Analysis and measurement of serotonin.

Serotonin, also known as 5-hydroxytryptamine, is an important signaling molecule in the central and peripheral nervous systems of humans. Acting through several receptor types, it helps regulate the normal functioning of the gastrointestinal tract, cardiovascular system and brain. Serotonin signaling has also been implicated in the etiology of several diseases, including depression, anxiety disorders, hypertension and irritable bowel syndrome. The present review focuses on the chemical analysis of serotonin in biological fluids and biomatrices and traces the development and application of early methods based on UV absorbance or fluorescence to more widely used current methods such as high-performance liquid chromatography coupled to mass spectrometry. A brief summary of the biochemistry, metabolism and physiological roles of serotonin is also presented.

Hepatic microsomal metabolism of BDE-47 and BDE-99 by lesser snow geese and Japanese quail.

In the present study, we investigated the oxidative biotransformation of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) and 2,2',4,4',5-pentabromodiphenyl ether (BDE-99) by liver microsomes from wild lesser snow geese (Chen caerulescens caerulescens) and domesticated Japanese quail (Coturnix japonica). Formation of hydroxy-metabolites was analyzed using an ultra-high performance liquid chromatography-tandem mass spectrometry-based method. Incubation of BDE-47 with avian liver microsomes produced sixteen hydroxy-metabolites, eight of which were identified using authentic standards. The major metabolites formed by liver microsomes from individual lesser snow geese were 4-hydroxy-2,2',3,4'-tetrabromodiphenyl ether (4-OH-BDE-42), 3-hydroxy-2,2',4,4'-tetrabromodiphenyl ether (3-OH-BDE-47), and 4'-hydroxy-2,2',4,5'-tetrabromodiphenyl ether (4'-OH-BDE-49). By comparison, 4-OH-BDE-42 and 4'-OH-BDE-49, but not 3-OH-BDE-47, were major metabolites of Japanese quail liver microsomes. Unidentified metabolites included monohydroxy- and dihydroxy-tetrabromodiphenyl ethers. Incubation of BDE-99 with avian liver microsomes produced seventeen hydroxy-metabolites, twelve of which were identified using authentic standards. The major metabolites formed by lesser snow goose liver microsomes were 2,4,5-tribromophenol, 3-OH-BDE-47, 4'-OH-BDE-49, 4-hydroxy-2,2',3,4',5-pentabromodiphenyl ether (4-OH-BDE-90), and 5'-hydroxy-2,2',4,4',5-pentabromodiphenyl ether (5'-OH-BDE-99). By comparison, the major metabolites produced by liver microsomes from Japanese quail included 6-hydroxy-2,2',4,4'-tetrabromodiphenyl ether (6-OH-BDE-47) and 2-hydroxy-2',3,4,4',5-pentabromodiphenyl ether (2-OH-BDE-123), but not 3-OH-BDE-47. Unidentified metabolites consisted of monohydroxy-pentabromodiphenyl ethers, monohydroxy-tetrabromodiphenyl ethers and dihydroxy-tetrabromodiphenyl ethers. Another difference between the two species was that formation rates of BDE-47 and BDE-99 metabolites were greater with liver microsomes from male than female Japanese quail, but a sex difference was not observed with lesser snow geese.

Editor's Highlight: Congener-Specific Disposition of Chiral Polychlorinated Biphenyls in Lactating Mice and Their Offspring: Implications for PCB Developmental Neurotoxicity.

Chiral polychlorinated biphenyl (PCB) congeners have been implicated by laboratory and epidemiological studies in PCB developmental neurotoxicity. These congeners are metabolized by cytochrome P450 (P450) enzymes to potentially neurotoxic hydroxylated metabolites (OH-PCBs). The present study explores the enantioselective disposition and toxicity of 2 environmentally relevant, neurotoxic PCB congeners and their OH-PCB metabolites in lactating mice and their offspring following dietary exposure of the dam. Female C57BL/6N mice (8-weeks old) were fed daily, beginning 2 weeks prior to conception and continuing throughout gestation and lactation, with 3.1 µmol/kg bw/d of racemic 2,2',3,5',6-pentachlorobiphenyl (PCB 95) or 2,2',3,3',6,6'-hexachlorobiphenyl (PCB 136) in peanut butter; controls received vehicle (peanut oil) in peanut butter. PCB 95 levels were higher than PCB 136 levels in both dams and pups, consistent with the more rapid metabolism of PCB 136 compared with PCB 95. In pups and dams, both congeners were enriched for the enantiomer eluting second on enantioselective gas chromatography columns. OH-PCB profiles in lactating mice and their offspring were complex and varied according to congener, tissue and age. Developmental exposure to PCB 95 versus PCB 136 differentially affected the expression of P450 enzymes as well as neural plasticity (arc and ppp1r9b) and thyroid hormone-responsive genes (nrgn and mbp). The results suggest that the enantioselective metabolism of PCBs to OH-PCBs may influence neurotoxic outcomes following developmental exposures, a hypothesis that warrants further investigation.

