Biotransformation of (-)-verbenone by human liver microsomes

The biotransformation of (-)-verbenone was investigated with human liver microsomes by using GC-MS. Regioselective biotransformation was observed when (-)-verbenone was incubated with the liver microsomes. (-)-10-Hydroxyverbenone was formed from (-)-verbenone of kinetic analysis showed that the Km and Vmax values for the hydroxylation of (-)-verbenone by liver microsomes from three human samples, HG-70, HG-56 and HG-23, were 1.1 mM and 4.8 nmol/min/nmol P450, 0.6 mM and 2.1 nmol/min/nmol P450, and 2.8 mM and 4.6 nmol/min/nmol P450, respectively.     

Characterisation of tolbutamide hydroxylase activity in the common brushtail possum, (Trichosurus vulpecula) and koala (Phascolarctos cinereus): inhibition by the eucalyptus terpene 1,8-cineole

Plant constituents such as terpenes are major constituents of the essential oil in Eucalyptus sp. 1,8-Cineole and p-cymene (Terpenes present in high amounts in Eucalyptus leaves) are potential substrates for the CYP family of enzymes. We have investigated tolbutamide hydroxylase as a probe substrate reaction in both koala and terpene pretreated and control brushtail possum liver microsomes and examined inhibition of this reaction by Eucalyptus terpenes. The specific activity determined for tolbutamide hydroxylase in the terpene treated brushtails was significantly higher than that for the control animals (1865+/-334 nmol/mg microsomal protein per min versus 895+/-27 nmol/mg microsomal protein per min). The activity determined in koala microsomes was 8159+/-370 nmol/mg microsomal protein per min. Vmax values and Km values for the terpene treated possum, control, possum and koala were 1932-2225 nmol/mg microsomal protein per min and 0.80 0.81 mM; 1406-1484 nmol/mg microsomal protein per min and 0.87-0.92 mM and 5895-6403 nmol/mg microsomal protein per min and 0.067-0.071 mM, respectively. Terpenes were examined as potential inhibitors of tolbutamide hydroxylase activity. 1,8-Cineole was found to be a competitive inhibitor for the enzyme responsible for tolbutamide hydroxylation (Ki 15 microM) in the possum. In koala liver microsomes stimulation of tolbutamide hydroxylase activity was observed when concentrations of cineole were increased. Therefore, although inhibition was observed, the type of inhibition could not be determined.     

Phenobarbital inducible UDP-glucuronosyltransferase is responsible for glucuronidation of 3'-azido-3'-deoxythymidine: characterization of the enzyme in human and rat liver microsomes

Glucuronidation by liver microsomes of 3'-azido-3'-deoxythymidine (AZT) was characterized in human and in various animal species. The glucuronide isolated by HPLC, was identified by mass spectrometry (fast atom bombardment, desorption in chemical ionization), and beta-glucuronidase hydrolysis. AZT glucuronidation reaction in liver microsomes of human and monkey proceeded similarly with an apparent Vmax of 0.98 nmol/min/mg protein and apparent Km of 13 mM. Oleoyl lysophosphatidylcholine activated more than twofold the formation of the glucuronide. Human kidney microsomes could also biosynthesize AZT glucuronide, although to a lower extent (six times less than the corresponding liver). Probenecid, which is administered to AIDS patients, decreased hepatic AZT glucuronidation in vitro (I50 = 1.5 mM), whereas paracetamol did not exert any effect at concentrations up to 21.5 mM. Morphine also inhibited the reaction (I50 = 2.7 mM). AZT glucuronidation presented the highest rate in human and in monkey (0.50 nmol/min/mg protein); pig and rat glucuronidated the drug two and three times less, respectively. In Gunn rat, the specific activity in liver microsomes was similar (0.18 nmol/min/mg protein) to that of the congenic normal strain; this suggests that an isozyme other than bilirubin UDP-glucuronosyltransferase catalyzed the reaction. In rats, AZT glucuronidation was stimulated fourfold by phenobarbital; 3-methylcholanthrene or clofibrate failed to increase this activity. This result was consistent with the bulkiness of the AZT molecule (thickness 6.7 A), which is a critical structural factor for glucuronidation of the drug by phenobarbital-induced isozymes. Altogether, the results strongly indicate that UDP-glucuronosyltransferase (phenobarbital inducible forms) is responsible for AZT glucuronidation.     

