Human Metabolic Interactions of Pesticides: Inhibition, Induction, and

Chapter 8

Human Metabolic Interactions of Pesticides: Inhibition, Induction, and Activation

Downloaded by UNIV OF ARIZONA on July 26, 2012 | http://pubs.acs.org Publication Date (Web): July 25, 2012 | doi: 10.1021/bk-2012-1099.ch008

Ernest Hodgson* and Andrew D. Wallace* Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina 27695 *E-mail: [email protected] (E.H.); [email protected] (A.D.W.)

The regulation of agrochemicals utilizes, among other data, hazard determination of single chemicals carried out in surrogate animals that show little genetic variation. Relevance to real world situations is questionable, since dose and species extrapolations require uncertainty factors and agrochemicals are typically used as mixtures. Human studies provide information on population variation, sub-groups at increased risk and interactions among agrochemicals and endogenous metabolites. How components in agrochemical mixtures affect each others toxicokinetics is largely unknown. Metabolism and metabolic interactions of agrochemicals have been investigated using human hepatocytes, human liver microsomes and recombinant human cytochrome P450 (CYP) isoforms. Potential for interactions based on CYP induction, cytotoxicity, inhibition and activation is apparent. For example, in human hepatocytes fipronil is a potent inducer at low doses but cytotoxic at higher doses. The role of the pregnane X receptor (PXR) has been demonstrated in induction by pesticides and, in microarray studies of chlorpyrifos (CPS) exposed human hepatocytes, it was shown that a number of genes were differentially expressed, including gene pathways regulating detoxication enzymes, retinol metabolism, and the cytoskeleton. Phosphorothioates are potent inhibitors of the CYP metabolism of steroid hormones as well as other pesticides, including carbaryl and fipronil and some diesel fuel components. Fipronil also inhibits testosterone metabolism. Expression of CYP2B6 with age and variation in chlorpyrifos metabolism is also discussed. © 2012 American Chemical Society In Parameters for Pesticide QSAR and PBPK/PD Models for Human Risk Assessment; Knaak, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Introduction While not specifically on the subject of physiologically based pharmacokinetic (PBPK) modeling, the intent of this chapter is to demonstrate why human studies are important in human health risk assessment of pesticides and in the development of human PBPK models of pesticide disposition. For some seven decades, the metabolism of pesticides, and their metabolic interactions, has been investigated using single chemicals in surrogate animals and the resultant literature is extremely large. See Hodgson for a recent summary. The regulation of agrochemicals utilizes hazard determination of single chemicals carried out in surrogate animals that show little genetic variation. Relevance to real world situations is questionable, because dose and species extrapolations require uncertainty factors and agrochemicals are typically used as mixtures. How components in agrochemical mixtures affect each others toxicokinetics is largely unknown. Ethical in vitro human metabolic studies have been possible only for the last decade or so and the literature in this regard is both small and inadequate (summarized in (2, 3)). In vitro human studies provide information on such aspects as the role of xenobiotic-metabolizing enzymes (XMEs), their isoforms and polymorphic variants, in detoxication and activation reactions, population variation, sub-groups at increased risk, and interactions among agrochemicals and endogenous metabolites. Some of these aspects cannot be studied in surrogate animals. Whether in surrogate animals or humans it is important to keep in mind that mode of action is not a single molecular event (the term mechanism of action is best used for such reactions) but a cascade of events, starting with exposure and ending either with detoxication and excretion or with the expression of a toxic endpoint (Figure 1). Metabolism, while important, is only one of the steps in this cascade. Metabolism and interactions of agrochemicals have been investigated using human liver microsomes and cytochrome P450 (CYP) isoforms and, to a lesser extent, by other human XMEs and cell fractions. Chlorpyrifos (CPS) (4–8), carbaryl (9), carbofuran (10), fipronil (11) and endosulfan (12–14) are metabolized by liver microsomes and liver CYPs, CYP2B6 and CYP3A4 being the most important. Permethrin is hydrolyzed by esterase(s) present in human liver cytosol and microsomes. Phosphorothioates are potent inhibitors of CYP metabolism of steroid hormones as well as other pesticides, including carbaryl and fipronil, and some diesel fuel components. Fipronil also inhibits testosterone metabolism. Expression of CYP2B6 variation in chlorpyrifos metabolism with age has been investigated (15, 16). In vitro metabolic studies, whether in surrogate animals or humans, are designed, initially, to demonstrate the potential for metabolism and/or interaction and, for that reason are conducted at substrate concentrations which permit the accurate measurement of metabolites. Following the demonstration of potential effect, the difficult question of the relevance at substrate concentrations likely to be present in vivo has to be addressed. With surrogate animals this can be addressed directly by the measurement of tissue residues over time following 116 In Parameters for Pesticide QSAR and PBPK/PD Models for Human Risk Assessment; Knaak, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


