Christopher S. Mazur

Human and Rat ABC Transporter Efflux of Bisphenol A and Bisphenol A Glucuronide: Interspecies Comparison and Implications for Pharmacokinetic Assessment

Significant interspecies differences exist between human and rodent with respect to absorption, distribution, and excretion of bisphenol A (BPA) and its primary metabolite, BPA-glucuronide (BPA-G). ATP-Binding Cassette (ABC) transporter enzymes play important roles in these physiological processes, and their enzyme localization (apical vs. basolateral) in the plasma membrane allows for different cellular efflux pathways. In this study, we utilized an ATPase assay to evaluate BPA and BPA-G as potential substrates for the human and rat ABC transporters: P-glycoprotein (MDR1), multidrug resistance-associated proteins (MRPs), and breast cancer-resistant protein (BCRP). Based on high ATPase activity, BPA is likely a substrate for rat mdr1b but not for human MDR1 or rat mdr1a. Results indicate that BPA is a potential substrate for rat mrp2 and human MRP2, BCRP, and MRP3. The metabolite BPA-G demonstrated the highest apparent substrate binding affinity for rat mrp2 and human MRP3 but appeared to be a nonsubstrate or potential inhibitor for human MRP2, MDR1, and BCRP and for rat mdr1a, mdr1b, and bcrp. Analysis of ABC transporter amino acid sequences revealed key differences in putative binding site composition that may explain substrate specificity. Collectively, these results suggest that in both rat and human, apical transporters efflux BPA into the bile and/or intestinal lumen. BPA-G would follow a similar pathway in rat; however, in human, due to the basolateral location of MRP3, BPA-G would likely enter systemic and portal blood supplies. These differences between human and rodent ABC transporters may have significant implications for interspecies extrapolation used in risk assessment.

Differences Between Human and Rat Intestinal and Hepatic Bisphenol-A Glucuronidation and the Influence of Alamethicin on In vitro Kinetic Measurements

The extent to which membrane-disrupting agents, such as alamethicin, may alter cofactor transport and influence in vitro kinetic measurements of glucuronidation is a major concern regarding the characterization and extrapolation of inter- and intraspecies pharmacokinetics of bisphenol A (BPA). An additional concern is the omission of a BPA intestinal metabolism component in current pharmacokinetic models used to assess oral exposure. In this study, BPA glucuronidation in native hepatic microsomes from female rat and female human liver displayed higher Vmax values than that in males. In the presence of alamethicin, all hepatic Vmax values increased; however, this increase was disproportionately greater in males and gender differences were no longer observed. Female rats exhibited a much higher Km than all other species and genders; the addition of alamethicin had little influence on Km values for any of the test systems. The dissimilar Km measured for female rat suggests that different UDP-glucuronosyltransferase (UGT) enzyme(s) are involved in BPA glucuronidation. The presence of different UGTs in female rat was confirmed using Hill coefficients measured from diclofenac-mediated chemical inhibition assays within hepatic microsomes and purified human UGT2B7 and UGT2B15. Mixed-gender human intestinal microsomes showed little BPA glucuronidation reactivity compared with those from male rat intestine. Male rat intestinal microsomes in the presence of alamethicin exhibited a Vmax that was nearly 30-fold higher than that for mixed human microsomes. The species and gender metabolic differences we observed between rat and human liver and intestine provide key information for delineating BPA pharmacokinetics needed for human health risk assessment.

Mechanistic investigation of the noncytochrome P450-mediated metabolism of triadimefon to triadimenol in hepatic microsomes.

Recently, much emphasis has been placed on understanding the toxic mode of action of the 1,2,4-triazole fungicides (i.e., conazoles) in an effort to improve and harmonize risk assessment. Relative to other conazoles, triadimefon is unique with respect to tumorigenesis in rodents, and it has been proposed that triadimefon does not share a common mechanism of toxicity with other conazoles. We postulate that one reason for this difference is that while many conazoles are metabolized via an oxidative P450-mediated pathway, triadimefon is not. In studies conducted with rat hepatic microsomes, triadimenol was identified as the major metabolite (approximately 80%) of triadimefon metabolism, and reduction of the carbonyl group in triadimefon occurred stereoselectively with preferential formation of the less toxic triadimenol B diastereomer. Using chemical inhibitors of P450s (i.e., clotrimazole and 1-aminobenzotriazole) and carbonyl reducing enzymes (i.e., glycyrrhetinic acid, quercitrin, and cortisone), both triadimefon depletion and triadimenol formation were found to be mediated by 11 beta-hydroxysteroid dehydrogenase type 1 (11 beta-HSD1). Studies examining NADPH production and inhibitor studies for glucose-6-phosphate translocation across the endoplasmic reticulum (ER) membrane implicated hexose-6-phosphate dehydrogenase (H6PDH) in the metabolism of triadimefon as well. These results ultimately associate triadimefon metabolism not only with steroidogenesis (i.e., 11 beta-HSD1) but carbohydrate metabolism (i.e., H6PDH) as well. Considering the impact of triadimefon on these biochemical pathways may help explain some of triadimefons unique toxicological effects relative to other conazole fungicides.

