Chromosomal aberrations in V79 cells induced by superoxide radical generated by the hypoxanthine-xanthine oxidase system

The effect of the superoxide radical, generated by the hypoxanthine-xanthine oxidase system, on chromosomal mutation was examined in Chinese hamster V79 cells. When cells were treated with this system for 1 h in Hanks' solution, the incidence of metaphases with chromosomal aberrations was increased with hypoxanthine at concentrations of 2.5 to 10 micrograms/ml. On the other hand, in Eagle's minimum essential medium (MEM) or MEM supplemented with 10% fetal bovine serum, only hypoxanthine at 5 micrograms/ml plus xanthine oxidase induced chromosomal aberrations and higher concentrations of hypoxanthine were cytotoxic to V79 cells. The increased frequency of chromosomal aberrations and the cytotoxicity of hypoxanthine plus xanthine oxidase were not affected by superoxide dismutase, but were strongly inhibited by catalase.     

Antimutagenic potential of Solanum lycocarpum against induction of chromosomal aberrations in V79 cells and micronuclei in mice by doxorubicin

Solanum lycocarpum A. St. Hil. (Solanaceae) is a hairy shrub or small much-branched tree of the Brazilian Cerrado. S. lycocarpum fruits are commonly used in traditional medicine in powder form or as folk preparations for the treatment of diabetes and obesity, as well as for controlling cholesterol levels. The aim of the present study was to chemically characterize the hydroalcoholic extract (SL) of S. lycocarpum by determination of total flavonoids and total poyphenols and quantification of steroidal alkaloids, as well as to evaluate its mutagenic and/or antimutagenic potential on V79 cells and Swiss mice using chromosomal aberrations and bone marrow micronucleus assays, respectively. Three concentrations of SL (16, 32, and 24?μg/mL) were used for the evaluation of its mutagenic potential in V79 cells and four doses (0.25, 0.50, 1.0, and 2.0?g/kg body weight) were used for Swiss mice. In the antimutagenicity assays, the different concentrations of SL were combined with the chemotherapeutic agent doxorubicin (DXR). HPLC analysis of SL gave contents of 6.57?%?±?0.41 of solasonine and 4.60?%?±?0.40 of solamargine. Total flavonoids and polyphenols contents in SL were 0.04 and 3.60?%, respectively. The results showed that not only SL exerted no mutagenic effect, but it also significantly reduced the frequency of chromosomal aberrations induced by DXR in both V79 cells and micronuclei in Swiss mice at the doses tested.     

Tartrazine-induced chromosomal aberrations in mammalian cells

Tartrazine (FD & C Yellow No.5) has been shown to induce chromosomal aberrations in fibroblast cells of Muntiacus muntjac in vitro. M. muntjac cells were exposed to various concentrations of tartrazine (in the 5-20 micrograms/ml range) and were evaluated for induced chromosomal aberrations after two different periods of culture. Total percentages of chromosomal aberrations were significantly increased above control levels in all experimental groups. The results suggest that further studies are needed to determine the potential mutagenic effects of tartrazine.     

二氯胺基酚对 V79 细胞 DNA 损伤效应的研究

我们采用单细胞凝胶电泳新技术和ＳＣＥ试验研究了８３－１除草剂的主要代谢产物２，４－二氯－６－胺基酚（ＤＣＡＰ）对Ｖ７９细胞的ＤＮＡ损伤作用。结果表明，ＤＣＡＰ浓度≥５０μｇ／ｍｌ时，细胞出现明显的ＤＮＡ迁移和姐妹染色单体交换（Ｐ＜０．０１），说明ＤＣＡＰ是ＤＮＡ损伤剂。结合前期研究结果推断，６位硝基还原成胺基是该除草剂在体内活化致癌的基本途径。结果还表明，单细胞凝胶电泳比ＳＣＥ试验更灵敏，可能是研究环境低剂量暴露的遗传危害最有效的手段。     

Diesel exhaust particulate matter dispersed in a phospholipid surfactant induces chromosomal aberrations and micronuclei but not 6-thioguanine-resistant gene mutation in V79 cells