treatment; with humans this is not possible and the question of relevance must be approached indirectly. However, the incorporation of kinetic data on human CYP isoforms by Foxenberg et al. (17), demonstrates how human studies may be used in the development of PBPK models of pesticide mixtures.

Figure 1. Mode of action of toxicants: a cascade of events. A brief summary of the possible events between absorption at portals of entry to the expression of a toxic endpoint and/or the excretion of metabolites.

If it is determined that the effective concentrations are low and the effect high as in the case of fipronil induction of CYP isoforms in human hepatocytes (18) or the inhibition of the metabolism of steroid hormones by human microsomes and/or recombinant human CYP isoforms by chlorpyrifos (19, 20), it can be assumed for the purpose of risk management that a risk to applicators exists. It might be noted in this regard that Di Consiglio et al. (21), have described a pesticide/clinical (organophosphorothionates/imipramine) drug interaction occurring at what they believe to be relevant doses. There are a number of possibilities to determine whether or not a particular potential interaction does in fact occur. For example urinary metabolites in applicators using a particular mixture could be compared to the metabolites found in those using either component alone. To summarize, aspects of importance in the study of the in vitro metabolism of pesticides in humans include: the XMEs, their isoforms and polymorphic variants and their variation within the human population; pesticide-pesticide substrate interactions; pesticide-endogenous metabolite substrate interactions; pesticide induction and the effect of pesticide and non-pesticide inducers on pesticide metabolism; cytotoxicity. 117 In Parameters for Pesticide QSAR and PBPK/PD Models for Human Risk Assessment; Knaak, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Importance of Human Studies in Risk Assessment and New Risk Assessment Paradigms: Extrapolation, Variation, and Mixtures Some types of human studies, primarily exposure and epidemiology, have been carried out, to advantage, on human populations for some time, others are relatively recent while others cannot be carried out to any extent. As detailed above, among recent human studies are those involving recombinant human XMEs, human cell fractions such as microsomes and cytosol as well as those involving human hepatocytes. Studies involving microsomes, cytosol and recombinant XMEs have demonstrated the identity of metabolites, particularly phase I metabolites, which isoforms are involved in their production and, in a small number of cases, the effect of single nucleotide polymorphisms (SNPs) (5, 6, 9, 10, 22–24) and the variation in metabolism within groups of humans (6, 10, 11, 16, 25–28). Human metabolic studies can contribute to human health risk assessment, as it is presently carried out, in several ways including defining human variation, providing insight into uncertainty factors, defining uncertainty factors, showing the potential for human-specific interactions and defining populations or individuals at increased risk. Most of these facets cannot be defined by the use of surrogate animals. Overall, it seems clear that human studies will become more important as new approaches (29) to human health risk assessment are implemented, inasmuch as these new molecular approaches stress the use of human and human-derived cell lines as well as recombinant human enzymes. However, since pesticides are commonly utilized as mixtures, usually with other pesticides, the more sophisticated, and more complex, PBPK models and human health risk assessments on single chemicals cannot take metabolic interactions between more than one pesticide into account. There are, however considerable efforts being made to develop theoretical and practical approaches to PBPK modeling and human health risk assessment of mixtures and the progress and problems are illustrated by, for example, the work of Clewell and associates (30–32) Few PBPK models of mixtures have involved pesticides or human data. However, the PBPK model for chlorpyrifos (33) was the template for later individual PBPK models for chlorpyrifos and parathion (8, 17) and a PBPK model for the binary mixture of chlorpyrifos and diazinon (34). Timchalk et al. (34), utilized a binary mixture of chlorpyrifos and diazinon, together with rat Vmax and Km values. The model assumed that chlorpyrifos was a substrate and diazinon the inhibitor or vice versa, that diazion was the substrate and chlorpyrifos was the inhibitor. The individual models of Foxenberg et al. (8, 17), incorporated human CYP Vmax and Km values into individual PBPK models for chlorpyrifos and parathion. All of these studies involve chemically closely related pesticides (OPs). Hopefully, future PBPK models will involve mixtures of chemically dissimilar pesticides since it is possible, based on studies described below (pesticide-pesticide inhibition), that interactions occur between chemicals as chemically dissimilar as chlorpyrifos and permethrin. Other aspects of the nature of the mixture tested will also be of importance. 118 In Parameters for Pesticide QSAR and PBPK/PD Models for Human Risk Assessment; Knaak, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

