Dicoumarol-sensitive glucuronidation of benzo(a)pyrene metabolites in rat liver microsomes

The effect of dicoumarol on glucuronidation of 3-OH-benzo(a)pyrene (BP) appears to be due to inhibition of UDPglucuronosyltransferase (UDPGT) and not to an inhibited DT-diaphorase (NAD(P)H:quinone oxidoreductase); to date the only enzyme known to be inhibited by dicoumarol. This dicoumarol-sensitive form of UDPGT does not seem to be identical to the major form catalyzing the glucuronidation of p-nitrophenol or methylumbelliferone, nor to the isozyme involved in the formation of phenolphthalein glucuronides. These conclusions are based on the following observations: In solubilized microsomes, devoid of DT-diaphorase, a 3-OH-BP glucuronidation activity is found which is very similar to that observed in microsomes before passing through an azodicoumarol Sepharose 6B column that binds more than 98% of DT-diaphorase; in the eluate from this column the inhibition by dicoumarol of 3-OH-BP glucuronidation is the same as in microsomes containing DT-diaphorase; other coumarin derivatives, which are either modified or substituted in the methylene bridge between the two coumarin entities in dicoumarol, are potent inhibitors of DT-diaphorase but not of UDPGT; a concentration of 10(-6) M dicoumarol is sufficient to inhibit 3-OH-BP glucuronidation 50%. In contrast, to inhibit glucuronidation of p-nitrophenol or methylumbelliferone the concentration of dicoumarol must be raised to the substrate level: i.e., 10(-4) M. Phenolphthalein glucuronidation is almost unaffected even by this high concentration of dicoumarol. The present investigation also reveals that DT-diaphorase and NADPH-cytochrome P-450 reductase can both catalyze the reduction of BP-3,6-quinone for the formation of BP-3,6-quinol glucuronides. In the eluate from the azodicoumarol Sepharose 6B column, no NADH-supported glucuronidation of BP-3,6-quinone can be detected unless DT-diaphorase is added. However, NADPH-supported formation of BP-3,6-quinol glucuronides can still be observed. The rate of the latter reaction is sufficient enough to allow studies on the effect of dicoumarol on BP-3,6-quinone glucuronidation. These results show that glucuronidation of BP-3,6-quinols is also catalyzed by a dicoumarol-sensitive UDPGT. However, not only is the formation of BP-3,6-quinol monoglucuronides inhibited by dicoumarol, but the conversion of monoglucuronides to diglucuronides is inhibited as well. The former reaction is inhibited 50% by 3.5 X 10(-6) M dicoumarol (close to the I50 for 3-OH-BP glucuronidation), whereas 10 times less dicoumarol (2 X 10(-7) M) is sufficient for 50% inhibition of the latter reaction.(ABSTRACT TRUNCATED AT 400 WORDS)     

Bilirubin mono- and di-glucuronide formation by purified rat liver microsomal bilirubin UDP-glucuronyltransferase.

Highly purified bilirubin UDP-glucuronyltransferase from Wistar-rat liver, when reconstituted with Gunn-rat liver microsomes (microsomal fraction), was able to catalyse the conversion of unesterified bilirubin into both bilirubin monoglucuronide and diglucuronide. Under zero-order kinetic conditions for monoglucuronide formation, the fraction of bilirubin diglucuronide formed by incubation of bilirubin with the reconstituted highly purified transferase accounted for 18% of total bilirubin glucuronides, which was only slightly lower than the fraction of diglucuronides (23% of total bilirubin glucuronides) formed by incubation with hepatic microsomes in the presence of UDP-N-acetylglucosamine or Lubrol. The reconstituted purified enzyme also catalysed the UDP-glucuronic acid-dependent conversion of bilirubin monoglucuronide into diglucuronide and, when bilirubin was incubated with UDP-glucose or UDP-xylose, the formation of bilirubin glucosides and xylosides respectively. These results suggest that a single microsomal bilirubin UDP-glycosyltransferase may be responsible for the formation of bilirubin mono- and di-glycosides.     

