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.

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.

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.

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.

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.

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.

3-ketocholanoic acid is the major in vitro human hepatic microsomal metabolite of lithocholic acid.

3alpha-Hydroxy-5 beta-cholan-24-oic (lithocholic) acid is a relatively minor component of hepatic bile acids in humans but is highly cytotoxic. Hepatic microsomal oxidation offers a potential mechanism for effective detoxification and elimination of bile acids. The aim of the present study was to investigate the biotransformation of lithocholic acid by human hepatic microsomes and to assess the contribution of cytochrome P450 (P450) enzymes in human hepatic microsomes using human recombinant P450 enzymes and chemical inhibitors. Metabolites were identified, and metabolite formation was quantified using a liquid chromatography/mass spectrometry-based assay. Incubation of lithocholic acid with human liver microsomes resulted in the formation of five metabolites, which are listed in order of their rates of formation: 3-oxo-5 beta-cholan-24-oic (3-ketocholanoic) acid, 3 alpha,6 alpha-dihydroxy-5 beta-cholan-24-oic (hyodeoxycholic) acid, 3 alpha,7 beta-dihydroxy-5 beta-cholan-24-oic (ursodeoxycholic) acid, 3 alpha,6 beta-dihydroxy-5 beta-cholan-24-oic (murideoxycholic) acid, and 3 alpha-hydroxy-6-oxo-5 beta-cholan-24-oic (6-ketolithocholic) acid. 3-Ketocholanoic acid was the major metabolite, exhibiting apparent K(m) and V(max) values of 22 muM and 336 pmol/min/mg protein, respectively. Incubation of lithocholic acid with a of human recombinant P450 enzymes revealed that all five metabolites were formed by recombinant CYP3A4. Chemical inhibition studies with human liver microsomes and recombinant P450 enzymes confirmed that CYP3A4 was the predominant enzyme involved in hepatic microsomal biotransformation of lithocholic acid. In summary, the results indicate that oxidation of the third carbon of the cholestane ring is the preferred position of oxidation by P450 enzymes for lithocholic acid biotransformation in humans and suggest that formation of lithocholic acid metabolites leads to enhanced hepatic detoxification and elimination.

Developmental expression and endocrine regulation of CYP1B1 in rat testis.

Mammalian testis expresses xenobiotic-metabolizing enzymes, including cytochrome P450 1B1 (CYP1B1), which catalyzes the bioactivation of procarcinogens and other chemicals. The factors that control testicular expression of CYP1B1 are largely not known. In the present study, we investigated the influence of age and pituitary, gonadal, and thyroid hormones on CYP1B1 expression in rat testis. Immunoblot analysis showed that testicular CYP1B1 protein was expressed at a level of 5.9+/-2.0 (mean+/-S.E.M.) pmol/mg microsomal protein in prepubertal 22-day-old rats, whereas it was 6.6-fold greater in pubertal rats (34 days old) and 9.6-fold greater in adult rats (84-91 days old). Hypophysectomy decreased testicular CYP1B1 protein levels by 69% in adult rats when compared with intact rats of the same age. Intermittent subcutaneous administration of growth hormone to hypophysectomized adult rats further decreased it by 63%. Luteinizing hormone (LH) and follicle-stimulating hormone increased CYP1B1 expression in hypophysectomized rats, but they did not restore protein levels to those in intact adult male rats. Prolactin treatment alone had no effect; however, it potentiated the increase in CYP1B1 mRNA and protein expression by LH. 3,5,3'-Triiodothyronine, but not thyroxine, resulted in a small increase in testicular CYP1B1 protein levels. Likewise, treatment of hypophysectomized rats with testosterone propionate elicited a small increase in CYP1B1 protein expression. In contrast, treatment of intact adult male rats with 17beta-estradiol benzoate decreased it by 91%. Overall, our findings indicate that rat testicular CYP1B1 protein expression is subject to developmental and endocrine control, with multiple hormones playing a role.

Identification of human hepatic cytochrome p450 enzymes involved in the biotransformation of cholic and chenodeoxycholic acid.