Diesel exhaust particulate material (DPM) was assayed for induction of chromosomal aberrations (CA), micronucleus (MN) formation, and 6-thioguanine-resistant (TG9 gene mutation in V79 cells as a dispersion in dipalmitoyl phosphatidylcholine (DPPC) in physiological saline, a simulated pulmonary surfactant. Filter-collected automobile DPM provided for the study was not organic solvent extracted, but was directly mixed into DPPC in saline dispersion as a model of pulmonary surfactant conditioning of a soot particle depositing in a lung alveolus. A statistically significant difference was found between treated and control groups at all concentrations tested in a CA assay. Assay for MN induction also gave a positive response: Above 50 microg/ml, the frequencies of micronucleated cells (MNC) were about 2 times higher than those in the control group. The forward gene mutation assay did not show a positive response when cells were treated with up to 136 microg DPM/ml for 24 h, as dispersion in DPPC in saline. Some comparison assays were run on direct dispersions of the DPM into dimethyl sulfoxide, with results equivalent to those seen with a DPPC-saline preparation: DPM in dimethyl sulfoxide (DMSO) was positive for MN induction but was negative for forward gene mutation in V79 cells. The positive clastogenicity results are consistent with other studies of DPM dispersed into DPPC-saline surfactant that have shown activity in mammalian cells for sister chromatid exchange, unscheduled DNA synthesis, and MN induction. The forward gene mutation negative results are consistent with studies of that assay applied to V79 cells challenged with DPM solvent extract.     

氟吗啉诱发V79和CHL细胞基因突变和染色体畸变的研究

目的 探讨氟吗啉的致突变性。方法 首先测定氟吗啉对V79和CHL的细胞毒性，然后在非代谢活化和代谢活化条件下，进行氟吗啉诱发V79细胞HGPRT基因突变试验和CHL细胞染色体畸变试验。结果 以氟吗啉500、100、20和4μg/mL浓度处理V79细胞，处理组诱变率与阴性对照组突变率比较，差异无显著性(P≥0．05)；以500、250、125和62．5μg／mL浓度处理CHL细胞24、48h后，在非代谢活化条件下，处理组染色体畸变均小于5％，但在代谢活化条件下，处理组染色体畸变率均大于5％，而且呈剂量．反应关系，通过G-显带发现断裂集中发生于4q上，是断裂热点。结论 可以认为应用氟吗啉100～200mg／L防治植物病害不会对人类健康造成危害，但是职业人群应注意防护。     

Hydrogen peroxide increases the activity of γ-glutamylcysteine synthetase in cultured Chinese hamster V79 cells

Hydrogen peroxide (H₂O₂) caused a rapid and a concentration-dependent increase in the activity of γ-glutamylcysteine synthetase (γ-GCS) in cultured Chinese hamster V79 cells. The increase in the activity was transient and declined rapidly during post-treatment incubation. Inhibition of protein synthesis by cycloheximide, chelation of divalent iron byo-phenanthroline, and scavenging of free radicals by butyl-4-hydroxyanisole failed to suppress the increase in activity of γ-GCS caused by H₂O₂. However, catalase completely inhibited the increase in the activity of the enzyme. H₂O₂ did not change the level of total glutathione (GSH+GSSG) but is oxidized GSH. The increased in levels of GSSG caused by H₂O₂ was enhanced byo-phenanthroline. These results suggest that the increase in activity of γ-GCS caused by H₂O₂ is not an inducible phenomenon, nor it is attributable to the action of free radicals generated by an iron-catalyzed Fenton reaction. Furthermore, the changes in levels of GSH and GSSG caused by H₂O₂ appear not to be responsible for the increase in activity of γ-GCS caused by the hydroperoxide. However, chemical reduction of the enzyme, the activity of which had been increased by H₂O₂, resulted in a decrease, in the activity, suggesting the involvement of oxidation of the enzyme in the increased activity of γ-GCS caused by H₂O₂. The results also suggest that the activity of γ-GCS in cultured V79 cells can be regulated by the cellular oxidation-reduction state.     

