Project Type: Core Research
- To examine the role of specific human xenobiotic metabolizing enzymes in the metabolism of selected pesticides important to agriculture.
- Establish methods for analytical identification of all test compounds and their metabolites.
- Determine metabolic pathways and rates of metabolism in human liver microsomes.
- Determine substrate specificity of recombinant CYP or FMO isoforms relative to the test compounds.
- Determine the importance of human polymorphisms in identifying populations and/or individuals at increased risk.
- Examine potential for adverse pesticide interactions by induction of metabolizing enzymes in human hepatocytes using branched DNA signal amplification technology.
Project Activities and Accomplishments:
The initial studies on this project developed analytical methods for several pesticides and their metabolites and provided information on their in vitro human metabolism. The pesticides included permethrin and other pyrethroids, fipronil, etc. These findings were combined our human metabolic studies of other pesticides such as chlorpyrifos, carbaryl and endosulfan to ascertain the potential for interactions, in humans, that need to be included in risk analysis for agricultural workers.
Comparative tests for cytotoxicity and apoptosis in the HepG2 cell line and human hepatocytes have been conducted, and the results published, using several pesticides including, fipronil, fipronil sulfone, endosulfan, deltamethrin, chlorpyrifos, cypermethrin, fenvalerate and permethrin. Fipronil and its primary metabolite, fipronil sulfone, have the greatest potential for cytotoxicity in human hepatocytes. Cytotoxicity and human hepatocyte induction studies using bDNA technology in combination with western blotting to document CYP induction by fipronil have also been published.
Studies on the metabolism of endosulfan using human liver microsomes demonstrated that endosulfan is metabolized in humans to endosulfan sulfate. A screen of 19 CYP isoforms demonstrated that CYP2B6 is almost exclusively responsible for the formation of this metabolite. It has been demonstrated that both endosulfan and endosulfan sulfate induce CYP3A4 and 2B6 in human hepatocytes via the pregnane X receptor. Because there are few specific probes for CYP2B6 activity, it was anticipated that endosulfan may be a good probe substrate for 2B6 activity in human samples and this has turned out to be the case.
Our previous studies indicated that CYP2B6 and 2C19 are important in the intoxication and detoxication of organophosphorus pesticides, respectively. We previously demonstrated that polymorphisms of CYP2C19 abolish detoxication of chlorpyrifos. A literature survey identified several polymorphisms of CYP2B6, several of which affect catalytic activity towards other xenobiotics. Three protocols have been tested to assay for these polymorphisms in CYP2B6 and 2C19. Preliminary tests with human liver samples have identified two polymorphisms found in the CYP CYP2B6*6 allele. This allele has been linked, by others, to reduced metabolism of other CYP2B6 substrates such as efavirenz and cyclophosphamide. Since CYP2B6 primarily activates chlorpyrifos to the more toxic oxon, the presence of this allele could be protective. In contrast the presence of the wild-type CYP2B6 allele could result in increased activation. After polymorphisms from liver samples have been identified we plan to prepare microsomes and test their ability to metabolize chlorpyrifos based on genotype. Several liver samples have been recently acquired from the National Disease Research Interchange to be utilized in these studies. More recent studies have utilized DNA from participants in the Agricultural Health Study.We have also acquired the A549 human lung adenocarcinoma cell line to test its cytotoxic and metabolic response to chlorpyrifos.The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was used to test exposure to chlorpyrifos and two metabolites, chlorpyrifos oxon and 3,5,6 trichloro-2-pyridinol. Only chlorpyrifos was found to have a significant effect. It is postulated that the cytotoxicity of chlorpyrifos in the A549 cell line is due in part to the metabolic production of the reactive sulfur during metabolism to chlorpyrifos oxon. The addition of nicotine was found to be protective against the cytotoxicity of chlorpyrifos. This protective effect was also found in the HepG2 cell line. We have prepared microsomes from the A549 cell line and will test their ability to metabolize chlorpyrifos. The observations that organthiophosphorus insecticides such as chlorpyrifos and others, made in our laboratory, inhibits the human metabolism of steroid hormones, including both testosterone and estradiol, is important in terms of risk analysis and its significance for agricultural workers. Consideration is currently being given to appropriate outreach material on this subject.
Annual Report 2006
Presentation: Human Metabolism of New and Emerging Pesticides