Metabolism of [6,7-3H, 35S] estradiol 17-sulfate in rats

To confirm whether or not the sulfo group of estradiol 17-sulfate (ES) is removed during in vivo metabolism in rats, the doubly labeled conjugate [6,7-3H, 35S] ES was injected into rats, and its biliary and urinary metabolites were determined by reverse isotope dilution method (RIDM). In male rats, the major radioactivity was detected in biliary disulfate fraction, which was composed of mainly ES and its two minor metabolites, 2-hydroxyestradiol 17-sulfate (2-OH-ES) and 2-methoxyestradiol 17-sulfate (2-MeO-ES). In female rats, in contrast, the radioactivity was dispersed into three fractions:biliary monosulfate, biliary disulfate, and urinary monosulfate fractions (Frs.) In both monosulfate Frs., 7beta-hydroxyestradiol 17-sulfate was detected as the major metabolite followed by 6alpha-, 6beta-, and 15beta-hydroxyestradiol 17-sulfates. Like male rats, 2-OH-ES and 2-Meo-ES as the minor products were detected in biliary disulfate fraction. The isotope ratios of ES and its metabolites in both sexes were essentially the same as that of the dose except that of 6alpha-hydroxylated metabolite, which may be derived from the loss of the tritium labeled at C6. These results confirm the occurrence of the direct metabolism of ES in rats.     

Synthesis of 6- and 7-hydroxyestradiol 17-sulfates: the potential metabolites of estradiol 17-sulfate by female rat liver microsomes.

The potential ring-B hydroxylated metabolites of estradiol 17-sulfate (1) by female rat liver microsomes were chemically prepared as authentic compounds. They are 6alpha- and 6beta-hydroxyestradiol 17-sulfates (7 and 9), and 7alpha- and 7beta-hydroxyestradiol 17-sulfates (12 and 16), whose synthetic procedures are described.     

The metabolism of estrone and estradiol-17β and their 3-sulfates by female guinea pig liver microsomes

The metabolism, by female guinea pig liver microsomes of estrogen 3-sulfates (estrone-3-sulfate and 17β-estradiol-3-sulfate) was compared to that of the unconjugated estrogens, estrone and estradiol-17β. Metabolites identified indicated that 16β-hydroxylated products (16β hydroxyestrone and 16 epiestriol) arose mainly from the free estrogens while 16α-hydroxy steroid sulfates (16α hydroxyestrone-3-sulfate and estriol-3-sulfate) were predominantly formed from the sulfated estrogens. These results show that the sulfate moiety at position 3 of the steroids directs 16-hydroxylation from the β to the α configuration.     

Metabolism and hepatotoxicity of the naturally occurring benzo[b]pyran precocene I

The in vitro metabolism of precocene I by liver microsomes from control and treated rats and the effects of precocene I on the function and histology of the rat liver were examined. The major metabolites (80-90% of total metabolites) from all microsomal preparations were the cis and trans 3,4-diols of precocene I produced with a cis/trans isomer ratio of 1:2. These diols appear to arise mainly by spontaneous hydrolysis of precocene I 3,4-oxide. (+)-(3R,4R)-cis- and (-)-(3R,4S)-trans-precocene I 3,4-diols were the predominant enantiomers of the 3,4-diol formed. The enantiomeric excess of these diols (2-50%) is dependent on the microsomal preparation, with microsomes from control rats exhibiting the highest stereoselectivity and microsomes from phenobarbital-treated rats the least. 6-Hydroxyprecocene I was the next major metabolite and was formed to the extent of 5% (control), 10% and 17% (phenobarbital and 3-methylcholanthrene treatment, respectively) of total metabolites. Treatment of rats with a single i.p. dose of precocene I (300 mg/kg) resulted in extensive hepatic damage as evidenced by a marked increase of plasma glutamic pyruvic transaminase levels and histologic observation in liver sections of severe centrolobular necrosis. Although phenobarbital treatment of rats increased the rate of liver microsomal metabolism of precocene I by approximately 50% (nmol products/nmol cytochrome P-450/min) compared to liver microsomes from control rats, hepatic damage caused by precocene I was not significantly affected. Depletion of glutathione levels in the rats with diethyl maleate prior to precocene I treatment dramatically increased the severity of hepatic insult, whereas treatment of the rats with the mixed function oxidase inhibitor piperonyl butoxide prior to treatment with precocene I blocked hepatic damage. Treatment of rats with cysteamine prior to treatment with precocene I protected the animals against the toxic effects. Neither cis nor trans precocene I 3,4-diol nor 3,4-dihydroprecocene I elicited impaired liver function or cellular damage. The above results are consistent with the view that precocene I 3,4-oxide is the metabolite responsible for the hepatotoxic effects observed when precocene I is injected into rats.     