Contrasting Influence of NADPH and a NADPH-regenerating System on the Metabolism of Carbonyl-Containing Compounds in Hepatic Microsomes.

Carbonyl containing xenobiotics may be susceptible to NADPH-dependent cytochrome P450 (P450) and carbonyl-reduction reactions. In vitro hepatic microsome assays are routinely supplied NADPH either by direct addition of NADPH or via an NADPH-regenerating system (NRS). In contrast to oxidative P450 transformations, which occur on the periphery of a microsome vesicle, intraluminal carbonyl reduction depends on transport of cofactors across the endoplasmic reticulum (ER) membrane into the lumen. Glucose 6-phosphate, a natural cofactor and component of the NRS matrix, is readily transported across the ER membrane and facilitates intraluminal NADPH production, whereas direct addition of NADPH has limited access to the lumen. In this study, we compared the effects of direct addition of NADPH and use of an NRS on the P450-mediated transformation of propiconazole and 11β-hydroxysteroid dehydrogenase type 1 (HSD1) carbonyl reduction of cortisone and the xenobiotic triadimefon in hepatic microsomes. Our results demonstrate that the use of NADPH rather than NRS can underestimate the kinetic rates of intraluminal carbonyl reduction, whereas P450-mediated transformations were unaffected. Therefore, in vitro depletion rates measured for a carbonyl-containing xenobiotic susceptible to both intraluminal carbonyl reduction and P450 processes may not be properly assessed with direct addition of NADPH. In addition, we used in silico predictions as follows: 1) to show that 11β-HSD1 carbonyl reduction was energetically more favorable than oxidative P450 transformation; and 2) to calculate chemical binding score and the distance between the carbonyl group and the hydride to be transferred by NADPH to identify other 11β-HSD1 substrates for which reaction kinetics may be underestimated by direct addition of NADPH.

In Vitro Metabolism of the Fungicide and Environmental Contaminant trans-bromuconazole and Implications for Risk Assessment

trans-Bromuconazole is a chiral chemical representative of a class of triazole derivatives known to inhibit specific fungal cytochrome P-450 (CYP) reactions. Kinetic measurements and delineation of metabolic pathways for triazole chemicals within in vitro hepatic microsomes are needed for accurate risk assessment and predictive in vivo physiological modeling. The studies described here were conducted with rat liver microsomes to determine Michaelis–Menten saturation kinetic parameters (V max and K M) for trans-bromuconazole using both substrate depletion and product formation reaction velocities. Kinetic parameters determined for trans-bromuconazole depletion at varying protein levels incubated at physiological temperature 37°C resulted in a K M value of 1.69 μM and a V max value of 1398 pmol/min/mg protein. The concomitant linear formation of two metabolites identified using liquid chromatography/time-of-flight mass spectrometry (LC/MS-TOF) and LC-MS/MS indicated hydroxylation of the trans-bromuconazole dichlorophenyl ring moiety. K M values determined for the hydroxylated metabolites were 0.87 and 1.03 μM, with V max values of 449 and 694 pmol/min/mg protein, respectively. Chemical inhibition assays and studies conducted with individual purified human recombinant enzymes indicated the CYP3A subfamily was primarily responsible for biotransformation of the parent substrate. Additionally, trans-bromuconazole was found to undergo stereoselective metabolism as evidenced by a change in the enantiomeric ratio (trans−/trans +) with respect to time.

H2 consumption during the microbial reductive dehalogenation of chlorinated phenols and tetrachloroethene.