3alpha,7alpha,12alpha-trihydroxy-5beta-cholan-24-oic (cholic) and 3alpha,7alpha-dihydroxy-5beta-cholan-24-oic (chenodeoxycholic) acids are the predominant hepatic and biliary bile acids of most mammalian species including humans. Cholic and chenodeoxycholic acids are synthesized from cholesterol and accumulate in the liver during cholestasis. Biotransformation by hepatic cytochrome P450 (P450) enzymes represents a potentially effective pathway for elimination of these lipid-soluble bile acids. We developed a liquid chromatography/mass spectrometry-based assay to identify and quantify the human hepatic microsomal metabolites of cholic acid and chenodeoxycholic acid, and using a panel of human recombinant P450 enzymes, we determined the P450 enzymes involved. Incubation of cholic acid with human hepatic microsomes and NADPH produced a single metabolite, 7alpha,12alpha-dihydroxy-3-oxo-5beta-cholan-24-oic (3-dehydrocholic) acid. Of the recombinant P450 enzymes tested, only CYP3A4 catalyzed 3-dehydrocholic acid formation. Similar experiments with chenodeoxycholic acid revealed the formation of 7alpha-hydroxy-3-oxo-5beta-cholan-24-oic acid and 3alpha,6alpha,7alpha-trihydroxy-5beta-cholan-24-oic (gamma-muricholic) acid as major metabolites and 3alpha-hydroxy-7-oxo-5beta-cholan-24-oic (7-ketolithocholic) acid and cholic acid as minor metabolites. Among the human recombinant P450 enzymes examined, CYP3A4 exhibited the highest rates of formation for 7alpha-hydroxy-3-oxo-5beta-cholan-24-oic acid and gamma-muricholic acid from chenodeoxycholic acid. Formation of 7-ketolithocholic acid and cholic acid from chenodeoxycholic acid has not been reported previously and could not be attributed to any of the recombinant P450 enzymes tested. In conclusion, the predominant pathway for the biotransformation of both cholic and chenodeoxycholic acids in human hepatic microsomes was oxidation at the third carbon of the cholestane ring. This study highlights a major role for CYP3A4 and suggests a possible route for the elimination of these two bile acids.

Biotransformation of lithocholic acid by rat hepatic microsomes: metabolite analysis by liquid chromatography/mass spectrometry.

Lithocholic acid is a lipid-soluble hepatotoxic bile acid that accumulates in the liver during cholestasis. A potential detoxification pathway for lithocholic acid involves hydroxylation by hepatic cytochrome P450 (P450) enzymes. The purpose of the present study was to identify the hepatic microsomal metabolites of lithocholic acid by liquid chromatography/mass spectrometry and to determine the P450 enzymes involved. Incubation of lithocholic acid with rat hepatic microsomes and NADPH produced murideoxycholic acid (MDCA), isolithocholic acid (ILCA), and 3-keto-5beta-cholanic acid (3KCA) as major metabolites and 6-ketolithocholic acid and ursodeoxycholic acid as minor metabolites. Experiments with hepatic microsomes prepared from rats pretreated with P450 inducers and with inhibitory antibodies indicated that CYP2C and CYP3A enzymes contribute to microsomal MDCA formation. Results obtained with a panel of recombinant P450 enzymes and CYP2D6 antiserum showed that CYP2D1 can also catalyze MDCA formation. Similar experimental evidence revealed that formation of 3KCA was mediated primarily by CYP3A enzymes. ILCA formation appeared to be catalyzed by a distinct pathway mediated largely by microsomal non-P450 enzymes. Based on the results obtained using lithocholic acid and 3KCA as substrates, a mechanism for the formation of ILCA involving a geminal diol intermediate is outlined. In conclusion, lithocholic acid was extensively metabolized by multiple P450 enzymes with the predominant biotransformation pathway being hydroxylation at the 6beta-position. This study provides an insight into possible routes of detoxification of lithocholic acid.

2,2',3,3',6,6'-Hexachlorobiphenyl (PCB 136) atropisomers interact enantioselectively with hepatic microsomal cytochrome P450 enzymes.

2,2',3,3',6,6'-Hexachlorobiphenyl (PCB 136) is a chiral and highly neurotoxic PCB congener of environmental relevance. (+)-PCB 136 was previously shown to be enriched in tissues from mice treated with racemic PCB 136. We investigated the spectral interactions of (+)-, (-)-, and (+/-)-PCB 136 with mouse and rat hepatic microsomal cytochrome P450 (P450) enzymes to test the hypothesis that enantioselective binding to specific P450 enzymes causes the enrichment of (+)-PCB 136 in vivo. Hepatic microsomes prepared from C57BL/6 mice or Long Evans rats treated with beta-naphthoflavone or 3-methylcholanthrene, phenobarbital, and dexamethasone (prototypical inducers of CYP1A, CYP2B, and CYP3A, respectively) were used to determine first, whether the (+)-PCB 136 atropisomer binds to hepatic microsomal P450 enzymes to a greater extent than does the (-)-PCB 136 atropisomer and second, whether P450 enzymes of one subfamily bind the two PCB 136 atropisomers more efficiently than do P450 enzymes of other subfamilies. Increasing concentrations of (+)-, (-)-, or (+/-)-PCB 136 were added to hepatic microsomes, and the difference spectrum and maximal absorbance change, a measure of PCB binding to P450 enzymes, were measured. A significantly larger absorbance change was observed with (+)-PCB 136 than with (-)-PCB 136 with all four hepatic microsomal preparations in mice and rats, indicating that (+)-PCB 136 interacted with microsomal P450 enzymes to a greater degree than did (-)-PCB 136. In addition, binding of the PCB 136 atropisomers was greatest in microsomes from PB-treated mice and rats and was inhibited by CYP2B antibodies, indicating the involvement of CYP2B enzymes. Together, these results suggest preferential binding of (+)-PCB 136 to P450 enzymes (such as CYP2B and CYP3A) in hepatic microsomes, an observation that may explain the enantioselective enrichment of the (+)-PCB 136 atropisomer in tissues of mice.

