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PubMed1996

Selective suppression of rat hepatic microsomal activity during chronic cyclosporine nephrotoxicity.

Cyclosporine is an immunosuppressant that undergoes extensive hepatic biotransformation to hydroxylated and demethylated metabolites. At present, the CYP3A gene family is thought to be the primary enzyme system responsible for cyclosporine metabolism. The effect of chronic cyclosporine therapy on the suppression of drug metabolism was studied in male and female rats maintained on a low-salt diet. After 28 days of subcutaneous cyclosporine dosing 15 mg/kg, cyclosporine-treated rats had significant renal dysfunction as compared with gender-matched control rats. Creatinine clearance in male cyclosporine-treated rats was reduced by 47% (P < .01) as compared with male controls. Female rats demonstrated a 38% (P < .01) decrease in creatinine clearance as a result of chronic cyclosporine therapy. Despite similar nephrotoxicity, female rats had whole blood cyclosporine levels 48% (P < .01) less than male rats. Immunoblot analysis of hepatic microsomal proteins indicated that chronic cyclosporine treatment decreased the protein levels of P450 3A2 in male rats. This loss was paralleled by reduced production of 6 beta-hydroxytestosterone, the primary product of P450 3A activity, by hepatic microsomes from cyclosporine-treated male rats by 76% (P < .001). In addition, cyclosporine treatment of male rats also reduced the formation of 2 alpha-hydroxytestosterone and 16 alpha-hydroxytestosterone by 81% (P < .01) and 84% (P < .001), respectively. At the end of the study period, steroid 5 alpha-reductase activity in control male rats was only 4% (P < .001) of female counter-parts; however, cyclosporine treatment increased steroid 5 alpha-reductase activity in male rats to 79% (P < .001) of female values. These alterations in testosterone metabolism are consistent with the suppression of the predominately male-associated P450 3A2, P450 2C11 and P450 2C13 isoforms. Levels of 6 alpha-hydroxytestosterone and 7 alpha-hydroxytestosterone were not statistically different between rat groups. Taken together, the steady-state blood levels and metabolism studies suggest that, after chronic cyclosporine treatment, isoforms other than those from the CYP3A family or unidentified members of the CYP3A family are likely responsible for cyclosporine metabolism.

Animals↗
PubMed1977

Peripheral and central androgenic stimulation of sexual behaviour of castrated male rats.

The effects of androgens on the maintenance and restoration of sexual behaviour (mounts, intromissions and ejaculations) of castrated male rats were studied. In the maintenance study the rats were treated during 5 weeks, starting one day following castration. Testosterone propionate maintained sexual behaviour at an almost normal level. The androgenoestrogen intermediate 19-hydroxytestosterone propionate was unable to prevent the decline in the number of ejaculations over the weeks although this hormone maintained the post-ejaculatory refractory period in those rats that ejaculated and also maintained normal sexual latencies. In the restoration study administration of testosterone propionate during 7 weeks to long-term castrated rats restored sexual behaviour to normal. 19-Hydroxytestosterone propionate treated rats displayed mounts but no other signs of sexual behaviour. The 5alpha-reduced androgen dihydrotestosterone propionate did not restore sexual behaviour. Testosterone propionate and dihydrotestosterone propionate stimulated peripheral target organs; 19-hydroxytestosterone propionate was ineffective in this respect. It has been suggested that testosterone might stimulate sexual behaviour in rats in two ways, i.e., via its aromatization to oestradiol in the brain, andy by stimulating growth of peripheral tissues via its 5alpha-reduction to dihydrotestosterone. In support for this view we have found that the combination of 19-hydroxytestosterone propionate and dihydrotestosterone propionate was effective in restoring the full pattern of sexual behaviour in castrated male rats.

Animals↗
PubMed2002

Characterization of testosterone metabolism and 7-hydroxycoumarin conjugation by rat and human liver slices after storage in liquid nitrogen for 1 h up to 6 months.

