Potential value of the comet assay and DNA adduct measurement in dab (Limanda limanda) for assessment of in situ exposure to genotoxic compounds (original) (raw)
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… Research/Reviews in …, 1998
Molecular epizootiological studies are increasingly being used to investigate environmental effects of genotoxic contaminants. The assessment of damage to DNA and linking the damage to subsequent molecular, cellular, or tissue-level alterations is a central component of such studies. Our research has focused on the refinement of the 32 P-postlabeling assay for measuring covalent DNA-xenobiotic adducts arising from exposure to polycyclic aromatic compounds, using DNA adducts as molecular dosimeters of genotoxic contaminant exposure in biomonitoring studies, and investigating the relationship of DNA adduct formation to toxicopathic liver disease, including neoplastic lesions. A combination of field and laboratory studies using the 32 P-postlabeling assay has shown that DNA adducts in marine fish are effective molecular dosimeters of genotoxic contaminant exposure. Investigations of the relationship of DNA adduct formation to neoplastic liver disease have shown that elevated levels of DNA adducts in certain fish species from contaminated coastal sites are associated with increased prevalences of toxicopathic hepatic lesions, including neoplasms, and that the ability to assess DNA damage has helped to explain, in part, species differences in lesion prevalence. Moreover, in a study of a site in Puget Sound contaminated with polycyclic aromatic compounds, we have shown, for the first time, that elevated levels of hepatic DNA adducts are a significant risk factor for certain degenerative and preneoplastic lesions occurring early in the Ž . histogenesis of hepatic neoplasms in feral English sole Pleuronectes Õetulus . These latter findings coupled with our current studies of mutational events in the K-ras proto-oncogene should provide further mechanistic substantiation that mutagenic events resulting from exposure to complex mixtures of genotoxic polycyclic aromatic compounds are involved in the etiology of hepatic neoplasia in English sole. q
Chemical carcinogenesis in feral fish: uptake, activation, and detoxication of organic xenobiotics
Environmental Health Perspectives, 1987
The high prevalence of liver neoplasms in English sole (Parophrys vetulus) and substantially lower prevalence of neoplasms in a closely related species, starry flounder (Platichthys stellatus) captured from industrialized waterways, provide a unique opportunity to compare biochemical processes involved in chemical carcinogenesis in feral fish species. Because levels of aromatic hydrocarbons (AHs) in urban sediments are correlated with prevalences of liver neoplasms in English sole, we have initiated detailed studies to evaluate the effects of endogenous and exogenous factors on uptake, activation and detoxication of carcinogenic AHs, such as benzo[a]pyrene (BaP), using spectroscopic, chromatographic, and radiometric techniques. The results obtained thus far show that sole readily takes up AHs associated with sediment from urban areas and that the presence of other xenobiotics, such as PCBs, in sediment increases tissue concentrations of BaP metabolites. Extensive metabolism of BaP occurred whether sole was exposed to this AH via sediment, per os, or intraperitoneally. Substantial modification of hepatic DNA occurred and persisted for a period of 2-4 weeks after a single exposure to BaP. The level of covalent binding of BaP intermediates to hepatic DNA was 10-fold higher in juvenile than adult sole and 90-fold higher in juvenile sole than in Sprague-Dawley rat, a species which is resistant to BaP-induced hepatocarcinogenesis. The level of chemical modification of hepatic DNA in juvenile flounder was 2-4 fold lower than that for juvenile sole and concentration of BaP 7,8-diol glucuronide in bile of sole was significantly higher than that in flounder bile, although the rate of formation of BaP 7,8-diol by hepatic microsomes was comparable for both species. Moreover, liver microsomes from both species, in the presence of exogenous DNA, metabolized BaP into essentially a single adduct, identified as (+)anti-7,8-diol-9,10-epoxy-7,8,9,10-tetrahydroBaP-dG. These results, along with our findings that hepatic GST activity in flounder was two times higher than in sole, demonstrate that microsomal metabolism of BaP does not accurately reflect the differences in the ability of these fish to form BaP-DNA adducts in vivo and also suggest that detoxication of reactive intermediates is an important factor in determining the levels of DNA modification by AHs and resulting toxic effects in feral fish.
