Progress 10/01/01 to 09/30/07
Outputs
Dr R. L. Rose, the investigator, was killed in an accident on 05/24/06 and the project should be terminated as of that date. The following report was written by Dr Ernest Hodgson. Studies of the inducing and hepatocytotoxic effects of the insecticide, fipronil, have been completed and a publication is currently in press. Fipronil is a potent inducer of CYP3A4 and other CYP isoforms in human hepatocytes at low doses while at somewhat higher doses this insecticide is cytotoxic. Previously completed studies demonstrated that endosulfan-alpha was metabolized in human liver primarily by CYP2B6 and CYP3A4 producing primarily a single product, endosulfan sulfate. These studies enabled the development of endosulfan-alpha as a simultaneous probe for CYP2B6 and CYP3A4 in human liver microsomes. Studies on endosulfan-alpha as an inducer of these two CYP isoforms via the pregnane-X receptor are being continued by Dr Andrew Wallace. Studies of inhibition of CYP isoforms by
chloropyrifos conducted in collaboration with Dr Ernest Hodgson have shown that this insecticide inhibits the metabolism of nonane, a diesel and jet fuel component, as well as fipronil, an insecticide. The inhibitory properties of chlorpyrifos are shared by other organphosphorus insecticides such as fonofos and phorate but chlorpyrifos is the most potent of those tested to date.
Impacts
These studies have demonstrated that pesticides are capable of inducing metabolic isoforms involved in their metabolism. Cytotoxic effects of fipronil to human hepatocytes have also been demonstrated at relatively low concentrations relative to other pesticides. Metabolic studies of endosulfan have indicated its potential utility as a probe substrate for CYP2B6; an important enzyme in pesticide and drug metabolism. The importance of CYP2B6 in metabolism of chlorpyrifos and related organophosphorus compounds has been established. Additional studies are needed to determine the influence that polymorphisms in this gene may play in some of the health effects observed in farmers and pesticide applicators who have higher than normal exposure levels to these compounds.
Publications
- K. A. Usmani, T. M. Cho, R. L. Rose and E. Hodgson. 2006. Inhibition of the human liver microsomal and human cytochrome P450 1A2 and 3A4 metabolism of estradiol by deployment-related and other chemicals. Drug Metabolism. Disp. 34: 1606-1614.
- R. C. T. Casabar, A. A. Wallace, E. Hodgson and R. L. Rose. 2006. Metabolism of endosulfan-alpha by human liver microsomes and its utility as a simultaneous probe for CYP2B6 and CYP3A4. Drug Metabol. Disp. 34: 1779-1785.
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Progress 10/01/04 to 09/30/05
Outputs
Comparative tests of the potential of pesticides to cause cytotoxicity, apoptosis and CYP inducibility have been conducted in human hepatocytes and the HepG2 cell line. Pesticides examined include fipronil, fipronil sulfone, endosulfan, deltamethrin, chlorpyrifos, cypermethrin, fenvalerate and permethrin. The greatest cytotoxic effects were observed with fipronil and its primary metabolite, fipronil sulfone. Fipronil also was demonstrated to induce CYP3A4, 1A1 and other important metabolism enzymes. Studies documenting fipronil induction and cytotoxicity effects have been prepared for publication. A second manuscript delineating results for these effects with respect to other pesticides examined is under development. 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. Collaborative studies with Andrew Wallace 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 is anticipated that endosulfan may be a good probe substrate for 2B6 activity in human samples. Our previous studies indicated the importance of CYP2B6 and 2C19 in intoxication and detoxication of organophosphorus pesticides. 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. Studies are underway to allow characterization of CYP2B6 polymorphisms in human liver samples. Several liver samples have been recently acquired from the National Disease Research Interchange to be utilized
in these studies. We have also acquired the A549 human lung adenocarcinoma cell line to test its cytotoxic and metabolic response to chlorpyrifos. The MTT 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. We have prepared microsomes from the A549 cell line and will test their ability to metabolize chlorpyrifos.