Metabolic fate of glutathione conjugate of benzo[a]pyrene-(7R,8S)-diol (9S,10R)-epoxide in human liver.

Benzo[a]pyrene-(7R,8S)-diol (9S,10R)-epoxide [(+)-anti-BPDE] is believed to be the activated form of the widely spread environmental pollutant benzo[a]pyrene. Glutathione (GSH) S-transferase (GST)-catalyzed conjugation of (+)-anti-BPDE with GSH is an important mechanism in its cellular detoxification. Here, we report that the GSH conjugate of (+)-anti-BPDE [(-)-anti-BPD-SG] is a potent inhibitor (K(i) 15 microM) of class Mu human GST isoenzyme, which, among human liver GSTs, is a highly efficient detoxifier of (+)-anti-BPDE. Thus, the inhibition of GST activity by (-)-anti-BPD-SG may hinder GSH conjugation of (+)-anti-BPDE, unless the conjugate is metabolized and/or eliminated. The results of the present study show that gamma-glutamyltranspeptidase (gamma-GT) can metabolize (-)-anti-BPD-SG at a rate of about 0.29 nmol/min/mg protein. Our studies also show that (-)-anti-BPD-SG is transported across the human canalicular liver plasma membrane (cLPM) in an ATP-dependent manner at a rate of about 0.33 nmol/min/mg protein. The ATP-dependent transport of (-)-anti-[(3)H]BPD-SG across human cLPM follows Michaelis-Menten kinetics (K(m) 84 microM; V(max) 0.33 nmol/min/mg). In conclusion, the results of the present study suggest that both gamma-GT-mediated metabolism and ATP-dependent canalicular transport may be important steps in overall detoxification of (+)-anti-BPDE in the human liver.     

Hepatic metabolism of artemisinin drugs--I. Drug metabolism in rat liver microsomes.

1. In this communication, metabolism of the semisynthetic antimalarial drugs of the artemisinin class (beta-arteether, beta-artelinic acid and dihydroartemisinin) in rat liver microsomes, is reported. 2. Dihydroartemisinin was the major early metabolite of arteether (57%) and artelinic acid (80%); in addition, arteether was hydroxylated in the positions 9 alpha- and 2 alpha- of the molecule. 3. Dihydroartemisinin was further metabolized by extensive hydroxylation of its molecule; we were able to identify four hydroxylated derivatives of DQHS, but not the exact positions of the hydroxyl groups. 4. The rates of NADPH-supported metabolism of arteether, artelinic acid and dihydroartemisinin in rat liver microsomes were: 4.0, 2.5 and 1.3 nmol/min/mg of microsomal protein, respectively. 5. The apparent affinity constants of arteether and artelinic acid for the microsomal metabolizing system, calculated from the rates of product formation, were 0.54 mM and 0.33 mM (for arteether) and 0.11 mM (for artelinic acid), respectively. The appearance of two affinity constants indicated that arteether was metabolized by two different isoenzymes of cytochrome P-450 in rat liver microsomes.     