Bulletin of The European Association of Fish Pathologists, 2006
Field and laboratory studies indicate a strong positive association between exposure to chemical pollutants in aquatic environments and development of neoplasia in fishes. This brief communication reviews some of the more important North American and European studies that have been conducted on this relationship. We then review work conducted on a small nonmigratory estuarine cyprinodontid teleost fish, the mummichog (Fundulus heteroclitus) in the industrialized Elizabeth River, Virginia USA. Histopathological surveys of mummichogs from variously degraded habitats indicate an association between PAH exposure and development of neoplasia. We have observed non-neoplastic lesions, preneoplasms and hepatic, biliary, exocrine pancreatic and vascular neoplasms in mummichogs inhabiting Virginia and Maryland estuaries. These lesions track a sediment PAH gradient with highest prevalence occurring in fish from PAI-I-contaminated sites in the industrialized portions of the Elizabeth River, Virginia. Liver metabolizing enzyme profiles typical of those observed in carcinogen-challenged laboratory rodents (e.g., depressed phase I enzymes, elevated phase II enzymes, elevated p-glycoprotein) provided additional support of a chemical etiology for the neoplasms occurring in these fish. Long-term laboratory exposure of mummichogs to creosote-contaminated sediments resulted in development of altered hepatocellular foci and hepatic neoplasms. Biomarkers of fish exposure and effects (e.g., CYPlA induction, bile FA C's, DNA adducts, liver histopathology) are presently used in the Puget Sound, Washington USA and Elizabeth River, Virginia USA to track long-term environmental recovery following habitat remediation.
The main goal of this study was to test the usefulness of the Comet assay in the PLHC-1 hepatoma fish cell line as a tool for detecting the presence of genotoxic compounds in contaminated marine sediments. The system has been tested using both model chemicals (benzo[a]pyrene (B[a]P) and ethyl methanesulfonate (EMS)) and extracts of sediment samples obtained with solvent dichloromethane/methanol. For all of the analysed sediment extracts as well as for the model chemicals a concentration dependent genotoxic effect was observed. The sediment with the highest observed genotoxic potential was additionally extracted using various solvents in order to test which class of compounds, according to their polarity, is most responsible for the observed genotoxic effect. Non-polar solvents (cyclohexane and dichloro-methane) yielded stronger genotoxic effect but the highest level of DNA damage was determined after exposure to sediment extract obtained with the solvent mixture dichloromethane/methanol which extracts a wide range of contaminants. Our results indicate that the PLHC-1 cell line is a suitable in vitro model in sediment genotoxicity assessment and encourage the use of fish cell lines as versatile tools in ecogenotoxicology.
Marine Pollution Bulletin, 2002
A protocol for detecting hepatic micronuclei in fish was performed to check genotoxic damage, as an indicator of environmental hydrocarbons exposure, in relation to the ''Haven'' oil spill. As target fish, we have chosen three demersal species with different habitats and feeding behaviour (i.e., Lepidorhombus boscii, Merluccius merluccius and Mullus barbatus) collected from two differently impacted areas and a control site. Additional analysis was performed by histological detection of hepatic tissue damages such as the presence of necrotic and tumour-like aspects. The three studied species showed different sensitivity to environmental pollutants exposure, L. boscii resulting the more sensitive in terms of both micronuclei incidence and tissue damage. The results of this study show that: (1) the micronucleus test could be an effective and fast method to detect oil pollution; (2) a clear response of L. boscii only to oil contamination for both micronucleus test and liver tissue alterations. Ó
Background: Presently, there are wide uses of detergents, which discharged to marine environment (Mediterrean sea). Detergents are organic chemical compounds, consisting of fatty acids, and different components of Alkyl Benzene, Sulphonates, Polyphosphates, Cellulose, Sucrinic acid, and Sodium Lauryl Sulfates. Further, even if at low concentrations, the synthetic detergents are considered to be toxic to marine biodiversity. Since, these surfactants are not completely degraded by waste water treatment. It has ability to damage the DNA and accumulated on fish and other marine organisms. Methods: Flow cytometry technique was applied, because it's accuracy for analysis of DNA contents. Hence, the present study evaluated the impact of sodium lauryl sulfate as synthetic detergent on the DNA of sardine fish as a marine biological indicator. Sardinella aurita was collected from clean and SLScontaminated sites. The flow cytometry technique was employed to assess levels of active (apoptosis) and passive (necrosis) cell death in both exposed and unexposed fishes to SLS. Result: Chemical detergent contains 120 ml (i.e., 20mg/L) of sodium lauryl sulfates. The result shows more distinct and significant differences of the early and late apoptosis/ necrosis of Sardine fish liver, compared with unexposed fish samples from cleaned reference site. Conclusions: The present data indicate that SLS was able to cause toxicity to fish. Data recorded revealed that toxicity as a significant accumulation of hepatic tissue cellular DNA during the G0-G1 in a significant way (P<0.05), compared with that of unexposed liver tissue. In the same way, there was an insignificant accumulation of hepatic cell's DNA in the S phase but not during the G2/M phase, finally there was a significant arrest / DNA accumulation during the PreG1-phase (P<0.05). In the meantime, accompanies of apoptotic profile of cells exposed to SLS showed a significant total apoptotic form significantly elevated compared with reference site.