Although the public at large tend to be exposed to complex mixtures of numerous chemicals in small amounts, in the occupational setting, including agriculture, workers are exposed to mixtures in which a small number of chemicals, in larger amounts, predominate. The studies refered to above (17, 34, 35) on the chlorpyrifos-diazinon mixture and chlorpyrifos and parathion individually, represent an initial approach to this problem. In future it will be important to select, on the basis of known use patterns, the most important binary mixtures and to develop PBPK and human health risk assessment models for these mixtures.


Importance of Human Studies in PBPK: Extrapolation, Variation, and Mixtures Physiologically based pharmacokinetics (PBPK), as applied to xenobiotics, is derived from intensive studies related primarily to the safety and efficacy of clinical drugs (e.g. (36)) and examples based on pesticides and other occupational chemicals are correspondingly fewer in number than those based on drugs. Regardless of the xenobiotic, however, a PBPK model for humans is derived from experimental data, extrapolation from surrogate animals and computer programs of greater or lesser complexity and the test of its utility is its ability to predict absorption, distribution, metabolism and excretion between different chemicals, individuals, and populations. It is axiomatic, therefore, that the greater the extent and accuracy of the experimental data, the greater will be the predictability. These points are emphasized and made clear in an extended discussion of PBPK modeling of inter-individual variability in the pharmacokinetics of environmental chemicals by Bois et al. (30), and, more specifically a discussion of modeling metabolic interactions between chemicals in chemical mixtures (31). A significant advance in how these models can be used is seen in the CYP-specific human PBPK/PD models developed by Foxenberg et al., (17), for chlorpyrifos and parathion. Using this model they predicted that individuals with higher levels of CYP2B6 and lower levels of CYP2C19 would have increased sensitivity to chlorpyrifos and parathion, based on blood acetycholinesterase inhibition. Such individuals would be, potentially, at greater risk of acute toxicity from chlorpyrifos and parathion and, based on this model, would be more sensitive to chlorpyrifos than parathion.

Induction, Cytotoxicity, Inhibition, and Enzyme Activation: Definitions and Examples Studies by our group have indicated potential for interactions based on cytochrome(s) P450 (CYPs) induction and/or inhibition as well as cytotoxicity. For example fipronil is a potent inducer at low doses but cytotoxic at higher doses in human hepatocytes. Chlorpyrifos is bioactivated by CYPs to the toxic chlorpyrifos-oxon (CPO) and the non-toxic trichloropyridinol (TCP). Biphasic interactions, first inhibition, followed by induction are also known, good examples being the insecticide synergist, piperonyl butoxide and other benzodioxole 119 In Parameters for Pesticide QSAR and PBPK/PD Models for Human Risk Assessment; Knaak, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

chemicals (37, 38). The demonstration of variation in metabolism and metabolite production by CYPs of fipronil and other pesticides raises the possibility that the extent and effect of pesticide interactions may also show wide variability.


