Biliary Metabolites Research Articles

Glucuronidation of N-hydroxy heterocyclic amines by human and rat liver microsomes.

The food-borne carcinogenic and mutagenic heterocyclic aromatic amines undergo bioactivation to the corresponding N-hydroxy (OH)-arylamines and the subsequent N-glucuronidation of these metabolites is regarded as an important detoxification reaction. In this study, the rates of glucuronidation for the N-OH derivatives of 2-amino-3-methylimidazo[4,5-f]-quinoline (IQ), 2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine (PhIP), 2-amino-6-methyl-dipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) by liver microsomal glucuronosyltransferase were compared to that of the proximate human urinary bladder carcinogen, N-OH-aminobiphenyl (N-OH-ABP) and the proximate rat colon carcinogen N-OH-3,2'-dimethyl-4-amino-biphenyl (N-OH-DMABP). Human liver microsomes catalyzed the uridine 5'-diphosphoglucuronic acid (UDPGA)-dependent glucuroidation of N-OH-IQ, N-OH-PhIP, N-OH-Glu-P-1 and N-OH-MeIQx at rates of 59%, 42%, 35% and 27%, respectively, of that measured for N-OH-ABP (11.5 nmol/min/mg). Rat liver microsomes also catalyzed the UDPGA-dependent glucuronidation of N-OH-PhIP, N-OH-Glu-P-1 and N-OH-IQ at rates of 30%, 20% and 10%, respectively of that measured for N-OH-DMABP (11.2 nmol/min/mg); activity towards N-OH-MeIQx was not detected. Two glucuronide(s) of N-OH-PhIP, designated I and II, were separated by HPLC. Conjugate II was found to be chromatographically and spectrally identical with a previously reported major biliary metabolite of PhIP in the rat, while conjugate I was identical with a major urinary metabolite of PhIP in the dog. Hepatic microsomes from rat, dog and human were found to catalyze the formation of both conjugates. The rat preferentially formed conjugate II (I to II ratio of 1:15), while the dog and human formed higher relative amounts of conjugate I (I to II ratio of 2.5:1.0 and 1.3:1.0 respectively). Fast atom bombardment mass spectrometry of conjugates I and II gave the corresponding molecular ions and showed nearly identical primary spectra. However, collision-induced spectra were distinct and were consistent with the identity of conjugates I and II as structural isomers. Moreover, the UV spectrum of conjugate I exhibited a lambda max at 317 nm and was essentially identical to that of N-OH-PhIP, while conjugate II was markedly different with a lambda max of 331 nm. Both conjugates were stable in 0.1 N HCl and were resistant to hydrolysis by rat, dog and human liver microsomal beta-glucuronidases.(ABSTRACT TRUNCATED AT 400 WORDS)

Separatory Determination of Biliary Metabolites of Equilin in Rat by High-Performance Liquid Chromatography

Abstract A method for the separation and determination of biliary metabolites of equilin in rat by high-performance liquid chro-matography with electrochemical detection has been developed. After extraction of equine estrogens in bile with a C18 bonded silica cartridge, conjugated metabolites were hydrolyz-ed with sulfatase containing β-glucuronidase and free estrogens liberated were then separated on a reversed phase C18 column, the limit of detection being in the range of 5–20 pg. The transformation of equilin administered orally into 2-methoxy derivatives of equilin, equilenin and their 17β-reduced metabolites is also discussed.

Identification of radiolabeled metabolites of nicotine in rat bile: Synthesis of S-(−)-nicotine N-glucuronide and direct separation of nicotine-derived conjugates using high-performance liquid chromatography

Four metabolites of nicotine, including two glucuronides, have been separated by high-performance liquid chromatography. This separation was applied to identification of biliary metabolites of radiolabeled nicotine by radiometric detection. S-(−)-Nicotine N-glucuronide was synthesized and used as a standard in method development.

The characterization of two biliary glutathione conjugates of amsacrine using liquid secondary ion mass spectrometry.

An additional biliary glutathione (GSH) conjugate of the anilinoacridine anti-tumour agent amsacrine (4'-(9-acridinylamino)methanesulphon-m-anisidide, NSC 249992) has been identified in bile collected from male Wistar rats by cannulation of the common bile duct and from male BDF1 mice by removal of the gall bladder after treatment with amsacrine. The presence of this conjugate, at the 6'-position of the anilino ring, has been confirmed by liquid secondary ion (LSI) mass spectrometric analysis of selected biliary metabolites separated by high-performance liquid chromatography. The two major metabolites each gave a daughter ion spectrum which was diagnostic for either 5'- or 6'-GSH conjugation. This pattern was confirmed by comparison with LSI mass spectral data obtained from authentic chemical standards formed on reaction of the quinone diimine derivative of amsacrine with methanethiol or mercaptoethanol.

