Hormonal activity of combinations of genistein, bisphenol A and 17β-estradiol in the female Wistar rat

Phytoestrogens have been described as weak estrogens,selective estrogen receptor mediators (SERMs) or to exhibit antiestrogenic properties. However, information about their activity in combination with xenoestrogens and 17β-estradiol in vivo, is limited. Therefore, the combinatory activity of the phytoestrogen genistein (Gen), the industrial chemical bisphenol A (BPA), and ethinylestradiol (EE) in ovariectomized Wistar rats was analyzed in this study. All compounds were administered orally on three consecutive days (EE at 30 μg, Gen at 100 mg and BPA at 200 mg per kg body weight per day). The pure antiestrogen fulvestrant (3 mg/kg) served as estrogen receptor (ER) antagonist control. Effects on uterine wet weight, height of the uterine epithelium, uterine clusterin (Clu) and complement C3 expression, and the height of the vaginal epithelium were examined. Treatment with Gen alone resulted in a moderate stimulation of uterine weight; in the vagina the height of the epithelium was strongly stimulated. BPA did not stimulate any of the above-mentioned parameters significantly. In combination with EE, Gen acted on most of the analyzed parameters in an additive manner, whereas BPA significantly antagonized the effects of EE on the uterine epithelium and uterine Clu expression. Given in combination with Gen, BPA was also able to antagonize the stimulatory effect of Gen on the uterine epithelium. In summary, our results demonstrate that Gen, in contrast to BPA, does not exhibit any antiestrogenic properties, even if given at high concentrations. The results of this study characterize BPA as a functional antiestrogen, very likely the result of a lack of ability to activate ER-mediated transactivation after binding to the receptor. This is not the case for Gen. Our results point to the involvement of complex molecular mechanisms in the action of Gen. These mechanisms, especially the role of ERβ have to be characterized in further investigations.     

Role of pregnane xenobiotic receptor in the midbrain ventral tegmental area for estradiol- and 3α,5α-THP-facilitated lordosis of female rats

Rationale

Progesterone and its metabolite, 5α-pregnan-3α-ol-20-one (3α,5α-THP), have actions in the ventral tegmental area (VTA) that are required for lordosis, a characteristic mating posture of female rodents. 17β-estradiol (estradiol) co-varies with progestogens over natural cycles, enhances production of 3α,5α-THP, and is required for successful reproductive behavior.

Objectives

A question of interest is the role of pregnane xenobiotic receptor (PXR), a nuclear receptor that regulates enzymes needed for the production of 3α,5α-THP, for estradiol-mediated lordosis. The hypothesis tested was that if PXR is involved in estradiol-mediated biosynthesis of 3α,5α-THP and reproductive behavior, knocking down expression of PXR in the VTA of estradiol-primed, but not vehicle-primed, rats should decrease lordosis and midbrain 3α,5α-THP; effects may be attenuated by 3α,5α-THP administered to the VTA.

Methods

Ovariectomized rats were administered subcutaneous injections of oil vehicle or estradiol. Rats were then administered PXR antisense oligonucleotides (PXR AS-ODNs; which are expected to locally knock down expression of PXR), or control (saline), infusions to the VTA. Rats were administered 3α,5α-THP or vehicle via infusions to the VTA. Reproductive behavior (paced mating task) of rats was determined in addition to exploratory (open field), affective (elevated plus maze), and pro-social (social interaction task) behavior.

Results

Reproductive behavior (i.e., increased lordosis) was enhanced with estradiol-priming and infusions of 3α,5α-THP to the VTA. Infusions of PXR AS-ODNs to the VTA attenuated responses in estradiol-, but not vehicle-, primed rats, compared to control infusions.

Conclusions

PXR may be involved in a neuroregulatory response involving biosynthesis of 3α,5α-THP in the midbrain VTA of estradiol-primed rats.     

Expression and Significance of IL-6, IL-17 and TGF-β in Cystitis Glandularis

目的:探讨白细胞介素(IL)-6、IL-17和转化生长因子(TGF)-β在腺性膀胱炎(CG)组织中的表达及其在CG发病及进展中的作用.方法:将15例正常膀胱黏膜定为对照(A)组,43例未复发CG定为治愈(B)组,13例复发和癌变的CG定为进展(C)组;检测3组IL-6、IL-17和TGF-β的表达情况,并分析这些因子与CG发病及进展的相关性.结果:A组、B组及C组IL-6、IL-17和TGF-β的阳性表达率差异均有统计学意义(P＜0.01).3种因子与CG发病(rs分别为0.431、0.545、0.354,P＜0.05)和进展(rs分别为0.426、0.333、0.288,P＜0.05)呈正相关;IL-17分别与IL-6、TGF-β呈正相关(rs分别为0.997、0.961,P＜0.05).结论:IL-6、IL-17和TGF-3可能在CG的发病及进展中起重要作用.     