Evaluation of hepatic biotransformation of polybrominated diphenyl ethers in the polar bear (Ursus maritimus).

Polar bears are at the top of the Arctic marine food chain and are subject to exposure and bioaccumulation of environmental chemicals of concern such as polybrominated diphenyl ethers (PBDEs), which were widely used as flame retardants. The aim of the present study was to evaluate the in vitro oxidative metabolism of 2,2',4,4'-tetrabrominated diphenyl ether (BDE-47) and 2,2',4,4',5-pentabrominated diphenyl ether (BDE-99) by polar bear liver microsomes. The identification and quantification of the hydroxy-brominated diphenyl ethers formed were assessed using an ultra-high performance liquid chromatography-tandem mass spectrometry-based method. Incubation of BDE-47 with archived individual liver microsomes, prepared from fifteen polar bears from northern Canada, produced a total of eleven hydroxylated metabolites, eight of which were identified using authentic standards. The major metabolites were 4'-hydroxy-2,2',4,5'-tetrabromodiphenyl ether and 5'-hydroxy-2,2',4,4'-tetrabromodiphenyl ether. Incubation of BDE-99 with polar bear liver microsomes produced a total of eleven hydroxylated metabolites, seven of which were identified using authentic standards. The major metabolites were 2,4,5-tribromophenol and 4-hydroxy-2,2',3,4',5-pentabromodiphenyl ether. Among the CYP specific antibodies tested, anti-rat CYP2B was found to be the most active in inhibiting the formation of hydroxylated metabolites of both BDE-47 and BDE-99, indicating that CYP2B was the major CYP enzyme involved in the oxidative biotransformation of these two congeners. Our study shows that polar bears are capable of forming multiple hydroxylated metabolites of BDE-47 and BDE-99 in vitro and demonstrates the role of CYP2B in the biotransformation and possibly in the toxicity of BDE-47 and BDE-99 in polar bears.

Involvement of Cytochrome P450 in Reactive Oxygen Species Formation and Cancer.

This review examines the involvement of cytochrome P450 (CYP) enzymes in the formation of reactive oxygen species in biological systems and discusses the possible involvement of reactive oxygen species and CYP enzymes in cancer. Reactive oxygen species are formed in biological systems as byproducts of the reduction of molecular oxygen and include the superoxide radical anion (∙O2-), hydrogen peroxide (H2O2), hydroxyl radical (∙OH), hydroperoxyl radical (HOO∙), singlet oxygen ((1)O2), and peroxyl radical (ROO∙). Two endogenous sources of reactive oxygen species are the mammalian CYP-dependent microsomal electron transport system and the mitochondrial electron transport chain. CYP enzymes catalyze the oxygenation of an organic substrate and the simultaneous reduction of molecular oxygen. If the transfer of oxygen to a substrate is not tightly controlled, uncoupling occurs and leads to the formation of reactive oxygen species. Reactive oxygen species are capable of causing oxidative damage to cellular membranes and macromolecules that can lead to the development of human diseases such as cancer. In normal cells, intracellular levels of reactive oxygen species are maintained in balance with intracellular biochemical antioxidants to prevent cellular damage. Oxidative stress occurs when this critical balance is disrupted. Topics covered in this review include the role of reactive oxygen species in intracellular cell signaling and the relationship between CYP enzymes and cancer. Outlines of CYP expression in neoplastic tissues, CYP enzyme polymorphism and cancer risk, CYP enzymes in cancer therapy and the metabolic activation of chemical procarcinogens by CYP enzymes are also provided.