Excretion balance and urinary metabolites of the S-enantiomer of indobufen in rats and mice

The excretion balance and urinary metabolites of the S-enantiomer of indobufen, (( S)2-[p-(1-oxo-2-isoindolinyl)-phenyl]butyric acid), a platelet aggregation inhibitor, were studied in rats and mice after oral administration. The urinary metabolic profile exhibited a marked species difference. The major metabolic pathway in the mouse was acyl glucuronidation followed by renal excretion, whereas in rat urine 5-hydroxylation and subsequent sulphation at the introduced hydroxyl group accounted for almost all recovered radioactivity. Indobufen glucuronide was the major biliary metabolite in the rat, while very little indobufen glucuronide was present in the urine of intact or bile ductcannulated rats. A marked dose-effect on the elimination and metabolism of S-indobufen was demonstrated in the rat. The recovery (% dose) of 5-hydroxyindobufen and its sulphate after the lower dose of the enantiomer (10mg/kg) was some 2.8-fold higher compared with the higher dose of 20 mg/kg.

Identification of multi-S-substituted conjugates of hydroquinone by HPLC-coulometric electrode array analysis and mass spectroscopy.

Chemical reaction of 1,4-benzoquinone with GSH gives rise to several multisubstituted hydroquinone (HQ)-GSH conjugates, each of which causes renal proximal tubular necrosis when administered to male Sprague-Dawley rats. In addition, HQ has recently been reported to be nephrocarcinogenic following long-term exposure in male rats. Since neither the mechanism nor the extent of HQ oxidation and thioether formation in vivo is known, we have assessed both the qualitative and quantitative significance of HQ-thioether formation in vivo and in vitro. HQ (1.8 mmol/kg, ip) was administered to AT-125-pretreated male Sprague-Dawley rats, and bile and urine samples were analyzed with a HPLC-coulometric electrode array system (CEAS) and by liquid chromatography (LC)/continuous-flow fast atom bombardment (CF-FAB) mass spectroscopy. Five S-conjugates of hydroquinone were identified in bile, and one S-conjugate was identified in urine. The major biliary S-conjugate identified was 2-glutathion-S-ylhydroquinone [2-(GSyl)HQ] (18.9 +/- 2.7 mumol). Additional biliary metabolites were 2,5-diglutathion-S-ylhydroquinone [2,5-(diGSyl)HQ] (2.2 +/- 0.6 mumol), 2,6-diglutathion-S-ylhydroquinone [2,6-(diGSyl)HQ] (0.7 +/- 0.3 mumol),2,3,5-triglutathion-S-ylhydroquinone [2,3,5-(triGSyl)HQ] (1.2 +/- 0.1 mumol), and 2-(cystein-S-ylglycyl)hydroquinone. 2-(N-Acetylcystein-S-yl)HQ was the only urinary thioether metabolite (11.4 +/- 3.6 mumol) identified. The quantity of S-conjugates excreted in urine and bile within 4 h of HQ administration [34.3 +/- 4.5 mumol (4.3 +/- 1.1% of dose)] appears sufficient to propose a role for such metabolites in HQ-mediated nephrotoxicity and nephrocarcinogenicity. Rat liver microsomes catalyzed the NADPH-dependent oxidation of HQ (300 microM), in the presence of GSH, to form 2-(GSyl)HQ,2,5-(diGSyl)-HQ, and 2,6-(diGSyl)HQ. A fraction of the microsomal oxidation of HQ appears to be catalyzed by cytochrome(s) P450, although the exact amount remains unclear. 2-(GSyl)HQ,2,5-(diGSyl)-HQ, and 2,6-(diGSyl)HQ (300 microM) also underwent NADPH-dependent oxidation and GSH conjugation in liver microsomes. The extent of the nonenzymatic oxidation of HQ and its GSH conjugates correlated, approximately, with their half-wave oxidation potentials.