Impacts
These studies have demonstrated that pesticides are capable of inducing metabolic isoforms involved in their metabolism. Cytotoxic effects of fipronil to human hepatocytes have also been demonstrated at relatively low concentrations relative to other pesticides. Metabolic studies of endosulfan have indicated its potential utility as a probe substrate for CYP2B6; an important enzyme in pesticide and drug metabolism. The importance of CYP2B6 in metabolism of chlorpyrifos and related organophosphorus compounds has been established. Additional studies are needed to determine the influence that polymorphisms in this gene may play in some of the health effects observed in farmers and pesticide applicators who have higher than normal exposure levels to these compounds.
Publications
- Rose, R.L., Tang, J., Choi, J., Cao, Y. Usmani, A., Cherrington, N., Hodgson, 2005. Pesticide metabolism in humans, including polymorphisms. Scand. J. Work Environ. Health 31 suppl 1:156-163.
- Hodgson, E. and Rose, R.L. 2005. Human metabolism and metabolic interactions of deployment-related chemicals. Drug Metabol. Rev. 1:1-39.
- Hodgson, E. and Rose, R.L. 2005. Toxicology of AHS important chemicals. J. Biochem. Molec. Toxicol. 19:180-181.
- Rose R.L. and Hodgson, E. 2005. Pesticide metabolism and potential for metabolic interactions. J. Biochem. Molec. Toxicol. 20:276-277.
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Progress 10/01/03 to 09/30/04
Outputs
We have performed a comparative in vitro metabolism study of carbofuran in human, rat, and mouse liver microsomes and characterized the enzymes invoved in this metabolic pathway. Carbofuran is metabolized by Cytochrome P450 (CYP) leading to the production of one major ring oxidative metabolite (3-hydroxycarbofuran) and two minor metabolites. The affinity of carbofuran for CYP enzymes involved in its oxidation is significantly less for human liver microsomes than for rat or mouse liver microsomes. Intrinsic clearance rate calculations indicate that human liver microsomes are 14-fold less efficient than rodent liver microsomes. A screen of 15 major human CYP isoforms for abilty to metabolize carbofuran indicated that CYP3A4 is the major isoform involved in humans. CYP1A2 and 2C19 are much less active while other human isoforms have minimal or no activity. In contrast with humans isoforms, members of the CYP2C family in rats are likely to have a major role in carbofuran
metabolism. The variation in carbofuran metabolism among 17 single-donor human samples was over 5-fold and was highly correlated with levels of CYP3A4 in these individuals (correlation coefficient = 0.96). Potential interactions between carbofuran with other pesticides as well as endogenous substrates were also explored. Ketoconazole, as specific inhibitor of CYP3A4, provided excellent inhibition of the formation of 3-hydroxycarbofuran (IC50 = 0.31 microM). Chlorpyrifos also was shown to be an irreversible non-competitive inhibitor of CYP3A4 activity, with an IC30 of 39 microM. Interactions between carbofuran and the endogenous substrates, testosterone and estradiol were also observed. In vitro incubations of carbofuran were demonstrated to slightly activate testosterone metabolism levels in both human liver microsomes and CYP3A4. No significant interactions between carbofuran and estradiol were observed. Previous work with permethrin indicated that microsomal and cytosolic esterases
played a prominent role in pyrethroid metabolism in human liver homogenates. Since ester hydrolysis is known to be inhibited by substrates which are capable of interaction with acetylcholinesterase enzymes, two known acetylcholinesterase inhibitory pesticides were tested for their ability to inhibit permethrin hydrolysis. Although chlorpyrifos did not directly inhibit permethrin hydrolysis, its oxidation product, chlorpyrifos oxon strongly and irreversibly inhibited permethrin hydrolysis at low concentrations. Carbaryl also was an effective inhibitor. Many efforts have been made to characterize the potential of pesticides to induce the induction of important metabolic enzymes in humans. Metabolic assays to demonstrate induction of specific isoforms using hepatocyte cultures have been developed to characterize CYP isoforms 1A1, 1A2, and 3A4. An assay which measures mRNA levels, the bDNA assay, has demonstrated that several pesticides have the ability to induce many CYP isoforms.
Western blotting and metabolic assays are being used to further validate these interesting results. Studies have also been initiated to document hepatocyte mortality and to study potential mechanisms involved in induction.