Differential inhibition of hepatic microsomal alkoxyresorufin O-dealkylation activities by tetrachlorobiphenyls.

Polychlorinated biphenyls (PCBs) elicit a spectrum of biochemical and toxic effects in exposed animals. In the present study, we assessed the effect of PCB structure, using four symmetrically-substituted PCBs, on cytochrome P450 (CYP)-mediated methoxy-, ethoxy- and benzyloxyresorufin O-dealkylase (MROD, EROD and BROD, respectively) activities. We found that 2,2',4,4'-tetrachlorobiphenyl (PCB 47), 2,2',5,5'-tetrachlorobiphenyl (PCB 52), 2,2',6,6'-tetrachlorobiphenyl (PCB 54) and 3,3',4,4'-tetrachlorobiphenyl (PCB 77) inhibited alkoxyresorufin O-dealkylase activities in hepatic microsomes from 3-methylcholanthrene (MC) or phenobarbital (PB)-treated rats. Measurement of the in vitro inhibitory potencies of the tetrachlorobiphenyls revealed that MROD, EROD and BROD activities were differentially inhibited and the degree of inhibition was determined by the chlorination pattern of the PCB. PCB 77 was more potent than PCB 47 or PCB 52 at inhibiting MROD and EROD activities in hepatic microsomes from MC-treated rats, while no inhibition of either activity was observed with PCB 54. In contrast, BROD activity measured in hepatic microsomes from PB-treated rats was inhibited by PCB 47, PCB 52 and PCB 54 but not by PCB 77. The mode of inhibition for each activity was also evaluated statistically. Inhibition of the alkoxyresorufin O-dealkylase activities could not be discerned in hepatic microsomes from corn oil-treated rats because the activities were inherently too low. No evidence for mechanism-based inhibition of MROD, EROD or BROD activities or an effect via CYP reductase was found. The results demonstrate that relatively coplanar PCBs such as PCB 77 preferentially inhibit EROD and MROD activities, whereas noncoplanar PCBs such as PCB 54 preferentially inhibit BROD activity.

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.

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.

Assessment of cytochrome P450 1A in harbour seals (Phoca vitulina) using a minimally-invasive biopsy approach.

Biomarkers of organochlorine exposure, such as the induction of cytochrome P450 1A (CYP1A), can be used to assess the impact of environmental contaminants on the health of free-ranging marine mammal populations. The objective of the present study was to measure CYP1A in skin and liver biopsies obtained from live harbour seals (Phoca vitulina). Twelve harbour seal pups, aged three to five weeks, were captured from the Fraser River estuary, British Columbia, Canada, and temporarily held in captivity. Skin ( approximately 60 mg) and liver ( approximately 40 mg) biopsies, obtained while seals were under general anaesthesia, yielded sufficient tissue for the measurement of CYP1A by immunoblot analysis and ethoxyresorufin O-deethylase activity. A short-term exposure experiment, in which harbour seals (n=3) were treated orally with beta-naphthoflavone (BNF), resulted in increased hepatic and cutaneous CYP1A protein levels, consistent with observations in other mammals. This study is the first to measure CYP1A in skin and liver biopsies from live harbour seals and to report in vivo BNF-associated CYP1A induction in a marine mammal. The results demonstrate that microsamples collected using minimally-invasive techniques can provide toxicologically-relevant information form marine mammals.

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.

Real-time polymerase chain reaction analysis of CYP1B1 gene expression in human liver.

Procarcinogen-activating cytochrome P450 (CYP) enzymes such as CYP1B1, CYP1A1, and CYP1A2 are considered to play an important role in chemical carcinogenesis. However, conflicting data exist with respect to CYP1B1 expression in human liver. In the present study, we measured CYP1B1 mRNA and protein expression in liver samples from 12 individuals (7 nonsmokers, 4 smokers, and 1 ex-smoker) and compared the levels to those of CYP1A1 and CYP1A2. As analyzed by real-time polymerase chain reaction, CYP1B1 mRNA was present in all samples and the inter-individual variability was 16-fold. The group mean level was 5-fold greater in smokers than nonsmokers (121 +/- 46 vs. 26 +/- 5 molecules/ng double-stranded DNA, p < 0.05). By comparison, CYP1A1 mRNA was detectable in samples from 4 of 7 nonsmokers, 3 of 4 smokers, and one ex-smoker, whereas CYP1A2 mRNA was detectable in samples from 5 nonsmokers, 4 smokers, and the ex-smoker. The mean levels of CYP1A1 and CYP1A2 mRNA were 4-fold and 9-fold greater, respectively, in smokers than nonsmokers, but the differences were not statistically significant. The inter-individual variability in CYP1A1 and CYP1A2 mRNA expression was 26-fold and 500-fold, respectively. Immunoblot analysis using several antibodies and with a larger panel (n = 27) of liver microsomes showed that CYP1A1 and CYP1B1 proteins were undetectable, whereas CYP1A2 was detectable in all samples and quantifiable in 24 of 27 samples. In summary, our novel finding indicates that CYP1B1 mRNA is expressed in human liver and the levels are increased in smokers, but the protein is undetectable.