Monooxygenase, peroxidase and peroxygenase properties and reaction mechanisms of cytochrome P450 enzymes.

This review examines the monooxygenase, peroxidase and peroxygenase properties and reaction mechanisms of cytochrome P450 (CYP) enzymes in bacterial, archaeal and mammalian systems. CYP enzymes catalyze monooxygenation reactions by inserting one oxygen atom from O2 into an enormous number and variety of substrates. The catalytic versatility of CYP stems from its ability to functionalize unactivated carbon-hydrogen (C-H) bonds of substrates through monooxygenation. The oxidative prowess of CYP in catalyzing monooxygenation reactions is attributed primarily to a porphyrin π radical ferryl intermediate known as Compound I (CpdI) (Por•+FeIV=O), or its ferryl radical resonance form (FeIV-O•). CYP-mediated hydroxylations occur via a consensus H atom abstraction/oxygen rebound mechanism involving an initial abstraction by CpdI of a H atom from the substrate, generating a highly-reactive protonated Compound II (CpdII) intermediate (FeIV-OH) and a carbon-centered alkyl radical that rebounds onto the ferryl hydroxyl moiety to yield the hydroxylated substrate. CYP enzymes utilize hydroperoxides, peracids, perborate, percarbonate, periodate, chlorite, iodosobenzene and N-oxides as surrogate oxygen atom donors to oxygenate substrates via the shunt pathway in the absence of NAD(P)H/O2 and reduction-oxidation (redox) auxiliary proteins. It has been difficult to isolate the historically elusive CpdI intermediate in the native NAD(P)H/O2-supported monooxygenase pathway and to determine its precise electronic structure and kinetic and physicochemical properties because of its high reactivity, unstable nature (t½~2 ms) and short life cycle, prompting suggestions for participation in monooxygenation reactions of alternative CYP iron-oxygen intermediates such as the ferric-peroxo anion species (FeIII-OO-), ferric-hydroperoxo species (FeIII-OOH) and FeIII-(H2O2) complex.

Regioselective Versatility of Monooxygenase Reactions Catalyzed by CYP2B6 and CYP3A4: Examples with Single Substrates.

Hepatic microsomal cytochrome P450 (CYP) enzymes have broad and overlapping substrate specificity and catalyze a variety of monooxygenase reactions, including aliphatic and aromatic hydroxylations, N-hydroxylations, oxygenations of heteroatoms (N, S, P and I), alkene and arene epoxidations, dehalogenations, dehydrogenations and N-, O- and S-dealkylations. Individual CYP enzymes typically catalyze the oxidative metabolism of a common substrate in a regioselective and stereoselective manner. In addition, different CYP enzymes often utilize different monooxygenase reactions when oxidizing a common substrate. This review examines various oxidative reactions catalyzed by a CYP enzyme acting on a single substrate. In the first example, 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), a halogenated aromatic environmental contaminant, was oxidatively biotransformed by human CYP2B6. Nine different metabolites of BDE-47 were produced by CYP2B6 via monooxygenase reactions that included aromatic hydroxylation, with and without an NIH-shift, dealkylation and debromination. In the second example, lithocholic acid (3α-hydroxy-5ß-cholan-24-oic acid), an endogenous bile acid, served as a substrate for human CYP3A4 and yielded five different metabolites via aliphatic hydroxylation and dehydrogenation reactions.

Levels of PBDEs in plasma of juvenile starlings (Sturnus vulgaris) from British Columbia, Canada and assessment of PBDE metabolism by avian liver microsomes.