Identification of a reactive intermediate of furazolidone formed by swine liver microsomes

Furazolidone (N-(5-nitro-2-furfurylidene)-3-amino-2-oxazolidone) is metabolized by swine liver microsomes under aerobic and anaerobic conditions (rate: 2.55 and 3.25 nmol/mg protein/min, respectively). Covalent binding to microsomal protein amounted aerobically to 0.29 nmol/mg protein/min. Of all amino acids tested, only addition of cysteine to the incubation mixture decreased microsomal protein binding of furazolidone, indicating that covalent binding may occur at protein thiol groups. Two known metabolites of furazolidone, 3-(4-cyano-2-oxobutylidene-amino)-2-oxazolidone and 2,3 dihydro-3-cyano-methyl-2-hydroxyl-5-nitro-1 alpha,2-di(2-oxo-oxazolidin-3-yl) iminomethyl-furo[2,3-b] furan, were minor metabolites. At least 50% of total metabolites is formed by swine liver microsomes via a reductive process of furazolidone as indicated by the formation of a furazolidone-mercaptoethanol conjugate after the addition of mercaptoethanol to the incubation mixture. The conjugate was identified as 3-(4-cyano-3-beta-hydroxyethylmercapto-2-oxobutylidene amino)-2-oxazolidone, indicating that the open-chain acrylonitrile-derivative is the reactive intermediate of furazolidone which also may be responsible for interaction with protein.     

Involvement of cytochrome P450 and the flavin-containing monooxygenase(s) in the sulphoxidation of simple sulphides in human liver microsomes

This study was conducted to examine the involvement of cytochrome P450 (CYP450) and the flavin-containing monooxygenase (FMO) in the sulphoxidation of ethyl methyl sulphide (EMS), 4-chlorophenyl methyl sulphide (CPMS) and diphenyl sulphide (DPS) in human liver microsomes from a phenotypic CYP2D6 extensive metabolizer. Human liver microsomes catalyzed the sulphoxidation of EMS, CPMS and DPS to their corresponding sulphoxides. Lineweaver-Burk plots for the sulphoxidation of EMS in human liver microsomes indicated that the apparent K(m) and V(max) were 1.53 +/- 0.07 mM and 1.11 +/- 0.25 nmoles/mg protein/min, respectively. The apparent K(m) and V(max) for the sulphoxidation of CPMS were 0.17 +/- 0.05 mM and 1.41 +/- 0.16 nmoles/mg protein/min, respectively. The apparent K(m) and V(max) for the sulphoxidation of DPS were 0.10 +/- 0.01 mM and 1.08 +/- 0.05 nmoles/mg protein/min, respectively. Methimazole noncompetitively inhibited the sulphoxidation of EMS, CPMS and DPS by human liver microsomes with K(i) values of 8.6 +/- 0.6, 5.7 +/- 0.4 and 6.6 +/- 0.5 mM, respectively. SKF525A noncompetitively inhibited the sulphoxidation of CPMS and DPS by human liver microsomes with K(i) values of 6.6 +/- 0.4 and 0.40 +/- 0.1 mM, respectively. The results suggest that FMO is involved in the sulphoxidation of EMS, CPMS and DPS while CYP450 is involved in the sulphoxidation of CPMS and DPS in human liver microsomes.     

Biochemical characterization of prostaglandin 19-hydroxylase of seminal vesicles

The microsomal fraction of homogenates of seminal vesicles of men and monkeys, Macaca fascicularis, were analyzed for prostaglandin (PG) 19-hydroxylase activity. The microsomes of the monkey seminal vesicles, supplemented with 1 mM NADPH, metabolized 0.2 mM PGE1 to 19-hydroxy-PGE1 at a mean rate of 0.26 nmol/min/mg of protein (with an apparent Km and an apparent Vmax of 40 microM and 0.30 nmol/min/mg of protein, respectively). The enzyme catalyzed the incorporation of atmospheric oxygen into the substrate. Substituting NADH for NADPH reduced the prostaglandin E1 19-hydroxylase activity to 40%. Carbon monoxide and proadifen (SKF 525A) inhibited the enzyme. Prostaglandin E2 (0.2 mM) was metabolized to 19-hydroxyprostaglandin E2 (0.2 nmol/min/mg of protein), but PGE1 was preferred as a substrate. Prostaglandin B1 was metabolized to 18-hydroxy-, 19-hydroxy-, and 20-hydroxyprostaglandin B1 at a combined rate of approximately 25% of prostaglandin E1. 19-Hydroxyprostaglandin B1 was the main product. The microsomes of human seminal vesicles metabolized 0.2 mM PGE2 to 19-hydroxy-PGE2 in the presence of 1 mM NADPH, while carbon monoxide inhibited this reaction. These results suggest that prostaglandin 19-hydroxylase of seminal vesicles might be a cytochrome P-450. The biosynthesis of 19-hydroxyprostaglandin E1 and 19-hydroxyprostaglandin E2 was also studied in vivo in man by analysis of the product/substrate ratios (i.e. 19-hydroxyprostaglandin E1/prostaglandin E1 and 19-hydroxyprostaglandin E2/prostaglandin E2) in a series of consecutive ejaculates, which were obtained during short intervals. There was a 10-fold interindividual difference in these ratios. Although the product/substrate ratios decreased, the 19-hydroxylation of E prostaglandins appeared to be efficient in vivo, which was in contrast to the rather slow biosynthesis in vitro.     

