Evaluation of antimutagenic effect of todralazine in cultured lymphocytes

Todralazine, an antihypertensive drug from the hydrazinophthalazine group, significantly decreased the activities of benzo[a]pyrene and mitomycin C in three short-term genotoxicity tests in human lymphocyte cultures. The thioguanine resistance test, the cytokinesis-blocked micronucleus assay and the sister chromatid exchange test were used to demonstrate the antimutagenicity of todralazine. Todralazine lowered the level of free radicals generated by human granulocytes in vitro in the presence of benzo[a] pyrene and also in the presence of the granulocyte activator and tumor promoter phorbol myristate acetate. These results, together with our previous data obtained in the standard bacterial Ames test, strongly suggest that todralazine is a good antimutagen in vitro and deserves further research on its inhibitory action on mutagenesis and carcinogenesis.     

Involvement of oxygen free radicals in the serum-mediated increase of benzoquinone genotoxicity

The genotoxicity of benzoquinone (BQ), a toxic benzene metabolite, is greatly enhanced by the presence of fetal calf serum (FCS) in the incubation medium. The FCS effect is abolished by heat denaturation of serum proteins and is slightly decreased by dialysis. In the present study, we have further investigated the serum effect on BQ genotoxicity by measuring DNA damage produced in peripheral blood mononuclear cells (PBMCs) using the Comet assay. We have also evaluated the effect of human serum and rat liver post-mitochondrial fraction (S9) on the DNA damage produced by BQ. Both human serum and a rat liver S9 enhanced the genotoxicity of BQ in a manner similar to FCS. Gel filtration experiments showed that all the enhancing activity of the serum eluted with the high molecular weight fractions, suggesting that low molecular weight serum constituents do not play an important role in modulating genotoxicity. The genotoxicity-enhancing activity of serum was inhibited by the iron chelator deferoxamine and by superoxide dismutase and catalase. Incubating PBMCs with BQ in the presence of FCS also resulted in the accumulation of intracellular peroxides as demonstrated by loading the cells with 2',7'-dichlorofluorescin diacetate and analyzing for peroxide formation by flow cytometry. These results indicate that oxygen free radicals are involved in the enhancement of BQ-induced DNA damage by serum. We hypothesize that enzyme activities that reduce BQ by transferring single electrons could be the source of the oxygen free radicals.     

Genotoxic and protective effects of hyperbaric oxygen in A549 lung cells

The human lung cell line A549 is frequently used as an in vitro model for studying lung toxicity and genotoxicity of environmental mutagens and carcinogens. Hyperbaric oxygen (HBO) treatment has been shown to be an excellent model for investigating the genetic consequences of oxidative stress in vitro and in vivo. We have now studied the genotoxic effects of and adaptive protection by HBO in A549 cells. Using the alkaline Comet assay, our results show that HBO exposure directly induces DNA damage but reduces the DNA-damaging effect of a second HBO treatment of the cells 24 h later. HBO pretreatment also reduces potassium chromate-induced genotoxicity in A549 cells. Heme oxygenase-1 (HO-1) protein level is increased 24 h after HBO exposure and inhibition of HO-1 activity by tin-mesoporphyrin increased HBO genotoxicity. These results are in accordance with previous findings suggesting an involvement of HO-1 in the adaptive protection. Our study indicates that A549 cells are an appropriate cell culture system for the evaluation of genetic effects induced by HBO in lung cells.     

Antimutagenic effects of natural and synthetic hormonal steroids

Among the steroid molecules, bile acids have been shown to have either a co- or an antimutagenic activity toward various direct and indirect acting mutagens in the Ames test. The present report extends such observations to other steroids having hormonal activity. The main effect of the active hormones is an inhibition of the genotoxicity of both direct and indirect acting mutagens. This effect is strictly structure-related. Moreover, both ethinyl oestradiol and mestranol, which are synthetic derivatives of beta-oestradiol largely used in contraceptive pills, are strong inhibitors of the mutagenicity, acting at nanomolar concentrations. The present article emphasizes the possible role of compounds with steroid structure as modulators of genotoxicity.     

