.11.1.9) activity was significantly reduced in liver and plasma of Se-deficient fish but there was no indication, from differential assay, of any non-Se-dependent glutathione peroxidase activity. Glutathione transferase (EC 2.5.1.18) activity was significantly increased in Se-deficient trout.     

Effect of selenium deficiency on hydroperoxide-stimulated release of glutathione from isolated perfused liver of rainbow trout (Salmo gairdneri)

1. Duplicate groups of rainbow trout (Salmo gairdneri) were each given partially purified diets which were either adequate or depleted in selenium for 40 weeks. 2. Although there was no significant difference in weight gain, liver Se concentration was significantly lower in fish given the deficient diet. 3. Glutathione (GSH) peroxidase (EC 1.11.1.9) activity was significantly reduced in liver of Se-deficient fish but a differential assay did not indicate the presence of a non-Se-dependent GSH peroxidase activity, although liver GSH S-transferase (EC 2.5.1.18) was significantly increased. 4. Perfusion of livers from trout given Se-adequate diets with t-butyl hydroperoxide (BuOOH) or hydrogen peroxide caused an increase in the rate of release of glutathione disulphide (GSSG) into the perfusate. 5. Perfusion of livers from Se-deficient trout with BuOOH or H2O2 did not result in any change in rate of release of GSSG into the perfusate. 6. These findings confirm the absence of any compensatory non-Se-dependent peroxidase activity in Se-depleted trout.     

Monoclonal antibody enzyme-linked immunosorbent assay to quantify vitellogenin for studies on environmental estrogens in the rainbow trout (Oncorhynchus mykiss)

Vitellogenin (VTG) induction has proved to be a valuable biomarker for assessing exposure to environmental estrogens in fish. The widespread use of VTG in this regard has lead to the need for standardized assays to quantify VTG, and monoclonal antibodies have the potential to help accomplish this. A VTG enzyme-linked immunosorbent assay (ELISA) was developed using a monoclonal antibody prepared against Atlantic salmon (Salmo salar) VTG (MAb BN-5) and its ability to quantify VTG in the rainbow trout (Oncorhynchus mykiss) compared with a rainbow trout vitellogenin (rt-VTG) ELISA that employed homologous polyclonal antibodies (PAb). In routine protocols, the working range of the homologous rt-PAb VTG ELISA was between 9 ng/ml and 70 ng/ml (80- 20% relative maximum binding [B/Bo]) with a 50% B/Bo of 25+/-0.9 ng/ml and inter- and intraassay variations at 50% B/Bo of 7% (n = 7) and 8% (n = 15), respectively. The working range of the MAb BN-5 VTG ELISA was between 60 ng/ml and 850 ng/ml (80-20% B/Bo) with a 50% B/Bo of 227+/-22 ng/ml and inter- and intraassay variations at 50% B/Bo of 5% (n = 10) and 9% (n = 12), respectively. In the routine protocols, detection limits for measurement of plasma VTG in rainbow trout (at 80% B/Bo; and given the requirement to dilute plasma to a minimum of 1:10 for the assays) were 90 ng/ml for the polyclonal rt-VTG assay and approximately 600 ng/ml in the monoclonal antibody assay. In juvenile female rainbow trout exposed to a series of doses of estradiol-17beta (E2) and 4-tert nonylphenol (4-NP), there were no differences in the vitellogenic responses measured in the PAb and MAb BN-5 VTG ELISAs. The monoclonal MAb BN-5 VTG ELISA is likely to be of considerable value for studies on environmental estrogens in juvenile female rainbow trout in standardized tests.     

Analysis of genotoxic effects of individual factors on chemical industry workers

The article provides data on the study of occupational and non-occupational factors relating to the prevalence of early spontaneous abortions in couples engaged in fertilizer production. A higher degree prevalence of early spontaneous abortions was detected in several groups affected by specific factors. As this degree of prevalence was found in the groups in which it was only the father that had been exposed to the specific hazardous factors, it was considered that the factors in question either hindered the mutagen tolerance or were capable of their own germ cell mutagen action. It was established that lowered mutagen tolerance related to early abortions was markedly higher in groups exposed to organic substances' compounds and industrial noise.     

