The human 8-oxoguanine DNA N-glycosylase 1 (hOGG1) DNA repair enzyme and its association with lung cancer risk

OBJECTIVE: The human 8-oxoguanine DNA N-glycosylase 1 gene encodes a DNA glycosylase that is involved in the base excision repair of 8-hydroxy-2-deoxyguanine from oxidatively-damaged DNA and expressed in lung tissue. The codon 326 polymorphism in the hOGG1 gene has been suggested to reduce DNA repair enzyme activity based on in vitro functional analysis. The goal of the present study is to determine whether the codon 326 polymorphism was significantly associated with alterations in individual risk for lung cancer. METHODS: To determine whether hOGG1 plays a role in risk for lung cancer, we measured the prevalence of the Ser326Cys polymorphism in incident lung cancer patients and matched non-cancer controls. hOGG1 genotyping was performed by PCR-restriction fragment length polymorphism analysis of genomic DNA isolated from 179 Caucasian lung cancer cases and 358 controls individually matched in a 1:2 ratio by race-, sex- and age (+/- 5 years). RESULTS: Significantly increased risk for lung cancer was observed for both the hOGG1 326 (odds ratio [OR] = 1.9, 95% confidence interval [CI] = 1.2-2.9) and hOGG1 326 genotypes (OR = 3.8, 95% CI = 1.4-10.6). The increased risk for lung cancer was observed for subjects with both the hOGG1 326 (OR = 1.7, 95% CI = 1.1-2.8) and hOGG1 326 genotypes (OR = 4.9, 95% CI = 1.5-16.1) in ever-smokers. A significant association was found between hOGG1 genotypes and lung cancer risk with a dose-dependent effect with smoking. Significantly increased risk for variant hOGG1 genotypes was observed for all non-small cell lung cancer patients. CONCLUSION: These results suggest that the hOGG1 Ser326Cys polymorphism plays an important role in the risk for lung cancer and is linked to exposure to tobacco smoke.     

Effects of an acute exposure to toluene on the DNA repair activity of the human 8-oxoguanine DNA glycosylase 1 (hOGG1) in healthy subjects

The structure and previous studies on the biotransformation of toluene lead to the suspicion that metabolites may be formed which preferentially react with strongly nucleophilic partners such as sulfhydryl groups of cysteines in proteins. Human 8-oxoguanine DNA glycosylase 1 removes the major oxidative DNA damage and possesses eight cysteines. Its potential inactivation may lead to accumulation of DNA damage by reactive oxygen species formed by exogenous agents or by ubiquitous endogenous processes. The goal of the present investigation was to study the in vivo effect in humans of an acute toluene exposure on hOGG1 activity. Twenty healthy, non-smoking males were exposed to 50 ppm toluene and to filtered air in an exposure chamber for 270 min, using a cross-over design. Before and 30 min after the end of exposure, blood samples were taken and toluene concentrations and the hOGG1 activity were measured. hOGG1 activity was determined in peripheral mononuclear blood cells. Thirty minutes after exposure to toluene, we found a median blood concentration of 0.25 mg toluene/l. Compared with the activity before exposure, upon exposure to toluene a statistically insignificant median increase of hOGG1 activity by +0.4% and upon exposure to air by +2.3% was determined. Thus, no reduction of the hOGG1 repair activity after acute exposure to 50 ppm toluene was observed.     

Cryopreservation of human blood for alkaline and Fpg-modified comet assay

The Comet assay is a reproducible and sensitive assay for the detection of DNA damage in eukaryotic cells and tissues. Incorporation of lesion specific, oxidative DNA damage repair enzymes (for example, Fpg, OGG1 and EndoIII) in the standard alkaline Comet assay procedure allows for the detection and measurement of oxidative DNA damage. The Comet assay using white blood cells (WBC) has proven useful in monitoring DNA damage from environmental agents in humans. However, it is often impractical to performance Comet assay immediately after blood sampling. Thus, storage of blood sample is required. In this study, we developed and tested a simple storage method for very small amount of whole blood for standard and Fpg-modified modified Comet assay. Whole blood was stored in RPMI 1640 media containing 10% FBS, 10% DMSO and 1?mM deferoxamine at a sample to media ratio of 1:50. Samples were stored at ?20?°C and ?80?°C for 1, 7, 14 and 28 days. Isolated lymphocytes from the same subjects were also stored under the same conditions for comparison. Direct DNA strand breakage and oxidative DNA damage in WBC and lymphocytes were analyzed using standard and Fpg-modified alkaline Comet assay and compared with freshly analyzed samples. No significant changes in either direct DNA strand breakage or oxidative DNA damage was seen in WBC and lymphocytes stored at ?20?°C for 1 and 7 days compared to fresh samples. However, significant increases in both direct and oxidative DNA damage were seen in samples stored at ?20?°C for 14 and 28 days. No changes in direct and oxidative DNA damage were observed in WBC and lymphocytes stored at ?80?°C for up to 28 days. These results identified the proper storage conditions for storing whole blood or isolated lymphocytes to evaluate direct and oxidative DNA damage using standard and Fpg-modified alkaline Comet assay.     