Functional characteristics of cytochromes P-4501A1 and P-4501A2 in rodent liver

Antibodies to mouse liver cytochrome P3-450 (anti-P3-450) and antibodies to rat liver cytochrome P-450d (anti-P-450d-c) inhibit the 0-deethylation of 7-ethoxyresorufin (ER) in liver microsomes of benz(a)pyrene-induced (BP) mice but do not inhibit the 0-deethylase activity in liver microsomes of BP-induced rats. Anti-P3-450 and anti-P-450c inhibit BP-hydroxylation in BP-induced mouse liver microsomes by 20%, but they do not inhibit this reaction at all in BP-induced rat liver microsomes. In a reconstituted monooxygenase system isolated cytochrome P3-450 metabolized 7-ER and BP. In contrast, its homologue, cytochrome P-450d, did not metabolize these substrates. The fraction containing cytochrome P1-450 metabolized 7-ER at a low rate and BP at a rate of 3.6 nmol product/min/nmol cytochrome. Western blot analysis with anti-P-450c + d revealed two bands in SDS-PAGE gels containing BP-induced mouse liver microsomes. The interaction of mouse liver BP-microsomes with anti-P3-450 and anti-P-450d-c was accompanied by the appearance of a single band (cytochrome P3-450).     

Functional characterization of human and cynomolgus monkey UDP-glucuronosyltransferase 1A6 enzymes

UDP-glucuronosyltransferase 1A6 (UGT1A6) is a major isoform in the human liver that glucuronidates numerous drugs, environmental chemicals and endogenous substrates. In this study, human and cynomolgus monkey UGT1A6 cDNAs (humUGT1A6 and monUGT1A6, respectively) were cloned, and the corresponding proteins were heterologously expressed in yeast cells to identify the functions of primate UGT1A6s. The enzymatic properties of UGT1A6 proteins were characterized by the kinetic analysis of serotonin (5-hydroxytryptamine, 5-HT) and 4-methylumbelliferone (4-MU) glucuronidation. humUGT1A6 and monUGT1A6 showed 96% identity in their nucleotide and amino acid sequences. Immunoblotting analysis using an antibody raised against human UGT1A6 showed that protein staining intensities were different between human and cynomolgus monkey UGT1A6 enzymes in microsomal fractions from livers and yeast cells, although both enzymes were detectable. The apparent K(m) value (15 mM) for 5-HT glucuronidation of cynomolgus monkey liver microsomes was significantly higher than that (8.6mM) of human liver microsomes, whereas V(max) values were lower in cynomolgus monkeys (2.8 nmol/min/mg protein) than in humans (8.6 nmol/min/mg protein). No significant species difference was observed in K(m) (approximately 90 microM) or V(max) (approximately 25 nmol/min/mg protein) values for liver microsomal 4-MU glucuronidation. In yeast cell microsomes, K(m) values (approximately 6mM) for 5-HT glucuronidation by recombinant UGT1A6s were similar, while a V(max) value (0.1nmol/min/mg protein) of monUGT1A6 was significantly lower than that (0.7 nmol/min/mg protein) of humUGT1A6. In 4-MU glucuronidation, both K(m) (210 microM) and V(max) (3.5 nmol/min/mg protein) values of monUGT1A6 were significantly higher than those of humUGT1A6 (K(m), 110 microM; V(max), 1.5nmol/min/mg protein). These findings suggest that the enzymatic properties of UGT1A6 were extensively different between humans and cynomolgus monkeys, although humUGT1A6 and monUGT1A6 showed high homology at the amino acid level. The information gained in this study should help with in vivo extrapolation and to assess the toxicity of xenobiotics.     