Genotoxic effects of benzyl isothiocyanate, a natural chemopreventive agent

Benzyl isothiocyanate (BITC) is contained in cruciferous plants which are part of the human diet. Numerous reports indicate that BITC prevents chemically induced cancer in laboratory animals and it has been postulated that BITC might also be chemoprotective in humans. On the other hand, evidence is accumulating that this compound is a potent genotoxin in mammalian cells by itself. To further elucidate the potential hazards of BITC, we investigated its genotoxic effects in different in vitro genotoxicity tests and in animal models. In in vitro experiments [differential DNA repair assay with Escherichia coli, micronucleus assay with human HepG2 cells and single cell gel electrophoresis (SCGE) assay with hepatocytes and gastrointestinal tract cells] pronounced dose-dependent genotoxic effects were found at low dose levels (相似文献   

The contribution of excision repair to the DNA effects seen in the alkaline single cell gel test (comet assay)

The alkaline single cell gel test (SCG test or comet assay)was used to study the contribution of excision repair activityto the observed DNA effect mutagen treatment. The cytotoxicityand genotoxicity of UV-irradiation and the chemical mutagens4-nitroquinoline-1-oxide (4NQO), benzo[a]pyrene (BP) and 7,12-dimethy-benz[a]anthracenentracene(DMBA) were compared in a normal human cell line (MRC5CV1) andan excision-deficient xeroderma pigment-osum (XP) cell line(XP12ROSV). The XP cells showed increased cell killing aftertreatment with all mutagens tested, but did not show a clearincrease in DNA migration in the comet assay. DNA effects inMRC5 cells were strongly enhanced by the repair inhibitor aphidicolin(APC), while under the same experimental conditions, APC hadon effect on the XP cell line. The enhancing effect of APC onDNA migration in MRC5 cells and the lack of effects in XP cellsindicate that the induced DNA effects of 4NQO, BP and DMBA inthe comet assay mainly represent the activity of an excisionrepair process. ¹To whom correspondence should be addressed     

The GreenScreen genotoxicity assay: a screening validation programme

A yeast (Saccharomyces cerevisiae) DNA repair reporter assay termed the GreenScreen assay (GSA) is described. This is a novel, cost-effective genotoxicity screen, developed to provide a pre-regulatory screening assay for use by the pharmaceutical industry and in other applications where significant numbers of compounds need to be tested. It provides a higher throughput and a lower compound consumption than existing eukaryotic genotoxicity assays and is sensitive to a broad spectrum of mutagens and, importantly, clastogens. We describe a simple, robust assay protocol and a validation study. The end-point of the test reflects the typically eukaryotic chromosomes and DNA metabolizing enzymes of yeast. The capacity for metabolic activation (MA) in yeast is limited compared with the mammalian liver or its extracts, but the assay does detect a subset of compounds that would require MA in existing genotoxicity tests. The GSA detects a different spectrum of compounds to bacterial genotoxicity assays and thus, together with an in silico structure-activity relationship (SAR) screen, and possibly a high throughput bacterial screen, would provide an effective preview of the regulatory battery of genotoxicity tests.     

Antimutagens and anticarcinogens: a survey of putative interceptor molecules

In this review recent publications are cited for a number of antimutagens. The molecules surveyed are potential or proven "desmutagens" or "interceptors." These are biologically prevalent or synthetic molecules that are most often small metabolites proficient in binding to, or reacting with, mutagenic chemicals and free radicals. Many of this class of "blocking agents" are "soft" and "hard" nucleophiles with consequently varying abilities to react with particular classes of electrophiles, the major classes of direct-acting mutagens. Although they serve as a first line of defense against mutagens and carcinogens, many interceptor molecules are under-investigated with regard to their spectra of activity and their possible relevance to prophylaxis or treatment of human disease states.     

Oxygen radicals potentiate the genetic toxicity of tobacco-specific nitrosamines

Tobacco-specific nitrosamines (TSNAs), nitrosonornicotine (NNN) and 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), are metabolites of nicotine and the major carcinogens in cigarette smoke. To evaluate the effect of oxygen radicals on TSNA-induced genetic damage, MRC-5 fetal human lung cells were exposed to NNN and NNK (5 mM) and DNA single-strand breaks measured. Both NNN and NNK produced a dose-dependent increase in strand breaks up to 10 mM which was cytotoxic. In combination with enzymatically-generated oxygen radicals, strand breakage increased by approximately 50% for both NNN and NNK. Oxygen radical scavengers (superoxide dismutase, catalase, mannitol) significantly reduced the DNA damage caused by both the TSNAs and TSNAs plus oxygen radicals, suggesting that the genotoxicity is radical-mediated. Because both superoxide dismutase and catalase were protective, the hydroxyl radical may be playing an important role in the mediation of the DNA damage observed.     