Twelve-gel slide format optimised for comet assay and fluorescent in situ hybridisation

The comet assay is widely used to measure DNA damage and repair in basic research, genotoxicity testing and human biomonitoring. The conventional format has 1 or 2 gels on a microscope slide, 1 sample per slide. To increase throughput, we have designed and tested a system with 12 smaller gels on one slide, allowing incubation of individual gels with different reagents or enzymes. Thus several times more samples can be analysed with one electrophoresis run, and fewer cells and smaller volumes of test solutions are required. Applications of the modified method include treatment with genotoxic agents at different concentrations; simultaneous analysis of different lesions using a range of enzymes; analysis of cell extracts for DNA repair activity; and fluorescent in situ hybridisation (FISH) to comet DNA with specific labelled probes.     

Evaluation of environmental waters using the comet assay in Tilapia rendalli

Testing for environmental pollutants is an ever-growing concern. Various tests in organisms have been utilized for the detection and identification of toxic substances in the air, water and soil. In the present study, we utilized the comet assay in Tilapia rendalli to conduct an environmental assessment of Lake Igapó II, a lake located in the metropolitan area of Londrina, PR-Brazil. The results demonstrated that samples from Lake Igapó II had a significantly greater number of comets, mainly in classes 2 and 3. The results suggest a genotoxicity of the aquatic environment at Lake Igapó II and that the comet assay in T. rendalli provides adequate sensitivity to be utilized as a tool in the monitoring of water pollution and environmental risk assessment.     

Sensitivity of the FPG protein towards alkylation damage in the comet assay

The comet assay (single cell gel electrophoresis) is widely used for the evaluation of DNA-damaging effects in genotoxicity testing and population monitoring. In its standard version at pH >13, DNA double strand breaks (DSB), DNA single strand breaks (SSB) and alkali-labile sites (ALS) lead to increased DNA migration. At reduced pH (12.5-12.1) the expression of ALS as SSB can be eliminated and the effect of SSB only can be identified. Specific endonucleases have been used to characterize specific classes of DNA damage. The formamido pyrimidine glycosylase (FPG) protein has been used to assess oxidative DNA base damage because it detects 8-OH guanine and other oxidatively damaged purines. Here, we show that the FPG protein also detects alkylation damage with high sensitivity in the comet assay. Human whole blood, isolated lymphocytes and V79 cells were treated with alkylating agents and post-incubated with FPG. FPG strongly enhanced MMS- and EMS-induced DNA damage but had no significant effect on ENU-induced DNA damage, indicating that the amount of N-7 guanine alkylation is responsible for the observed effect. Reducing the pH during alkali unwinding and electrophoresis to 12.5 to avoid the contribution of ALS to the comet assay effects, strongly decreased the sensitivity of the comet assay with and without FPG treatment and prevented DNA migration. We conclude that enhanced DNA effects in the comet assay by FPG after exposure to genotoxins with unknown mode of action should not directly be regarded as evidence for the presence of oxidative damage. Furthermore, reducing the pH leads to a considerable loss in sensitivity and should not be used in biomonitoring and other applications which require a sensitive protocol.     

DNA damage measured by comet assay and 8-OH-dG formation related to blood chemical analyses in aged rats