Sulfatides of Mycobacterium tuberculosis. Synthesis of the core alpha,alpha-trehalose 2-sulfate

alpha,alpha-Trehalose 2-sulfate, the core carbohydrate of sulfatides of Mycobacterium tuberculosis, and the 3-sulfate isomer were synthesized by sulfation of 4,6:4',6'-di-O-benzylidene-alpha,alpha-trehalose with pyridine-sulfur trioxide complex to give the 2- and 3-sulfates, which were separated by column chromatography. The ammonium 2-sulfate salt wa was identical with the natural product obtained from the principal sulfatide (SL-I) of M. tuberculosis.     

Chondroitin sulfates of the epiphysial cartilages of different mammals.

1. The distribution chondroitin 4- and 6-sulfates in the epiphysial cartilages of several mammals are reported. 2. Chondroitin 6-sulfate is present in higher relative proportion in articular surfaces of young and adult epiphysial cartilages in most of the mammals studied. 3. Exception to this was found in some species of the order Rodentia in which chondroitin 4-sulfate was almost the only chondroitin present in young and adult cartilages. 4. These and other results suggest that chondroitin 4-sulfate may be an important component for the calcification process, whereas chondroitin 6-sulfate seems to be related to the integrity of the articular surfaces.     

The physiological role of estradiol 17-sulfate during pregnancy

To clarify the physiological role of estradiol 17-sulfate (ES) during pregnancy, experiments were conducted and the following results were obtained: (1) serum or urinary ES levels rose as a function of gestational age; (2) placental microsomes showed fairly high 2- and 4-hydroxylase activity for ES; and (3) the catechol products, 2- and 4-hydroxy-ES, had a strong inhibitory effect upon the in vitro production of lipid peroxides. These results suggest that ES actsas a precursor to the catechol metabolites which maintain normal gestation. This is coincident with the negative correlation of serum levels in ES and lipid peroxides observed in late pregnancy.     

Cytochrome P-450-dependent metabolism of xenobiotics. A comparative study of rat hepatic and plant microsomal metabolism

1. A comparison was made between rat hepatic and plant microsomal cytochrome P-450 and cytochrome P-450 linked enzymic activities. 2. The results show that, compared with plant microsomes, rat hepatic microsomal protein concentrations were 165-fold higher, and rat hepatic cytochrome P-450 concentration were 32-fold higher. 3. Rat hepatic Cytochrome P-450 linked enzyme activities were 1765-fold and 25-fold greater when compared with plant microsomes using aldrin and biphenyl as substrates, respectively. 4. Rats metabolised biphenyl to 2- and 4-hydroxybiphenyl, whereas plants produced only the latter metabolite. 5. Pretreatment of rats and plant tissues with biphenyl, Aroclor 1248 and the sodium salt of phenobarbital increased significantly the microsomal protein concentrations, and enzyme activities linked to cytochrome P-450. 6. Unlike rat microsomes, those of plants were unable to metabolise halosubstituted biphenyls at measurable rates.     

High-performance liquid chromatographic method for measurement of cytochrome P450-mediated metabolism of 7-ethoxy-4-trifluoromethylcoumarin

An HPLC method for analysis of deethylation of 7-ethoxy-4-trifluoromethylcoumarin (ETFMC), a substrate of various enzymes of the cytochrome P450 superfamily, was developed. ETFMC was incubated at 37°C with human hepatic microsomes or microsomes prepared from a lymphoblastoid cell line that expresses human CYP2B6. Under these conditions, the highly fluorescent metabolite 7-hydroxy-4-trifluoromethylcoumarin (HTFMC) is formed. The metabolite was analyzed by reversed-phase HPLC with fluorescence detection. The limits of detection of the metabolite were 5.0 fmol per injection, a sensitivity at least one order of magnitude greater than the standard method, which does not involve HPLC. This method will be of great utility when quantities of microsomal protein from cell lines expressing human CYP enzymes are limited.     