Aluminium interfere with vitellogenesis in the estradiol-17β-administered immature rockfish,Sebastes schlegeli

Effect of aluminum (al) on the plasma vitellogenin (VTG), alkaline-labile phosphorus (ALPP), Calcium (Ca), glutamate pyruvate transaminase (GPT) and hepatosomatic index (HSI) were examined in the estradiol-17β (E₂)-administered immature rockfish, Sebastes schlegeli. Fish were intraperitoneally injected with E2 (5 mg/kg body weight (B.W.)) and/or Al (0, 0.1, 1, 5 and 10 mg/kg B.W.) and then plasma were extracted at 7 days. Thick VTG band was detected at a molecular weight position of about 170 kDa in SDSPAGE using the plasma of E₂-administered rockfish. This band was reduced by the administration of Al to rockfish. Al reduced the concentrations of ALPP and Ca in a concentration-dependent way and a significant reduction occurred at Al concentrations greater than 5 mg/kg and 10 mg/kg, respectively. The concentration of GPT was increased in concentrationdependent way at all the group of Al-administered rockfish. HSI was reduced in a concentration-dependent way. These results suggest that Al interfere with E₂-induced vitellogenesis by being toxic to hepatocyte.     

The enterohepatic circulation of the metabolites of 17α-ethynyl[3H]estradiol in the rat

1. The biliary metabolites of 17α-ethynyl[³H]estradiol (³H-EE₂) and their enterohepatic circulation were studied in female rats.

2. Rapid and extensive elimination of radioactivity in bile followed i.v. administration of ³H-EE₂. The metabolites consisted largely of β-glucuronides of 2-hydroxy-EE₂ and, to a lesser extent, 2-methoxy-EE₂.

3. Intraduodenally infused radiolabelled biliary metabolites underwent enterohepatic circulation; 15.6% (mean±S.D., n = 4) of the ³H was excreted in the bile of the recipient rats in 5.5?h. The metabolites excreted by the recipients were qualitatively and quantitatively similar to the infused metabolites.

4. The 1(4)-glutathione adduct of 2-hydroxy-EE₂ was synthesized, but was not observed in either the donors' or recipients' bile.     

17β estradiol induced ROS generation,DNA damage and enzymatic responses in the hepatic tissue of Japanese sea bass

The importance of endocrine disrupting chemicals and their effects on fish has been documented in recent years. However, little is known about whether the estrogenic compound 17β estradiol (E2) causes oxidative stress in the hepatic tissue of fish. Therefore, this work tested the hypothesis that E2 might cause oxidative stress in the Japanese sea bass Lateolabrax japonicus liver. To test this hypothesis, its effects on reactive oxygen species (ROS) production, DNA damage, antioxidants and biotransformation enzyme were investigated in two different size groups (fingerling and juvenile groups) following 30 days exposure. Results showed that there was a good relationship between the E2 exposure concentration, plasma E2 level and ROS generation. In addition ROS production correlated negatively with 7-ethoxyresorufin-O-deethylase activity and positively with DNA damage and lipid peroxidation (LPO). Antioxidant enzymes such as superoxide dismutase and catalase did not show any significant relation with ROS, LPO and DNA damage. In contrast, glutathione mediated enzymes showed a good relationship with the above parameters suggesting that the glutathione system in fish might be responsible for protection against the impact of E2 and also indicating a possible adaptive response during exposure periods. In addition, it was observed that fingerling was more susceptible to E2 exposure than juvenile fish. The present study provided strong evidence that the ROS level increased significantly in the liver of E2 exposed fish, and that ROS might serve as a biomarker to indicate estrogen contamination.     