To evaluate the effects of aging on DNA damage, spontaneous and chemical-induced DNA damage and its repair were examined using comet assays at pH 9, 12.1 and 13, and an 8-OH-dG assay in the liver and kidney of young (9-week-old) and aged (20-month-old) rats. Additionally, blood chemistry was examined to investigate any correlation between vital functions and age-dependent DNA damage. DNA migration at pH 13 and 8-OH-dG levels increased in the liver and/or kidney of aged rats, but DNA migration did not increase at pH 9 or 12.1; that is, alkali-labile sites and 8-OH-dG were concomitantly accumulated in aged rats. These results suggest that 8-OH-dG production caused by reactive oxygen species exceeded glycosylation and that the glycosylation activity is far more than the AP endonucleation in aged rats. Methyl methanesulfonate (MMS, 80 mg/kg, i.p.) increased DNA migration at pH 12.1 and 13 in the liver and kidney at 3 and 24 hr after treatment in young and aged rats. The DNA damage in aged rats was less and decreased more slowly compared with young rats. The pictures of MMS-induced DNA migrations at pH 12.1 and 13 were very similar to each other. These results suggest that the adduct glycosylation and repair of the single-strand breaks (SSBs) of aged rats are less than those of young rats, although AP endonucleation is sufficient to remove the AP sites. N-nitrosodiethylamine (160 mg/kg, i.p.) increased DNA migration at pH 12.1 and 13 in the liver and kidney at 3 and 24 hr in young rats and at pH 12.1 and 13 in the kidney at 24 hr in aged rats. These results showed that SSBs were predominantly detected as chemical-induced DNA damage and DNA repairs such as N-glycosylase, DNA polymerase and DNA ligase, and that the metabolic activation declined in aged rats. Aspartate aminotransferase, alanine aminotransferase, total bilirubin, total cholesterol, total protein, globulin, creatinine and chloride age-dependently increased and alkaline phosphates, albumin/globulin ratio, inorganic phosphorus and potassium age-dependently decreased, and these changes were correlated with the DNA migration at pH 13 and/or 8-OH-dG. These results suggest that the activity of DNA repair and metabolic activation enzymes declines in aged rats and that the accumulation of spontaneous DNA damage may affect vital functions.     

Oxygenated water does not induce genotoxic effects in the comet assay

Drinking of oxygenated water (i.e. water with increased concentration of physically dissolved oxygen) is said to improve oxygen availability of the body and will do the consumer good. However, increased oxygen concentrations can also lead to an increased production of reactive oxygen species (ROS). If antioxidant defences are not completely efficient, ROS can cause cell injury including DNA damage. We therefore investigated whether drinking of oxygenated water can lead to increased DNA damage in peripheral blood cells of test subjects. We also tested whether direct exposure of V79 Chinese hamster cells to oxygenated medium or oxygenated Hank's solution for various time periods induces DNA damage. Induction of DNA damage was measured with the alkaline comet assay (single cell gel electrophoresis). The comet assay, in particular the modification with FPG post-treatment for the determination of oxidative DNA base damage, has been proven to be extremely sensitive for the detection of oxygen-induced DNA damage. However, both the in vivo and the in vitro studies with the comet assay in the absence and presence of FPG post-treatment did not provide evidence for a genotoxic effect of oxygenated water.     

hOGG1 promoter methylation,hOGG1 genetic variants and their interactions for risk of coal-borne arsenicosis: A case-control study

To identify the effect of hOGG1 methylation, Ser326Cys polymorphism and their interactions on the risk of coal-borne arsenicosis, 113 coal-borne arsenicosis subjects and 55 reference subjects were recruited. Urinary arsenic contents were analyzed with ICP-MS. hOGG1 methylation and Ser326Cys polymorphism was measured by mehtylation-specific PCR and restriction fragment length polymorphism PCR in PBLCs, respectively. The results showed that the prevalence of methylated hOGG1 and variation genotype (326 ^Ser/Cys & 326 ^Cys/Cys) were increased with raised levels of urinary arsenic in arsenicosis subjects. Increased prevalence of methylated hOGG1 and variation genotype were associated with raised risk of arsenicosis. Moreover, the results revealed that variant genotype might increase the susceptibility to hOGG1 methylation. The interactions of methylated hOGG1 and variation genotype were also found to contribute to increased risk of arsenicosis. Taken together, hOGG1 hypermethylation, hOGG1 variants and their interactions might be potential biomarkers for evaluating risk of coal-borne arsenicosis.     