Impacts
These studies have defined metabolic pathways of metabolism for several commonly used insecticides in humans. Not only have the metabolic products of metabolism been defined, but also the enzymes responsible for metabolism have been elucidated. An understanding of these processes will enable future studies involving pesticide interactions with metabolic processes and provide information which can be used in pesticide risk assessment.
Publications
- K.A. Usmani, E.D. Karoly, E. Hodgson, and R.L. Rose. 2004. In vitro sulfoxidation of thioether compounds by human cytochrome P450 and flavin-containing monooxygenase isoforms with particular reference to the CYP2C subfamily. Drug Metab. Dispos. 32:333-339.
- J. Tang, K.A. Usmani, E. Hodgson, and R.L. Rose. 2004. In vitro metabolism of fipronil by human and rat cytochrome P450 and its interactions with testosterone and diazepam. Chemico-Biol. Interact. 147:319-329.
- K.A. Usmani, E. Hodgson, and R.L.Rose. 2004. In vitro metabolism of carbofuran by human, mouse, and rat cytochrome P450 and interactions with chlorpyrifos, testosterone, and estradiol. Chemico-Biol. Inter. 150:221-232.
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Progress 10/01/02 to 09/30/03
Outputs
We have examined the in vitro human metabolism of several pesticides including fipronil, acetamiprid, phorate, disulfoton, sulprofos, and methiocarb and characterized the cytochrome P450 (CYP) and flavin-containing monooxygenase (FMO) isoforms involved in their metabolism. For each of these pesticides, the metabolic route in human liver microsomes was determined and the specific metabolizing enzyme isoforms determined. For some of these pesticides, the potential to induce or inhibit human CYP isoforms has also been determined. Fipronil is metabolized by human liver microsomes to fipronil sulfone. A screen of 10 CYP isoforms demonstrated that most of the product is the result of CYP3A4 oxidation, although CYP3A5 and CYP2C19 are also able to produce this product. Other CYP isoforms examined had little or no activity toward fipronil. Ketoconazole, a well known CYP3A4 inhibitor specifically inhibits fipronil metabolism in human liver microsomes, further demonstrating the
role of CYP3A4. Oxidative activity towards fipronil in 19 single-donor human liver microsomes also correlates with their ability to oxidize testosterone. Fipronil was also demonstrated to significantly inhibit testosterone hydroxylation activity as well as diazepam hydroxylase activity. Interactions between fipronil and other CYP3A4 substrates display activation and region-selectivity effects, suggesting that CYP3A4 has multiple binding sites. Several organophosphate insecticides have a sulfur atom that is susceptible to oxidative attack, resulting in the production of sulfoxides and sulfones. The discovery that CYP2C family members could produce the sulfoxide product from phorate prompted a detailed study of several insecticides containing the appropriate functional group to study this in greater detail. Four thioether pesticides were examined; three organophosphates (phorate, disulfoton, sulprofos) and one carbamate (methiocarb). Metabolism of each of these pesticides to the
sulfoxide product was demonstrated in human liver microsomes and shown to be predominantly CYP driven (85-90%) compared to FMO driven (10-15%). Sixteen different CYP isoforms (including some polymorphic forms) were examined for their potential to produce the sulfoxidation product. Although several could produce this product, the predominant isoforms involved belonged to the CYP2C family. Other families with prominent ability included CYP1A2, 3A4, and 2B6. Use of specific chemical inhibitors further indicated CYP2C family involvement. Results of this study indicated that the substrate specificity of the CYP2C family may be more extensive than is currently believed and that some reevaluation of the current structure-activity relationships for the CYP2C family may be necessary. In related studies using human hepatocytes we have demonstrated that pesticides can induce and/or inhibit many CYP isoforms involved in their metabolism. These studies, involving bDNA assays, western blotting and
specific substrates have shown that pesticides have the potential for dramatic induction of some CYP isoforms. The implications of such induction on metabolism of other pesticides and/or drugs will require further study.
Impacts
These studies have defined metabolic pathways of metabolism for several commonly used insecticides in humans. Not only have the metabolic products of metabolism been defined, but also the enzymes responsible for metabolism have been elucidated. An understanding of these processes will enable future studies involving pesticide interactions with metabolic processes and provide information which can be used in pesticide risk assessment.