Effect of five dietary antimutagens on the genotoxicity of six mutagens in the microscreen prophage-induction assay

Dietary antimutagens have been studied extensively in the last two decades, using mainly bacterial and mammalian cells. These studies have shown that certain dietary antimutagens, acting individually or as mixtures, are useful in counteracting the effects of certain mutagens and/or carcinogens to which humans are commonly exposed. However, there are some inconsistencies among publications using different bioassays. The general purpose of the research presented here was to conduct a comparative study of the antigenotoxic activity of five dietary antimutagens against six mutagens, using three rather different short-term tests: the Microscreen prophage-induction assay, the Tradescantia micronucleus test, and the Salmonella/mammalian microsome test. In this study I report the results with the Microscreen prophage-induction assay. The antimutagens selected were chlorophyllin, beta-carotene, and vitamins A, C, and E. The mutagens selected were 2-aminoanthracene, benzo[a]pyrene, 2-nitrofluorene, toxaphene, dichlorvos, and nitrofen. The results show that chlorophyllin and beta-carotene inhibited the genotoxicity of all six mutagens; vitamin E inhibited all except dichlorvos; and vitamins C and A inhibited 2-aminoanthracene, benzo[a]pyrene, 2-nitrofluorene, and nitrofen.     

Comparison of in vitro micronucleus and gene mutation assay results for p53-competent versus p53-deficient human lymphoblastoid cells

The high frequency of false or irrelevant positive results in in vitro mammalian cell genotoxicity tests is a critical concern for regulators. Here, we tested whether such results may be due to the mammalian cells used in the tests being deficient in p53, which is involved in the maintenance of genomic stability. We compared the in vitro responses of two human lymphoblastoid cell lines derived from the same progenitor cell-p53-competent (TK6) and p53-deficient (WTK-1) cells-in a micronucleus (MN) test and a thymidine kinase gene (TK) mutation assay. We tested 14 chemicals including three mutagens and 11 clastogens and spindle poisons. The three mutagens evoked clear positive responses in both assays in both cell lines. The responses to the clastogens and spindle poisons, on the other hand, depended on the assay endpoint and/or the cell line. Most of clastogens and spindle poisons were positive in the MN test in both cell lines. In the TK mutation assay, on the other hand, WTK-1 cells but not TK6 cells detected spindle poisons, which may have been due to the disturbance of the spindle checkpoint and lack of apoptosis in the p53-deficient cells. Some chemicals that induced chromosome aberrations in rodent cells were negative in both TK6 and WTK-1 cells, indicating that a species-specific factor rather than p53 status was associated with the response. In conclusion, the p53 status did not seriously influence the MN test results but it did influence the TK mutation assay results.     

Use of a high-throughput umu-microplate test system for rapid detection of genotoxicity produced by mutagenic carcinogens and airborne particulate matter

In the present study, we developed a rapid umu-microplate test system that uses the nitroreductase- and O-acetyltransferase-overproducing Salmonella typhimurium strain NM3009 and the O-acetyltransferase-overproducing S. typhimurium strain NM2009 to detect genotoxic activity in small volume samples. The assay was used to test the genotoxicity of several standard mutagens and environmental samples. Exponentially growing cultures of NM3009, NM2009, and the parental strain TA1535/pSK1002 were incubated in 96-well microplates with test chemicals both in the presence and in the absence of rat liver S9. The relative beta-galactosidase activities were then determined colorimetrically using either chlorophenol red-beta-D-galactopyranoside (CPRG) or O-nitrophenyl-beta-D-galactopyranoside (ONPG) as a measure of umuC gene induction activity. The sensitivities of NM3009 without S9 mix and NM2009 with S9 mix to nitroarenes and aromatic amines were up to 24- to 75-fold higher than those of the parent strain. Induction of umuC gene expression was detected more readily with CPRG than ONPG. The umu-microplate assay also detected genotoxicity in organic extracts of particulate matter from air samples collected in Osaka City, Japan. The pattern of the responses suggested that the genotoxic activity of the particulate extract was due primarily to nitrated polycyclic aromatic hydrocarbons. Our results indicate that the umu-microplate assay may be a useful way of carrying out rapid screens for genotoxicity in small-volume environmental samples.     