In this study, the levels of polybrominated diphenyl ethers (PBDEs), HO-PBDEs, and bromophenols were monitored in starling chick plasma samples collected in Delta (British Columbia, Canada) close to the Vancouver municipal landfill and in Glen Valley, a rural area in British Columbia. The in vitro formation of hydroxylated metabolites of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) and 2,2',4,4',5-pentabromodiphenyl ether (BDE-99) was also investigated using starling chick liver microsomes. Total PBDE plasma levels were approximately 60 times higher in starling chicks from Delta than from Glen Valley, suggesting that the Delta site is a major source of PBDEs for the local population of starlings and that PBDEs previously measured in starling eggs are bioavailable to chicks. In both locations, BDE-47 and BDE-99 were the two major congeners present at similar concentrations, suggesting contamination with the Penta-BDE mixture. Among the several possible hydroxylated metabolites of PBDEs monitored in starling plasma, only 2,4,5-tribromophenol was detected and its levels did not exceed 18±7 pg/mL. Also, several hydroxylated metabolites of BDE-47 and BDE-99 were formed by starling chick liver microsomes, but in low amounts. Therefore, our data consistently suggest that oxidative metabolism of PBDEs in starling chicks proceeds at low rate in vivo and in vitro. In conclusion, the landfill located in Delta is a relevant source of bioavailable PBDEs for the local starling population. Because of the limited ability of starling chicks to metabolize PBDEs, these compounds are likely to bioaccumulate in starlings over time. The possible toxicological implications of PBDEs bioaccumulation in starlings are currently unknown and require further research.

Localization of cytochrome P450 and related enzymes in adult rat testis and downregulation by estradiol and bisphenol A.

There is a growing body of evidence that exposure to endocrine disrupting chemicals and to estrogenic compounds in particular can affect the testis and male fertility. In the present study, the constitutive expression of steroidogenic and non-steroidogenic cytochrome P450 (CYP) and related enzymes in adult rat testis, and their regulation by estradiol and bisphenol A, were investigated. CYP1B1, CYP2A1, NADPH-cytochrome P450 oxidoreductase (POR) and microsomal epoxide hydrolase (mEH) proteins, together with CYP17A1 and 3ß-hydroxysteroid dehydrogenase (HSD3B), were detected by immunoblot analysis in testicular microsomes prepared from untreated adult Sprague Dawley rats. In contrast, CYP1A, CYP2B, CYP2E, CYP2D, CYP2C, CYP3A, and CYP4A enzymes were not detected. Immunofluorescence staining of cryosections of perfusion-fixed testes showed that CYP1B1, CYP2A1, CYP17A1, and HSD3B were expressed exclusively or mainly in interstitial cells, whereas mEH and POR protein staining was detected both in interstitial cells and in seminiferous tubules. Testicular CYP1B1 and CYP2A1 protein levels were decreased following treatment of adult rats with estradiol benzoate at 0.004, 0.04, 0.4, or 4 µmol/kg/day or bisphenol A at 400 or 800 µmol/kg/day, for 14 days, whereas expression of HSD3B was unaffected. Testicular CYP17A1, POR, and mEH protein expression was also downregulated at the three highest dosages of estradiol benzoate and at both dosages of bisphenol A. The present study is the first to establish the cellular localization of CYP1B1, mEH, and POR in rat testis and to demonstrate the suppressive effect of bisphenol A on testicular CYP1B1, CYP2A1, mEH, and POR protein levels.

Hepatic bile acid metabolism and expression of cytochrome P450 and related enzymes are altered in Bsep (-/-) mice.

The bile salt export pump (BSEP/Bsep; gene symbol ABCB11/Abcb11) translocates bile salts across the hepatocyte canalicular membrane into bile in humans and mice. In humans, mutations in the ABCB11 gene cause a severe childhood liver disease known as progressive familial intrahepatic cholestasis type 2. Targeted inactivation of mouse Bsep produces milder persistent cholestasis due to detoxification of bile acids through hydroxylation and alternative transport pathways. The purpose of the present study was to determine whether functional expression of hepatic cytochrome P450 (CYP) and microsomal epoxide hydrolase (mEH) is altered by Bsep inactivation in mice and whether bile acids regulate CYP and mEH expression in Bsep (-/-) mice. CYP expression was determined by measuring protein levels of Cyp2b, Cyp2c and Cyp3a enzymes and CYP-mediated activities including lithocholic acid hydroxylation, testosterone hydroxylation and alkoxyresorufin O-dealkylation in hepatic microsomes prepared from female and male Bsep (-/-) mice fed a normal or cholic acid (CA)-enriched diet. The results indicated that hepatic lithocholic acid hydroxylation was catalyzed by Cyp3a/Cyp3a11 enzymes in Bsep (-/-) mice and that 3-ketocholanoic acid and murideoxycholic acid were major metabolites. CA feeding of Bsep (-/-) mice increased hepatic Cyp3a11 protein levels and Cyp3a11-mediated testosterone 2ß-, 6ß-, and 15ß-hydroxylation activities, increased Cyp2b10 protein levels and Cyp2b10-mediated benzyloxyresorufin O-debenzylation activity, and elevated Cyp2c29 and mEH protein levels. We propose that bile acids upregulate expression of hepatic Cyp3a11, Cyp2b10, Cyp2c29 and mEH in Bsep (-/-) mice and that Cyp3a11 and multidrug resistance-1 P-glycoproteins (Mdr1a/1b) are vital components of two distinct pathways utilized by mouse hepatocytes to expel bile acids.

