Andrew D. Wallace

Human hepatic CYP2B6 developmental expression: the impact of age and genotype.

Although CYP2B6 is known to metabolize numerous pharmaceuticals and toxicants in adults, little is known regarding CYP2B6 ontogeny or its possible role in pediatric drug/toxicant metabolism. To address this knowledge gap, hepatic CYP2B6 protein levels were characterized in microsomal protein preparations isolated from a pediatric liver bank (N=217). Donor ages ranged from 10 weeks gestation to 17 years of age with a median age of 1.9 months. CYP2B6 levels were measured by semi-quantitative western blotting. Overall, CYP2B6 expression was detected in 75% of samples. However, the percentage of samples with detectable CYP2B6 protein increased with age from 64% in fetal samples to 95% in samples from donors >10 years of age. There was a significant, but only 2-fold increase in median CYP2B6 expression after the neonatal period (birth to 30 days postnatal) although protein levels varied over 25-fold in both age groups. The median CYP2B6 level in samples over 30 postnatal days to 17 years of age (1.3 pmol/mg microsomal protein) was lower than previously reported adult levels (2.2-22 pmol/mg microsomal protein), however, this likely relates to the median age of these samples, i.e., 10.3 months. CYP2B6 expression did not vary significantly by gender. Furthermore, CYP2B6 levels did not correlate with CYP3A4, CYP3A5.1 or CYP3A7 activity, consistent with different mechanisms controlling the ontogeny and constitutive expression of these enzymes and the lack of significant induction in the pediatric samples.

Metabolism of endosulfan-alpha by human liver microsomes and its utility as a simultaneous in vitro probe for CYP2B6 and CYP3A4.

Endosulfan-α is metabolized to a single metabolite, endosulfan sulfate, in pooled human liver microsomes (Km = 9.8 μM, Vmax = 178.5 pmol/mg/min). With the use of recombinant cytochrome P450 (P450) isoforms, we identified CYP2B6 (Km = 16.2 μM, Vmax = 11.4 nmol/nmol P450/min) and CYP3A4 (Km = 14.4 μM, Vmax = 1.3 nmol/nmol P450/min) as the primary enzymes catalyzing the metabolism of endosulfan-α, although CYP2B6 had an 8-fold higher intrinsic clearance rate (CLint = 0.70 μl/min/pmol P450) than CYP3A4 (CLint = 0.09 μl/min/pmol P450). Using 16 individual human liver microsomes (HLMs), a strong correlation was observed with endosulfan sulfate formation and S-mephenytoin N-demethylase activity of CYP2B6 (r2 = 0.79), whereas a moderate correlation with testosterone 6 β-hydroxylase activity of CYP3A4 (r2 = 0.54) was observed. Ticlopidine (5 μM), a potent CYP2B6 inhibitor, and ketoconazole (10 μM), a selective CYP3A4 inhibitor, together inhibited approximately 90% of endosulfan-α metabolism in HLMs. Using six HLM samples, the percentage total normalized rate (% TNR) was calculated to estimate the contribution of each P450 in the total metabolism of endosulfan-α. In five of the six HLMs used, the percentage inhibition with ticlopidine and ketoconazole in the same incubation correlated with the combined % TNRs for CYP2B6 and CYP3A4. This study shows that endosulfan-α is metabolized by HLMs to a single metabolite, endosulfan sulfate, and that it has potential use, in combination with inhibitors, as an in vitro probe for CYP2B6 and 3A4 catalytic activities.

Endosulfan induces CYP2B6 and CYP3A4 by activating the pregnane X receptor.