Cytochrome P450 enzymes involved in the metabolism of tetrahydrocannabinols and cannabinol by human hepatic microsomes

In this study, tetrahydrocannabinols (THCs) were mainly oxidized at the 11-position and allylic sites at the 7alpha-position for Delta(8)-THC and the 8beta-position for Delta(9)-THC by human hepatic microsomes. Cannabinol (CBN) was also mainly metabolized to 11-hydroxy-CBN and 8-hydroxy-CBN by the microsomes. The 11-hydroxylation of three cannabinoids by the microsomes was markedly inhibited by sulfaphenazole, a selective inhibitor of CYP2C enzymes, while the hydroxylations at the 7alpha-(Delta(8)-THC), 8beta-(Delta(9)-THC) and 8-positions (CBN) of the corresponding cannabinoids were highly inhibited by ketoconazole, a selective inhibitor of CYP3A enzymes. Human CYP2C9-Arg expressed in the microsomes of human B lymphoblastoid cells efficiently catalyzed the 11-hydroxylation of Delta(8)-THC (7.60 nmol/min/nmol CYP), Delta(9)-THC (19.2 nmol/min/nmol CYP) and CBN (6.62 nmol/min/nmol CYP). Human CYP3A4 expressed in the cells catalyzed the 7alpha-(5.34 nmol/min/nmol CYP) and 7beta-hydroxylation (1.39 nmol/min/nmol CYP) of Delta(8)-THC, the 8beta-hydroxylation (6.10 nmol/min/nmol CYP) and 9alpha,10alpha-epoxidation (1.71 nmol/min/nmol CYP) of Delta(9)-THC, and the 8-hydroxylation of CBN (1.45 nmol/min/nmol CYP). These results indicate that CYP2C9 and CYP3A4 are major enzymes involved in the 11-hydroxylation and the 8-(or the 7-) hydroxylation, respectively, of the cannabinoids by human hepatic microsomes. In addition, CYP3A4 is a major enzyme responsible for the 7alpha- and 7beta-hydroxylation of Delta(8)-THC, and the 9alpha,10alpha-epoxidation of Delta(9)-THC.     

Stimulation of CTP: phosphocholine cytidylyltransferase and phosphatidylcholine synthesis by calcium in rat hepatocytes

The effects of Ca2+, ionophore A23187, and vasopressin on CTP:phosphocholine cytidylyltransferase were investigated. Cytidylyltransferase is present in the cytosol and in a membrane-bound form on the microsomes. Digitonin treatment caused release of the cytosolic form rapidly. Addition of 7 mM Ca2+ to hepatocyte medium resulted in a 3-fold decrease in cytidylyltransferase released by digitonin treatment (1.7 +/- 0.1 nmol/min per mg compared to 5.1 +/- 0.2 nmol/min per mg in the control). Verapamil, a calcium channel blocker, partially overcame this effect of Ca2+. Ionophore A23187 and vasopressin both mimicked the effect of Ca2+ and resulted in a decrease in cytidylyltransferase release (2.4 +/- 0.1 nmol/min per mg and 2.5 +/- 0.2 nmol/min per mg, respectively) compared to control (3.4 +/- 0.1 nmol/min per mg). In agreement with the digitonin experiments, incubation with 7 mM Ca2+ resulted in a decrease in cytidylyltransferase in the cytosol (from 4.0 to 1.2 mol/min per mg) and a corresponding increase in the microsomes (from 0.6 to 2.4 nmol/min per mg). Verapamil partially blocked this translocation caused by Ca2+. Ionophore A23187 and vasopressin also caused translocation of the cytidylyltransferase from the cytosol to the microsomes. The addition of Ca2+ also resulted in an increase in PC synthesis. With 7 mM Ca2+ in the medium, the label associated with PC increased to 3.8 +/- 0.1.10(6) dpm/dish from 2.7 +/- 0.1.10(6) dpm/dish after 10 min. PC degradation was also affected, since 7 mM Ca2+ in the medium resulted in an increase in LPC formation both in the cell and the medium. We conclude that high concentrations of calcium in the hepatocyte medium can cause a stimulation of CTP:phosphocholine cytidylyltransferase and PC synthesis in cultured hepatocytes.     