PLOS ONE, 2020
The research objective was to study the presence of DNA damages in haddock exposed to petrogenic or pyrogenic polyaromatic hydrocarbons (PAHs) from different sources: 1) extracts of oil produced water (PW), dominated by 2-ring PAHs; 2) distillation fractions of crude oil (representing oil-based drilling mud), dominated by 3-ring PAHs; 3) heavy pyrogenic PAHs, mixture of 4/5/6-ring PAHs. The biological effect of the different PAH sources was studied by feeding juvenile haddock with low doses of PAHs (0.3-0.7 mg PAH/kg fish/ day) for two months, followed by a two-months recovery. In addition to the oral exposure, a group of fish was exposed to 12 single compounds of PAHs (4/5/6-ring) via intraperitoneal injection. The main endpoint was the analysis of hepatic and intestinal DNA adducts. In addition, PAH burden in liver, bile metabolites, gene and protein expression of CYP1A, GST activity, lipid peroxidation, skeletal deformities and histopathology of livers were evaluated. Juvenile haddock responded quickly to both intraperitoneal injection and oral exposure of 4/ 5/6-ring PAHs. High levels of DNA adducts were detected in livers three days after the dose of the single compound exposure. Fish had also high levels of DNA adducts in liver after being fed with extracts dominated by 2-ring PAHs (a PW exposure scenario) and 3-ring PAHs (simulating an oil exposure scenario). Elevated levels of DNA adducts were observed in the liver of all exposed groups after the 2 months of recovery. High levels of DNA adduct were found also in the intestines of individuals exposed to oil or heavy PAHs, but not in the PW or control groups. This suggests that the intestinal barrier is very important for detoxification of orally exposures of PAHs.
Toxicology and applied …, 1992
English sole (Parophtys vetulus) and starry flounder (Pfatichthys stelfatus) are closely related benthic fish which show substantial differences in prevalences of contaminant-associated hepatic neoplasms and putatively preneoplastic foci of cellular alteration when captured from estuaries containing a variety of organic chemical contaminants, including polycyclic aromatic hydrocarbons (PAH) and chlorinated hydrocarbons. Because PAH are strongly implicated as causative agents in the etiology of these lesions, several of the hepatic enzymes involved in activation and detoxication of PAH were studied in these two species. Hepatic aryl hydrocarbon hydroxylase (AHH), epoxide hydrolase (EH), and glutathione Stransferase (GST) activities were measured in animals sampled from both contaminated and reference areas. English sole, the species showing higher prevalences of contaminant-associated hepatic lesions, had higher (l-to 2-fold) hepatic activities of AHH and lower activities of EH (O.&fold) and GST (1%fold) than those of starry flounder, regardless of site of capture. These results are largely consistent with increased activation and decreased detoxication of PAH by English sole in comparison to starry flounder. Both laboratory and field data suggested that the observed species differences in enzyme activities were constitutive and not related to differential exposure to contaminants. There were also substantial differences between these species with respect to expression of GST isoenzymes, in that starry flounder expressed two highly anionic GST isoenzymes which did not correspond to any GST isoenzymes expressed in English sole liver; a previous study in an elasmobranch fish showed that an anionic GST was most active toward PAH oxides. These differences in enzyme activities and isoenzyme profiles suggest a toxicological basis which may help to explain, at least in part, the differences in prevalences of contaminant-associated liver neoplasms between these two Species. 0 1992 Academic Press, 1~.