Publications
- J. Choi, R.L. Rose, and E. Hodgson. 2002. In vitro human metabolism of permethrin: the role of human alcohol and aldehyde dehydrogenases. Pestic. Biochem. Physiol. 73:117-128.
- K.A. Usmani, R.L. Rose, and E. Hodgson. 2003. Inhibition and activation of the human liver and human cytochrome P450 3A4 metabolism of testosterone by deployment-related chemicals. Drug Metabol. Disp. 31:384-391.
- K.A. Usmani, E.D. Karoly, E. Hodgson, and R.L. Rose. 2004. In vitro sulfoxidation of thioether compounds by human cytochrome P450 and FMO isoforms with particular reference to the CYP2C subfamily. Drug Metabol. Disp. (in press).
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Progress 10/01/01 to 09/30/02
Outputs
Metabolic pathways involved in the metabolism of pesticides including chlorpyrifos, carbaryl, permethrin and the insect repellant DEET had previously been determined. With the identification of common metabolic pathways for several of these chemicals, it was important to examine the potential interactions between pesticides and enzymes involved in their metabolism as well as on metabolism of endogenous chemicals such as testosterone. Chlorpyrifos, an organophosphate insecticide, is activated to chlorpyrifos-oxon through a CYP-catalyzed desulfuration reaction. One of the products of this reaction is a highly reactive sulfur atom which is believed to bind to the heme iron of CYP and inhibit its activity. CYP2B6 is the isoform most responsible for production of its neurotoxic metabolite, chlorpyrifos-oxon, although other CYPs including the predominant CYP3A4 are also involved. Since CYP2B6 was also a major factor in the metabolism of carbaryl, the effect of chlorpyrifos
on carbaryl metabolism was investigated. Pre- and/or co-incubations of chlorpyrifos with carbaryl in human liver microsomes resulted in significant inhibition of the production of carbaryl methylol, while the production of other metabolites was less affected. This result was demonstrated to be primarily due to direct interactions with CYP2B6. Because the first step in permethrin metabolism involves esterases, pesticides known to inhibit esterases were examined for their potential to interfere with permethrin metabolism in vitro. In these studies, chlorpyrifos-oxon completely inhibited permethrin metabolism at low concentrations, with a Ki of 20 nM. Although chlorpyrifos did not significantly inhibit permethrin metabolism, pre-incubation with chlorpyrifos did, further implicating the role of chlorpyrifos-oxon. Carbaryl was also capable of significant inhibition at relatively low concentrations, however, carbaryl did not completely inhibit permethrin metabolism. The combined inhibition
results with chlorpyrifos and carbaryl indicate that the esterase(s) involved in permethrin metabolism are likely to be B-esterases, and that more than one esterase is involved. Potential interactions between pesticides and testosterone metabolism by human liver microsomes were also explored. Pesticides tested for their effects on testosterone metabolism included chlorpyrifos, chlorpyrifos-oxon, permethrin, phorate, fonofos, fipronil, imidacloprid, and deltamethrin. Other chemicals tested were the insect repellant DEET and the neuroprotective agent administered to troops during the Gulf War, pyridostigmine bromide. Significant inhibition of testosterone metabolism was limited primarily to the three organophosphates tested, chlorpyrifos, phorate and fonofos. Chlorpyrifos-oxon also had a significant inhibitory potential though much less than the parent pesticide, chlorpyrifos. The majority of the inhibitory potential of these pesticides is the direct result of their inhibition of
CYP3A4, the major testosterone-metabolizing enzyme. In similar studies, the conversion of testosterone to estradiol, catalyzed primarily by CYP19, was not affected by the pesticides examined.
Impacts
Agricultural chemicals have often been implicated as potential causative agents for increased cancer rates among farmers and other occupationally exposed workers. Similarly, pesticides along with other deployment related chemicals are thought to have played a role in Gulf War Syndrome. Although pesticides are thoroughly evaluated individually in rodent models, little is actually known about their metabolism in humans and the potential interactions of pesticides with one another or with other chemicals to which humans are exposed. These studies have identified important human metabolic pathways for several pesticides and demonstrated that pesticides can inhibit metabolism of both exogenous and endogenous chemicals. Further characterization of these types of pesticide interactions will be necessary to identify populations or individuals who may have greater risks to pesticide exposure.