Biotransformation of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) by human liver microsomes: identification of cytochrome P450 2B6 as the major enzyme involved.

Polybrominated diphenyl ethers (PBDEs) were widely used flame retardants that have become persistent environmental pollutants. In the present study, we investigated the in vitro oxidative metabolism of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), a major PBDE detected in human tissue and environmental samples. Biotransformation of BDE-47 by pooled and individual human liver microsomes and by human recombinant cytochrome P450 (P450) enzymes was assessed using a liquid chromatography/tandem mass spectrometry-based method. Of the nine hydroxylated metabolites of BDE-47 produced by human liver microsomes, seven metabolites were identified using authentic standards. A monohydroxy-tetrabrominated and a dihydroxy-tetrabrominated metabolite remain unidentified. Kinetic analysis of the rates of metabolite formation revealed that the major metabolites were 5-hydroxy-2,2',4,4'-tetrabromodiphenyl ether (5-OH-BDE-47), 6-hydroxy-2,2',4,4'-tetrabromodiphenyl ether (6-OH-BDE-47), and possibly the unidentified monohydroxy-tetrabrominated metabolite. Among the human recombinant P450 enzymes tested, P450 2B6 was the most active enzyme in the formation of the hydroxylated metabolites of BDE-47. Moreover, the formation of all metabolites of BDE-47 by pooled human liver microsomes was inhibited by a P450 2B6-specific antibody and was highly correlated with P450 2B6-mediated activity in single donor liver microsomes indicating that P450 2B6 was the major P450 responsible for the biotransformation of BDE-47. Additional experiments involving the incubation of liver microsomes with individual monohydroxy-tetrabrominated metabolites in place of BDE-47 demonstrated that 2,4-dibromophenol was a product of BDE-47 and several primary metabolites, but the dihydroxy-tetrabrominated metabolite was not formed by sequential hydroxylation of any of the monohydroxy-tetrabrominated metabolites tested. The present study provides a comprehensive characterization of the oxidative metabolism of BDE-47 by human liver microsomes and P450 2B6.

Oxidative metabolism of BDE-99 by human liver microsomes: predominant role of CYP2B6.

Hydroxylated polybrominated diphenyl ethers (PBDEs) have been found in human serum, suggesting that they are formed by in vivo oxidative metabolism of PBDEs. However, the biotransformation of 2,2',4,4',5-pentabromodiphenyl ether (BDE-99), a major PBDE detected in human tissue and environmental samples, is poorly understood. In the present study, the oxidative metabolism of BDE-99 was assessed using pooled and single-donor human liver microsomes, a panel of human recombinant cytochrome P450 (CYP) enzymes, and CYP-specific antibodies. Hydroxylated metabolites were quantified using a liquid chromatography/tandem mass spectrometry-based method. In total, 10 hydroxylated metabolites of BDE-99 were produced by human liver microsomes. Six metabolites were identified as 2,4,5-tribromophenol (2,4,5-TBP), 4-OH-BDE-90, 5'-OH-BDE-99, 6'-OH-BDE-99, 4'-OH-BDE-101, and 2-OH-BDE-123 using authentic standards. Three monohydroxy- and one dihydroxy-pentabrominated metabolites were unidentified. Rates of formation of the three major metabolites (2,4,5-TBP, 5'-OH-BDE-99, and 4'-OH-BDE-101) by human liver microsomes ranged from 24.4 to 44.8 pmol/min/mg protein. Additional experiments demonstrated that the dihydroxylated metabolite was a primary metabolite of BDE-99 and was not produced by hydroxylation of a monohydroxy metabolite. Among the panel of recombinant CYP enzymes tested, formation of all 10 hydroxylated metabolites was catalyzed solely by CYP2B6. A combined approach using antibodies to CYP2B6 and single-donor liver microsomes expressing a wide range of CYP2B6 levels confirmed that CYP2B6 was responsible for the biotransformation of BDE-99. Collectively, the results show that the oxidative metabolism of BDE-99 by human liver microsomes is catalyzed solely by CYP2B6 and is an important determinant of the toxicity and bioaccumulation of BDE-99 in humans.