Exposure to 17α-ethynylestradiol causes dose and temporally dependent changes in intersex,females and vitellogenin production in the Sydney rock oyster

Although mounting evidence suggests exposure to estrogenic contaminants increases vitellogenin production in molluscs, demonstration of dose–response relationships and knowledge of the temporal nature of the vitellogenin response with continual exposure is currently lacking for biomarker utility. To address this knowledge gap, adult Sydney rock oysters, Saccostrea glomerata, were exposed to a range of environmentally relevant concentrations of 17α-ethynylestradiol (EE2) (0, 6.25, 12.5, 25 or 50 ng/l) in seawater under laboratory conditions. Vitellogenin induction and gonadal development was assessed following 4, 21 and 49 days exposure to EE2. Vitellogenin was found to increase in a dose dependent manner with EE2 exposure for females (4 and 49 days) and males (4 and 21 days). Histological examination of gonads revealed a number of individuals exhibited intersex (ovotestis) in 50 ng/l EE2 (after 21 days) and in 6.25 and 12.5 ng/l EE2 (after 49 days). Furthermore, a significant shift towards females was observed following 49 days exposure at 50 ng/l EE2 suggesting estrogenic exposure is capable of facilitating a progression for protandric males from male-intersex-female gametal status. Increases in female vitellogenin (4 days) were predictive of later increases in female developmental stages at 21 days and increases in oocyte area following 49 days. Male vitellogenin (4 days) was predictive of decreased male percentages and lower male developmental stages at 49 days. Vitellogenin in S. glomerata is a predictive biomarker of estrogenic exposure and effect if sampled soon after exposure and at the commencement of a gonadal development cycle.     

Study of the genotoxic and cytotoxic effects of the α-, β-, and γ- Hexachlorocyclohexane isomers in human lymphocyte cells using the cytokinesis-block micronucleus assay

The genotoxic potential of hexachlorocyclohexane (HCH) isomers (α-, β-, and γ-) which are organochlorine pesticides was tested in peripheral blood lymphocyte cultures from two donors by using the cytokinesis-block micronucleus assay. Micronucleus (MN) frequency, binucleated cells with micronucleus (BNMN), and cytokinesis-blocked proliferation index (CBPI) were determined as genotoxic and cytotoxic endpoints. At the concentration ranges tested (12.5–100?μg.L?^?1), all HCH isomers induced dose-dependent cytotoxic effects, γ-HCH being the most toxic. This isomer was also able to induce significant increase in MN frequency and BNMN cells indicating a genotoxic potential at 50 and 100?μg.L?^?1. The genotoxic test of β-HCH showed a positive induction of MN and BNMN cells at the highest concentration of 100?μg.L?^?1 and a significant cytotoxicity at 50?μg.L?^?1. Under the experimental condition used, α-HCH was unable to induce any significant increase in MN frequency confirming that α-HCH is a non-genotoxic agent.     

Metabolism in vivo of 3,4,3′,4′-tetrachlorobiphenyl and toxicological assessment of the metabolites in rats

1. Metabolism in vivo of 3,4,3′,4′-tetrachlorobiphenyl (TCB) and toxicological assessment of the metabolites were investigated in the rat.

2. Four metabolites were isolated from faeces of rats dosed with 3,4,3′,4′-TCB. Two were identified as 5-hydroxy-3,4,3′,4′-TCB and a chlorine-shift metabolite, 4-hydroxy-3,5,3′,4′-TCB, by comparison of melting points, chromatographic mobilities and spectral features with those of the synthetic samples. A dihydroxy-TCB and monohydroxy-trichlorobiphenyl were also indicated by mass spectrometry to be excreted in faeces as minor metabolites.

3. Faecal excretion of unchanged 3,4,3′,4′-TCB, 5-hydroxy-3,4,3′,4′-TCB and 4-hydroxy-3,5,3′,4′-TCB was 0.8%, 19.6% and 11.6% of dose, respectively, in 5 days after i.p. injection of 3,4,3′,4′-TCB at a dose of 50mg/kg.

4. From the inability to cause the liver hypertrophy and thymus atrophy, both monohydroxy-metabolites of 3,4,3′,4′-TCB are much less toxic than the parent 3,4,3′,4,-TCB. In addition, these phenolic metabolites did not induce the activities of benzo[a]pyrene hydroxylase and DT-diaphorase, whereas 3,4,3′,4′-TCB greatly induced these activities. These results indicated that unlike PCB congeners with phenobarbital-type inducing ability, 3,4,3′,4′-TCB, a prototype of 3-methylcholanthrene-type inducers. is detoxified by metabolic hydroxylation.     