Validity of chromosome analysis and bleomycin sensitivity assay in the prevention of head and neck cancer in Hungary

Because of unfavourable cancer mortality statistics of Hungary, the search of different biomarkers is one of the most important demands of the national primary cancer prevention programme. The aim of this study was to clarify the usefulness of bleomycin sensitivity assay elaborated in the USA, and to find whether it serves under our environmental conditions as a biomarker of individual sensitivity and risk for head and neck cancer, beside chromosomal aberration analysis. The test reflecting mutagen sensitivity is based on the mean values of chromatid breaks induced by bleomycin in vitro in a single lymphocyte (break/cell = b/c). Since cancer formation is influenced by environmental mutagens, in contrast to others, their 111 head and neck cancer patients were matched not only with 230 healthy controls (106 nonsmokers and 124 smokers), but also with 44 strong alcoholic and smoking patients with liver diseases whose lifestyle did not differ from that of the cancer patients. According to the results of conventional chromosome analysis, the aberrant cell frequency was the highest in the cancer patients (3.34%), while in the alcoholics (2.73%) and healthy smokers (2.88%) the values were similar. Thus, the genetic instability occurring in the form of elevated rate of spontaneous chromosomal aberrations was mostly expressed in head and neck cancer patients. Mutagen sensitivity measured by the b/c values of bleomycin assay was significantly higher in both the cancer (1.16 b/c) and the alcoholic patients (1.34 b/c) compared with the controls (1.0 b/c). The bleomycin sensitivity assay, therefore, seems to be the biomarker not only of cancer, but also the disease of the same etiology such as alcohol-related liver disease. However the method is not suitable for the assessment of individual cancer risk because of the high variability of b/c values in each group, and their considerable overlapping with the controls. It can also be supported with extremely high mutagen sensitivity of Hungarian controls (63 and 67%), which is three-fold of US values (23%). The bleomycin sensitivity assay is not a selective biomarker if comparing to the controls, probably due to the action of more complex exposures under Hungarian environmental conditions. When estimating cancer risk, the results of conventional chromosome analysis offer more information than bleomycin sensitivity assay.     

Spontaneous and induced sister chromatid exchanges in lymphocytes of healthy persons

Spontaneous and mitomycin C-induced sister chromatid exchanges (SCEs) in lymphocytes were analyzed in 46 healthy persons. Our results suggest that the frequency of spontaneous SCE in lymphocytes is useful for evaluation of the biological effects of environmental mutagens, and that the induced-SCE test with a mutagen and lymphocytes may be used to detect persons with a high sensitivity to the mutagen.     

某市饮用水有机提取物对小鼠肝细胞DNA损伤的研究

目的研究某市生活饮用水有机提取物对小鼠肝原代细胞所致的DNA损伤作用。方法采用小鼠肝原代细胞彗星试验分别对某市南郊水厂的水源水、出厂水、自来水的有机提取物的致突变性进行检测。结果该市水源水、出厂水和自来水对小鼠肝原代细胞均产生了不同程度的DNA损伤作用,但出厂水和自来水引起肝细胞DNA损伤作用的阈值更低(在本研究为100m1)。结论该市生活饮用水的水源水和氯化消毒后饮用水都受到不同程度的有机致突变物的污染,氯化消毒后饮用水比水源水的致突变性更强,可能是由氯化消毒副产物的致突变性所致。     

Vitellogenin Induction in Mucus from Brook Trout (Salvelinus fontinalis)

Induction of vitellogenin (VTG) is widely used as a biomarker of exposure of male or immature fish to chemicals that are agonists of the estrogen receptor (i.e., xenoestrogens). Analysis of VTG in samples of epidermal mucosa collected from fish is a non-invasive method for evaluating whether wild fish are exposed to xenoestrogens. In this study, the mean levels of VTG in the mucus of immature brook trout (Salvelinus fontinalis) collected from the Credit River in Ontario, Canada downstream of aging residential septic systems and in an agricultural watershed were 0.67 ng per mg protein, which was significantly elevated relative to the mean VTG levels of 0.22 ng per mg protein in the mucus of immature brook trout collected from a less impacted site. To validate the mucus assay, immature brook trout were exposed in the laboratory to 17α-ethinylestradiol (EE2) at nominal concentrations of 10, 50 and 100 ng/L and VTG levels in mucus from these fish showed a concentration-dependent increase relative to fish from the control treatment. This study illustrates the utility of this non-lethal method for assessing whether wild fish have been exposed in situ to xenoestrogens. Exposures to xenoestrogens from non-point sources may be impacting brook trout populations in urban watersheds in southern Ontario.

     

冷培养对金属化合物枯草杆菌致变试验结果的影响

目的：研究枯草杆菌芽孢重组修得试验中增加冷培养对金属化合物和间接致变物DNA损伤试验的影响。方法：比较直接培养法与增加4℃ 24h冷培养对测试结果的作用。观察指标：最小抑菌浓度（MIC）和敏感性。结果：增加冷培养对大多数金属化合物测试结果无影响，而对K2Cr2O4、CoCl2、CoSO4及2种间接致突变物2-AF、AFB反而提高了MIC，降低了敏感性。此结果与Kada的报道不尽一致，对其原因加以探讨。结论：枯草杆菌芽孢重组修复试验增加冷培养，降低了部分金属致突变物和间接致变物敏感性。     