Genotoxicity of environmental agents assessed by the alkaline comet assay

Generation of DNA damage is considered to be an important initial event in carcinogenesis. A considerable battery of assays exists for the detection of different genotoxic effects of compounds in experimental systems, or for investigations of exposure to genotoxic agents in environmental or occupational settings. Some of the tests may have limited use because of complicated technical setup or because they only are applicable to a few cell types. The single cell gel electrophoresis (comet) assay is technically simple, relatively fast, cheap, and DNA damage can be investigated in virtually all mammalian cell types without requirement for cell culture. The aim of this thesis was to evaluate the comet assay as a genotoxicity test in genetic toxicology of environmental agents, encompassing both experimental animal models and biomonitoring. The comet assay detects strand breaks (SB). The cells are embedded in agarose and lysed, generating nucleus-like structures in the gel (referred to as nucleoids). Following alkaline electrophoresis, the DNA strands migrate toward the anode, and the extent of migration depends on the number of SB in the nucleoid. The migration is visualized and scored in a fluorescence microscope after staining. Broad classes of oxidative DNA damage can be detected as additional SB if nucleoids are incubated with bacterial DNA glycosylase/endonuclease enzymes. Oxidized pyrimidines and purines can be detected by incubation with endonuclease III and formamidopyrimidine DNA glycosylase, respectively. The animal experimental studies indicated that the comet assay was able to detect genotoxic effects of diesel exhaust particles in lung tissue, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-induced DNA damage in colon epithelial cells and liver tissue, and benzene-induced damage in bone marrow and liver cells. The strength of the comet assay was further outlined by application of repair enzymes, indicating no oxidative DNA base damage following IQ treatment. High levels of oxidative DNA lesions were detected after exposure to benzene or X-ray irradiation. The comet assay did not detect DNA damage in colon or liver following ingestion of diets containing of high contents of animal fat or sucrose, although other indices of DNA damage were found. Determined from the results of a large Japanese study, the discrimination between carcinogens and non-carcinogens appears to be similar between the comet assay and alkaline elution, which also detects SB. This suggests that the comet assay is a reliable genotoxicity test in animal experimental systems. In the biomonitoring studies, we investigated the effect of common exposures and lifestyle factors (rather than effects of known carcinogens) on the level of oxidative DNA damage in mononuclear blood cells of humans. In the first study, based on repeated measurements, it was shown that interindividual variation and seasonal variation were major determinants for the basal level of SB, whereas no effect of age, exercise, or antioxidant intake could be detected. The effect of exercise was further investigated under both normoxic and hypoxic circumstances, showing a strong effect of hypoxia, and only effect of exercise in terms of SB in hypoxia. In a placebo-controlled parallel dietary fruit and vegetable (or the corresponding amount of antioxidants) intervention study, no effects of the level of oxidative DNA damage or sensitivity to hydrogen peroxide were observed. Although this may seem in contrast to other antioxidant intervention studies, a critical literature survey of antioxidant intervention studies on oxidative DNA damage suggested that well-controlled studies tended to show no effect of antioxidant supplementation. In summary, the aggregated data from the publications included in this thesis, and other publications encompassing the comet assay, indicate that the comet assay is a reliable method for detection of DNA damage in tissues of experimental animals. Although not all types of genotoxic exposures should be expected to result in DNA damage in mononuclear blood cells, the comet assay seems to be a valuable tool for detection of genotoxic exposure in humans. The comet assay indicates that DNA damage is abundant in mammalian cells and affected by lifestyle and many environmental exposures, including diet, exercise, hypoxia, and sunlight.     

Overestimation of nanoparticles-induced DNA damage determined by the comet assay

The increasing use of engineered nanoparticles (NPs) in a wide range of commercial products raises concern about the possible risks that NPs pose to human health. Many aspects of the interaction between living cells and NPs are still unclear, and a reliable assessment of NP genotoxicity would be important. One of the most common tests used for genotoxicity is the comet assay, a sensitive method measuring DNA damage in individual cells. The assay was originally developed for soluble molecules, but it is also used in the assessment of genotoxicity of NPs. However, concerns have been raised recently about the reliability of this test in the case of NPs, but no conclusive results have been presented. Using nuclei isolated from human epithelial cells incubated with NPs, we obtained clear evidence of overestimation of NP genotoxicity by the comet assay in the case of CeO₂, TiO_2, SiO₂, and polystyrene NPs. Removal of the NPs in the cytoplasm was effective in eliminating this genotoxicity overestimation (ex post damage) and determining the actual damage produced by the NPs during incubation with the cells (ex ante damage). This method could improve significantly the determination of NP genotoxicity in eukaryotic cells.     