Effects of propolis crude hydroalcoholic extract on chromosomal aberrations induced by doxorubicin in rats

Propolis has been reported to display a broad spectrum of biological activities such as anticancer, antioxidant, anti-inflammatory, antibiotic and antifungal properties, among others. There is great interest not only in the determination of the chemical composition of propolis but also in the understanding of the mechanisms related to its therapeutic actions. In this respect, the aim of the present investigation was to study the influence of both simultaneous (6, 12 and 24 mg/kg b. w.) and subacute (12 mg/kg b. w.) treatment with a crude hydroalcoholic extract of propolis on the frequency of chromosome aberrations induced by the chemotherapeutic agent doxorubicin (DXR) in Wistar rat bone marrow cells. HPLC analysis of the crude extract allowed the quantification of the phenolic compounds: caffeic acid, P-coumaric acid, aromadendrin 4'-methyl ether, 3-prenyl- P-coumaric acid (drupanin), isosakuranetin, kaempferide, 3,5-diprenyl- P-coumaric acid (artepellin C), baccharin and 2,2-dimethyl-6-carboxyethenyl-2 H-1-benzopyran. A total of 100 metaphase cells/animal were analyzed for chromosome aberration frequency and 1000 cells/animal were counted to obtain the mitotic index. The results showed that the dose of 12 mg propolis/kg b. w., administered either as a single dose or as subacute treatment, caused a statistically significant decrease in the frequency of chromosome damage induced by DXR compared to the group treated only with DXR. This reduction might be, in part, due to the presence of phenolic compounds in the studied propolis, which are able to capture free radicals produced by chemotherapeutic agents such as DXR.     

Induction of apoptosis by Phenothiazine derivatives in V79 cells.

Phenothiazine derivatives chlorpromazine (cpz) and trifluoperazine (tfp) were found to induce apoptosis, abnormal cell cycle and expression of p53 in Chinese hamster lung fibroblast V79 cells. Both the drugs can induce apoptosis when cells are treated with drug at a concentration of 10 microg/ml within 4 h, as detected by propidium iodide staining and DNA fragmentation analysis. Flow cytometric analysis revealed that the apoptotic response is mediated by a loss of G(1) population of cells. In Western blot analysis, p21 is induced and p53 is accompanied by additional bands. Also indirect immunolabeling of single cells revealed that p21 is accumulated from cytoplasm into nucleus after the drug treatment and the intensities of p53 increased. Our findings demonstrate for the first time that phenothiazine derivatives, in addition to their cytotoxic effects, could induce apoptosis, an observation that has important clinical implications.     

Oxidative DNA lesions in V79 cells mediated by pentachlorophenol metabolites

 Incubation of the pentachlorophenol (PCP) metabolites, tetrachloro-p-benzoquinone (chloranil, TCpBQ), tetrachloro-p-hydroquinone (TCpHQ) and tetrachloro-o-benzoquinone (TCoBQ) with V79 Chinese hamster cells led to a significant enhancement of the amount of 8-hydroxydeoxyguanosine (8-OH-dG) in DNA. With PCP itself and the metabolite tetrachloro-o-hydroquinone (TCoHQ) no distinct induction of this lesion could be observed. The average yields of 8-OH-dG were about 2–2.5 times above background levels. In addition, TCpBQ and TCpHQ were able to generate DNA single-strand breaks, while PCP, TCoHQ and TCoBQ failed to induce this lesion. All incubations were performed for 1 h without exogenous metabolic activation and concentrations were 25 μM of the respective agent. It is concluded that these metabolites may contribute to the carcinogenicity of PCP observed in mice, by generating reactive oxygen species (ROS) through their redox cycling properties. Received: 20 September 1995/Accepted: 14 November 1995     