Biliary secretion and enterohepatic recycling of fenbendazole metabolites in sheep

Fenbendazole (FBZ) was administered intraruminally at 5.0 mg/kg, containing a trace of [14C]-FBZ, to sheep fitted with a permanent bile duct cannula and the behaviour of FBZ and its metabolites examined in bile and plasma. Of the administered radiolabelled dose, 47% was secreted in bile of which 34% was accounted for as conjugated and 4% as unconjugated (free) metabolites. Hydroxylated oxfendazole (OH.OFZ) was the major biliary metabolite contributing 66%, and hydroxy-FBZ (OH.FBZ) 27%, of the total metabolites characterized. Small amounts of OFZ and hydroxy FBZ sulphone (OH.FBZ.SO2) were also present in bile. The rapid appearance of OH.OFZ in bile, even before maximum concentrations of OFZ occurred in plasma, indicated that sulphoxidation and hydroxylation was the major route of FBZ metabolism. Following intraduodenal infusion of free biliary metabolites, FBZ and its metabolites rapidly appeared in bile indicating absorption from the small intestine. When conjugated metabolites were infused they continued to appear in bile for a further 15-20 h after cessation of infusion indicating that absorption of hydroxylated metabolites occurred largely after bacterial deconjugation in the large intestine. Approximately 40% of biliary metabolites were estimated to undergo enterohepatic reabsorption but they contributed minimally to the metabolite content in plasma. It is suggested that during the process of recycling-biliary metabolites make substantial contact with parasites in the mucosa of the small and large intestine thereby contributing to the anti-helminthic activity of FBZ.

Partial contribution of biliary metabolites to nephrotoxicity, renal content and excretion of [14C]hexachloro-1,3-butadiene in rats.

Male Sprague Dawley rats with cannulated bile duct (BDC rats) received 100 or 200 mg kg-1 labelled hexachloro-1,3-butadiene ([14C]HCBD) by gavage 1 h (BDC1 rats) or 24 h (BDC24 rats) after surgical cannula implantation. Twenty-four hours after treatment with HCBD, rats were examined histochemically and biochemically for kidney damage. Urine, faeces, liver and kidney radioactivities were also measured in 24-h samples. Results were compared with those obtained from non-cannulated (NC) rats. Bile-duct cannulation did not completely protect against HCBD-induced kidney damage. The 24-h [14C] urinary excretion and tissue content was 30-50% lower in BDC rats compared to NC rats and correlated well with the toxicity findings. BDC1 rats appeared to be much more resistant to HCBD treatment than BDC24 rats. Since faecal [14C] radioactivity extractable by diethyl ether at neutral pH in BDC1 rats was twice that measured in BDC24 rats, the greater resistance was attributed to a higher deficiency in the gastrointestinal absorption of unchanged HCBD. The present results reveal that the biliary metabolites of HCBD are not solely responsible for kidney toxicity as previously assumed. They suggest a sinusoidal efflux of the HCBD conjugates from the liver.

Biliary metabolites of levonorgestrel in rats

The metabolism of levonorgestrel (LNG) in the bile following oral administration of the drug was examined in female rat. 1) Within 48 h after administration of 14C-labelled LNG (LNG-14C), 67-82% of the radioactivity was excreted into the bile. 2) Almost all the metabolites in the bile were conjugated with glucuronic acid or sulfuric acid and only a small amount of the unchanged compound was found. 3) After treatment of these metabolites in the bile with beta-glucuronidase and arylsulfatase, more than ten aglycones were detected on TLC. Three main aglycones, M1, M2 and M3, were isolated. They accounted for 68.0, 0.8 and 11.5% of the radioactivity excreted into the bile, respectively. 4) The structures of M1 and M2 were assumed to be 13-ethyl-18,19-dinor-5 alpha,17 beta-pregn-20-yne-3 alpha,17- diol and 13-ethyl-18,19-dinor-5 beta,17 beta-pregn-20-yne-3 alpha,17-diol, respectively, by NMR and LC/MS analyses, and confirmed by direct comparison with respective authentic samples. M3 was assigned to be 13-ethyl-18,19-dinor-5 alpha,17 beta-pregn-20-yne-3 alpha,16 beta,17-triol by NMR, LC/MS and GC/MS analyses and acetonide derivation. 5) Isolation of the glucuronide metabolite, M4, from the bile, was achieved by column chromatography using Amberlite XAD-2 and Sephadex LH-20. Hydrolysis of this compound with beta-glucuronidase released M1 and glucuronic acid. After M4 was converted to an acetylated-methyl ester derivative, the definite structural assignment of M4 was established to be M1-3-O-yl glucuronic acid by NMR analysis. The NOE effect and the value of the corresponding coupling constant of the anomeric proton showed that the glucoside moiety was in the beta configuration. These findings suggested that LNG was predominantly converted to 5 alpha-reduced metabolites and that the 5 beta-metabolite accounted for less than 1% of the total metabolites in female rats. These metabolites were excreted as glucuronides into the bile.