Utility of in vitro test methods to assess the activity of xenoestrogens in fish

The results of the present study have demonstrated the utility of an estrogen receptor (ER) competitive ligand-binding assay, a hepatocyte vitellogenin (VTG) induction bioassay, and an ER reporter gene bioassay in characterizing the activity of model estrogen agonists (17beta-estradiol [E2], ethynylestradiol, and nonylphenol) and antagonists (tamoxifen and ZM 189154) in rainbow trout (Oncorhynchus mykiss). The in vitro results were validated against in vivo trout waterborne exposures to E2 and tamoxifen. The results showed that all three in vitro assays were capable of detecting the hormonal activities of the selected model compounds in a dose-dependent manner, with the exception of nonylphenol in the ER reporter gene bioassay. However, the relative potency rankings of the model compounds were not consistent between these assays, which suggests that the relative potencies obtained within assays may have limited predictive value between assays. Discrepancies in potencies most likely can be attributed to the different levels of cellular organization in each assay. In addition to model compounds, we also evaluated the responses of the ER-binding assay and the hepatocyte VTG induction bioassay to complex mixtures associated with endocrine effects in fish, specifically extracts of pulp mill effluent. Of the 14 pulp mill effluent extracts tested, only six showed activity in both assays, whereas the remaining eight samples showed activity in only one of the two assays. The hepatocyte VTG induction bioassay consistently showed that the pulp mill effluent extracts were antiestrogenic, which to our knowledge has not been reported in previous studies. Collectively, these results suggest that a combination of in vitro assays that depend on differing endpoints is required to identify potential xenoestrogens and to characterize their modes of action.     

类黄酮和维生素的抗突变研究

目的 :　比较类黄酮、维生素和咖啡碱等对不同致突变物的抗突变活性特征、作用及影响。方法 :　用改进型的 Ames试验方法、鼠伤寒沙门氏菌 (Salmonella tyhimurim)的变异种 TA系列菌株、直接致突变物的 1 -硝基芘 (1 -NP)和间接致突变物的色氨酸热解产物 (Trp-P-1 )对测试物进行抗突变效果的比较。结果 :　植物性食物中富含的黄烷醇 (儿茶素 )类、黄酮醇类、酚酸类对Trp-P-1比 1 -NP的抗突变活性高 ;维生素 C、B族和 A对 1 -NP的抗突变性高于类黄酮物质 ,而对Trp-P-1的抗突变性则较低 ,但均有抗突变的剂量效应关系 ;咖啡碱与儿茶素合并使用时对 Trp-P-1的致突变有拮抗现象作用。结论 :　类黄酮对抗突变有较好的作用 ;维生素有辅助和互补作用 ;而咖啡碱的抗突变性不明显     

Comparative analysis of estrogenic activity in sewage treatment plant effluents involving three in vitro assays and chemical analysis of steroids

In this study, we assessed and compared the suitability of three in vitro screening tools for the measurement of estrogenic activity in sewage treatment plant effluents (STPEs). These assays were the yeast estrogen screen (YES), production of zona radiata proteins (ZRPs) in trout hepatocytes, and the induction of reporter gene expression in the transfected rainbow trout gonad cell line RTG-2. Data obtained with the YES were additionally compared with calculated estrogenicity, based on steroid analysis data of the effluents. For comparison purposes, the response of the in vitro systems toward the estrogenic chemicals beta-estradiol, ethinyl estradiol, bisphenol-A, nonylphenol, and octylphenol was assessed. All three assays showed sensitivities in the same order of magnitude in response to the steroid compounds tested, with ZRP production being the least sensitive. Regarding the estrogenic environmental chemicals tested, the RTG-2 assay was more than an order of magnitude more sensitive than the other two assays. Despite their different sensitivities toward selected test chemicals, the three in vitro systems indicated estrogenic activity in the same concentration range for the tested STPEs. Calculated estrogenicity (chemical analysis) and measured estrogenicity (YES) were of the same order of magnitude for the STPEs tested. The present study indicates that all three in vitro systems, with the yeast-based system being the easiest and most robust, are applicable for the screening of estrogenic activity in effluent samples.     

电镀厂废水对蚕豆根尖细胞遗传学毒性的研究

目的　研究电镀厂的生产用水、未处理的生产废水以及已处理水对蚕豆胚根根尖的细胞遗传学毒性效应。方法　采用蚕豆根尖细胞的微核试验方法和染色体畸变试验方法 ,以电镀厂的生产用水、未处理的生产废水以及已处理水为诱变剂 ,测定蚕豆根尖细胞的微核率和染色体畸变率。结果　诱发蚕豆根尖细胞的微核率和染色体畸变率的大小均依次为未处理的生产废水 >已处理水 >生产用水。结论　该电镀厂的未处理的生产废水以及已处理水对植物根尖细胞具有遗传毒性效应 ,且能诱导细胞产生多种类型的染色体畸变