Inhibition of DNA replication by ozone in Chinese hamster V79 cells

DNA replication in Chinese hamster lung fibroblasts, line V79, was depressed in a dose-dependent manner over an ozone concentration range of 1-10 ppm. When the cells were exposed for 1 h at concentrations up to 6 ppm, the rate of DNA replication, as measured by [3H]thymidine incorporation, declined further during a 3-h period immediately following exposure. At higher ozone concentrations, at which more than 99.9% of the cells were killed, no further decline in DNA replication was seen beyond that immediately following exposure. Cultures exposed for 1 h to 10 mM ethyl methanesulfonate or to 10 J/m2 of ultraviolet (UV) light showed a similar progressive decline in the rate of DNA replication. The inhibition of DNA replication by ozone resembled that seen after exposure of cells to chemical mutagens or radiation and did not resemble the inhibition produced by metabolic poisons. The results may indicate that ozone or its reaction products interact directly with DNA in a way that inhibits replication.     

Induction of the bystander effect in Chinese hamster V79 cells by actinomycin D

Bystander effect (BE) can be induced by ionizing radiation and chemicals, including alkylating agents. Ionizing radiation mostly induces the bystander effect by causing double-strand DNA breakage in the exposed cells. However, the chemical-induced bystander effect is poorly studied. Here we chose actinomycin D (ACTD), a genotoxic chemotherapeutic drug, to investigate whether it could cause bystander effect in Chinese hamster V79 cells. Results are that (1) ACTD induced apoptosis in V79 cells and an optimal apoptosis model in V79 cells was established with ACTD (4 mg/L, 1 h); (2) using apoptosis rate, chromosome aberration, and ultrastructure changes as endpoints of bystander effect, ACTD could induce bystander effect in V79 cells; (3) as in the exposed cells, ACTD mainly induced apoptosis in bystander V79 cells cultured in different period conditioned medium; (4) the strongest bystander effect was induced by 4 h conditioned medium collected from cells treated with ACTD. It suggests that ACTD could cause BE through the medium soluble factors excreted from exposed cells during apoptosis and ACTD-induced BE was a novel quantitative and kinetic response.     

The repair of DNA damage induced in V79 mammalian cells by the nitroimidazole-aziridine, RSU-1069. Implications for radiosensitization

The induction and repair of single (ssb) and double (dsb) strand breaks in DNA under aerobic or hypoxic conditions have been determined using sucrose sedimentation techniques following incubation of V79 mammalian cells with RSU-1069 or misonidazole, representative of a conventional 2-nitroimidazole radiosensitizer, for 1-1.5 hr at either 293 or 277 degrees K and subsequent irradiation at 277 degrees K. In all cases, the dose dependences for the induction of strand breaks are linear and consistent with an enhancement in the yield of DNA damage induced by the 2-nitroimidazoles under hypoxic conditions. With RSU-1069 at 293 degrees K, the dose dependence of ssb is displaced reflecting DNA damage induced during pre-incubation. From these dependences, it is evident that the enhanced radiosensitization by RSU-1069 may not be accounted for in terms of accumulation of the agent at DNA. From the repair studies, DNA breaks induced by RSU-1069 in the absence of radiation have been shown to persist for at least 3 hr. With a combination of RSU-1069 and radiation under hypoxic conditions, the repair timescale of the induced breaks is significantly longer and an increase in the residual yields of both ssb and dsb (at 2-3 hr) was observed when compared with the observation in the presence of misonidazole or oxygen. From these studies, it is inferred that the enhanced radiosensitization of RSU-1069 at 293 degrees K is a consequence of the formation of non-repairable DNA damage together with a modification of the repairability of the radiation-induced DNA breaks.     