Ontogenesis of phase I hepatic drug metabolic enzymes in sheep.

Cytochrome P450 (CYP) enzymes are important for the metabolism of many drugs. While there is information on their identity and ontogeny in humans and rodents, similar data in sheep are lacking. In the present study, cDNA sequences of several CYP enzymes (CYP2A6, CYP2C19, CYP2D6) were cloned by rapid amplification of cDNA ends. In adult, newborn and fetal sheep the mRNA and protein levels of these CYPs and the regulatory factor, hepatic nuclear factor 4α (HNF4α) were determined in liver samples using real-time PCR and western blotting. The effect of antenatal glucocorticoid on these enzymes was also studied by i.v. infusion of cortisol (0.45 mgh(-1); 80 h) to another group of fetuses. The mRNA and protein levels of the CYPs and HNF4α were low or absent in the fetus, followed by increasing levels in the newborn and adult. Fetal cortisol administration significantly increased the mRNA and protein levels of CYP2D6. Moreover, the correlation observed between the CYP and HNF4α mRNA levels suggests a possible regulatory role for this transcription factor. The findings suggest that fetal and newborn lambs have a low ability to metabolise drugs that are substrates of these enzymes, and that this ability increases with advancing postnatal age, similar to the situation in humans.

The monooxygenase, peroxidase, and peroxygenase properties of cytochrome P450.

This review examines the monooxygenase, peroxidase, and peroxygenase properties of cytochrome P450 (P450)1 enzymes and their mechanisms of action in archaeal, bacterial, and mammalian systems. In the P450 catalytic cycle, a transient iron oxo monooxygenating species is generated that reacts with substrate to produce a monooxygenated product. We describe results of early investigations that endeavored to trap and detect this elusive monooxygenating species, as well as results of experiments that attempted to generate and characterize this active oxidant spectroscopically after reacting ferric P450 enzymes with peroxy compounds (e.g. peroxides, peracids) or single oxygen atom donors (e.g. periodate, iodosobenzene). Surrogate oxidants were able to promote P450-catalyzed monooxygenations in a manner similar to that of O2/NAD(P)H, suggesting involvement of a common transitory monooxygenating species in the two pathways. This common P450 oxidant was characterized as a porphyrin radical iron(IV) oxo complex and assigned a Compound I structure (Por+FeIV=O) exhibiting a formal FeV oxidation state. Other reactive oxidants, such as the ferric oxenoid complex (PorFeIII=O), ferryloxy radical species (PorFeIV-O·), and perferryloxo entity (PorFeV=O), were also proposed to function as P450 monooxygenating species. We also discuss the possible involvement of the ferriperoxo (PorFeIII-OO-) and ferrihydroperoxo (PorFeIII-OOH) species as alternative oxidants in P450-mediated monooxygenation reactions.

2,2',3,3',6,6'-Hexachlorobiphenyl (PCB 136) is enantioselectively oxidized to hydroxylated metabolites by rat liver microsomes.