免疫荧光染色微核试验检测非整倍体诱变剂

采用免疫荧光染色技术显示微核中的着丝粒，发现纺锤体毒物秋水仙碱诱发Ｖ７９，ＣＨＬ双核细胞徽核着丝粒阳性率分别在７６Ｖ和６６％以上，长春新碱诱发ＢＡＬＢ／ｃ小鼠骨髓红细胞微核着丝粒阳性率在７０％以上；而染色体断裂剂丝裂霉素Ｃ诱发Ｖ７９，ＣＨＬ双核细胞微核着丝粒阳性率分别低于２７％和２１％，环磷酞胺诱发ＢＡＬＢ／ｃ小鼠骨髓红细胞微核着丝粒阳性率低于１５％表明将免疫荧光染色技术与微核试验相结合，计数微核着丝粒阳性率，能够区分微核主要来源于染色体断裂剂引起的无着丝粒断片或纺锤体毒物造成的滞留在胞质中的整条染色体，从而达到简便、快速检测非整倍体诱变剂的目的．     

Evaluation of environmental genotoxicity by comet assay in Columba livia

The concentrations of recognized or suspected genotoxic and carcinogenic agents found in the air of large cities and, in particular, developing countries, have raised concerns about the potential for chronic health effects in the populations exposed to them. The biomonitoring of environmental genotoxicity requires the selection of representative organisms as “sentinels,” as well as the development of suitable and sensitive assays, such as those aimed at assessing DNA damage. The aim of this study was to evaluate DNA damage levels in erythrocytes from Columba livia living in the metropolitan area of Monterrey, Mexico, compared with control animals via comet assay, and to confirm the results via Micronuclei test (MN) and DNA breakage detection–fluorescence in situ hybridization (DBD–FISH). Our results showed a significant increase in DNA migration in animals from the area assayed compared with that observed in control animals sampled in non-contaminated areas. These results were confirmed by MN test and DBD–FISH. In conclusion, these observations confirm that the examination of erythrocytes from Columba livia via alkaline comet assay provides a sensitive and reliable end point for the detection of environmental genotoxicants.     

DNA damage in workers exposed to lead using comet assay

Lead (Pb) is a ubiquitous and toxic metal. Secondary Pb recovery unit workers are prone to possible occupational Pb exposure. Hence, this investigation was conducted to assess the genotoxic effect of Pb exposure in these workers. In the study, 45 workers were monitored for DNA damage in blood leucocytes. Simultaneously 36 subjects were used as control group in this study. All the subjects were estimated for Pb content in whole blood by ICP-MS. The alkali single cell gel electrophoresis assay (comet assay) was adopted for detecting the DNA damage. The air inside the premises of the unit had Pb concentrations of 4.2 microg/m(3). The level of DNA damage was determined as the percentage of cells with comets. The mean Pb content was found to be significantly higher in the study group (248.3 microg/l) when compared with the controls (27.49 microg/l). Significantly more cells with DNA damage (44.58%) were observed in the study group than in the control persons (21.14%). Smoking had a significant effect on DNA damage in the control group whereas an insignificant effect was noticed in the exposed workers. Study as well as the control group failed to show a significant effect on DNA damage with age (P>0.05). Pb content and years of exposure significantly correlated with DNA damage in the study group (r=0.602, r=0.690; P<0.01). The increased levels of DNA damage observed in the exposed workers, justifies the use of the comet assay for the evaluation of genotoxic effects in humans exposed to Pb.     

大鼠体内彗星试验方法的建立与应用研究

目的 建立大鼠肝细胞和外周血淋巴细胞的体内彗星试验方法 ,并应用该方法对遗传毒性阳性物甲磺酸乙酯(EMS)和环磷酰胺(CPA)进行检测。方法选择SD雄性大鼠为实验动物,分别给予不同剂量EMS和CPA。给药结束后,采集外周血样本和摘取动物左侧肝叶组织样本,铺制成单细胞凝胶玻片。玻片经裂解、解旋、电泳、中和、脱水等步骤后,得到可观察的实验标本。染色标本,并在40×荧光显微镜下观察拍照,使用专业分析软件对单细胞电泳图像进行分析和测定。结果成功建立了基于大鼠肝细胞和外周血淋巴细胞的体内彗星试验方法。经该方法检测,EMS结果为阳性,CPA结果为阴性,EMS适合作为本方法的阳性对照。结论成功建立大鼠体内彗星试验方法 ,并可应用于遗传毒性检测。     