Ultrastructural and histological characterization of hepatic neoplasms in mummichog Fundulus heteroclitus inhabiting a creosote-contaminated environment : Wolfgang K. Vogelbein. Virginia Institute of Marine Science, The College of William and Mary, Gloucester Point, Virginia 23062, USA

We surveyed four Chesapeake Bay tributaries for skin and liver tumors in brown bullhead (Ameiurus nebulosus). We focused on the South River, where the highest skin tumor prevalence (53%) in the Bay watershed had been reported. The objectives were to 1) compare tumor prevalence with nearby rivers (Severn and Rhode) and a more remote river (Choptank); 2) investigate associations between tumor prevalence and polynuclear aromatic hydrocarbons (PAHs) and alkylating agents; and 3) statistically analyze Chesapeake Bay bullhead tumor data from 1992 through 2008. All four South River collections exhibited high skin tumor prevalence (19% to 58%), whereas skin tumor prevalence was 2%, 10%, and 52% in the three Severn collections; 0% and 2% in the Choptank collections; and 5.6% in the Rhode collection. Liver tumor prevalence was 0% to 6% in all but one South River collection (20%) and 0% to 6% in the three other rivers. In a subset of samples, PAH-like biliary metabolites and 32P-DNA adducts were used as biomarkers of exposure and response to polycyclic aromatic compounds (PACs). Adducts from alkylating agents were detected as O6-methyl-2′-deoxyguanosine (O6Me-dG) and O6-ethyl-2′-deoxyguanosine (O6Et-dG) modified DNA. Bullheads from the contaminated Anacostia River were used as a positive control for DNA adducts. 32P-DNA adduct concentrations were significantly higher in Anacostia bullhead livers compared with the other rivers. We identified alkyl DNA adducts in bullhead livers from the South and Anacostia, but not the Choptank. Neither the PAH-like bile metabolite data, sediment PAH data, nor the DNA adduct data suggest an association between liver or skin tumor prevalence and exposure to PACs or alkylating agents in the South, Choptank, Severn, or Rhode rivers. Logistic regression analysis of the Chesapeake Bay database revealed that sex and length were significant covariates for liver tumors and length was a significant covariate for skin tumors.

Identification of four biliary metabolites of the diterpene sclareol in the laboratory rat

1. Ag.l.c. method was developed to determine sclareol (1) and its microbial metabolites: 3-keto-sclareol (2), 2 alpha-hydroxysclareol (3), 3 alpha-hydroxysclareol (4), 3 beta-hydroxysclareol (5), 18-hydroxysclareol (6), and 2 alpha, 18-dihydroxysclareol (7) in both microbial cultures and biological fluids of the laboratory rat. 2. Metabolism of the diterpene (1) was studied in the laboratory rat. This in vivo study was facilitated by the availability of microbial metabolites of sclareol as reference standards, and the g.l.c. assay for sclareol and its metabolites in biological fluids. 3. Following i.v. treatment (100 mg/kg), the disappearance of (1) from rat plasma was rapid and biphasic. No microbial metabolites of sclareol were detectable in plasma. 4. Sclareol (1) and its microbial metabolites were not detected in rat urine following either i.v. or oral treatments; unchanged (1) was excreted in rat faeces to the extent of 9% of an oral dose in 48 h. 5. Following i.v. treatment, 0.02% dose was recovered in bile as unchanged (1). Four biliary metabolites of (1) (0.4% dose) were identified as (2), and (4)-(6) based on g.l.c.-mass spectrometry and comparisons with reference standards. All four biliary metabolites of (1) in rat have been identified as microbial metabolites of (1).