Enzymatic conversion of bilirubin monoglucuronide to diglucuronide by rat liver plasma membranes.

Formation of bilirubin monoglucuronide from unconjugated bilirubin requires a microsomal enzyme, UDP-glucuronate glucuronyltransferase (EC 2.4.1.17). Conversion of bilirubin monoglucuronide to bilirubin diglucuronide, the major bilirubin conjugate in bile, was studied in subcellular fractions of rat liver. The highest specific activity for bilirubin diglucuronide formation occurred in a fraction highly enriched in plasma membranes. Studies of reaction stoichiometry and utilization of UDP-D-[14C]glucuronic acid revealed that conversion of bilirubin monoglucuronide to bilirubin diglucuronide is not catalyzed by UDP-glucuronyltransferase, and results from transglucuronidation of bilirubin monoglucuronide, with formation of bilirubin diglucuronide and unconjugated bilirubin. When unconjugated bilirubin was infused intravenously into rats at rates exceeding the maximal hepatic excretory capacity, bilirubin monoglucuronide accumulated in serum and bilirubin diglucuronide was found exclusively in bile as the predominant bilirubin metabolite. These results suggest that formation of bilirubin diglucuronide occurs at the surface membrane of the liver cell. Conversion of bilirubin monoglucuronide to bilirubin diglucuronide may play a role in the transport of bilirubin glucuronides from liver to bile.     

Purification and partial characterization of rat liver bilirubin glucuronoside glucuronosyltransferase.

Bilirubin glucuronoside glucuronosyltransferase (EC 2.4.1.95) converts bilirubin monoglucuronide to bilirubin diglucuronide and is concentrated in plasma membrane-enriched fractions of rat liver homogenates. The enzyme was purified 2,000-fold to homogeneity from rat liver. The pI of the enzyme is 7.9 +/- 0.2. The enzyme has a molecular weight of 160,000 and is an oligomer of 28,000 dalton subunits. Km for purified enzyme was 35 microM and Vmax was 2.2 mumol of bilirubin diglucuronide formed/min/mg of protein. Freshly biosynthesized bilirubin monoglucuronide was injected intravenously into homozygous Gunn rats which had bile duct cannulation. Gunn rats lack UDP-glucuronate glucuronyltransferase activity (EC 2.4.1.17), have normal bilirubin glucuronoside glucuronosyltransferase activity, cannot form bilirubin monoglucuronide in vitro or in vivo, and do not excrete bilirubin glucuronides after intravenous injection of unconjugated bilirubin. Within 1 h, approximately 75% of the injected conjugated bilirubin was recovered in bile, of which 20% consisted of bilirubin diglucuronide. These results indicate that bilirubin glucuronide glucuronosyltransferase catalyzes conversion of bilirubin monoglucuronide to diglucuronide in vivo.     

Microsomal conjugation and oxidation of bilirubin

Bilirubin diglucuronide and bilirubin monoglucuronide are formed on incubation of microsomal preparations from rat liver with bilirubin and UDPglucuronate. Microsomal diglucuronide formation is a two-step reaction: first monoglucuronide is formed and this is subsequently converted to diglucuronide. Both steps require UDPglucuronate and have a similar pH optimum at pH 7.8. Albumin inhibits the conversion of monoto diglucuronide. Factors favouring diglucuronide formation are: (a) low bilirubin concentration; (b) relatively high UDPglucuronate concentration; (c) complete removal of UDPglucuronyltransferase latency. For the latter, trypsin-treatment appeared superior over digitonin or UDP-N-acetylglucosamine. Trypsin-treatment had to be done under strictly anaerobic conditions. If trypsin treatment was done under aerobic conditions, reactive molecules were formed which initiated the rapid oxidation of bilirubin and its glucuronides. Microsomal oxidation of bilirubin and glucuronides also occurred in untreated and digitonin-treated microsomes and was stimulated by NADPH and by the cytochrome P-450 inhibitor, metyrapone. This suggests that lipid peroxides act as initiators of bilirubin oxidation. Indirect evidence was found that trypsin inactivates nucleotide pyrophosphatase. This is an active UDPglucuronate-consuming enzyme in microsomal preparations which must be inactivated before meaningful kinetic studies can be done. With trypsin-treated microsomal preparations the Vmax for bilirubin monoglucuronide formation was 1.7 X 10(-9) mol . mg protein-1 . min-1 and KUDPglucuronatem 43 X 10(-6) M. For bilirubin diglucoronide formation the apparent Vmax was 0.7 X 10(-9) mol . mg protein-1 . min-1 and the apparent KUDPglucuronate m 1.0 X 10(-3) M.     

