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.

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 Agency's 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.

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 V(max) values than that in males. In the presence of alamethicin, all hepatic V(max) values increased; however, this increase was disproportionately greater in males and gender differences were no longer observed. Female rats exhibited a much higher K(m) than all other species and genders; the addition of alamethicin had little influence on K(m) values for any of the test systems. The dissimilar K(m) 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 V(max) 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 approach to understanding the toxicity of the azole fungicide triadimefon to a nontarget aquatic insect and implications for exposure assessment.

Mechanistic and stereoselective based in vitro metabolism assays were utlilized to gain insight into the toxic mode of action of the 1,2,4-triazole fungicide, triadimefon, with black fly (Diptera: Simuliidae) larvae. Based on results from enzyme inhibitor studies, the metabolism of triadimefon in black fly larvae microsomes was found to occur predominantly via an oxidative P450-mediated pathway; triadimenol was formed via the stereoselective reduction of the prochiral carbonyl group of triadimefon. The relatively minor contribution of carbonyl reduction suggests that triadimefon may inhibit ecdysone 20-monooxygenase and disrupt insect molting hormone biosynthesis. 48-h LC50 tests for triadimefon and triadimenol with black fly larvae yielded median values (with 95% confidence intervals) of 6.1 (5.8-6.4) and 22.3 (20.3-24.1) mg/L respectively. The exposure of black fly larvae to sublethal concentrations of triadimefon resulted in increased microsomal P450 activity and affected the microsomal rates of both triadimefon depletion and triadimenol formation. In contrast to trout, black fly larvae produced a higher fraction of the more toxic triadimenol stereoisomers, which may explain in part why triadimefon exhibited a significantly greater toxicity with black fly larvae than trout. These results illustrate that while LC50 tests conducted with commercial triadimenol would presumably expose each organism to the same relative abundance of the four triadimenol stereoisomers, LC50 tests with triadimefon ultimately expose each organism to a unique set of triadimenol stereoisomers depending upon the organism's stereoselective metabolism.

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 11 beta-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 11 beta-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 11 beta-HSD1 substrates for which reaction kinetics may be underestimated by direct addition of NADPH.

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 triadimefon's unique toxicological effects relative to other conazole fungicides.

Cross-species comparison of conazole fungicide metabolites using rat and rainbow trout (Onchorhynchus mykiss) hepatic microsomes and purified human CYP 3A4.

Ecological risk assessment frequently relies on cross-species extrapolation to predict acute toxicity from chemical exposures. A major concern for environmental risk characterization is the degree of uncertainty in assessing xenobiotic biotransformation processes. Although inherently complex, metabolite identification is critical to risk assessment since the product(s) formed may pose a greater toxicological threat than the parent molecule. This issue is further complicated by differences observed in metabolic transformation pathways among species. Conazoles represent an important class of azole fungicides that are widely used in both pharmaceutical and agricultural applications. The antifungal property of conazoles occurs via complexation with the cytochrome P450 monooxygenases (CYP) responsible for mediating fungal cell wall synthesis. This mode of action has cause for concern regarding the potential adverse impact of conazoles on the broad spectrum of CYP-based processes within mammalian and aquatic species. In this study, in vitro metabolic profiles were determined for thirteen conazole fungicides using rat and rainbow trout (Oncorhynchus mykiss) liver microsomes and purified human CYP 3A4. Results showed that 10 out of the 13 conazoles tested demonstrated identical metabolite profiles among rat and trout microsomes, and these transformations were well conserved via both aromatic and aliphatic hydroxylation and carbonyl reduction processes. Furthermore, nearly all metabolites detected in the rat and trout microsomal assays were detected within the human CYP 3A4 assays. These results indicate a high degree of metabolic conservation among species with an equivalent isozyme activity of human CYP 3A4 being present in both the rat and trout, and provides insight into xenobiotic biotransformations needed for accurate risk assessment.

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 (Vmax and KM) 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 degrees C resulted in a KM value of 1.69 microM and a Vmax 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. KM values determined for the hydroxylated metabolites were 0.87 and 1.03 microM, with Vmax 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.

Enantioselective microbial transformation of the phenylpyrazole insecticide fipronil in anoxic sediments.

Fipronil, a chiral insecticide, was biotransformed initially to fipronil sulfide in anoxic sediment slurries following a short lag period. Sulfidogenic or methanogenic sediments transformed fipronil with half-lives of approximately 35 and 40 days, respectively. In all microbially active sediment slurries tested, the transformation of fipronil to fipronil sulfide was enantioselective. In the sulfidogenic sediment slurry, the enantiomeric fraction (EF) of fipronil decreased from an initial racemic EF value of 0.46 to a value of 0.22 during the incubation period of active fipronil transformation, indicating preferential transformation of the S-(+)-enantiomer. A previously unidentified product, 5-amino1-[2,6-dichloro-4-(trifluoromethyl)-phenyl]-4-(trifluoromethylthio)-1-H-pyrazole-3-carboxyamide, or fipronil sulfide-amide, was detected in the sulfidogenic slurries and coincided with the loss of fipronil sulfide. Biota from methanogenic freshwater sediment slurries also transformed fipronil enantioselectively but with a preference for the R-(-)-enantiomer. In all microbially inhibited (autoclaved) sediment slurries tested, no changes in the enantiomeric fractions of fipronil were observed and only low levels (< 5% of the added fipronil) of the fipronil sulfide metabolite were detected. In defined (model) chemical experiments, solutions of pyrite (FeS2) and iron sulfide (FeS) non-enantioselectively transformed fipronil primarily to either 2,6-dichloro-4-(trifluoromethyl)-aniline or to fipronil sulfide and fipronil amide, respectively. This report provides the first experimental evidence of enantioselective microbial transformation of fipronil in a natural environment (soil, water, and sediment) as well as identification of a novel fipronil biotransformation product.

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

Competition for molecular hydrogen exists among hydrogen-utilizing microorganisms in anoxic environments, and evidence suggests that lower hydrogen concentrations are observed with more energetically favorable electron-accepting processes. The transfer of electrons to organochlorines via reductive dehalogenation reactions plays an important role in hydrogen dynamics in impacted systems. We studied the flux of aqueous hydrogen concentrations in methanogenic sediment microcosms prior to and during reductive dehalogenation of a variety of substituted chlorophenols (CP) and tetrachloroethene (perchloroethylene, PCE). Mean hydrogen concentrations during reductive dehalogenation of 2,4-CP, 2,3,4-CP, and PCP were 3.6 nM, 4.1 nM, and 0.34 nM, respectively. Sediment microcosms that were not dosed with chlorophenols yet were actively methanogenic maintained a significantly higher mean hydrogen concentration of 9.8 nM. During active PCE dehalogenation, sediment microcosms maintained a mean hydrogen concentration of 0.82 nM. These data indicate that during limiting hydrogen production, the threshold ecosystem hydrogen concentration is controlled by microbial populations that couple hydrogen oxidation to thermodynamically favorable electron accepting reactions, including reductive dehalogenation of chloroaromatic and chloroaliphatic compounds. We also present revised estimates for the Gibbs free energy available from the reductive dehalogenation of a variety of substituted chlorophenols based on recently published values of vapor pressure, solubility, and pKa for these compounds.