Testosterone metabolism by cytochrome P-450 isozymes RLM3 and RLM5 and by microsomes. Metabolite identification

Testosterone metabolism by cytochrome P-450 isozymes RLM3 and RLM5 in a reconstituted system and by rat liver microsomes was examined. Eleven metabolites were detected. Two of these, found in spots 2 and 4 of a thin layer plate, were only formed by the rat liver microsomes and may represent reductive metabolites of testosterone. A number of monohydroxy metabolites were conclusively identified by gas chromatography-mass spectrometry. These include the 2-, 6 beta-, 7 alpha-, and 16 alpha-hydroxy isomers. Liver microsomes formed the 2 alpha- and 2 beta-epimers in a 1:2 ratio and both co-chromatographed with a third reduced metabolite in thin layer plate spot 4. In contrast with RLM5 about 90% of the 2-hydroxy isomer was the 2 alpha-epimer. RLM3 did not perform the 2-hydroxylation in detectable amounts. The 6 beta-isomer was a major metabolite of RLM3 and microsomes, but a minor product of metabolism by RLM5. In contrast, the 7 alpha-isomer was a minor metabolite of RLM3, was not formed by RLM5, and was a major microsomal metabolite. Hydroxylation at position 16 alpha was a major activity of RLM5 and the heterogeneous microsomal cytochromes, but with RLM3 it was a minor reaction. One new metabolite was found which appeared to be hydroxylated in the D-ring, had a mass spectrum different from both 16 alpha- and 16 beta-hydroxytestosterone, and was tentatively identified as a 15-hydroxy isomer. In agreement with the literature, androstene-3,17-dione was found to be an oxidative metabolite of testosterone by both microsomes and purified cytochrome P-450. It was a major metabolite of RLM5 but was not produced by RLM3. Studies with 18O2 and H218O conclusively show that oxidation of testosterone at C-17 does not involve transient incorporation of an oxygen atom in this position. A mechanism is suggested whereby cytochrome P-450 acts as a peroxidase in the formation of androstenedione.     

The distribution of chondroitin sulfates in articular and growth cartilages of human bones.

The relative contents of chondroitin 4- and 6-sulfates in cartilages of different human bones are reported. Articular and vertebral body cartilages contain almost exclusively chondroitin 6-sulfate, whereas growth and subarticular cartilages contain nearly equal amounts of chondroitin 4-sulfate and chondroitin 6-sulfate. Adult cartilages, where the calcification process is complete, contain only chondroitin 6-sulfate. These results that chondroitin 4-sulfate may be an important component for the calcification process, whereas chondroitin 6-sulfate seems to be related to the integrity of the articular surfaces. A chemical defect of chondroitin 6-sulfate in a new mucopolysaccharidosis, characterized by platyspondyly and irregularities of articular surfaces, is in agreement with these results.     

Microsomal metabolism of carbon tetrachloride: participation of pyridine nucleotide synergism

The role of pyridine nucleotide synergism in CCl4 metabolism was evaluated for its potential contribution to enhanced lipid peroxidation. Male Sprague-Dawley rats receiving either no treatment (control) or treatment with phenobarbital (PB) were used to prepare hepatic microsomes. Metabolism was evaluated in the presence and absence of an NADPH generator system and in the presence and absence of NADH. The generator system produced a greater extent of metabolism for both control and PB microsomes. NADH-catalyzed CCl4 metabolism occurred to a similar extent in control and PB microsomes, amounting to 9-10% and 5-6% of the NADPH rate in control and PB microsomes, respectively. Synergism by NADH occurred at the lowest concentrations of NADPH, apparently decreasing the Km for NADPH and having little effect on the Vmax. Addition of NAD+ produced synergism, as did the addition of 5' AMP, an inhibitor of nucleotide pyrophosphatase. Thus, the synergistic increase in CCl4 metabolism produced by NADH may occur in part from an increased availability of NADPH, as a result of decreased degradation, rather than by electron donation from NADH.     