The enzyme-catalyzed formation of bilirubin diglucuronide by a solubilized preparation from cat liver microsomes

When bilirubin monoglucoronide is incubated with a preparation from the 105 000 × g-supernatant of deoxycholate-treated cat liver microsomes, bilirubin diglucuronide is formed. This is an UDPglucuronate-dependent reaction whereby bilirubin IXα monoglucuronide is stoichiometrically converted into bilirubin IXα diglucuronide.The pH optimum for the conversion of bilirubin into bilirubin monoglucuronide lies between pH 8.0 and pH 8.8. For the conversion of mono- into diglucuronide two optima were found, one at about pH 6.5 and another at pH 8.1.When incubation was performed at pH 6.5 and the enzyme protein concentration was lowered, bilirubin monoglucuronide started to isomerise. As a result of this isomerisation bilirubin diglucuronide is also formed. Diglucuronide formation according to this mechanism however, can be clearly differentiated from the enzyme-catalyzed diglucuronide formation.By the formation of bilirubin monoglucuronide, one monoglucuronide isomer is preferentially synthesized.The alkaline-labile bilirubin conjugates in the bile of cats and rats have mainly the IXα isomeric structure. This suggests that in these animals bilirubin diglucuronide is formed enzymically as the bilirubin moiety of diglucuronide, formed by means of the isomerisation reaction, has predominantly the XIIα structure.     

Direct high-performance liquid chromatography determination of propofol and its metabolite quinol with their glucuronide conjugates and preliminary pharmacokinetics in plasma and urine of man

Propofol (P) is metabolized in humans by oxidation to 1,4-di-isopropylquinol (Q). P and Q are in turn conjugated with glucuronic acid to the respective glucuronides, propofol glucuronide (Pgluc), quinol-1-glucuronide (Q1G) and quinol-4-glucuronide (Q4G). Propofol and quinol with their glucuronide conjugates can be measured directly by gradient high-performance liquid chromatographic analysis without enzymic hydrolysis. The glucuronide conjugates were isolated by preparative HPLC from human urine samples. The glucuronides of P and Q were present in plasma and urine, P and Q were present in plasma, but not in urine. Quinol in plasma was present in the oxidised form, the quinone. Calibration curves of the respective glucuronides were constructed by enzymic deconjugation of isolated samples containing different concentrations of the glucuronides. The limit of quantitation of P and quinone in plasma are respectively 0.119 and 0.138 μg/ml. The limit of quantitation of the glucuronides in plasma are respectively: Pgluc 0.370 μg/ml, Q1G 1.02 μg/ml and Q4G 0.278 μg/ml. The corresponding values in urine are: Pgluc 0.264 μg/ml, Q1G 0.731 μg/ml and Q4G 0.199 μg/ml. A pharmacokinetic profile of P with its metabolites is shown, and some preliminary pharmacokinetic parameters of P and Q glucuronides are given.     

Comparison of hepatic, renal and intestinal bilirubin UDP-glucuronyl transferase activities in rat microsomes

1. Bilirubin UDP-glucuronyltransferase activity and its dependence on substrate concentrations in rat liver, renal cortex and intestinal mucosa microsomes were studied. 2. Bilirubin monoglucuronide synthesis from unconjugated bilirubin was a higher capacity, lower affinity step in comparison with bilirubin diglucuronide formation in the three tissues tested. 3. Bilirubin glucuronide formation in liver microsomes showed a higher capacity but a lower affinity than extrahepatic ones. Renal cortex and intestinal mucosa exhibited similar kinetics parameters. 4. In vitro bilirubin glucuronidation in renal cortex and intestinal mucosa was quantitatively important as compared with the hepatic one.     