Publications
- Usmani, K.A., R.L. Rose, J.A. Goldstein, W.G. Taylor, A.M. Brimfield, and E. Hodgson. 2002. In vitro human metabolism and interactions of the repellent, N,N-diethyl-M-toluamide. Drug Metabol. Disp. 30:289-294.
- Tang, J., Y. Cao, R.L. Rose, and E. Hodgson. 2002. In vitro metabolism of carbaryl by human cytochrome P450 and its inhibition by chlorpyrifos. Chemico-Biol. Interac. 141:229-241.
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Progress 10/01/00 to 09/30/01
Outputs
The oxidative metabolism of two pesticides (carbaryl and permethrin) and one insect repellant (DEET) have been explored using a combination of human, rat and mouse liver microsomes with a major focus on human metabolism. In humans, carbaryl is readily metabolized to three major metabolites; 5-hydroxy carbaryl, 4-hydroxy carbaryl, and carbaryl methylol. Each of these metabolites may be produced by several cytochrome P450 (CYP) isoforms. Of thirteen cDNA expressed CYP isoforms examined, only one failed to produce any metabolite. Isoforms with the greatest metabolic capacity included CYPs 1A1, 1A2, 2B6, 2C19 and 3A4. Co-incubations of carbaryl and the organophosphate, chlorpyrifos, demonstrated significant inhibition of the formation of the carbaryl methylol metabolite. This was shown to be the result of direct inhibition of the CYP2B6 isoform. In vitro metabolism of DEET by human, rat and mouse hepatic microsomes produced two predominant products; a ring hydroxymethyl
metabolite, BALC, and an N-deethylated metabolite, ET. Both products were formed by human liver microsomes, though activities were less than in rats and mice. Among twelve CYP human isoforms screened, four displayed detectable activity for the production of the BALC metabolite (1A2, 2B6, 2D6, and 2E1). Formation of the ET metabolite, depended on an entirely different set of CYPs, including 3A4, 3A5, 2A6 and 2C19. Studies were also designed to examine potential interactions between pesticides and DEET in mice. In these studies, DEET was demonstrated to induce the CYP2B family, resulting in a 2.4-fold increase in the production of chlorpyrifos-oxon following microsomal exposure to chlorpyrifos. Preincubations of human CYP2B6 with chlorpyrifos completely inhibited the formation of the BALC metabolite from DEET, while having no significant effect on production of the ET metabolite. Several other mixture combinations were explored. Preliminary studies of permethrin metabolism have resulted
in the description of a better method for analysis of permethrin and three major metabolites. Use of an acidified mobile phase was demonstrated to be an important factor in the analysis of phenoxybenzoic acid, which is ionized at neutral pH. Human liver microsomes produce phenoxybenzyl alcohol and phenoxybenzoic acid following incubation with trans-permethrin. Production of these metabolites was demonstrated to be primarily the result of alcohol and aldehyde dehydrogenase enzymes. Work to characterize specific isoforms responsible in permethrin metabolism is in progress.
Impacts
Health hazards associated with pesticide use can be better predicted as we gain a better understanding of human pesticide metabolism. These studies have demonstrated that although many metabolites may be formed by a variety of CYP isoforms, that some isoforms more specifically produce certain metabolites. They also show that pesticides (and/or repellants such as DEET) are capable of inducing or inhibiting enzymes involved in metabolism. Further characterization of these types of pesticide interactions will be necessary to identify populations or individuals who may have greater risks to pesticide exposure.
Publications
- J. Tang, Y. Cao, R.L. Rose, A.A. Brimfield, D. Dai, J.A. Goldstein, and E. Hodgson. 2001. Metabolism of chlorpyrifos by human cytochrome P450 isoforms and human, mouse, and rat liver microsomes. Drug Metab. Disp. 29:1201-1204.
- D. Dai, J. Tang, R. Rose, E. Hodgson, R.J. Bienstock, H.W. Mohrenweiser and J.A. Goldstein. 2001. Identification of variants of CYP3A4 and characterization of their abilities to metabolize testosterone and chlorpyrifos. J. Pharm. Exptl. Therap. 299:825-831.
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