7 alpha-hydroxylation of 27-hydroxycholesterol in human liver microsomes.

Human liver microsomes were found to catalyze 7 alpha-hydroxylation of 27-hydroxycholesterol at a rate of up to about 0.2 nmol/mg protein per min. The product of the reaction, 5-cholestene-3 beta, 7 alpha, 27-triol, was identified by means of combined gas chromatography-mass spectrometry. Liver microsomes from two patients with an upregulated cholesterol 7 alpha-hydroxylase, did not have higher 7 alpha-hydroxylase activity towards 27-hydroxycholesterol than those from untreated patients, suggesting that the 7 alpha-hydroxylase active towards 27-hydroxycholesterol is not the same as that active towards cholesterol. The mitochondrial fraction of liver from untreated patients and patients treated with cholestyramine, had negligible 7 alpha-hydroxylase activity towards 27-hydroxycholesterol less than 0.01 nmol/mg protein per min). The results are in accord with the possibility that there is a pathway to bile acids in human liver in which the first step is a 27-hydroxylation of cholesterol.     

Cocaine metabolism in human fetal and adult liver microsomes is related to cytochrome P450 3A expression

Cocaine N-demethylation to norcocaine was studied in human liver microsomes of different ages. Norcocaine was formed at a considerable rate in fetal (45.4+/-18.2 nmol/mg x hour, n = 8) and adult specimens (82.0+/-46.6 nmol/mg x hour, n = 15), p = 0.04 (Mann-Whitney). Furthermore, the apparent Km values in fetal specimens (0.57 and 0.48 mM, n = 2) showed a higher affinity compared with those of adults (mean value 2.7 (1.8-4.25) mM, n = 4). Estimated enzyme metabolic clearance with respect to P450 total content was higher in fetal than in adult liver microsomes (2.22 ml/nmol P450 x hour, and 0.18 (0.14-0.23) ml/nmol P450 x hour, respectively). Several drugs, known to be CYP3A substrates, were used as potential inhibitors of cocaine metabolism. Midazolam, ergotamine and erythromycin showed strong inhibition (approx. 70 %) when used at concentrations of 500 microM (midazolam, erythromycin) or 200 microM (ergotamine). The metabolism of 1 mM cocaine correlated strongly with immunodetected CYP3A protein determined by Western blotting in both fetal (r = 0.89, p = 0.19) and adult specimens (r = 0.82, p < 0.01) . These findings further support CYP3A as a major catalyst of norcocaine formation in human liver microsomes. These results are important given the potential risk of toxicity to the foetus of maternal cocaine abuse during pregnancy. Although the high Km values found in adult livers reduce the importance of this enzyme pathway in cocaine detoxication, this pathway would emerge as significant in circumstances of CYP3A induction and/or drug interactions leading to potential liver toxicity in chronic cocaine abusers.     