Developmental exposure to multiple ortho-substituted polychlorinated biphenyls (PCBs) causes adverse neurodevelopmental outcomes in laboratory animals and humans by mechanisms involving the sensitization of Ryanodine receptors (RyRs). In the case of PCB 136, the sensitization of RyR is enantiospecific, with only (-)-PCB 136 being active. However, the role of enantioselective metabolism in the developmental neurotoxicity of PCB 136 is poorly understood. The present study employed hepatic microsomes from phenobarbital (PB)-, dexamethasone (DEX)- and corn oil (VEH)-treated male Sprague-Dawley rats to investigate the hypothesis that PCB 136 atropisomers are enantioselectively metabolized by P450 enzymes to potentially neurotoxic, hydroxylated PCB 136 metabolites. The results demonstrated the time- and isoform-dependent formation of three metabolites, with 5-OH-PCB 136 (2,2',3,3',6,6'-hexachlorobiphenyl-5-ol) being the major metabolite. The formation of 5-OH-PCB 136 increased with the activity of P450 2B enzymes in the microsomal preparation, which is consistent with PCB 136 metabolism by rat P450 2B1. The minor metabolite 4-OH-PCB 136 (2,2',3,3',6,6'-hexachlorobiphenyl-4-ol) was produced by a currently unidentified P450 enzyme. An enantiomeric enrichment of (-)-PCB 136 was observed in microsomal incubations due to the preferential metabolism of (+)-PCB 136 to the corresponding 5-OH-PCB 136 atropisomer. 4-OH-PCB 136 displayed an enrichment of the atropisomer formed from (-)-PCB 136; however, the enrichment of this metabolite atropisomer did not affect the enantiomeric enrichment of the parent PCB because 4-OH-PCB 136 is only a minor metabolite. Although the formation of 5- and 4-OH-PCB 136 atropisomers increased with time, the enantioselective formation of the OH-PCB metabolites resulted in constant enantiomeric enrichment, especially at later incubation times. These observations not only demonstrate that the chiral signatures of PCBs and their metabolites in wildlife and humans are due to metabolism by P450 enzymes but also suggest that the enantioselective formation of neurotoxic PCB 136 metabolites, such as 4-OH-PCB 136, may play a role in the developmental neurotoxicity of PCBs.

Estradiol-mediated suppression of CYP1B1 expression in mouse MA-10 Leydig cells is independent of protein kinase A and estrogen receptor.

Estrogens have multifaceted roles in mammalian testis. In the present study, we focused on estradiol as a potential regulator of testicular cytochrome P450 1B1 (CYP1B1) expression and investigated the possible mechanisms involved in the estradiol-mediated suppression. CYP1B1 protein levels were measured in the testes of rats that were treated with 17ß-estradiol benzoate (1.5 mg/kg) at different stages of development. In addition, CYP1B1 mRNA levels were measured in mouse MA-10 Leydig tumor cells treated with (a) various concentrations of 17ß-estradiol benzoate, (b) 17ß-estradiol benzoate in the presence of exogenous luteinizing hormone (LH), or (c) 17ß-estradiol benzoate in the presence of ICI 182,780, a competitive steroidal antagonist of estrogen receptors (ERs). Treatment of neonatal, pubertal, or adult rats with 17ß-estradiol benzoate was associated with a reduction of approximately 90% in testicular CYP1B1 protein content compared to age-matched controls. Treatment of MA-10 cells with 17ß-estradiol benzoate (10-500 nM) produced a concentration- and time-dependent decrease in CYP1B1 mRNA levels, but had no effect on LH receptor mRNA levels or on protein kinase A (PKA) activity. However, 17ß-estradiol benzoate (10-500 nM), regardless of the concentration tested, failed to attenuate the LH-elicited increase in CYP1B1 mRNA or PKA activity in MA-10 cells that were co-treated with LH and estradiol. Similarly, ICI 182,780 (10-1000 µM) did not reverse the suppressive effect of estradiol on CYP1B1 mRNA expression in MA-10 cells co-treated with estradiol and ICI 182,780. The results indicate that downregulation of testicular CYP1B1 by estradiol was independent of PKA activity and was not mediated by ERs in MA-10 cells.

Comparative oxidative metabolism of BDE-47 and BDE-99 by rat hepatic microsomes.