Endosulfan is an organochlorine pesticide commonly used in agriculture. Endosulfan has affects on vertebrate xenobiotic metabolism pathways that may be mediated, in part, by its ability to activate the pregnane X receptor (PXR) and/or the constitutive androstane receptor (CAR) which can elevate expression of cytochrome P450 (CYP) enzymes. This study examined the dose-dependency and receptor specificity of CYP induction in vitro and in vivo. The HepG2 cell line was transiently transfected with CYP2B6- and CYP3A4-luciferase promoter reporter plasmids along with human PXR (hPXR) or hCAR expression vectors. In the presence of hPXR, endosulfan-alpha exposure caused significant induction of CYP2B6 (16-fold) and CYP3A4 (11-fold) promoter activities over control at 10 microM. The metabolite endosulfan sulfate also induced CYP2B6 (12-fold) and CYP3A4 (6-fold) promoter activities over control at 10 microM. In the presence of hCAR-3, endosulfan-alpha induced CYP2B6 (2-fold) promoter activity at 10 microM, but not at lower concentrations. These data indicate that endosulfan-alpha significantly activates hPXR strongly and hCAR weakly. Using western blot analysis of human hepatocytes, the lowest concentrations at which CYP2B6 and CYP3A4 protein levels were found to be significantly elevated by endosulfan-alpha were 1.0 microM and 10 microM, respectively. In mPXR-null/hPXR-transgenic mice, endosulfan-alpha exposure (2.5mg/kg/day) caused a significant reduction of tribromoethanol-induced sleep times by approximately 50%, whereas no significant change in sleep times was observed in PXR-null mice. These data support the role of endosulfan-alpha as a strong activator of PXR and inducer of CYP2B6 and CYP3A4, which may impact metabolism of CYP2B6 or CYP3A4 substrates.

Phthalate Induction of CYP3A4 is Dependent on Glucocorticoid Regulation of PXR Expression

Cytochrome P450 3A4 (CYP3A4) is responsible for oxidative metabolism of more than 60% of all pharmaceuticals. CYP3A4 is inducible by xenobiotics that activate pregnane X receptor (PXR), and enhanced CYP3A4 activity has been implicated in adverse drug interactions. Recent evidence suggest that the widely used plasticizer, di-2-ethylhexyl phthalate (DEHP), and its primary metabolite mono-2-ethylhexyl phthalate (MEHP) may act as agonists for PXR. Hospital patients are uniquely exposed to high levels of DEHP as well as being administered glucocorticoids. Glucocorticoids positively regulate PXR expression in a glucocorticoid receptor (GR)-mediated mechanism. We suggest that the magnitude of CYP3A4 induction by phthalates is dependent on the expression of PXR and may be significantly higher in the presence of glucocorticoids. DEHP and MEHP induced PXR-mediated transcription of the CYP3A4 promoter in a dose-dependent fashion. Coexposure to phthalates and dexamethasone (Dex) resulted in enhanced CYP3A4 promoter activity; furthermore, this induction was abrogated by both the GR antagonist RU486 and GR small interfering ribonucleic acid. Dex induced PXR protein expression in human hepatocytes and a liver-derived rat cell line. CYP3A4 protein was highly induced by Dex and DEHP coadministration in human hepatocyte cultures. Finally, enhanced 6beta-hydroxytestosterone formation in Dex and phthalate cotreated human hepatocytes confirmed CYP3A4 enzyme induction. Concomitant exposure to glucocorticoids and phthalates resulting in enhanced metabolic activity of CYP3A4 may play a role in altered efficacy of pharmaceutical agents. Understanding the role of glucocorticoid regulation of PXR as a key determinant in the magnitude of CYP3A4 induction by xenobiotics may provide insight into adverse drug effects in a sensitive population.

DNA methylation alterations in response to pesticide exposure in vitro

Although pesticides are subject to extensive carcinogenicity testing before regulatory approval, pesticide exposure has repeatedly been associated with various cancers. This suggests that pesticides may cause cancer via nonmutagenicity mechanisms. The present study provides evidence to support the hypothesis that pesticide‐induced cancer may be mediated in part by epigenetic mechanisms. We examined whether exposure to seven commonly used pesticides (i.e., fonofos, parathion, terbufos, chlorpyrifos, diazinon, malathion, and phorate) induces DNA methylation alterations in vitro. We conducted genome‐wide DNA methylation analyses on DNA samples obtained from the human hematopoietic K562 cell line exposed to ethanol (control) and several organophosphate pesticides (OPs) using the Illumina Infinium HumanMethylation27 BeadChip. Bayesian‐adjusted t‐tests were used to identify differentially methylated gene promoter CpG sites. In this report, we present our results on three pesticides (fonofos, parathion, and terbufos) that clustered together based on principle component analysis and hierarchical clustering. These three pesticides induced similar methylation changes in the promoter regions of 712 genes, while also exhibiting their own OP‐specific methylation alterations. Functional analysis of methylation changes specific to each OP, or common to all three OPs, revealed that differential methylation was associated with numerous genes that are involved in carcinogenesis‐related processes. Our results provide experimental evidence that pesticides may modify gene promoter DNA methylation levels, suggesting that epigenetic mechanisms may contribute to pesticide‐induced carcinogenesis. Further studies in other cell types and human samples are required, as well as determining the impact of these methylation changes on gene expression. Environ. Mol. Mutagen. 2012.

