HEPATOTOXICITY OF ATRAZINE AND ITS DEGRADATION PRODUCTS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Atrazine is one of the herbicides that are most heavily used in the world. The proposed research is focused on studying the hepatotoxicity of atrazine and its degradation products. In the funding period from 6/06 to 5/07, we studied the metabolism of atrazine in rat liver microsome with or without the presence of iron (III) by using an advantageous HPLC-ESI-MS/MS method. After incubation for 12 hours, metabolites were extracted by trichloroacetic acid + 50% methanol. Four phase I metabolites, i.e. deethylatrazine, deisopropylatrazine, hydroxyatrazine, and deisopropylhydroxyatrazine were always detected no matter Fe(III) presented or not. The concentrations of deethylatrazine and deisopropylatrazine were approx. 50-times higher than those of hydroxyatrazine and deisopropylhydroxyatrazine. In the presence of Fe(III), another metabolite, i.e. didealkylatrazine was identified in the incubation solutions. The concentration of didealkylatrazine was found to increase with the increase in Fe(III) concentration. To the best of our knowledge, this was the first study of atrazine degradation products in this biological in vitro system. In addition, we developed an HPLC-MS/MS method for the determination of 4-hydroxy-nonenal (4-HNE), a specific biomarker for lipid peroxidation. The method was highly selective and very sensitive with an LOD of 10 ng /mL 4-HNE in rat liver microsome incubation solution. Three peer-reviewed publications from this group are acknowledging the RCMI grant (12RR13459) in this funding period. Next year, we plan to study the effects of the identified degradation products of atrazine on NADPH induced lipid peroxidation in rat liver microsome, and compare the results with those from atrazine, thus obtaining the relative toxicity of the degradation products against their parent compound, atrazine. Both TBAS assay and the advantageous HPLC-MS/MS assay for 4-HNE will be used to assess the oxidative stress status. In addition, the potential structure-activity relationship (SAR) will be explored using computational methods including the semi empirical AM1.