Competition for molecular hydrogen exists amonghydrogen-utilizing microorganismsin anoxic environments, and evidence suggeststhat lower hydrogen concentrations areobserved with more energetically favorableelectron-accepting processes. The transferof electrons to organochlorines via reductivedehalogenation reactions plays an importantrole in hydrogen dynamics in impacted systems. Westudied the flux of aqueous hydrogenconcentrations in methanogenic sediment microcosmsprior to and during reductivedehalogenation of a variety of substituted chlorophenols(CP) and tetrachloroethene(perchloroethylene, PCE). Mean hydrogen concentrationsduring reductive dehalogenationof 2,4-CP, 2,3,4-CP, and PCP were 3.6 nM, 4.1 nM,and 0.34 nM, respectively. Sedimentmicrocosms that were not dosed with chlorophenolsyet were actively methanogenicmaintained a significantly higher mean hydrogenconcentration of 9.8 nM. Duringactive PCE dehalogenation, sediment microcosmsmaintained a mean hydrogenconcentration of 0.82 nM. These data indicate thatduring limiting hydrogen production,the threshold ecosystem hydrogen concentration iscontrolled by microbial populationsthat couple hydrogen oxidation to thermodynamicallyfavorable electron acceptingreactions, including reductive dehalogenationof chloroaromatic compounds. Wealso present revised estimates for the Gibbsfree energy available from the reductivedehalogenation of a variety of substitutedchlorophenols based on recently publishedvalues of vapor pressure, solubility, and pKafor these compounds.

P-glycoprotein inhibition by the agricultural pesticide propiconazole and its hydroxylated metabolites: Implications for pesticide–drug interactions

The human efflux transporter P-glycoprotein (P-gp, MDR1) functions as an important cellular defense system against a variety of xenobiotics; however, little information exists on whether environmental chemicals interact with P-gp. Conazoles provide a unique challenge to exposure assessment because of their use as both pesticides and drugs. Propiconazole is an agricultural pesticide undergoing evaluation by the U.S. Environmental Protection Agencys Endocrine Disruptor Screening Program. In this study, the P-gp interaction of propiconazole and its hydroxylated metabolites were evaluated using MDR1-expressing membrane vesicles and NIH-3T3/MDR1 cells. Membrane vesicle assays demonstrated propiconazole (IC50,122.9μM) and its metabolites (IC50s, 350.8μM, 366.4μM, and 456.3μM) inhibited P-gp efflux of a probe substrate, with propiconazole demonstrating the strongest interaction. P-gp mediated transport of propiconazole in MDR1-expressed vesicles was not detected indicating propiconazole interacts with P-gp as an inhibitor rather than a substrate. In NIH-3T3/MDR1 cells, propiconazole (1 and 10μM) led to decreased cellular resistance (chemosensitization) to paclitaxel, a chemotherapeutic drug and known MDR1 substrate. Collectively, these results have pharmacokinetic and risk assessment implications as P-gp interaction may influence pesticide toxicity and the potential for pesticide-drug interactions.

Inhibition of the Human ABC Efflux Transporters P-gp and BCRP by the BDE-47 Hydroxylated Metabolite 6-OH-BDE-47: Considerations for Human Exposure

High body burdens of polybrominated diphenyl ethers (PBDEs) in infants and young children have led to increased concern over their potential impact on human development. PBDE exposure can alter the expression of genes involved in thyroid homeostasis, including those of ATP-binding cassette (ABC) transporters, which mediate cellular xenobiotic efflux. However, little information exists on how PBDEs interact with ABC transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). The purpose of this study was to evaluate the interactions of 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) and its hydroxylated metabolite 6-OH-BDE-47 with P-gp and BCRP, using human MDR1- and BCRP-expressing membrane vesicles and stably transfected NIH-3T3-MDR1 and MDCK-BCRP cells. In P-gp membranes, BDE-47 did not affect P-gp activity; however, 6-OH-BDE-47 inhibited P-gp activity at low µM concentrations (IC50 = 11.7 µM). In BCRP membranes, BDE-47 inhibited BCRP activity; however, 6-OH-BDE-47 was a stronger inhibitor [IC50 = 45.9 µM (BDE-47) vs. IC50 = 9.4 µM (6-OH-BDE-47)]. Intracellular concentrations of known P-gp and BCRP substrates [(3H)-paclitaxel and (3H)-prazosin, respectively] were significantly higher (indicating less efflux) in NIH-3T3-MDR1 and MDCK-BCRP cells in the presence of 6-OH-BDE-47, but not BDE-47. Collectively, our results indicate that the BDE-47 metabolite 6-OH-BDE-47 is an inhibitor of both P-gp and BCRP efflux activity. These findings suggest that some effects previously attributed to BDE-47 in biological systems may actually be due to 6-OH-BDE-47. Considerations for human exposure are discussed.