Somatic mutation, DNA damage and cytotoxicity induced by benzo[a]pyrenedione/benzo[a]pyrenediol redox couples in cultured mammalian cells

BP-3,6-dione was found to be mutagenic, cytotoxic and to induce DNA damage in a transformed line of Syrian hamster fibroblasts at low concentrations, 2 micrograms/ml and less. Inhibition of sulfate and glucuronic acid conjugating enzymes with salicylamide potentiated the above effects of BP-3,6-dione. Diminishing cellular capacity to scavenge superoxide anion radicals also potentiated the mutagenic and cytotoxic action of the dione. The presence of dicumarol, a specific inhibitor of the two-electron reduction of quinones by DT-diaphorase, afforded some protection against cytotoxicity. The results indicate that BP-3,6-dione undergoes two-electron reduction to an unstable hydroquinone, BP-3,6-diol, or one-electron reduction to a semiquinone radical intermediate and that both of these reduced forms undergo rapid univalent oxidation to generate active reduced oxygen species. The data are consistent with the hypothesis that active oxygen species generated by BP-dione/BP-diol redox cycling are responsible, at least in part, for the mutagenic and cytotoxic effects observed with BP-3,6-dione.     

Metabolism of benzo[a]pyrene and persistence of DNA adducts in the brown bullhead (Ictalurus nebulosus)

• 1.1. The in vitro metabolism of [³H]benzo[a]pyrene (BP) and [¹⁴C]benzo[a]pyrene-7,8-dihydrodiol (BP-7,8-diol) by liver of brown bullhead (Ictalurus nebulosus) was characterized, as was the formation and persistence of BP-DNA adducts in vivo.
• 2.2. Compared to rat liver microsomes, bullhead liver microsomes produced relatively larger amounts of BP-7,8-diol (predominantly the [−] enantiomer) and smaller amounts of BP-4,5-diol.
• 3.3. BP phase I metabolites were efficiently converted by freshly isolated bullhead hepatocytes to conjugates, predominantly glucuronides.
• 4.4. BP-7,8-diol was metabolized by hepatocytes 4-fold more rapidly than was BP and was converted to approximately equal amounts of glucuronides, glutathione conjugates and sulfates.
• 5.5. BP-DNA adducts formed in bullhead liver with a lag time of several days and maximum adduct formation at 25–30 days. The major adduct was anti-BPDE-deoxyguanosine.

     

Androgen glucuronides: I. Direct formation in rat accessory sex organs

A simple one-step procedure is described on the isolation of androgen glucuronides from various rat tissues. This procedure uses polyacrylamide gel electrophoresis, and permits a quantitative isolation of a single band containing the total androgen glucuronides without the contamination of free androgens and androgen sulfates. This procedure was used to determine the ability of various tissues of the rat to form androgen glucuronides directly when they were incubated with 1,2-[³H]-testosterone (0.1 μM) $\text{in}$$\text{vitro}$. Of eleven organs studied, only the accessory sex organs (ventral prostate, seminal vesicle, and coagulating gland), liver, and kidney were capable of forming androgen glucuronides. At the end of a one-hour incubation period, approximately 1% of the total radiolabeled steroids in the prostatic tissue minces were in the form of glucuronide conjugates. The predominant androgen glucuronide formed in the accessory sex organs was 5α-androstane-3α,17β-diol 17β-d-glucuronide. This is in contrast to the rat liver and kidney in which testosterone glucuronide was the predominant conjugate.A similar amount of labeled glucuronide conjugates was formed from either [³H]-testosterone, [³H]-dihydrotestosterone or [³H]-androstenedione, whereas negligible amount of steroid conjugates was formed from [³H]-cortisol. The formation of androgen glucuronides requires metabolically active tissues; furthermore, the conjugation process was inhibited by the antiandrogen, cyproterone acetate, or by metabolic inhibitors, such as oligomycin or N-ethylmaleimide.     

Chemical structures critical for the induction of FMN-dependent NADH-quinone reductase in Escherichia coli.