The distributions of chondroitin sulfates in articular and growth cartilages of human bones

The relative contents of chondroitin 4- and 6-sulfates in cartilages of different human bones are reported. Articular and vertebral body cartilages contain almost exclusively chondroitin 6-sulfate, whereas growth and subarticular cartilages contain nearly equal amounts of chondroitin 4-sulfate and chondroitin 6-sulfate. Adult cartilages, where the calcification process is complete, contain only chondroitin 6-sulfate. These results suggest that chondroitin 4-sulfate may be an important component for the calcification process, whereas chondroitin 6-sulfate seems to be related to the integrity of the articular surfaces. A chemical defect of chondroitin 6-sulfate in a new mucopolysaccharidosis, characterized by platyspondyly and irregularities of articular surfaces, is in agreement with these results.     

Intestinal and hepatic microsomal metabolism of testosterone and progesterone by a 3 alpha-hydroxysteroid dehydrogenase to the 3 alpha-hydroxy derivatives in the channel catfish,Ictalurus punctatus

Intestinal or hepatic microsomes from channel catfish converted [4-14C]-testosterone to three major metabolites: 6 beta-hydroxytestosterone, androstenedione and a third metabolite. Formation of the unknown metabolite required NADPH as cofactor. When incubated with 200 microM testosterone, the rate of formation of the unknown metabolite was 265+/-158 pmol/(min mg) protein (mean+/-S.D.) in microsomes from the proximal intestine, 515+/-93 pmol/(min mg) protein in distal intestine and 226+/-42 pmol/(min mg) protein in hepatic microsomes. Comparison of the chromatographic and spectral properties of the unknown metabolite with those of authentic testosterone derivatives showed that this metabolite corresponded to 4-androstene-3 alpha,17 beta-diol. No 3 alpha-reduced metabolite was formed in incubations of testosterone with catfish intestinal cytosol. Testosterone was reduced to 5 alpha-dihydrotestosterone primarily in the cytosolic fraction and not in microsomes. Incubation of progesterone with intestinal microsomes resulted in the formation of a metabolite with properties similar to that of the 3 alpha-reduced testosterone, and this metabolite was identified by co-chromatography with authentic standard as 3 alpha-reduced progesterone. Thus, 3 alpha-hydroxysteroid dehydrogenase is an important pathway in intestinal microsomes of the channel catfish.     

Metabolism of benzo[a]pyrene VI. Stereoselective metabolism of benzo[a]pyrene and benzo[a]pyrene 7,8-dihydrodiol to diol epoxides

(±)-7β,8α-Dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-1) and (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-2) are highly mutagenic diol epoxide diastereomers that are formed during metabolism of the carcinogen (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene. Remarkable stereoselectivity has been observed on metabolism of the optically pure (+)- and (?)-enantiomers of the dihydrodiol which are obtained by separation of the diastereomeric diesters with (?)-α-methoxy-α-trifluoromethylphenylacetic acid. The high stereoselectivity in the formation of diol epoxide-1 relative to diol epoxide-2 was observed with liver microsomes from 3-methylcholanthrene-treated rats and with a purified cytochrome P-448-containing monoxygenase system where the (?)-enantiomer produced a diol epoxide-2 to diol epoxide-1 ratio of 6 : 1 and the (+)-enantiomer produced a ratio of 1 : 22. Microsomes from control and phenobarbital-treated rats were less stereospecific in the metabolism of enantiomers of BP 7,8-dihydrodiol. The ratio of diol epoxide-2 to diol epoxide-1 formed from the (?)- and (+)-enantiomers with microsomes from control rats was 2 : 1 and 1 : 6, respectively. Both enantiomers of BP 7,8-dihydrodiol were also metabolized to a phenolic derivative, tentatively identified as 6,7,8-trihydroxy-7,8-dihydrobenzo[a]pyrene, which accounted for ～30% of the total metabolites formed by microsomes from control and phenobarbital-pretreated rats whereas this metabolite represents ～5% of the total metabolites with microsomes from 3-methylcholanthrene-treated rats. With benzo[a]pyrene as substrate, liver microsomes produced the 4,5-, 7,8- and 9,10-dihydrodiol with high optical purity (>85%), and diol epoxides were also formed. Most of the optical activity in the BP 7,8-dihydrodiol was due to metabolism by the monoxygenase system rather than by epoxide hydrase, since hydration of (±)-benzo[a]pyrene 7,8-oxide by liver microsomes produced dihydrodiol which was only 8% optically pure. Thus, the stereospecificity of both the monoxygenase system and, to a lesser extent, epoxide hydrase plays important roles in the metabolic activation of benzo[a]pyrene to carcinogens and mutagens.     