Exposure–response analyses of blood pressure and heart rate changes for methylphenidate in healthy adults

The aim of the study was to evaluate the exposure–response (E–R) relationships of blood pressure (BP) and heart rate (HR) changes in healthy adults taking methylphenidate (MPH). Intensive time profiles of BP and HR from healthy adults in placebo and MPH treatment arms of seven clinical trials from the FDA internal database were utilized for this analysis. The analysis model contains a circadian component for placebo effect and an E–R component to describe drug effect. Internal validation was performed using goodness-of-fit plots and visual predictive check. A meta-database based on a systemic literature search was constructed and used for external validation of the developed models. We found that circadian models could quantify the time profiles of BP/HR in placebo arms. Linear models could describe the correlations between MPH concentrations, and BP/HR changes. The BP and HR changes were highly dependent on the shapes of MPH pharmacokinetic (PK) profiles without an apparent time delay. MPH has the greatest effect on HR, followed by systolic BP, and diastolic BP. Internal validation revealed that the developed models could adequately describe the circadian rhythms of HR and BP in placebo arms and the E–R relationships of MPH. External validation showed the models had good predictive capability of the literature data. In conclusion, the developed models adequately characterized the circadian rhythm and the MPH induced effects on BP and HR. The changes in BP and HR were highly correlated with MPH blood levels with no apparent delay. The time courses of BP and HR are similar to the MPH PK profiles. As a result, the immediate-release formulation may yield larger maximum BP and HR effect than the extended-release formulation under similar dose.     

GC-MS method for the simultaneous determination of β-blockers, flavonoids, isoflavones and their metabolites in human urine

A sensitive and selective method based on gas chromatography hyphenated to mass spectrometry (GC-MS) for the screening of 23 different compounds including β-blockers, flavonoids, isoflavones and metabolites in human urine sample was developed and validated. The present paper reports, for the first time, the method for the simultaneous determination of β-blockers, isoflavones, flavonoids and metabolites in human urine samples. When flavonoids are ingested in combination with drugs that have a narrow therapeutic range, interactions between flavonoids and drugs should be investigated.Substances of interest were extracted from urine samples by solid-phase extraction (SPE) employing a mixture of tert-butyl methyl ether:methanol:formic acid (4.5:4.5:1; v/v/v) as a mobile phase and Oasis HLB (Waters) as a stationary phase. Before extraction, urine samples were incubated with β-glucuronidase/sulfatase in order to achieve enzymatic hydrolysis. Before GC-MS analysis the analytes had to be derivatized with N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) into their trimethylsilyl derivatives by incubating for 60 min at 60 °C. Statistical central composite design and response surface analysis were used to optimize the derivatization reagent. These multivariate procedures were efficient in determining the optimal separation condition, using peak areas as responses.The calibration curves were indicative of high linearity (r² ≥ 0.9992) in the range of interest for each analyte. LODs (S/N = 3) ranged between 0.6 and 9.7 ng/ml. Intra-day and inter-day precision (CV, %) was less than 4.96%, accuracy between 0.01 and 4.98% and recovery was found in the range from 70.20 to 99.55%.The developed method can be applied to the routine determination of examined compounds’ concentrations in human urine. Moreover the method is suitable for detecting pharmaceutical compounds containing β-blockers, isoflavones and flavonoids in urine after administration to humans.     

Influence of polymorphisms and TNF and IL1β serum concentration on the infliximab response in Crohn’s disease and ulcerative colitis

Aim

Inflammatory bowel diseases (IBD), such as Crohn’s disease (CD) and ulcerative colitis (UC), are partially attributable to an increased secretion of proinflamatory cytokines, such as tumour necrosis factor (TNF) and interleukin-1β (IL1β), which play essential roles in the disease pathogenesis and are target molecules for specific therapy. Given the inter-individual variability in the response to the anti-TNF monoclonal antibody infliximab, the aim of our study was to explore the predictive value of TNF and/or IL1β as surrogate markers of infliximab response.

Methods

Serial serum concentrations of TNF and IL1β and TNF promoter region and IL1B polymorphisms were determined in 47 patients (29 CD and 18 UC) receiving infliximab and correlated with treatment response.

Results

Baseline serum concentrations of TNF and IL1β were higher in UC patients than in CD patients (p?=?0.0097 and?0.0024, respectively). CD patients showing <0.64 pg/ml IL1β at baseline were more frequently responders than non-responders (p?=?0.036), and the C allele of the IL1B polymorphism was associated with higher IL1β serum concentrations (p?=?0.026) and with poorer clinical remission after 14 weeks of infliximab treatment. No significant association was found between serum TNF concentration or TNF polymorphism and patient response to infliximab.