An FMN-dependent NADH-quinone reductase is induced in Escherichia coli by growing the cells in the presence of menadione (2-methyl-1,4-naphthoquinone). Since the properties of induced enzyme are very similar to those of NAD(P)H: (quinone-acceptor) oxidoreductase (EC 1.6.99.2), known as DT-diaphorase, from animal cells, structural requirements of quinone derivatives as an inducer of NADH-quinone reductase in E. coli were examined. Among quinone derivatives examined, it was found that 2-alkyl-1,4-quinone structure with C-3 unsubstituted or substituted with Br is critical as a common inductive signal. Michael reaction acceptors which have been reported to be strong inducers of DT-diaphorase in mouse hepatoma cells were not always effective inducers in E. coli. However, several compounds, such as 2-methylene-4-butyrolactone, methylacrylate and methyl vinyl ketone, showed a slight inductive activity. The efficient inducers of NADH-quinone reductase in E. coli contain 1,4-quinone structure as a part of the inductive signal. These compounds belong to Michael acceptors and are likely to conjugate with thiol compounds such as glutathione.     

Molecular characterization of NAD(P)H:quinone oxidoreductase of tobacco leaves

Summary The NAD(P)H:quinone oxidoreductase activity of tobacco leaves is catalyzed by a soluble flavoprotein [NAD(P)H-QR] and membrane-bound forms of the same enzyme. In particular, the activity associated with the plasma membrane cannot be released by hypoosmotic and salt washing of the vesicles, suggesting a specific binding. The products of the plasma-membrane-bound quinone reductase activity are fully reduced hydroquinones rather than semi-quinone radicals. This peculiar kinetic property is common with soluble NAD(P)H-QR, plasma-membrane-bound NAD(P)H:quinone reductase purified from onion roots, and animal DT-diaphorase. These and previous results demonstrate that soluble and plasma-membrane-bound NAD(P)H:quinone reductases are strictly related flavo-dehydrogenases which seem to replace DT-diaphorase in plant tissues. Following purification to homogeneity, the soluble NAD(P)H-QR from tobacco leaves was digested. Nine peptides were sequenced, accounting for about 50% of NAD(P)H-QR amino acid sequence. Although one peptide was found homologous to animal DT-diaphorase and another one to plant monodehydroascorbate reductase, native NAD(P)H-QR does not seem to be structurally similar to any known flavoprotein.Abbreviations MDAR monodehydroascorbate reductase - PM plasma membrane - NAD(P)H-QR NAD(P)H:quinone oxidoreductase - DPI diphenylene iodonium - DQ duroquinone - CoQ₂ coenzyme Q₂     

Benzo[a]pyrene metabolism in rat fetal hepatocytes culture. Improved methodology and effect of substrate concentration

The different characteristics of benzo[a[pyrene (BP) metabolism in primary fetal rat liver cell culture have been investigated. We have determined the extent of the in vivo [3H]BP metabolism by measuring all of the metabolites retained in the cell and excreted into the culture medium. The extent of the conjugation as well as the nature of the conjugates was established and the pattern of these metabolites analyzed by high performance liquid chromatography (HPLC). The fetal hepatocytes very actively metabolize BP and readily excrete in the culture medium all the produced metabolites in the form of sulfate and glucuronide conjugates. The relative proportion of those compounds varies as a function of the substrate concentration added to the cell culture, the higher the BP concentration, the more glucuronide conjugates. The HPLC analysis of the metabolites shows that BP-1,6-quinone and -3,6-quinone are the major excreted products, indicating the probable existence of an active 6 hydroxylation reaction in the fetal hepatocytes. On the other hand, the pattern of the different metabolites is influenced by the BP concentration. At low BP doses (0.8 microM), the relative amount of polar metabolites is twice as high and that of primary phenols twice as low, when compared to those produced by cells treated with 80 microM BP. The AHH activity drastically modifies the overall rate of the BP metabolism but does not affect the qualitative pattern of the excreted metabolites. The overall metabolism of [3H]BP by the cell culture can easily be estimated by measuring the release of the tritiated water from the substrate into the culture medium.     