Induction Induction is the process that generally involves transcriptional activation of XMEs genes leading to an increase in mRNA and subsequently a greater amount of an enzyme following exposure to an inducing agent. The increase in the amount of XMEs by xenobiotics is of particular concern in studies of pesticide interactions. Increasing the amount of an enzyme can occur by decreasing the degradation rate and/or increasing the synthesis rate, although increasing the synthesis rate is the most common mechanism for induction by xenobiotics. Coordinate (pleiotypic) induction refers to the induction of multiple enzymes by a single inducing agent (e.g., phenobarbital induction of several of the cytochrome P450-dependent monooxygenases). Depending upon the principal activity of the XME induced, induction may give rise to a decrease (detoxication) or an increase (activation) in the effect of a toxicant and is not specific with regard to toxicants metabolized, a xenobiotic frequently inducing the metabolism of other xenobiotics. The stimulatory effect of xenobiotics on liver microsomal enzymes was first reported in the 1950s, these early studies being summarized by the landmark review of Conney (39) and since then has been extensively investigated in surrogate animals. Among the numerous reviews, recent ones with emphasis on pesticides include those of Hodgson and Meyer (40, 41) Induction as it relates to pesticides: induction by a pesticide that affects the metabolism of other pesticides; induction by a pesticide that affects the metabolism of endogenous metabolites; induction by non-pesticidal xenobiotics that affects the metabolism of pesticides. There are many examples of all of these, summarized in detail in Hodgson and Meyer (41), although there are few mechanistic studies involving pesticides and few studies involving human or human-derived cell types. In studies using human hepatocytes, the induction of CYPs by fipronil is a good example, with CYP1A1 and CYP3A4 being maximally induced at the mRNA by doses of 25 μM and 1 μM (42). Increases in CYP3A4 protein and activity levels were also observed and reached maximal levels at 1 μM. Chlorpyrifos significantly induced mRNA levels of CYP1A1, CYP1A2, CYP3A4, and CYP1B1, and to a lesser extent CYP2B6 and CYP2A6 in studies using human hepatocytes (18). Increases in CYP3A4 and CYP1A1 protein levels were also observed in chlorpyrifos exposed human hepatocytes. Endosulfan exposure of human hepatocytes leads to significant increases in CYP2B6 protein levels at 1 μM and CYP3A4 protein levels at 10 μM (43). Nuclear Receptors The induction of XMEs by pesticides involves the activation of ligand dependent transcription factor receptors. For example, a pesticide can act as a ligand for the pregnane X receptor (PXR, NR1I2) transcription factor, which is highly expressed in tissues such as the liver and intestine (Figure 2). PXR, and 120 In Parameters for Pesticide QSAR and PBPK/PD Models for Human Risk Assessment; Knaak, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


other similar receptors, contain closely related DNA binding domains (DBDs) and ligand binding domains (LBD). The activated PXR then translocates to the nucleus and binds to specific response elements in the promoters of XME genes, such as CYP3A4, leading to increased transcription and accumulation of CYP3A4 mRNA and protein (44). PXR is a unique receptor in that it has a very flexible LBD and thus can bind a structurally diverse array of pesticides and other xenobiotics. Besides PXR, the constitutive androstane receptor (CAR) and other receptors have been implicated in ability of pesticides to induce XMEs (45, 46).

Figure 2. PXR mechanism of action. A summary of processes involved in the induction of CYP3A4 by inducers acting through the pregnane X receptor (PXR). RXR = retinoid X receptor. Recent studies of the induction of CYPs by pesticides has established that a number of pesticides induce CYP3A4 or CYP2B6 promoter activities in a PXR dependent manner (43, 47). Pyrethroids, firponil, chlorpyrifos (CPS), and endosulfan have been shown to induce the activity of CYP3A4 or CYP2B6 promoter reporter plasmids that were transient co-transfected into HepG2 cells with a human PXR expression plasmid. When CYP reporter plasmids were transfected without a PXR expression plasmid, promoter activities were not induced by these pesticides. Significant inductions were seen at doses of 10 μM of these pesticides and at 1 μM for endosulfan. Further, the primary metabolite of endosulfan produced in the human liver, enodosulfan sulfate , also induced CYP3A4 and CYP2B6 promoter activities. Induction of metabolism was tested in vivo using wild type, mPXR null mice, and humanized PXR transgenic mice to investigate the importance of PXR and the species specificity in the response to endosulfan exposure. Wild type and humanized PXR mice exposed to endosulfan displayed a decreased sleep time after being administered the sedative 121 In Parameters for Pesticide QSAR and PBPK/PD Models for Human Risk Assessment; Knaak, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