Conclusion

This is the first study evaluating the pharmacogenetic role of the rs1143634 polymorphism of IL1B and TNF polymorphisms in infliximab-treated IBD patients. We found an association between the rs1143634 C allele and higher serum IL1β concentrations and a lower response to infliximab treatment in CD patients that warrants the interest of future studies in larger and independent series.     

Do multidrug resistance-associated protein-1 and -2 play any role in the elimination of estradiol-17 beta-glucuronide and 2,4-dinitrophenyl-S-glutathione across the blood-cerebrospinal fluid barrier?

The purpose of this study was to examine the role of multidrug resistance-associated protein-1 and -2 (Mrp1 and Mrp2) in the efflux transport of organic anions across the blood-cerebrospinal fluid (CSF) barrier. The CSF concentration of estradiol-17beta-glucuronide (E(2)17betaG) and 2,4-dinitrophenyl-S-glutathione (DNP-SG) in the CSF after intracerebroventricular and intravenous injection were compared between wild-type and Mrp1 gene knockout mice. There was no significant difference in the apparent CSF elimination rate constants of E(2)17betaG (0.158 and 0.145 min(-1)) and DNP-SG (0.116 and 0.0779 min(-1)) between wild-type and Mrp1 knockout mice, respectively. After intravenous administration of E(2)17betaG, its brain-to-serum and CSF-to-serum concentration ratios in Mrp1 knockout mice were not significantly different from those in the wild-type. Results from in vivo and in vitro studies using Eisai hyperbilirubinemic rats, in which Mrp2 is hereditarily deficient, were similar to those using normal rats. Quantitative polymerase chain reaction (PCR) showed that the expression level of Mrp4 and Mrp5 was several times higher than that of Mrp1, whereas the expression levels of Mrp2, Mrp3, and Mrp6 were negligible or low. Therefore, Mrp4 and Mrp5 may contribute to the efflux transport of E(2)17betaG and DNP-SG from the CSF.     

Catechol metabolites of zeranol and 17β-estradiol: a comparative in vitro study on the induction of oxidative DNA damage and methylation by catechol-O-methyltransferase

The current study examines whether the occupation of firefighting contributes to exposure to polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and dioxin-like polychlorinated biphenyls (PCBs). We compared serum dioxin concentrations and congener profiles of current firefighters (n=13) with those of men who have ceased employment as firefighters (n=17) and with men employed in occupations other than firefighter (n=10). We found that compared to former or non-firefighters, current firefighters have higher levels of dioxins primarily due to the contribution of PCBs and to a lesser extent PCDFs. PCDFs were significantly higher in former firefighters compared to non-firefighters (p<0.05). Comparisons with studies performed by other investigators suggest that local environmental conditions contribute to some of the elevation of PCBs. The congeners 1,2,3,4,6,7,8-heptachlorodibenzodioxin and PCB-114 were significantly higher in current firefighters when compared to former or non-firefighters. Moreover, levels of these congeners were inversely correlated with years since employed as firefighter (Spearman r=-0.610, p=0.009 and Spearman r=-0.53, p=0.03, respectively). The classes of dioxins show an overall decline with years since employed as firefighters, this decline is most evident with PCDDs (Spearman r=-0.46, p=0.06). Together, the combination of evidence supports firefighting as a source of exposure to dioxins.     

Differences in the biotransformation of a 17β-hydroxylated steroid,trenbolone acetate,in rat and cow

1. The metabolism of trenbolone acetate, 17β-acetoxyestra-4,9,11-trien-3-one (TBA), an anabolic compound used as a growth promoter, was compared in rat and cow.

2. [6,7-³H] TBA was injected i.v. into rats and a heifer, and bile was collected for 24?h. In both species, the bile was the major route of excretion. TBA undergoes an extensive hydrolysis to 17β-hydroxyestra-4,9,11-trien-3-one and the unchanged compound was not detected, but subsequent major metabolic pathways are different in the two species.

3. In the rat, oxidation of the 17β-hydroxyl to the 17-oxo group and hydroxylation in the 16α-position are the major routes. The three major metabolites are 17β-hydroxyestra-4,9,11-trien-3-one, 16α,17β-dihydroxyestra-4,9,11-trien-3-one and 16α-hydroxyestra-4,9,11-trien-3,17-dione.

4. In the heifer, 17α-epimerization is the major pathway and the main metabolite is the 17α-hydroxyestra-4,9,11-trien-3-one.