Vitamin K1 hydroquinone formation catalyzed by DT-diaphorase

Vitamin K₁ hydroquinone has been identified as a metabolite of vitamin K₁ biotransformation catalyzed by highly purified DT-diaphorase (NAD(P)H dehydrogenase, EC 1.6.99.2) isolated from livers of 3-methylcholanthrene induced rats. The hydroquinone was sufficiently stable to permit enzymatic reactions to be conducted under an atmosphere of air and quantitation of hydroquinone by high performance liquid chromatography. Based on kinetic data reported here, warfarin and probably dicoumarol at therapeutic levels do not appreciably affect DT-diaphorase catalyzed vitamin K hydroquinone formation.     

Flavonoid glucuronides with anti-leukaemic activity from Polygonum amphibium L

Introduction –  The chemical and pharmaceutical studies carried out on species from Polygonum L. genus showed biological activity both of the extracts and the components isolated from them. These results were the impulse to examine Polygonum amphibium L. Objective –  The aim of this study was the isolation of active components from methanol extract and the determination of their cytotoxic effect on human leukaemic cell lines. Methodology  – Three flavonoid components from butanol soluble fractions of methanol extract by CC and PC preparative chromatography were isolated. Their structures were established on the basis of ¹H, ¹³C and correlation (DEPT, H‐H, COSY, HMQC, HMBC) NMR, UV and FAB‐MS spectroscopic techniques. The evaluation of the anti‐leukaemic activities of 1 and 2 against Jurkat and HL60 cell lines was carried out in vitro using annexin V fluorescence assay. Results  – Two new flavonoid glucuronides, quercetin‐3‐O‐β‐glucuronide ( 1 ) and quercetin‐3‐O‐α‐rhamnosyl‐(1 → 2)‐β‐glucuronide ( 2 ), and kaempferol‐3‐O‐α‐rhamnosyl‐(1 → 2)‐β‐glucuronide ( 3 ), were isolated from Polygonum amphibium L. It was demonstrated that the glucuronides of quercetin are able to induce apoptosis in the tested human leukaemic cells. These compounds penetrate through cytoplasm to the cellular nucleus of the cultured cells, and give intensive apoptotic responses in the stimulated leukaemic cells. The number of apoptotic cells increased with the concentration (1 nm to 10 µm ) of 1 or 2 and periods of exposure (1–3 days). Conclusion  – Compounds 1 and 2 may be considered good candidates for leukaemia chemotherapeutic agents. Copyright © 2008 John Wiley & Sons, Ltd.     

Study of bilirubin metabolism by high-performance liquid chromatography: stability of bilirubin glucuronides

The stabilities of bilirubin (BR) glucuronide, monoglucuronide (BMG), and diglucuronide (BDG) were studied under various conditions by HPLC. In aqueous media, BMG showed a pronounced lability and was easily transformed into equimolar BDG and BR. It was proved by direct analysis of tetrapyrrole isomers that BDG and BR were formed from dipyrrole exchange of BMG molecules. All reducing agents examined (sodium ascorbate, cysteine, GSH, dithiothreitol, NADH, and NADPH) suppressed the transformation of BMG into BDG and BR. Bovine serum albumin and rat liver cytosol fractions also stabilized BMG strongly. BDG was fairly stable in aqueous media as compared with BMG. When BMG was incubated both with and without liver plasma membranes (N2 fraction) from Wistar rats, the formation rates of BDG and BR in both incubation mixtures were exactly the same. The composition of BDG and BR isomers was the same in both mixtures. Also, heat denaturation of the plasma membranes did not affect formation rates. Moreover, the reaction was completely inhibited by sodium ascorbate. These findings indicate that rat liver plasma membranes have no enzyme activity for BDG formation from BMG.     

Separation and characterization of isoforms of DT-diaphorase from rat liver cytosol.