tribromoethanol, indicating induction of CYP3A. The importance of PXR was demonstrated, as in studies using mPXR-null mice no significant difference was seen in sleep times between the control and the endosulfan exposed mice. Although CYP1A1 and CYP1A2 are also induced by CPS there has been, as yet, no evidence for the involvement of the aryl hydrocarbon receptor (AhR) in this induction. It may be noted, however, that at least two classes of chemicals, benzodioxoles (methylenedioxyphenyl) (35) and acenaphthylene (48) have been shown to induce Cyp1a2 without the involvement of the AhR.


Microarray Studies To investigate variation in humans from chlorpyrifos (CPS) exposure, hepatocytes were exposed to vehicle control or CPS, RNA was prepared, and microarray analysis performed (Affymetrix Gene Chip Human Genome Arrays) to identify regulated genes and pathways regulated in different individuals. Principle component analysis (PCA) was done to cluster samples by subject, exposure, and duration of exposure, which revealed clear separation of samples (Figure 3). Analysis determined that 314 genes were differentially expressed, with 49 induced genes, and 265 repressed compared to their controls. Further analysis of the differentially regulated genes was done to identify specific biological pathways impacted by CPS exposure utilizing the Kyoto encyclopedia of genes and genomes (KEGG) bioinformatics resource. Pathways that were impacted included genes important in retinol metabolism, regulation of the actin cytoskeleton, and xenobiotic metabolism.

Cytotoxicity The importance of cell viability assays should be included in assessments of pesticides when conducting studies of induction of XMEs using human hepatocytes and in human cell line reporter assays determining receptor activation by pesticides. For example, in PXR reporter assays, cytotoxicity can negatively impact PXR studies (49). In studies using human hepatocytes and the HepG2 human liver cell line, fipronil cytotoxicity was measured by adenylate kinase assays, as adenylate kinase is released into the medium from damaged cells. Caspase3/7 activity was also measured, which assesses caspase activity associated with apoptosis (Table 1). In these studies of fipronil significantly elevated adenylate kinase levels were seen at 0.5 μM in HepG2 cells and 25 μM in human hepatocytes. Caspase3/7 activity was elevated at 0.1 μM and 25 μM. In studies of chloropyrifos, exposure resulted in increased adenylate kinase activity in a dose-dependent manner which peaked at 50 μM in HepG2 cells and 12.5 μM in human hepatocytes. Caspase3/7 activity peaked at 12.5 μM in both cell types activity. Endosulfan exposure resulted in increased adenylate kinase activity at 50 μM in HepG2 cells and at 6.25 μM in human hepatocytes. Exposure to endosulfan increased caspase 3/7 levels in both cell types at the 1μM dose, which peaked at 12.5 μM and 50 μM. 122 In Parameters for Pesticide QSAR and PBPK/PD Models for Human Risk Assessment; Knaak, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Figure 3. Microarray analysis of chlorpyrifos exposed human hepatocytes. Hepatocytes from two individuals were exposed to solvent control (Con) or 50 uM chlopyrifos (CPS) for 12 or 24 hours. Principle component analysis (PCA) of gene expression data was done to cluster samples by subject, exposure, and duration of exposure.

Table 1. Fipronil Cytotoxicity: Human Hepatocytes and HepG2 Cells Cell Type

Threshold

Maximum

HepG2

Human Metabolic Interactions of Pesticides: Inhibition, Induction, and

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