Oxidative dehalogenation of 2-fluoro-17α-ethynyloestradiol in vivo: A distal structure-metabolism relationship of 17α-ethynylation

Metabolic activation to catechols and their oxidation products is variously considered to contribute to the genotoxic, cytotoxic, transforming and tumour-promoting activities of exogenous steroidal oestrogens. 2-Fluoro-17α-ethynyloestradiol (2-FEE 2) was synthesized as a prototype of pharmacologically active derivatives of 17β-oestradiol which are resistant to metabolic activation in vivo. It possessed high affinity for the rat uterine oestrogen receptor and was oestrogenic in rats. Biliary metabolites of [6,7- 3H]2-FEE 2 (0.73 μmol/kg, 157 μg/kg, i.v.) in female rats were characterized: 87% of the radiolabel was excreted, principally as 2-FEE 2 glucuronide, over 6hr. Although 2-nuoro-17β-oestradiol is not metabolized to C-2 oxygenated products in vivo, 2-FEE 2 underwent rapid and appreciable oxidative defluorination. 2-Hydroxy-17α-ethynyloestradiol and 2-methoxy-17α-ethynyl-oestradiol represented, respectively, 8% and 13% of the dose. Fluorination nevertheless restricted C-2 oxygenation to ca. 28% of that which 17α-ethynyloestradiol undergoes in female rats. C-4 oxygenation of 2-FEE 2, resulting in catechol formation, occurred but to a lesser extent ( ca. 12% of dose). None of the major and identified minor biliary metabolites was a product of metabolic activation at the ethynyl function. A mechanistic rationalization of the long range enhancement by 17α-ethynylation of oxidative defluorination at C-2 is presented.

The metabolism of 2- and 4-fluoro-17β- oestradiol in the rat and its implications for oestrogen carcinogenesis

2-Fluoro-[6,7- 3H]17β-oestradiol ([ 3H]2-FE 2) and 4-fluoro-[6, 7- 3H]17β-oestradiol([ 3H]4-FE 2) were synthesized by the fluorination and reduction of [ 3H]oestrone and purified by HPLC. [ 3H]2-FE 2 and [ 3H]4-FE 2 (72.5 μg/kg; 0.25 μmol/kg) were administered i.v. to anaesthetized female and male Wistar rats (N = 4) with biliary cannulae. Bile was collected for 6 hr. Female rats administered [ 3H]2-FE 2 excreted 85% of the dose into bile over 6 hr whilst male rats excreted 77%. After the administration of [ 3H]4-FE 2, female and male rats excreted 72 and 83% of dose into bile over 6 hr, respectively. The biliary metabolites were glucuronides in all cases. The principal metabolite of [ 3H]2-FE 2 liberated from biliary conjugates by β-glucuronidase was 2-fluoroestrone in both female rats (64% of dose) and male rats (57%). No 2-hydroxylated, i.e. oxidatively defluorinated, metabolites were detected in either sex. In contrast, 2-hydroxylation of [ 3H]4-FE 2 did occur, but only in female rats: 2-hydroxy-4-fluoro-oestrone (22%) and 2-methoxy-4-fluoroestrone (17%) were identified as biliary aglycones. However, the major metabolite was 4-fluoroestrone (4FE 1]; 38%). In male rats, 4-FE 1 and 4-fluoro d-ring-oxygenated products were the principal biliary aglycones. The differences in metabolism between the two fluoro analogues and oestradiol are discussed with particular reference to the possible involvement of 2- and 4-hydroxy (catechol) oestrogens in oestrogen toxicity.

Metabolism of 1,2,4-trichlorobenzene in rats: examination of thiol formation.

1. More than 60% of oral doses of 14C-1,2,4-trichlorobenzene (ca. 21 mg/kg) administered to rats were excreted in bile as S-trichlorophenyl-mercapturic acid pathway metabolites. 2. The biliary metabolites were ultimately excreted in urine mainly as the isomeric mercapturic acids. 3. An acetylated glutathione conjugate was isolated as a major metabolite in bile (8% dose). The acetyl group was shown by mass spectrometry to be on the glutamyl moiety. 4. A glutamylcysteine conjugate of trichlorobenzene was also isolated from bile as a major metabolite (8% dose). 5. Trichlorothiophenols were deduced not to be intermediates or end-products of enzymic metabolism of trichlorobenzene in rats because 14C-2,4,5-trichlorothiophenol dosed i.p. to rats was excreted as the S-glucuronide (17% dose) and as S-(methylsulphonyl-dichlorophenyl)-mercapturic acid (36% dose).

Intestinal metabolism of mephentermine and its biliary metabolites in male Wistar rats.