Rats were treated with 3-methylcholanthrene (MC) and DT-diaphorase from liver was partially purified on an azodicoumarol-Sepharose 6B column and applied to an FPLC-chromatofocusing column in order to resolve isoforms. Six peaks showing significant DT-diaphorase activity were eluted from this column with a pH gradient between 7.30 to 4.80. The amino acid compositions of the two major peaks (II and VIb) were found to be nearly identical, suggesting existence of isoforms rather than isozymes of DT-diaphorase. The isoforms of DT-diaphorase showed broad substrate specificities towards four different quinones (menadione, vitamin K-1, benzo(a)pyrene 3,6-quinone and cyclized-dopamine ortho-quinone), although quantitative differences in the specific activities were also found. All isoforms are glycoproteins but contain different carbohydrates. Thus isoform II reacts with biotinylated lectins which are specific for N-acetylgalactosamine, mannose, fucose and galactosyl(beta-1,3)N-acetylgalactosamine, while isoform VIb reacts only with biotinylated lectins specific for mannose and N-acetylgalactosamine. Separation of DT-diaphorase isoforms from control rat liver cytosol using FPLC-chromatofocusing revealed that the induction of the isoforms is not uniform, since isform II was not found and the major isoform was composed of three peaks, whereas the major isoform of DT-diaphorase from liver cytosol of rats treated with 3-methylcholanthrene was composed of only two peaks.     

Metabolism of benzo[a]pyrene and persistence of DNA adducts in the brown bullhead (Ictalurus nebulosus).

1. The in vitro metabolism of [3H]benzo[a]pyrene (BP) and [14C]benzo[a]pyrene-7,8-dihydrodiol (BP-7,8-diol) by liver of brown bullhead (Ictalurus nebulosus) was characterized, as was the formation and persistence of BP-DNA adducts in vivo. 2. Compared to rat liver microsomes, bullhead liver microsomes produced relatively larger amounts of BP-7,8-diol (predominantly the [-] enantiomer) and smaller amounts of of BP-7,8-diol (predominantly the [-] enantiomer) and smaller amounts of BP-4,5-diol. 3. BP phase I metabolites were efficiently converted by freshly isolated bullhead hepatocytes to conjugates, predominantly glucuronides. 4. BP-7,8-diol was metabolized by hepatocytes 4-fold more rapidly than was BP and was converted to approximately equal amounts of glucuronides, glutathione conjugates and sulfates. 5. BP-DNA adducts formed in bullhead liver with a lag time of several days and maximum adduct formation at 25-30 days. The major adduct was anti-BPDE-deoxyguanosine.     

Degradation of hemin IX and synthetic hemins to α-bilirubins and their conjugates in the isolated perfused rat liver

Hemin IX was perfused through rat liver of a normal, untreated animal. Its degradation products, collected in the bile fluid over a period of 90 min, were found to consist of the bilirubin IX-α diglucuronide (56%), the mixture of bilirubin IX-α monoglucuronides (42%), and free bilirubin IX-α (2%). When the synthetic hemin XIII $\text{2}$ was perfused with the same technique, it was found to be degraded in the same way. The bile fluid contained the diglucuronide of bilirubin XIII-α $\text{10}$ (55%), the monoglucuronide of bilirubin XIII-α $\text{9}$ (43%) and the free bilirubin XIII-α 8 (2%). Similar results were obtained when the iron 1,4-di(β-hydroxyethyl)-2,3,5,8-tetramethyl-6,7-di(β-carboxyethyl) porphyrin $\text{3}$ was perfused; the diglucuronide of the α-bilirubin $\text{11}$ comprised 65% of the excreted bile bilirubins, the monoglucuronide was 25% of the total and the free α-bilirubin $\text{11}$ 10% of the total. Perfusion of hematohemin gave 58% of the diglucuronide of α-hematobilirubin, as well as 40% of the monoglucuronides, and 2% of the free α-hematobilirubin. The simultaneous perfusion of hematohemin and of hemin IX produced an inhibition of the degradation of the hemin IX, while hematohemin was degraded as described above. It was concluded that the normal rat liver is prepared to dispose of exogenously added hemins by their oxidation to α-biliverdins, reduction of the latter to the corresponding α-bilirubin and excretion of their conjugated derivatives through the bile duct.