Genotoxicity of glyphosate assessed by the comet assay and cytogenetic tests

It was evaluated the genotoxicity of glyphosate which up to now has heterogeneous results. The comet assay was performed in Hep-2 cells. The level of DNA damage in the control group (5.42±1.83 arbitrary units) for tail moment (TM) measurements has shown a significant increase (p<0.01) with glyphosate at a range concentration from 3.00 to 7.50mM. In the chromosome aberrations (CA) test in human lymphocytes the herbicide (0.20-6.00mM) showed no significant effects in comparison with the control group. In vivo, the micronucleus test (MNT) was evaluated in mice at three doses rendering statistical significant increases at 400mg/kg (13.0±3.08 micronucleated erythrocytes/1000 cells, p<0.01). In the present study glyphosate was genotoxic in the comet assay in Hep-2 cells and in the MNT test at 400mg/kg in mice. Thiobarbituric acid reactive substances (TBARs) levels, superoxide dismutase (SOD) and catalase (CAT) activities were quantified in their organs. The results showed an increase in these enzyme activities.     

Assessment of genotoxic effects of benzyl derivatives by the comet assay

In this study, different concentrations of four benzyl derivatives (benzyl alcohol, benzyl acetate, benzoic acid and benzaldehyde) used as flavour ingredients were investigated for genotoxicity in in vitro. By taking blood from two healthy people comet assay was carried on to investigate the potential health damages of benzyl derivatives. For the evaluation of genotoxic effects, the tail moment and % tail DNA in the treated chemicals were compared to the solvent control, which is distilled water. The alkaline comet assay showed significantly increased tail moment and % tail DNA at 25 and 50 mM concentrations of benzyl alcohol. Benzyl acetate increased both % tail DNA and tail moment at 50 mM concentrations. While % tail DNA was statistically increased at 10 mM and higher concentrations, tail moment has significant difference at 10 and 25 mM concentrations of benzaldehyde. Benzoic acid has apoptotic effects at the concentrations higher than 5 mM, for this reason we tested concentrations less than 5 mM (0.05, 0.1, 0.5, 1 and 5 mM). Only the highest concentration of benzoic acid increased both tail moment and % tail DNA.     

Genotoxicity of cadmium chloride in the marine gastropod Nerita chamaeleon using comet assay and alkaline unwinding assay

This paper presents an evaluation of the genotoxic effects of cadmium chloride (CdCl₂) on marine gastropod, Nerita chamaeleon following the technique of comet assay and the DNA alkaline unwinding assay (DAUA). In this study, the extent of DNA damage in gill cells of N. chamaeleon was measured after in vivo exposure to four different concentrations (10, 25, 50, and 75 µg/L) of CdCl₂. In vitro exposure of hydrogen peroxide (H₂O₂; 1, 10, 25, and 50 µM) of the gill cells showed a significant increase in the percentage tail DNA, Olive tail moment, and tail length (TL). Significant changes in percentage tail DNA by CdCl₂ exposure were observed in all exposed groups of snails with respect to those in control. Exposure to 75 µg/L of CdCl₂ produced significant decrease in DNA integrity as measured by DAUA at all duration with respect to control. In vivo exposure to different concentrations of CdCl₂ (10, 25, 50, and 75 µg/L) to N. chamaeleon showed considerable increase in DNA damage as observed by both alkaline comet assay and the DAUA. The extent of DNA damage in marine gastropods determined by the application of alkaline comet assay and DAUA clearly indicated the genotoxic responses of marine gastropod, N. chamaeleon to a wide range of cadmium concentration in the marine environment. © 2013 Wiley Periodicals, Inc. Environ Toxicol 30: 177–187, 2015.     

Genotoxic effects of 8-hydroxylquinoline in loach (Misgurnus anguillicaudatus) assessed by the micronucleus test,comet assay and RAPD analysis

This study was a preliminary step in evaluating the genotoxic effects of 8-hydroxylquinoline (8-HOQ) in loach (Misgurnus anguillicaudatus) using the micronucleus, comet and RAPD assays. In the micronucleus test and comet assay, the micronuclei rate (%) and three comet parameters (trailing rate, tail length and tail moment) in treated fish increased with increasing 8-HOQ concentration and treatment time. These results showed that exposure to 8-HOQ significantly induced genetic toxicity in loach blood cells. A subsequent RAPD assay also showed that 8-HOQ induced a genotoxic effect in loach. Among the 23 tested RAPD primers, 11 primers produced unique polymorphic band patterns and generated RAPD profile variations that displayed the band intensity, disappearance of bands and appearance of new bands of amplified DNA in the 8-HOQ-treated genomic DNA samples. In addition, the variation in RAPD profiles was time- and concentration-dependent. These results suggested that 8-HOQ is potentially harmful to fish and may be a toxic contaminant in the aquatic environment.