1. Intestinal metabolites produced in the incubation (0-24 h) of mephentermine (MP), phentermine (Ph), N-hydroxymephentermine (N-hydroxy-MP), N-hydroxyphentermine (N-hydroxy-Ph), p-hydroxymephentermine (p-hydroxy-MP) and p-hydroxyphentermine (p-hydroxy-Ph) with male Wistar rat intestinal contents under N2 were examined by g.l.c. and g.l.c.-electron impact (EI) mass spectrometry. Metabolites produced in the anaerobic incubation of bile from rats given MP, with the intestinal contents were also examined. In addition, urinary and biliary metabolites of p-hydroxy-MP and p-hydroxy-Ph dosed orally to rat were examined. 2. Metabolites in the anaerobic incubation of N-hydroxy-MP and N-hydroxy-Ph were MP and Ph, and Ph, respectively. No metabolites were detected in the incubation of MP, Ph, p-hydroxy-MP and p-hydroxy-Ph. 3. p-Hydroxy-MP and p-hydroxy-Ph (major), and MP and Ph (minor) were detected when bile from rats given MP was incubated with intestinal contents. 4. Unchanged p-hydroxy-MP, and conjugates of p-hydroxy-MP and p-hydroxy-Ph, were detected in the 24-h urine of rats dosed with p-hydroxy-MP, which accounted for about 3, 72 and 1% dose, respectively. Unchanged p-hydroxy-Ph and conjugated p-hydroxy-Ph were detected in the 24-h urine of rats dosed with p-hydroxy-Ph, which accounted for about 4 and 68% dose, respectively. 5. Conjugated p-hydroxy-MP and conjugated p-hydroxy-Ph, which accounted for about 3% doses, were detected in the 24-h bile of rats dosed with p-hydroxy-MP and p-hydroxy-Ph.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of calcium antagonist Ro 40-5967: a case history of the use of diode-array u.v. spectroscopy and thermospray-mass spectrometry in the elucidation of a complex metabolic pathway.

1. The calcium antagonist, Ro 40-5967, is metabolized to a multitude of products by the rat and drug-related material is excreted predominantly via the bile. 2. Diode-array u.v. spectroscopy, following reverse phase h.p.l.c. separation of the partially purified metabolites, has been used to classify these compounds into six spectral classes which have been correlated with different metabolic reactions. 3. Connection of a mass spectrometer directly to the h.p.l.c. equipment by a thermospray interface, produced useful mass spectra. These, together with the u.v. spectra, enabled the structures of many metabolites to be elucidated. 4. Confirmation of structural assignments was provided by n.m.r. spectra of the major metabolites. 5. Major metabolic pathways included N-demethylation (16% of the biliary metabolites), hydrolysis of the ester side-chain (32%), hydroxylation at 4- (19%) and 5- (29%) positions of the benzimidazole ring, aromatization of the tetrahydronaphthyl system (26%), loss of the benzimidazole (15%) and glucuronidation of hydroxyl groups (81%).

Evidence of polycyclic aromatic hydrocarbon (PAH) exposure in fish from the Antarctic peninsula

PAH exposure was assessed in fish from Arthur Harbor, Antarctica and sites remote from human activities by measuring the concentrations of polycyclic aromatic hydrocarbon (PAH) metabolites in bile and PAH in tissues and stomach contents. The highest concentrations of biliary metabolites and tissue PAH were present in fish captured near an Argentine supply ship ( Bahia Paraiso) that ran aground in Arthur Harbor in 1989. The presence of biliary PAH metabolites in fish collected near Palmer Station, a small US scientific station in Arthur Harbor, suggests low level exposure to contaminants. However, PAH tissue concentrations in fish from Palmer Station were similar to those from remote sites. Both tissue PAH and metabolite concentrations were low in fish captured at sites remote from human activities. The confirmation of PAH metabolites in the bile of fish from Arthur Harbor is direct evidence that the presence of humans has resulted in Antarctic fish being exposed to petroleum-derived PAH.