The metabolism of estradiol 17-sulfate by pheochromocytoma tissue

The metabolism of estradiol 17-sulfate by subcellular localization enzymes of pheochromocytoma tissue obtained from a 41-year old female was investigated. In any incubations under the presence of NADH and NADPH, metabolites hydroxylated at the C-2, C-4, C-6 beta, C-7 alpha and C-7 beta positions were produced. These hydroxylations are considered to occur without cleavage of the sulfate group. The 2-hydroxylation at the substrate concentration of 100 microM by mitochondria, microsomes and cytosol fractions occurred at rates of 141, 222 and 167 pmol/mg protein/30 min, respectively; the corresponding rates for the 4-hydroxylation were 24, 40 and 38 pmol/mg protein/30 min. Mitochondrial 2- and 4-hydroxylations were enhanced by addition of calcium ion (Ca2+) into the incubation medium.     

Metabolism of [4-14C]estrone in hamster and rat hepatic and renal microsomes: species-, sex- and age-specific differences.

The metabolism of [4-14C]estrone (E1) was examined in liver and kidney microsomes of adult castrated male and ovariectomized female hamsters and rats and in neonatal and immature hamster renal microsomes. In castrated male hamster liver microsomes, E1 was metabolized extensively to six major metabolites; 15 beta-hydroxyestrone, 7 alpha-hydroxyestrone, 6 alpha-hydroxyestrone, 6 beta-hydroxyestrone, 2-hydroxyestrone, and delta(9,11)-dehydroestrone, and a nonpolar fraction. Six minor metabolites of E1 were also detected. In contrast, kidney microsomes derived from castrated male hamsters metabolized E1 to mainly 17 beta-estradiol, 2- and 4-hydroxyestrone, 6 alpha-hydroxyestrone, 6 beta-hydroxyestrone and one monohydroxyestradiol metabolite. However, 16 alpha-hydroxyestrone was not detected. A variable, but low amount of estriol was also found. Interestingly, the quantity of 2-hydroxyestrone found in kidney microsomes of the hamster represented 26% of the total amount of metabolites formed, whereas in liver microsomes, only 9% of the overall metabolism resulted in the formation of 2-hydroxyestrone. The ability of kidney microsomes of female ovariectomized hamsters and two different rat strains to metabolize E1 was 5.9- and 9.4-fold lower, respectively, compared to renal microsomes of male castrated hamsters. The onset of oxidative metabolism in newborn hamster kidneys during development was also assessed. The results indicate that the oxidative metabolism of [14C]E1 in renal microsomes of newborn hamsters was 20-fold less than in kidney microsomes of adult hamsters. While catechol E1 metabolites were essentially negligible in hamster kidneys of these ages, it was evident that the conversion of E1 to estradiol via 17 beta-hydroxysteroid dehydrogenase resembles levels seen in the adult animals. Between the age of one and two months, the male hamster kidney exhibited the capacity to metabolize E1 at levels seen in fully mature adult hamsters.     

