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Molecular dosimetry of DNA adducts in the medaka small fish model
1Department of Microbiology, Pathology, and Parasitology, College of Veterinary Medicine, North Carolina State University, Raleigh 27606, USA. MAC_LAW@NCSU.EDU
Abstract:
Small fish models are being used with increasing frequency for carcinogenicity testing and comparative cancer research in the US, Canada and Europe. However, there is a need to further define the early biochemical events of carcinogenesis in these species. Identification and quantitation of DNA adducts can integrate all of the various factors involved in chemical exposure, uptake, distribution and biotransformation of a putative carcinogen. In the present study, Japanese medaka (Oryzias latipes) were exposed to the alkylating agent, diethylnitrosamine (DEN), in the ambient water. Liver DNA was analyzed for O6-ethylguanine (O6EG), O4-ethylthymidine (O4ET) and O2-ethylthymidine (O2ET) by the immuno-slot-blot technique, using monoclonal antibodies against each adduct of interest. While fish exposed to 10 p.p.m. DEN had liver DNA adduct concentrations at or only slightly higher than background levels, those exposed to 100 p.p.m. DEN averaged 34 and 53 pmol O6EG/micromol guanine, 15 and 41 pmol O2ET/micromol thymidine and 2 and 6 pmol O4ET/micromol thymidine at 0 and 24 h post-exposure, respectively. The results of this study show that, under these short-term exposure conditions, ethyl-DNA adducts appear to accumulate in medaka liver tissue in a sublinear (i.e. non-linear) fashion after aqueous exposure to DEN. Thus, critical DNA repair enzymes such as O6-alkylguanine DNA alkyltransferase, which are relatively efficient at lower carcinogen levels, are probably saturated at the 100 p.p.m. concentration level of DEN.
Insights
Japanese medaka exposed to diethylnitrosamine (DEN) showed accumulating ethyl-DNA adducts, indicating potential saturation of DNA repair enzymes at higher carcinogen levels. This research aids understanding of early carcinogenesis in fish models.
Area of Science:
- Environmental Toxicology
- Molecular Carcinogenesis
- Aquatic Toxicology
Background:
- Small fish models are increasingly utilized for carcinogenicity testing and comparative cancer research.
- Defining early biochemical events of carcinogenesis in these species is crucial.
- DNA adduct analysis integrates chemical exposure, uptake, distribution, and biotransformation of carcinogens.
Purpose of the Study:
- To investigate the early biochemical events of carcinogenesis in Japanese medaka.
- To quantify specific ethyl-DNA adducts (O6-ethylguanine, O4-ethylthymidine, O2-ethylthymidine) following diethylnitrosamine (DEN) exposure.
- To assess the accumulation patterns of these adducts in medaka liver DNA.
Main Methods:
- Japanese medaka (Oryzias latipes) were exposed to varying concentrations of diethylnitrosamine (DEN) in ambient water.
- Liver DNA was isolated and analyzed for O6-ethylguanine (O6EG), O4-ethylthymidine (O4ET), and O2-ethylthymidine (O2ET).
- The immuno-slot-blot technique utilizing monoclonal antibodies was employed for adduct quantification.
Main Results:
- Fish exposed to 100 p.p.m. DEN showed significant levels of ethyl-DNA adducts at 0 and 24 hours post-exposure.
- Average adduct concentrations at 100 p.p.m. DEN included 34-53 pmol O6EG/micromol guanine, 15-41 pmol O2ET/micromol thymidine, and 2-6 pmol O4ET/micromol thymidine.
- Ethyl-DNA adducts accumulated in a sublinear (non-linear) fashion in medaka liver tissue under short-term exposure conditions.
Conclusions:
- Short-term aqueous exposure to DEN leads to sublinear accumulation of ethyl-DNA adducts in Japanese medaka liver.
- The observed accumulation pattern suggests saturation of critical DNA repair enzymes, such as O6-alkylguanine DNA alkyltransferase, at higher DEN concentrations (100 p.p.m.).
- These findings contribute to understanding the early biochemical mechanisms of chemical carcinogenesis in fish models.