Effect of antioxidant flavonoids and a food mutagen on lymphocytes of a thalassemia patient without chelation therapy in the Comet assay

Thalassemia remains a significant health problem in Europe, the Middle East, and Asia. In such patients, generally high iron levels make free oxygen radicals accessible, for example, through Fenton-type chemistry, and generate superoxide and hydroxyl radicals. Increased oxygen radical capacity is known to be associated with cancer and ageing. It was shown in previous studies that peripheral blood lymphocytes from a sickle/beta thal double heterozygote-sickle phenotype, thalassemia patient, not yet on chelation therapy, were more sensitive to the effects of oxygen radicals and iron salts than lymphocytes from normal controls. Iron overload in thalassemia patients can result from dietary absorption. It was considered that with other dietary agents, such as food mutagens and flavonoids, the thalassemia patient might also show increased sensitivity to the effects of these agents. The present study, therefore, compared the effects of the food mutagen/carcinogen, 3-amino-1-methyl-5H-pyrido(4,3-b)indole (Trp-P-2), in fresh or frozen normal human peripheral lymphocytes with frozen lymphocytes from the same thalassemia patient. The lymphocytes from the thalassemia patient showed an approximately two-fold increase in sensitivity. When a combination of Tryp-P-2, with either quercitin or kaempferol, was compared in frozen lymphocytes and lymphocytes from the thalassemia patient, a two-fold increase in sensitivity was also maintained. Responses to Trp-P-2 were reduced to untreated control levels at the highest doses of quercitin and kaempferol, and were highly significantly different by comparison with Trp-P-2 alone (P<0.001). The flavonoids acted in an antigenotoxic/antioxidant manner. Sensitivity was slightly increased with kaempferol by comparison with quercitin. At low concentrations of the flavonoids there was some evidence of an exacerbation of response, perhaps due to a switch to pro-oxidant status. This exacerbation of response at low doses of flavonoids has been seen in earlier studies with normal lymphocytes. Teratogenesis Carcinog. Mutagen. 21:165-174, 2001.     

Protective effects of a mixture of antioxidant vitamins and minerals on the genotoxicity of doxorubicin in somatic cells of Drosophila melanogaster

Antioxidant vitamins are able to deactivate highly bioactive molecules, such as free radicals, that are generated during cellular biochemical processes. Doxorubicin (DXR) is a cancer chemotherapeutic agent that generates free radicals as a byproduct. In the present study, the Drosophila melanogaster somatic wing spot test was used to evaluate the effects of a mixture of vitamins (Vitamins C, E, and beta-carotene) and minerals (copper, selenium, and zinc), commercially known as Vitergan Zinc Plus, on the genotoxicity of DXR in standard and high-bioactivation crosses of flies. 12.5, 25, and 50 mg/ml of the vitamin/mineral mixture by itself was nongenotoxic in the trans-heterozygous descendants of both crosses, while the mixture produced a significant reduction in the genotoxicity produced by 0.125 mg/ml DXR in the trans-heterozygous descendants of both crosses. The protective effect was observed when the larvae received either pre- or cotreatments of the multivitamin/mineral (MV) mixture. The results indicate that, under these experimental conditions, the MV mixture is not genotoxic; however, it protects against the genotoxic effects of the chemotherapeutic free-radical generator DXR.     

An evaluation of the CHO/HGPRT mutation assay involving suspension cultures and soft agar cloning: results for 33 chemicals

The Chinese hamster ovary cell assay (CHO), which measures forward mutation of the HGPRT locus, is used in several laboratories for the detection of mutagens. A procedure involving treatment of CHO cells in suspension culture and mutant selection in soft agar cloning has been developed (Oberly TJ, Bewsey BJ, Probst GS (1987): Mutat Res 182:99-111). In order to evaluate the effectiveness of these modifications, 33 chemicals representing six chemical classes were tested, and the results were compared to findings obtained in other tests for genotoxicity at Lilly Research Laboratories (LRL). A positive response was obtained with 21 chemicals, all of which are recognized mutagens. Of the 12 compounds that produced negative results, 4 were considered to be mutagens and/or carcinogens. Twelve of the compounds mentioned in this report have been previously tested in the CHO/HGPRT assay by other laboratories, and the results showed strong agreement between laboratories. These findings support the conclusion that the use of suspension cultures and soft agar cloning in the CHO assay provides a sensitive test for the identification of mutagens and is a viable alternative to the traditional monolayer procedure of O'Neill et al. (O'Neill JP, Couch DB, Machanoff R, San Sebastian JR, Brimer PA, Hsie AW (1977): Mutat Res 45:103-109).     

