DNA Adducts In Fish Research Articles

Dredging Associated Effects: Maternally Transferred Pollutants and DNA Adducts in Feral Fish

This study reports on the bioavailability, maternal transfer, and genotoxicity in feral fish of organic sediment pollutants. Northern pike (Esox lucius) and perch (Perca fluviatilis) were caught in a polluted bay before and during dredging activities and from reference areas. Polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs) were analyzed in ovulating eggs to investigate if the bay sediment posed a threat to early life-stages of fish. On the basis of previous investigations in this area, the level of exposure via maternal transfer and diffusive uptake of water-borne pollutants after hatch is likely sufficient to cause abnormalities in early life-stages of fish. During dredging, hepatic DNA adducts were elevated in adult fish, demonstrating an increased release of genotoxic compounds, which may contribute to adverse effects in aquatic organisms for several years. Although no substantial increase of maternally transferred pollutants were observed during dredging, this is the first time a correlation between hepatic DNA adducts in fish and pollutant burden in their eggs is demonstrated. Our findings underline the importance of combining chemical and toxicological methods as well as a need for greater emphasis on other polycyclic aromatic compounds in environmental risk evaluations.

DNA adducts in fish following an oil spill exposure

On 12 December 1999, one third of the load of the Erika tanker, amounting to about 10,000 t crude oil flowed into sea waters close to the French Atlantic Coast. This oil contained polycyclic aromatic compounds (PAC) that are known to be genotoxic. Genotoxic effects induce DNA adducts formation, which can thus be used as pollution biomarkers. Here, we assessed the genotoxic impact of the “Erika” oil spill by DNA adducts detection in the liver of immature fishes (Solea solea) from four locations of the French Brittany coasts. Two months after the spill, a high amount of DNA adducts was found in samples from all locations, amounting to 92–290 DNA adduct per 109 nucleotides. Then total DNA adduct levels decreased to reach about 50 adducts per 109 nucleotides nine months after the spill. In vitro experiments using human cell cultures and fish liver microsomes evidence the genotoxicity of the Erika fuel. They also prove the formation of reactive species able to create DNA adducts. Furthermore, in vitro and in vivo DNA adducts fingerprints are similar, thus confirming that DNA adducts are a result of the oil spill.

DNA adducts in perch ( Perca fluviatilis) from a creosote contaminated site in the River Ångermanälven, Sweden

Levels of hepatic DNA adducts, analysed by 32P-postlabelling, histopathological lesions and organosomatic indices, were measured in perch ( Perca fluviatilis). The fish were caught at five sites in the river Ångermanälven, Sweden: one site known to be contaminated with creosote, sites downstream from the contaminated site and a reference site upstream. Perch were also caught at a long-distance reference site. The level of DNA adducts in fish from the creosote-contaminated site was 6.8±4.1 nmol mol −1 nucleotides compared to 0.21±0.21 nmol mol −1 nucleotides in fish from the long-distance reference site. The adduct level was also significantly increased compared to adduct levels in fish from the local reference site. Hepatocellular degeneration and macrophage aggregates were observed but did not correlate with a specific site in the river. No effects on organosomatic indices that correlated with distance from the contaminated site were observed. In the laboratory, perch were exposed to an organic solvent extract prepared from sediment collected at the creosote-contaminated site or to benzo(a)pyrene (BaP) by oral administration. Perch treated with the extract had adduct patterns very similar to those observed in perch from the contaminated field site. Two diagonal zones including 10–12 adduct spots were observed on the autoradiograms. One adduct was tentatively identified by co-chromatography with a 32P-labelled standard of 7 R,8 S,9 S-trihydroxy, 10 R-( N 2-deoxyguanosyl 3′-phosphate)-7,8,9,10-tetrahydrobenzo(a)pyrene (BaPDE-dG-3′p) in two different solvent systems. Autoradiograms derived from the BaP-exposed perch showed one major adduct and several less intense spots. The major BaP adduct had the same chromatographic properties as the standard BaPDE-dG-3′p adduct. This study shows that 32P-postlabelling analysis of DNA adducts in perch can be used as a sensitive indicator of exposure to genotoxic polycyclic aromatic hydrocarbons, and that comparison of DNA adduct patterns of fish exposed in the field and laboratory can be used to determine the responsible source of pollutants.

DNA adducts in perch, Perca fluviatilis, from a creosote contaminated site and in perch exposed to an organic solvent extract of creosote contaminated sediment

DNA adducts were analysed by 32P-postlabelling in the liver of wild perch, Perca fluviatilis, from a site in the river Angermanälven, Sweden, whose sediments are contaminated with creosote. The level of DNA adducts in fish from the creosote contaminated site was 6.82 ± 4.08 nmol mol −1 nucleotides compared to 0.21 ± 0.20 nmol mol −1 nucleotides in fish from a reference site. Two diagonal zones including 10–12 adduct spots were observed on autoradiograms derived from perch from the contaminated site, while only very faint adduct spots were observed on autoradiograms derived from the reference site. In the laboratory, groups of perch were exposed to an organic solvent extract prepared from the creosote contaminated sediment or to benzo(a)pyrene by oral administration. Perch treated with the extract had adduct patterns very similar to those observed in perch from the contaminated field site. One minor adduct was identified by co-chromatography with a 32P-labelled standard of 7R,8S,9S-trihydroxy, 10R(N 2deoxyguanosyl-3′-phosphate)-7,8,9,10-tetrahydrobenzo(a)pyrene (BaPDE-dG-3′p). Autoradiograms derived from the B(a)P exposed fish showed one major adduct and several less intense spots. The major B(a)P-adduct had the same chromatographic properties as the standard BaPDE-dG-3′p-adduct.