Genome-wide study of DNA methylation alterations in response to diazinon exposure in vitro

Pesticide exposure has repeatedly been associated with cancers. However, molecular mechanisms are largely undetermined. In this study, we examined whether exposure to diazinon, a common organophosphate that has been associated with cancers, could induce DNA methylation alterations. We conducted genome-wide DNA methylation analyses on DNA samples obtained from human hematopoietic K562 cell exposed to diazinon and ethanol using the Illumina Infinium HumanMethylation27 BeadChip. Bayesian-adjusted t-tests were used to identify differentially methylated gene promoter CpG sites. We identified 1069 CpG sites in 984 genes with significant methylation changes in diazinon-treated cells. Gene ontology analysis demonstrated that some genes are tumor suppressor genes, such as TP53INP1 (3.0-fold, q-value <0.001) and PTEN (2.6-fold, q-value <0.001), some genes are in cancer-related pathways, such as HDAC3 (2.2-fold, q-value=0.002), and some remain functionally unknown. Our results provided direct experimental evidence that diazinon may modify gene promoter DNA methylation levels, which may play a pathological role in cancer development.

Human variation in CYP-specific chlorpyrifos metabolism.

Chlorpyrifos, an organophophorothioate insecticide, is bioactivated to the neurotoxic metabolite, chlorpyrifos-oxon (CPO) by cytochromes P450 (CYPs). To determine the variability in chlorpyrifos bioactivation, CPO production by human liver microsomes from 17 individual donors was compared relative to phenotype and genotype. CPO production varied over 14-fold between individuals in incubations utilizing 20 μM chlorpyrifos as substrate, while CPO production varied 57-fold in incubations with 100 μM chlorpyrifos. For all but two samples, the formation of the less toxic metabolite, 3,5,6-trichloro-2-pyridinol (TCP), was greater than CPO production. TCP production varied 9-fold in incubations utilizing 20 μM chlorpyrifos as substrate and 19-fold using 100 μM chlorpyrifos. Chlorpyrifos metabolism by individual human liver microsomes was significantly correlated with CYP2B6, CYP2C19 and CYP3A4 related activity. CPO formation was best correlated with CYP2B6 related activity at low (20 μM) chlorpyrifos concentrations while CYP3A4 related activity was best correlated with CPO formation at high concentrations (100 μM) of chlorpyrifos. TCP production was best correlated with CYP3A4 activity at all substrate concentrations of chlorpyrifos. The production of both CPO and TCP was significantly lower at a concentration of 20 μM chlorpyrifos as compared to 100 μM chlorpyrifos. Calculations of percent total normalized rates (% TNR) and the chemical inhibitors ketoconazole and ticlopidine were used to confirm the importance of CYP2B6, CYP2C19, and CYP3A4 for the metabolism of chlorpyrifos. The combination of ketoconazole and ticlopidine inhibited the majority of TCP and CPO formation. CPO formation did not differ by CYP2B6 genotype. Individual variations in CPO production may need to be considered in determining the risk of chlorpyrifos poisoning.

Lysine 419 Targets Human Glucocorticoid Receptor for Proteasomal Degradation

Glucocorticoid receptors (GRs) are members of a highly conserved family of ligand dependent transcription factors which following hormone binding undergo homologous down-regulation reducing the levels of receptor protein. This decline in human GR (hGR) is due in part to a decrease in protein receptor stability that may limit cellular responsiveness to ligand. To examine the role of the proteasome protein degradation pathway in steroid-dependent hGR responsiveness, we utilized the proteasomal inhibitors MG-132, beta-lactone, and epoxomicin. HeLa cells and COS cells were treated with proteasome inhibitors in the presence of the GR agonist dexamethasone (Dex), or were pretreated with proteasomal inhibitor and then Dex. Dexamethasone induced glucocorticoid responsive reporter activity significantly over untreated controls, whereas cells treated with proteasomal inhibitors and Dex together showed 2-3-fold increase in activity. Protein sequence analysis of the hGR protein identified several candidate protein degradation motifs including a PEST element. Mutagenesis of this element at lysine 419 was done and mutant K419A hGR failed to undergo ligand dependent down-regulation. Mutant K419A hGR displayed 2-3-fold greater glucocorticoid responsive reporter activity in the presence of Dex than wild type hGR. These differences in transcriptional activity were not due to altered subcellular localization, since when the mutant K419A hGR was fused with the green fluorescent protein (GFP) it was found to move in and out of the nucleus similarly to wild type hGR. Together these results suggest that the proteasome and the identified PEST degradation motif limit steroid-dependent human glucocorticoid receptor signaling.