Supplementation of melatonin protects human lymphocytes in vitro from the genotoxic activity of melphalan

Melatonin (MLT) is a natural oncostatic factor of the human body as well as an antioxidant thus protects the nuclear DNA from oxidative damage. It also has the ability to reduce the side effects of various drugs when used as a combination therapy. The anti-neoplastic agent melphalan (MEL), which encompasses a number of side effects, is a strong alkylating agent and a potent inducer of sister chromatid exchanges (SCEs). The aim of the current in vitro study was to investigate the ability of MLT to reduce the genotoxic effect of MEL on normal human cultured peripheral lymphocytes. Cells were treated with both agents at various concentrations (MLT 100, 200 and 400 microM and MEL 330, 490 and 650 nM) and incubated for 72 h prior harvesting. The levels of cytostaticity, cytotoxicity and genotoxicity were qualitatively evaluated using the proliferation rate index, the mitotic index and the SCE methodology, respectively. Our results demonstrated the protective effect of MLT on cells treated with MEL in vitro. The greatest protective effect of MLT at 100 and 400 microM was illustrated against 330 nM of MEL in comparison with all other doses of MEL. These observations imply that MLT may be proved useful in reducing some of the toxic effects associated with certain classes of chemotherapeutic agents and other chemical and physical mutagens and carcinogens, acting both as an antioxidant-radical scavenger and a protective mechanism against cellular damage due to exposure to free radical-producing agents. It is essential to investigate substances with protective properties which are normally produced from the human body.     

Protection of renal cells against free radical damage in vitro. A morphologic and functional study on human and rabbit kidney cells

The morphologic and functional effects of free radicals on renal cells in vitro were investigated, as well as the possibility of avoiding them by pretreatment with scavenger enzymes or a xanthine oxidase inhibitor. Cultured human kidney cells, incubated together with a free radical-generating system, with and without protective agents, were examined by light and scanning electron microscopy. The vimentin filament structure of the incubated cells was visualized by immunofluorescence. The membrane function was studied in human kidney cells by using a dye exclusion test and in rabbit kidney slices by determination of the sodium-potassium pump activity. Exposure of the cells to free radicals caused rapid development of severe morphologic lesions, including extensive cytoskeletal disorganization. After pretreatment, only a few cells had similar, although less severe, lesions. The results of the dye exclusion test and indirect evaluation of the sodium-potassium pump activity did not indicate any major damage to the cell membranes after exposure to free radicals.     

Genotoxicity of 2-nitro-7-methoxy-naphtho[2,1-b]furan (R7000): a case study with some considerations on nitrofurantoin and nifuroxazide

Two nitrofurans present broad-spectrum antimicrobial properties and some of them are used in human and veterinary medicine. Most of these molecules are mutagens and some of them were reported as carcinogens. Due to its extreme mutagenic potency in bacteria, the nitronaphtho derivative 2-nitro-7-methoxy-naphtho[2,1-b]furan (R7000) was used as a tool to analyze the mechanism of the genotoxic action of this family of chemicals. In the present paper, we review essential data on the genotoxicity of R7000 and briefly discuss the case of nitrofurantoin and nifuroxazide, two nitrofurans, still in use as urinary and gastrointestinal disinfectants.     

Ames试验在羟基磷灰石人工骨材料评价中的应用

目的通过鼠伤寒沙门氏菌回复突变试验(Ames试验)探讨羟基磷灰石人工骨材料的遗传毒性特征,以评价该材料的潜在致突变性。方法选择生理盐水(SC)和二甲基亚砜(DMSO)两种溶剂对试验样品浸提,采用平板掺入法,计数TA97、TA98、TA100和TA102菌株在活化和非活化条件下的回变菌落数,以检测其致突变比值。结果试验阴性对照和阳性对照结果有效,试验成立。采用SC或DMSO浸提,浸提原液均未引起试验菌株回变菌落数超过阴性对照的2倍,即致突变比值MR均<2。结论在本实验条件下,试验样品SC浸提原液及DMSO浸提原液对试验菌株无诱变性。