Inositol 1,4,5-trisphosphate-triggered Ca2+ release from bovine adrenal medullary secretory vesicles

The effect of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and calcium ionophore A23187 on Ca2+ release from bovine adrenal medullary secretory vesicles and microsomes was examined. Ins(1,4,5)P3 released 3.5 nmol of Ca2+/mg protein from secretory vesicles and 1.5 nmol of Ca2+/mg protein from microsomes as measured by a Ca2(+)-selective electrode. However, A23187 promoted Ca2+ uptake into vesicles while releasing Ca2+ from microsomes. Ins(1,4,5)P3-induced Ca2+ release from secretory vesicles was rapid, but the released Ca2+ was absorbed within 3 min during which the Ins(1,4,5)P3-releasable pools were refilled. The in situ calcium content of secretory vesicle measured by atomic absorption spectrometry was 112 +/- 6.3 nmol/mg protein indicating the potential importance of secretory vesicles as an intracellular Ca2+ store. The high Ca2(+)-buffering capacity of secretory vesicles is presumed to be due to the high Ca2(+)-binding capacity of chromogranin A, the major intravesicular protein, which has calsequestrin-like properties.     

Evidence for the presence of active cytochrome P450 systems in Schistosoma mansoni and Schistosoma haematobium adult worms

Extracts of the adult worms of both Schistosoma mansoni and Schistosoma haematobium can metabolise some typical P450 substrates but to differing degrees. S. mansoni worm extracts displayed a approximately 12-fold higher specific activity for an aminopyrine substrate than rat liver microsomes. At 4 mM substrate concentration the demethylation reaction with N-nitrosodimethylamine (NDMA) (5 nmol HCHO/mg protein/min) was only half that of rat liver microsomes, whereas in extracts of S. haematobium, no detectable activity was found towards NDMA. Using ethylmorphine as substrate the demethylation activity of S. mansoni extracts (1.82 nmol HCHO/mg protein/min) was 5.5-fold lower than that of rat liver microsomes. Benzphetamine demethylase activity was also readily detectable in S. mansoni worm extracts at 6.79 nmol HCHO/mg protein/min compared with 10.20 nmol HCHO/mg protein/min in the case of rat liver microsomes. When aniline was used as substrate, surprisingly, no activity was found in worm extracts of either S. mansoni or S. haematobium, whereas rat liver microsomes showed high activity towards this amine. The anti-P450 2E1 and 2B1/2 cross-reacted with both worm homogenates and gave a specific band corresponding to a protein of molecular weight of approximately 50.0 kDa. A study with anti-P450 IVA antibody revealed that while this protein was strongly expressed in S. haematobium worm extracts, no immunoreactivity was observed with extracts of S. mansoni. Immunoblotting analyses with anti-P450 IIIA and P450 1A1 did not detect immunoreactive protein in either S. mansoni or S. haematobium.     

Catalytic activity of isolated cytochromes P-450d and P-450c

Cytochrome P-450d was isolated from isosafrol-induced rat liver microsomes by affinity chromatography on 1.8-diaminooctyl-Sepharose 4B and chromatography on hydroxylapatite using a linear potassium phosphate gradient (45-250 mM). The enzyme has a molecular mass of 54 kDa, CO-maximum 448 nm is characterized by a high spin state; the rate of 4-aminobiphenyl hydroxylation is 54 nmol/min/nmol of cytochrome P-450d (37 degrees C), those, of 7-ethoxyresorufin O-deethylation and benz (a) pyrene oxidation are 1 nmol/min/nmol of cytochrome P-450d (22 degrees C) and 2 nmol/min/nmol of cytochrome P-450d (37 degrees C), respectively. The properties of cytochrome P-450d were compared to those of cytochrome P-450c isolated from 3-methylcholanthrene-induced rats. The yield of these cytochromes under the conditions used (10% P-450d from isosafrol-induced microsomes and 15% P-450c from 3-methylcholanthrene-induced microsomes) was relatively high. Antibodies to cytochromes P-450d and P-450c were obtained. Using rocket immunoelectrophoresis the percentage of these hemoprotein forms in 3-methylcholanthrene-induced (P-450d-20%, P-450c-70%) and isosafrol-induced rat liver microsomes (P-450d-50%, P-450c-15%) was determined.     