Acrylamide catalytically inhibits topoisomerase II in V79 cells

The vinyl monomer acrylamide is characterized by the presence of an α,β-unsaturated carbonyl group that makes it reactive towards thiol, hydroxyl or amino groups and towards the nucleophilic centers in DNA. The ability of acrylamide to chemically modify protein thiols has prompted us to consider topoisomerase II as one possible target of acrylamide, since agents targeting protein sulfhydryl groups act as either catalytic inhibitors or poisons of topoisomerase II. Nuclear extracts from V79 Chinese hamster cells incubated with acrylamide reduced topoisomerase II activity as inferred by an inability to convert kinetoplast DNA to the decatenated form. Nuclear extracts incubated with acrylamide pre-incubated with DTT converted kinetoplast DNA to the decatenated form, suggesting that acrylamide influences topoisomerase II activity through reaction with sulfhydryl groups on the enzyme. Furthermore, acrylamide did not induce the pBR322 DNA cleavage, as assessed by cleavage assay; thus, it cannot be regarded as a poison of topoisomerase II. As a catalytic inhibitor, acrylamide antagonizes the effect of etoposide, a topoisomerase II poison, as determined by clonogenic assay in V79 cells. This antagonism is confirmed by band depletion assay, from which it can be inferred that acrylamide reduces the level of catalytically active cellular topoisomerase II available for the action of etoposide.     

V79—6TG^s和V79—6TG^r细胞对诱变剂敏感性比较

为探讨细胞先前发生的突变对诱变剂敏感性的影响，在Ｖ７９细胞基因突变试验中用６－ＴＧ选择了抗６－ＴＧ的突变细胞。应用ＳＣＥ和微核试验比较了Ｖ７９－６ＴＧｓ（野生型细胞）和Ｖ７９－６ＴＧｒ（突变型细胞）对ＭＮＮＧ，ＭＭＳ和ＥＭＳ的敏感性。结果表明，Ｖ７９－６ＴＧｒ细胞对上述３种烷化剂较Ｖ７９－６ＴＧｓ敏感。结论指出，Ｖ７９－６ＴＧｒ细胞对诱变剂的敏感性增高可能与先前发生的ｈｐｒｔ基因突变有关。     

Induction of micronuclei in V79 cells by the anabolic doping steroids tetrahydrogestrinone and trenbolone

The synthetic steroid tetrahydrogestrinone is a new “designer drug” and was recently detected to be illegally used in sports. It is chemically closely related to trenbolone that is known as an animal growth promoter. The potencies of trenbolone, tetrahydrogestrinone and testosterone to induce micronuclei in V79 cells in vitro were determined. CREST analysis was employed to differentiate between aneugenic or clastogenic mechanisms. Cytotoxicity and an influence on the cell cycle were assessed in parallel. Incubations with testosterone, at concentrations between 3 and 300 μM, failed to induce micronuclei. By contrast, tetrahydrogestrinone and trenbolone increased the rate of micronuclei significantly, up to a doubling of the micronuclei rate of untreated controls. Tetrahydrogestrinone and trenbolone displayed a bell-shaped dose-response curve, with maximal effects observed at 3 and 30 μM, respectively. The micronuclei induced by tetrahydrogestrinone and trenbolone were predominantly kinetochor (CREST) positive, pointing to an aneugenic mode of action. This may be related to the specific structure of both molecules with a system of activated double bonds. As the genotoxic effect of tetrahydrogestrinone at a chromosomal level appears at a low concentration range, it cannot be ruled out that tetrahydrogestrinone presents a genotoxic hazard on a chromosomal level under conditions of its current misuse in sports.     

Changes in antioxidant defense systems induced by thiram in V79 Chinese hamster fibroblasts.