5. In both species, estra-4,9,11-trien-3,17-dione and the other metabolites, resulting either from hydroxylation in 1, 2, 6β, 16α or 16β positions, or from aromatization of the A ring, were minor products.

6. Overall, 60% of the 3-oxotriene structures identified in the rat bile were 17β-hydroxylated and the remainder were 17-keto metabolites, whereas in the heifer bile 90% were 17α-hydroxylated compounds.

7. Thus, in bovine species, the major pathway is similar to those of testosterone or 17β-estradiol which are mainly excreted as their 17α-epimers. This epimerization strongly decreases the biological potency, as with the natural 17β-hormones, and leads to detoxication of tissue residues.     

Naltrexone and β-funaltrexamine antagonism of the antinociceptive and response rate-decreasing effects of morphine, dezocine, and d-propoxyphene

  Rationale: Patterns of competitive and insurmountable antagonism provide important data to guide the classification and characterization of different types of opioid agonists as well as infer the mechanism of action for agonists. Objective: Experiments with the competitive antagonist, naltrexone, and the insurmountable antagonist, β-funaltrexamine (β-FNA), were conducted to determine whether the antinociceptive and rate-decreasing effects of the opioid agonists dezocine and d-propoxyphene are 1) mediated through μ opioid receptors in rats, and 2) differ from morphine with respect to relative efficacy. Methods: The rat tail-withdrawal assay was used to measure antinociception and a fixed ratio 20 (FR20) schedule of food delivery was used to measure rate suppression. Results: Naltrexone (0.01–1.0 mg/kg) was approximately equipotent as an antagonist of the antinociceptive and rate-decreasing effects of both morphine and dezocine and as an antagonist of the antinociceptive effects of d-propoxyphene. Naltrexone failed to block the rate-decreasing effects of d-propoxyphene. β-FNA (5 and 10 mg/kg) also antagonized the antinociceptive and rate-decreasing effects of morphine and dezocine as well as the antinociceptive effects of d-propoxyphene. β-FNA failed to produce a dose-dependent antagonism of the rate-decreasing effects of d-propoxyphene. Conclusions: These data suggest that the antinociceptive effects of morphine, dezocine, and d-propoxyphene and the rate-decreasing effects of morphine and dezocine are mediated through μ opioid receptors. Overall, high doses of β-FNA produced a greater degree of antagonism of the behavioral effects of dezocine than morphine or d-propoxyphene, confirming other reports that dezocine is a lower efficacy agonist than morphine. Additionally, the degree of antagonism produced by β-FNA was greater for the antinociceptive effects of all three compounds than for the rate-decreasing effects. Received: 14 August 1998 / Final version: 4 December 1998     

Metabolism of benzbromarone in man: structures of new oxidative metabolites, 6-hydroxy- and 1″-oxo-benzbromarone,and the enantioselective formation and elimination of 1″-hydroxybenzbromarone

1. The uricosuric drug benzbromarone is extensively metabolized in man and two main metabolites are formed: the previously characterized 1″-hydroxybenzbromarone (metabolite M₁) and an arylhydroxybenzbromarone (metabolite M₂) of unknown structure. A dimethyl derivative was isolated from urine after methylation and was characterized by gas chromatography-mass spectrometry (g.l.c.-m.s.) and high resolution nuclear magnetic resonance spectroscopy as 4″-O-methyl-6-methoxybenzbromarone; the structure of M₂ therefore is 6-hydroxybenzbromarone.

2. A minor metabolite was similarly characterized as 1″-oxobenzbromarone by comparison with authentic synthetic samples and is a product of biodegradation and not an artifact derived from the in vitro oxidation of 1″-hydroxybenzbromarone. Further minor metabolites were detected and were provisionally characterized by g.l.c.-m.s. after derivatization and include: 2″-hydroxybenzbromarone (an isomer of 1″-hydroxybenzbromarone); 1″,6-dihydroxybenzbromarone; dihydroxy-aryl-benzbromarone; and two structure isomers of 6-hydroxybenzbromarone. Debrominated metabolites were not detectable.

3. Benzbromarone is hydroxylated in vivo at the prochiral centre C1″to 1″-hydroxybenzbromarone; analysis of 1″-hydroxybenzbromarone from plasma and urine extracts by?h.p.l.c. using a chiral column revealed that two peaks were eluted which showed a mean enantiomeric ratio of 2.1 for plasma and 7.3 for urine; these data demonstrate that the formation and elimination of this metabolite is enantioselective; the absolute configuration of the 1″-chiral centre is presently unknown.