The proximate carcinogen trans-3,4-dihydroxy-3,4-dihydro-dibenz[c,h]acridine is oxidized stereoselectively and regioselectively by cytochrome 1A1, epoxide hydrolase and hepatic microsomes from 3-methylcholanthrene-treated rats.

Metabolism of the proximate carcinogen trans-3,4-dihydroxy-3,4-dihydrodibenz[c,h]acridine has been examined with rat liver enzymes. The dihydrodiol is metabolized at a rate of 2.4 nmol/nmol of cytochrome P450 1A1/min with microsomes from 3-methylcholanthrene-treated rats, a rate more than 10-fold higher than that observed with microsomes from control or phenobarbital-treated rats. Major metabolises consisted of a diastereomeric pair of bis-dihydrodiols (68-83%), where the new dihydrodiol group has been introduced at the 8,9-position, tetraols derived from bay region 3,4-diol-1,2-epoxides (15-23%), and a small amount of a phenolic dihydrodiol(s) where the new hydroxy group is at the 8,9-position of the substrate. A highly purified monooxygenase system reconstituted with cytochrome P450 1A1 and epoxide hydrolase (17 nmol of metabolites/nmol of cytochrome P450 1A1/min) gave a metabolite profile very similar to that observed with liver microsomes from 3-methylcholanthrene-treated rats. Study of the stereoselectivity of these microsomes established that the (+)-(3S,4S)-dihydrodiol gave mainly the diol epoxide-1 diastereomer, in which the benzylic 4-hydroxyl group and epoxide oxygen are cis. The (-)-(3R,4R)-dihydrodiol gave mainly diol epoxide-2 where these same groups are trans. The major enantiomers of the diastereomeric bis-dihydrodiols are shown to have the same absolute configuration at the 8,9-position. Correlations of circular dichroism spectra suggest this configuration to be (8R,9R). The (8R,9S)-oxide may be their common precursor.     

Biosynthesis of [3H]7 alpha-hydroxy-, 7 beta-hydroxy-, and 7-oxo-dehydroepiandrosterone using pig liver microsomal fractions

Current research on dehydroepiandrosterone (DHEA) is limited due to lack of radiolabeled metabolites. We utilized pig liver microsomal (PLM) fractions to prepare [(3)H]-labeled 7 alpha-hydroxy-DHEA (7 alpha-OH-DHEA), 7 beta-hydroxy-DHEA (7 beta-OH-DHEA), and 7-oxo-DHEA substrates from 50 microM [1,2,6,7-(3)H]DHEA (specific radioactivity 60-80 mCi/mmol). The metabolites were separated by preparative thin-layer chromatography (TLC) using ethyl acetate:hexane:glacial acetic acid (18:8:3 v:v:v) as the mobile phase, extracted with ethyl acetate, and dried under a stream of nitrogen. Metabolites assayed by TLC and gas chromatography-mass spectrometry were observed to be pure. In the presence of an reduced nicotinamide adenine dinucleotide phosphate (NADPH)-regenerating system initiated with 1 mM NADPH alone, 1 mg/ml PLM produced 7 alpha-OH-DHEA with minor amounts of 7-oxo-DHEA (68 and 14 nmol/2h/2 ml, respectively; 82% conversion), while in the presence of 1mM NADPH and 1 mM oxidized nicotinamide adenine dinucleotide phosphate (NADP(+)), more 7-oxo-DHEA than 7 alpha-OH-DHEA (58 and 11 nmol/2 ml/120 min, respectively; 69% conversion) was formed. When longer reaction times were used with NADPH and NADP(+), a mixture of 7 alpha-OH-DHEA, 7 beta-OH-DHEA, and 7-oxo-DHEA was produced (19,14, and 35 nmol/180 min/2 ml, respectively; 62% conversion). Using pig liver microsomes, the radiolabeled metabolites of DHEA can be prepared in stable, pure form at 10mM concentrations and >0.5 mCi/mmol levels of radioactivity for biochemical studies.