Pyrethroids: Cytotoxicity and Induction of CYP Isoforms in Human Hepatocytes

Deltamethrin [(S)-alpha-cyano-3-phenoxybenzyl-cis-(1 R,3R)-3(2,2-dibromovinyl)(2,2-dimethyl-cyclopropane-carboxylate] and permethrin [3-phenoxybenzyl(1RS)-cis,trans-3-(2,2-dichlorovinyl)-2,2-dimethyl-cyclopropanecarboxylate] are pyrethroid insecticides used in agriculture, public health and military deployments. Pyrethroids are known to be capable of inducing cytochrome P450 (CYP) 2B1/2B2, CYP1A1 and overall CYP content in rat liver. The objectives of this study were to evaluate the potential of deltamethrin and permethrin to cause cytotoxicity and to induce CYP isoforms in human hepatocytes. Permethrin and deltamethrin showed dose-dependent effects on adenylate kinase activity in HepG2 cells, in which 50 and 100 microM doses, respectively, induced a 3-5 fold increase in activity, and also induced adenylate kinase activity in primary human hepatocytes. An approximately 3-fold induction was noted at 200 microM deltamethrin and a 4-fold induction at 100 microM permethrin. Cytotoxicity was noted in HepG2 cells following 48-72 h exposure to 100 or 200 microM deltamethrin and permethrin, respectively. Dose-dependent induction of caspase-3/7 was initiated by 12.5 microM deltamethrin or by 3.125 microM permethrin. Actinomycin D, a positive control for induction of caspase 3/7, induced caspase-3/7, an effect completely abrogated by the specific inhibitor Z-DEVD-FMK. At 100 microM deltamethrin 2-3 fold induction of CYP1A1 and CYP2B6 mRNA was observed, while at the same time an approximately 25-fold induction of CYP3A4 was noted. Permethrin-mediated CYP induction was much less potent, 4-fold or less for CYP1A1, CYP3A4, CYP3A5, CYP2B6 and CYP2A6. It has also been shown that these pyrethroids are ligands for the pregnane X receptor (PXR).

Interactions of the crustacean nuclear receptors HR3 and E75 in the regulation of gene transcription

Endocrine signal transduction occurs through cascades that involve the action of both ligand-dependent and ligand-independent nuclear receptors. In insects, two such nuclear receptors are HR3 and E75 that interact to transduce signals initiated by ecdysteroids. We have cloned these nuclear receptors from the crustacean Daphnia pulex to assess their function as regulators of gene transcription in this ecologically and economically important group of organisms. Both nuclear receptors from D. pulex (DappuHR3 (group NR1F) and DappuE75 (group NR1D)) exhibit a high degree of sequence similarity to other NR1F and NR1D group members that is indicative of monomeric binding to the RORE (retinoid orphan receptor element). DappuE75 possesses key amino acid residues required for heme binding to the ligand-binding domain. Next, we developed a gene transcription reporter assay containing a luciferase reporter gene driven by the RORE. DappuHR3, but not DappuE75, activated transcription of the luciferase gene in this system. Co-transfection experiments revealed that DappuE75 suppressed DappuHR3-dependent luciferase transcription in a dose-dependent manner. Electrophoretic mobility shift assays confirmed that DappuHR3 bound to the RORE. However, we found no evidence that DappuE75 similarly bound to the response element. These experiments further demonstrated that DappuE75 prevented DappuHR3 from binding to the response element. In conclusion, DappuHR3 functions as a transcriptional activator of genes regulated by the RORE and DappuE75 is a negative regulator of this activity. DappuE75 does not suppress the action of DappuHR3 by occupying the response element but presumably interacts directly with the DappuHR3 protein. Taken together with the previous demonstration that daphnid HR3 is highly induced by 20-hydroxyecdysone, these results support the premise that HR3 is a major component of ecdysteroid signaling in some crustaceans and is under the negative regulatory control of E75.