The role of antioxidant defence systems in protection against oxidative damage of lipids and proteins induced by fungicide thiram during in vitro exposure was investigated in cultured Chinese hamster V79 cells with normal, depleted, and elevated glutathione (GSH) levels. We analyzed the catalytic activities of superoxide dismutases (SOD1 and SOD2), Se-dependent and Se-independent glutathione peroxidases (GSH-Px), glutathione reductase (GR), and catalase (CAT), as well as total glutathione/glutathione disulfide ratio (GSH(total)/GSSG). Thiram treatment resulted in an increase in activities of SOD1, Se-dependent GSH-Px, and GR at the highest tested dose (150 microM). On the contrary, inhibition of CAT and Se-independent GSH-Px activities, and no significant changes in the level of SOD2 activity was observed at any tested doses (100-150 microM). GSH(total)/GSSG ratio in the 100 microM thiram treated cells was not significantly changed comparing to the control, despite significant decrease of GSH total (50%). In 150 microM thiram treated cells the ratio falls to 43% of control value. Pretreatment with l-buthionine sulfoximine (L-BSO), an inhibitor of GSH synthesis, significantly enhanced decrease in CAT and Se-independent GSH-Px activities, as well as GSH(total)/GSSG ratio, and reduced Se-dependent GSH-Px activity, following exposure to thiram. Simultaneously, L-BSO pretreatment enhanced increase in SOD1 activity, and had no effect on SOD2, following thiram exposure. Pretreatment with N-acetyl cysteine (NAC), a GSH precursor, prevented enzymatic changes in CAT, Se-dependent GSH-Px, GR, SOD1 activities, and significantly decreased SOD2 activity following exposure to thiram. GSH(total)/GSSG ratio was restored to the control value. This study suggests that following the changes in antioxidant defense systems thiram can act through the production of free radicals.     

Cytotoxicity of prodigiosin and benznidazole on V79 cells

The cytotoxicity of prodigiosin, an antibiotic and potential trypanocide produced by Serratia marcescens, and Benznidazole, a trypanocidal drug, were assayed on V79 fibroblast cell line. Three independent endpoints for cytotoxicity were evaluated; namely, the nucleic acid content (NAC), MTT reduction and neutral red uptake (NRU). IC(50) values of 1-20 microM were obtained for prodigiosin in the NRU, MTT and NAC tests. Prodigiosin had greater trypanocidal activity (IC(50)=5 microM) than Nifurtimox (IC(50)=150 microM) a known trypanocide drug used in Chagas' disease therapy. Benznidazole was less toxic (IC(50)=2000 microM) than prodigiosin (IC(50)=1-20 microM) in V79 cells based on the MTT and NAC assays. Benznidazole stimulated the NRU until 2 mM. Indeed, the cell viability measured with the NRU was higher at all concentrations of benznidazole tested than that measured by MTT reduction and NAC assays.     

Chromosomal aberrations induced in mouse bone marrow cells by municipal landfill leachate

The cytogenetic damage induced by municipal landfill leachate was studied using chromosomal aberration (CA) in mouse bone marrow assay. Results show that leachate samples collected in different seasons decreased the mitotic index (MI), and caused significant increases of CA frequencies in treatment concentration (Chemical oxygen demand (COD) measured by the method of potassium dichromate oxidation, COD(Cr))-dependent manners. Compared with the negative control, reductions of the MI of 54 and 38% were detected for the highest leachate concentration (COD(Cr) 320mg/L) in mouse bone marrow treated with both samples. The frequencies of CA increased significantly with increasing concentrations of sample 1 from COD(Cr) 40 to 320mg/L, and from 80 to 320mg/L after exposure to sample 2. In addition, a seasonal difference of MI and CA frequencies induced by leachate was observed. The results confirm that leachate is a genotoxic agent in mammalian cells, and imply that exposure to leachate in aquatic environment may pose a potential genotoxic risk to mammals and humans. The results suggest that the CA in mouse bone marrow bioassay is efficient in genotoxicity studies of leachate on mammals, and that there appears to be a correlation between the genotoxicity in mammal system and the chemical measurement (COD(Cr)) of leachate. The results also indicate that different discharge guidelines and environmental quality standards should be established for leachates discharged from landfills to aquatic environment in different seasons.     

Inhibition of bleomycin-induced DNA strand breaks in V 79 Chinese hamster cells by the antioxidant propylgallate

Bleomycin induced DNA single-strand breaks in Chinese hamster V 79 cells which were detected by the alkaline filter elution assay. In the presence of propylgallate, an antioxidant, the amount of DNA single-strand breaks was significantly reduced. The production of DNA strand breaks by methylnitronitrosoguanidine used as a positive control was not influenced by propylgallate. It is suggested that propylgallate inhibits the generation of DNA single-strand breaks by trapping reactive oxygen species produced by the bleomycin-iron(II) complex.