Biotransformation of the hexachlorobutadiene metabolites 1-(glutathion-S-yl)-pentachlorobutadiene and 1-(cystein-S-yl)-pentachlorobutadiene in the isolated perfused rat liver

1. The first step in the bioactivation of the nephrotoxin hexachlorobutadiene is the biosynthesis of 1-(glutathion-S-yl)-1,2,3,4,4-pentachloro-1,3-butadiene (GPCB). GPCB formed in the liver is secreted into bile, may be reabsorbed in the gut, intact or after hydrolysis to 1-(cystein-S-yl)-1,2,3,4,4-pentachloro-1,3-butadiene (CPCB), and undergo enterohepatic circulation or translocation to the kidney. Hepatic uptake and metabolism of GPCB and CPCB may thus influence the disposition of these S-conjugates. We therefore studied the metabolism and uptake of CPCB and GPCB in the isolated perfused rat liver. 2. Dose-dependent uptake of GPCB and CPCB from the perfusion medium by isolated perfused liver was demonstrated; CPCB is cleared from the perfusion medium to a much higher extent than GPCB. 3. GPCB and CPCB are intensively biotransformed to biliary metabolites. These metabolites were identified by thermospray mass spectrometry as products of the conjugation reaction of GPCB and CPCB with glutathione and subsequent hydrolysis of the glutathione moieties. 4. Hepatic biosynthesis of 1-(N-acetyl-L-cystein-S-yl)-1,2,3,4,4-pentachloro- 1,3-butadiene from CPCB was only a very minor pathway in GPCB and CPCB metabolism in liver. 5. The results indicate that hepatic biosynthesis of mercapturic acids may not contribute to the disposition of S-conjugates formed from hexachlorobutadiene in vivo and that GPCB may be, at least in part, delivered intact to the kidney.

Intrahepatic conversion of a glutathione conjugate to its mercapturic acid. Metabolism of 1-chloro-2,4-dinitrobenzene in isolated perfused rat and guinea pig livers.

Because of the low hepatic activity of gamma-glutamyl-transferase in the rat, the liver is generally considered to play only a minor role in the degradation of glutathione conjugates, a limiting step in mercapturic acid formation. Recent findings indicate, however, that the liver has a prominent role in glutathione catabolism, particularly in species other than rat. To examine the contributions of liver to mercapturic acid biosynthesis, mercapturate formation was compared in isolated perfused livers from rats and guinea pigs dosed with either 0.3 or 3.0 mumol of 1-chloro-2,4-dinitrobenzene (CDNB). Chemically synthesized glutathione conjugate, mercapturic acid, and intermediary metabolites of CDNB were used as standards in the high performance liquid chromatography analysis of bile and perfusate samples. Biliary excretion accounted for almost all of the recovered metabolites. A marked species difference was observed in the pattern of CDNB metabolism. Rat livers dosed with 0.3 mumol of CDNB excreted 55% of total biliary metabolites as the glutathione conjugate and 8.2% as the mercapturic acid, whereas guinea pig livers excreted only 4.8% as the glutathione conjugate and 47% as the mercapturate. Mercapturic formation was also dose-dependent, with a larger fraction formed at the 0.3- versus the 3.0-mumol dose (8.2 versus 3.7% in the rat; 47 versus 19% in the guinea pig). Hepatic conversion of the glutathione conjugate to the mercapturic acid was markedly inhibited in both species after retrograde intrabiliary infusion of acivicin, an inhibitor of gamma-glutamyltransferase activity. These findings provide direct evidence for intrahepatic biosynthesis of mercapturic acids. Thus, glutathione conjugates synthesized within hepatocytes are secreted into bile and broken down to cysteine conjugates; the latter are then presumably reabsorbed by the liver, N-acetylated to form the mercapturic acid and re-excreted into bile.

Metabolism of cannabidiol by the rat

Metabolites of CBD excreted into the bile and perfusion fluid were examined in a rat liver perfusion preparation. Metabolites were extracted with ethyl acetate and identified by GC/MS as TMS derivatives. Four mono- and five di-hydroxy metabolites were identified with major sites of metabolic attack being at C-7 and C-4". A hydroxy-ketone was detected but not fully identified. All biliary metabolites were conjugated with glucuronic acid. Urinary metabolites were studied in rats with samples taken at times to 25 h after drug administration. Unmetabolized CBD and 13 metabolites were identified by GC/MS. Major metabolites were acids with beta-oxidation being a prominent pathway. The 6- and 7-hydroxy derivatives of 4",5"-bis,nor-CBD-3"-oic acid were the most abundant compounds but substantial concentrations of the di-acids, CBD-5",7-dioic acid and 4",5"-bis,nor-CBD-3",7-dioic acid were present. Concentrations of the more highly oxidized metabolites increased with time.