Conversion of 1 nitropyrene by Brown trout Salmo trutta and turbot Scophthalamus maximus to DNA adducts detected by 32P postlabelling

The ability of the common aquatic contaminant 1-nitropyrene to form DNA adducts in fish was investigated in vitro and in vivo using Brown trout (Salmo trutta) and turbot (Scophthalmus maximus)in comparison to the Wistar rat. In vitro studies used Brown trout (control and induced (50 mg kg-1-naphthoflavone (NF), i.p. 3 day pre-treatment single injection)) and induced rat (PB; 0 1% w/v for 7 days in drinking water, NF; 80 mg kg-1, single injection 2 days prior to sacrifice). Hepatic 9000 g supernatant (S9 fractions) were incubated for 2 hours (at 25 C for fish and 37 C for rat) with calf thymus DNA (1mg) and 1-NP (100 M). With all S9 fractions the presence of three distinct 1-NP-related DNA adducts was detected using the butanol enrichment procedure of the 32Ppostlabelling assay. A greater level of DNA adducts was observed with the uninduced compared to the induced trout S9 (37, 12 and 8 fold greaterfor adducts in chromatograph areas 1-3 respectively) suggesting the enhancement of detoxification pathways with respect to bulky adducts following NF pre-treatment. DNA adduct levels in the induced rat consistently demonstrated approximately two-fold higher levels as compared to the induced fish, reflecting the lower protein levels in the S9 fraction of Brown trout (42 and 22 mg ml-1 for rat and fish respectively). Turbot, rat and Brown trout (uninduced and induced (NF; 50 mg kg-1; i.p. single injection 3 days prior)) were dosed with 100 mg kg-1 1-NP (i.p. single injection, 24 hours). Liver DNA from both turbot and rat exhibited a 1- NP related adduct spot which was similar in position to that of area 1 in the incubations with S9 from rat and Brown trout. However, in contrast to the in vitro studies no 1-NPrelated adducts were found in liver DNA from induced and uninduced Brown trout. This study highlights the potential, in a marine and a freshwater fish, for 1-NP metabolism to reactive of binding to DNA. However, activation of 1-NP was more optimal in the S9-mediated system, possibly reflecting the influence of detoxification systems.

32P-postlabeling analysis of aromatic DNA-adducts in liver and brain of wild brook trout ( Salvelinus fontinalis)

Several studies have reported the absence of DNA-adducts in fish collected from unpolluted areas. We determined DNA-adduct levels in liver and brain tissue of wild brook trout ( Salvelinus fontinalis) from remote areas of Newfoundland and sub-arctic Labrador, that have no known input of anthropogenic contamination. Varying levels of adducts were detected in both liver and brain, at all locations. Hepatic adduct levels were generally higher than those in brain tissue. Tissue-specific differences in adduct levels and the possible effects of long range transport of air pollutants (LRTAP) are discussed.

Comparative analysis of aromatic DNA adducts in fish from polluted and unpolluted areas by the32P-postlabeling analysis

The {sup 32}P-postlabeling technique, developed by Randerath and his colleagues, is a highly sensitive assay to detect the damaged DNA as DNA-carcinogen adduct. Consequently, measurement of this DNA carcinogen adduct concentrations in target tissues of organisms may provide a key biologic end-point of exposure to environmental carcinogens. In this study the authors analyze the hepatic DNA from a bottom-feeding fish, tilapia (Tilapia mossambica), sampled from the down stream Damsui River where fish are exposed to high levels of polycyclic aromatic hydrocarbons (PAH) in sediment and from the upstream Fe-Tsui reservoir where the water has been used as the source of drinking water for Taipei Metropolitan area. In doing so, they will be able to determine whether this {sup 32}P-postlabeling analysis of hepatic DNA from the bottom-feeding tilapia can be used to monitor large molecule aromatic carcinogens in the environment.

32P-postlabeling detection of DNA adducts in fish from chemically contaminated waterways

Fish were collexted from sites in the chemically-contaminated Buffalo River, New York, and the Detroit River, Michigan. The sediments of these rivers have high levels of chemical contaminants, including polycyclic aromatic hydrocarbons (PAHs), and fish from these locations have high prevalences of liver cancer. To determine chemical-DNA interactions and a possible role for chemicals as a cause of the observed tumors, DNA was isolated from livers and was enzymatically digested to normal and adducted nucleotides. The DNA digests were enriched for hydrophobic, bulky adducts, either by preparative reverse phase high pressure liquid chromatography, or by selective nuclease P1 dephosphorylation of normal nucleotides. DNA-chemical adducts were then quantitated by 32P-postlabeling analysis. Regardless of the adduct enrichment procedure, the chromatograms derived from DNA of fish from polluted areas showed a diffuse, diagonal radioactive zone consisting, at least in part, of multiple overlapping discrete adduct spots. The behavior of the adducts in the diagonal radioactive zone and of their unlabeled precursors is consistent with their identification as nucleotide adducts of a variety of bulky, hydrophobic, aromatic genotoxic compounds. Analysis of bile demonstrated recent exposure to multi-ringed aromatic compounds.