Benzanthrone: a new substrate for hepatic microsomal cytochrome P-450

The metabolism of benzanthrone, a commonly used dy intermediate, by rat hepatic microsomes was investigated using thin layer chromatography (TLC) analysis. Incubation of benzanthrone with hepatic microsomes in the presence of NADPH generating system produced at least seven fluorescent metabolites on TLC plates. TLC spots numbered II, III, IV, V and VI were the major metabolites obtained from hepatic microsomes with the Rf values of 0.53, 0.45, 0.38, 0.33 and 0.26, respectively. Metabolites VII and VIII were faint bands with Rf values of 0.08 and 0.04, respectively. Preincubation of hepatic microsomes with either 1-benzyl-imidazole (10(-4)M) or SKF-525 A (10(-4)M) or metyrapone (10(-3)M) or flushing with carbon monoxide substantially inhibited the benzanthrone metabolism. alpha-Naphtho-flavone (10(-4)M) did not cause any change in hepatic microsomal metabolism of benzanthrone. Oral administration of benzanthrone to animals yielded at least six urinary metabolites. TLC spots numbered II, III, IV, V and VI in the urine were same as those of hepatic microsomal metabolites. However, one of the urinary metabolite numbered IX which stays at the origin of TLC plate with the Rf value of 0.05 may be a conjugate. Our results suggest that benzanthrone acts as a substrate for hepatic heme protein, cytochrome P-450 and that some of the metabolites are excreted in urine.     

The characteristics of the microsomal hydroxylation of tolbutamide

The in vitro metabolism of tolbutamide to the hydroxymethyl derivative was studied using hepatic microsomal homogenates. The hydroxymethyl metabolite was quantitated by HPLC. The hepatic microsomal hydroxylase was completely inhibited by carbon monoxide and was NADPH dependent. Metyrapone, alpha-naphthoflavone, phenelzine, mercuric chloride, and nitrogen significantly inhibited the reaction indicating the involvement of the cytochrome P-450 monooxygenase. Species variation showed that the order of hepatic microsomal activity was rat greater than rabbit much greater than guinea pig much greater than mouse and hamster. The reaction increased with time up to 40 min and followed Michaelis-Menten kinetics in rat liver microsomes with apparent Km and Vmax values of 224.4 microM and 359.9 pmol.mg-1.min-1, respectively. The reaction was induced by phenobarbital but was depressed after pretreatment with 3-methylcholanthrene and isosafrole. However, expression of the hydroxylase activity per nanomoles of cytochrome P-450 showed that the activity was much higher in liver microsomes of isosafrole pretreated rats. These results indicate the involvement of different isozymes of cytochrome P-450 in the microsomal hydroxylation of tolbutamide.     

Expression of four phenobarbital-inducible cytochrome P-450s in liver, kidney, and lung of rats

Specific antibodies were prepared against cytochromes P450 PB-1, PB-2, PB-4, and PB-5 purified from hepatic microsomes of male rats treated with phenobarbital. With these antibodies, the levels of these four cytochrome P450s in hepatic, renal, and pulmonary microsomes of male rats that were untreated, treated with phenobarbital, or treated with 3-methylcholanthrene were examined. P450 PB-1 and PB-2 were present in moderate amounts in hepatic microsomes of untreated male rats and were induced 2- to 3-fold with phenobarbital. Also, the expression of these forms was suppressed by 3-methylcholanthrene. These forms were not detected in the renal or pulmonary microsomes of untreated rats or rats treated with phenobarbital or 3-methylcholanthrene. P450 PB-4 and PB-5 were found in the hepatic microsomes of untreated male rats at a low level but were induced with phenobarbital more than 50-fold. P450 PB-4 and PB-5 were not detected in renal microsomes; only P450 PB-4 or a closely related form was present in the pulmonary microsomes of untreated male rats, and its level was not changed by phenobarbital treatment. The constitutive presence of P450 PB-4 in pulmonary microsomes was confirmed by the investigation of testosterone metabolism. Purified P450 PB-4 had high testosterone 16 alpha- and 16 beta-hydroxylation activity in a reconstituted system. The testosterone 16 beta-hydroxylation activity of hepatic microsomes was induced with phenobarbital, and more than 90% of the testosterone 16 beta-hydroxylation activity of hepatic microsomes from rats treated with phenobarbital was inhibited by anti-P450 PB-4 antibody.(ABSTRACT TRUNCATED AT 250 WORDS)