Polybrominated diphenyl ethers (PBDEs) are flame-retardant chemicals that have become ubiquitous environmental pollutants. 2,2',4,4'-Tetrabromodiphenyl ether (BDE-47) and 2,2',4,4',5-pentabromodiphenyl ether (BDE-99) are among the most prevalent PBDEs detected in humans, wildlife, and abiotic environmental matrices. The purpose of this study was to investigate the oxidative metabolism of BDE-47 and BDE-99 in rat hepatic microsomes by comparing metabolite formation rates, kinetic parameters associated with metabolite formation, and the effects of prototypical cytochrome P450 (CYP) inducers. The CYP enzymes involved were also identified. Incubation of BDE-47 with hepatic microsomes from phenobarbital-treated rats generated a total of five hydroxylated (OH-BDE) metabolites, among which 4'-hydroxy-2,2',4,5'-tetrabromodiphenyl ether (4'-OH-BDE-49) and 3-hydroxy-2,2',4,4'-tetrabromodiphenyl ether (3-OH-BDE-47) were the major metabolites, as identified using authentic standards and quantified by liquid chromatography/mass spectrometry. Incubations of BDE-99 with hepatic microsomes from dexamethasone-treated rats produced a total of seven hydroxylated metabolites, among which 4-hydroxy-2,2',3,4',5-pentabromodiphenyl ether (4-OH-BDE-90) and 6'-hydroxy-2,2',4,4',5-pentabromodiphenyl ether (6'-OH-BDE-99) were the major metabolites. Although the overall rate of oxidative metabolism of BDE-99 by hepatic microsomes was greater than that of BDE-47, para-hydroxylation involving a National Institutes of Health shift mechanism represented a major metabolic pathway for both PBDE congeners. Among the rat recombinant CYP enzymes tested, CYP2A2 and CYP3A1 were the most active in BDE-47 and BDE-99 metabolism, respectively. However, CYP1A1 exhibited the highest activity for 4'-OH-BDE-49 and 6'-OH-BDE-99 formation, and CYP3A1 exhibited the highest activity for 3-OH-BDE-47 and 4-OH-BDE-90 formation. Collectively, the results demonstrate that oxidative metabolism of BDE-47 and BDE-99 is mediated by distinct but overlapping sets of CYP enzymes and represents a key process that determines the bioaccumulation of BDE-47 and BDE-99 in mammals.

Ontogenesis of UDP-glucuronosyltransferase enzymes in sheep.

UDP glucuronosyltransferases (UGTs) are important for the metabolism of many drugs. To understand the ontogeny of these enzymes in sheep (Ovis aries), knowledge of their expression levels at different developmental stages is important. cDNA sequences of six UGTs (UGT1A1, UGT1A3, UGT1A4, UGT1A6 and UGT1A9) were cloned in sheep liver by rapid amplification of cDNA ends. The mRNA and protein levels of the enzymes in sheep liver (adult n=4; newborn n=3; fetus n=3) were determined using PCR and Western blots. mRNA levels in adult were very high followed by newborn and then fetus. However, no protein bands were detected on the Western blots of sheep hepatic microsomal proteins using several different antibodies against human UGTs. The effect of antenatal glucocorticoid administration was also studied by infusion of cortisol (0.45mg/h; 80h) to another group of fetuses (n=5). This did not result in any significant changes in fetal mRNA levels. However, the correlation observed between the UGT enzymes and hepatocyte nuclear factor 4α (HNF4α) suggests a possible regulatory role for this transcription factor in sheep. We postulate that fetal and newborn lambs may have a reduced ability to metabolize drugs that are substrates of these enzymes.

Regulation of cytochrome P450 1B1 expression by luteinizing hormone in mouse MA-10 and rat R2C Leydig cells: role of protein kinase A.

In the present study, we investigated the signaling pathway involved in luteinizing hormone (LH)-mediated regulation of testicular CYP1B1 in mouse MA-10 and rat R2C Leydig cells. CYP1B1 mRNA and protein levels were measured in MA-10 and R2C cells treated with LH and protein kinase activators or inhibitors. Treatment with LH or 8-bromo-cAMP, a protein kinase A (PRKA) activator, increased CYP1B1 expression and PRKA activity in a concentration-dependent manner in both cell lines, albeit to different extents. Treatment with 8-(4-chlorophenylthio)adenosine-3',5'-cyclic monophosphorothioate, Rp-isomer, a PRKA inhibitor, decreased basal CYP1B1 expression and attenuated LH-elicited increases in CYP1B1 mRNA and protein levels and PRKA activity. In contrast, treatment with a protein kinase G activator or an inhibitor of protein kinase C had no effect on basal or LH-induced CYP1B1 expression in MA-10 or R2C cells. Collectively, the results identify PRKA as the major signaling pathway involved in the LH-mediated regulation of testicular CYP1B1 expression in Leydig tumor cells.