1. Slices of human and rat liver were cryopreserved in 18% dimethyl sulphoxide (DMSO) and subsequently stored in liquid nitrogen for periods up to as long as 6 months. After thawing, the metabolism of testosterone to hydroxylated products and conjugation of 7-hydroxycoumarin were investigated. 2. Rat liver slices stored in liquid nitrogen for 6 months exhibited rates of formation of 7alpha-, 6beta- 16alpha- and 2alpha-hydroxytestosterone, and of androstenedione that did not differ significantly from those observed with fresh slices. 3. No formation of 2alpha-hydroxytestosterone was detected with slices of human liver. However, in contrast with the rat, human slices produced 2beta-hydroxytestosterone. The rates of formation of 7alpha-, 6beta-, 16alpha- and 2beta-hydroxytestosterone and of androstenedione by human liver slices after 6 months of storage in liquid nitrogen were 82, 71, 236, 66 and 92%, respectively, of the corresponding rates by fresh slices. 4. The rates of sulphation and glucuronidation of 7-hydroxycoumarin by slices from rat liver were 97 and 119%, respectively, of the corresponding fresh values after 6 months of storage in liquid nitrogen. 5. 7-Hydroxycoumarin glucuronidation by human liver slices was 53% of the corresponding fresh values after 6 months of storage. However, human slices showed little or no capacity to conjugate 7-hydroxycoumarin with sulphate. 6. It was demonstrated that slices of both human and rat liver can be cryopreserved and stored in liquid nitrogen for at least 6 months without major changes in their rates of metabolism of testosterone to its hydroxylated products and of 7-hydroxycoumarin conjugation. These findings further emphasize that cryopreservation of liver slices can be an effective tool in the use of biological material of limited availability.

Androstenedione↗
PubMed1995

Species and sex differences of testosterone and nifedipine oxidation in liver microsomes of rat, dog and monkey.

1. Species and sex differences in testosterone hydroxylation and nifedipine oxidation in liver microsomes from rat, dog and monkey have been investigated. 2. The formation of 2 alpha-, 2 beta-, 6 beta-, and 16 alpha-hydroxytestosterone and androstenedione in the male rat was higher than that in the female rat. Microsomes prepared from the male rat oxidized nifedipine about eight times faster than did those from the female rat. In contrast, marked sex-related differences were not seen in the dog and monkey. 3. Nifedipine oxidase activity in rat, dog and monkey correlated significantly with the activities for both testosterone 2 beta-hydroxylation and 6 beta-hydroxylation, suggesting the involvement of P4503A isozymes in these reactions. The ratios of formation of the 2 beta- to 6 beta-hydroxytestosterone in male rat and monkey were 0.17 and 0.18 respectively, whereas that in dog was 0.46. The corresponding activity ratios catalysed by P450DPB-1, a P4503A isoform purified from dog liver microsomes, was 0.36. 4. The formation of 16 beta-hydroxytestosterone was higher than that of the 16 alpha-hydrolated metabolite in liver microsomes from monkey, whereas 16 alpha-hydroxytestosterone was the predominant metabolite in the rat and dog, indicating species differences in stereoselectivity at the 16-position.

Animals↗
PubMedDate not supplied

An unusual metabolite of testosterone. 17 beta-Hydroxy-4,6-androstadiene-3-one.

A testosterone metabolite, 17 beta-hydroxy-4,6-androstadiene-3-one, possessing an absorbance maximum at 284 nm, was formed during incubation of testosterone with liver microsomes from dexamethasone-treated rats. The metabolite was identified by HPLC, UV spectroscopy, and thermospray liquid chromatography/mass spectrometry. The formation of this metabolite by rat liver microsomes required NADPH and oxygen and was inhibited markedly by SKF 525-A, 2,4-dichloro-6-phenylphenoxyethylamine, or CO/O2 (8:2, v/v), but not by cyanide, an inhibitor for stearyl-CoA desaturase. Pretreatment of rats with phenobarbital, pregnenolone 16 alpha-carbonitrile, and dexamethasone enhanced the formation of this metabolite in parallel with the increase in formation of 6 beta-hydroxytestosterone (r2 = 0.99). Although 16-methylprogesterone, a known 6 beta-hydroxylase inhibitor, competitively inhibited the formation of the metabolite and 6 beta-hydroxytestosterone by liver microsomes from dexamethasone-treated rats, the metabolite was not formed from either 6 beta-hydroxytestosterone or 7-hydroxytestosterone during incubation with liver microsomes. These findings are consistent with the view that cytochrome P-450 isozymes that catalyze 6 beta-hydroxylation of steroids in rat liver microsomes also catalyze the dehydrogenation of testosterone to form a double bond between the C-6 and C-7 positions.

Animals↗
PubMed2004

Quantitative analysis of eight testosterone metabolites using column switching and liquid chromatography/tandem mass spectrometry.

The rate at which testosterone is metabolized to different singly hydroxylated metabolites has been widely used as an in vitro marker for activity of different CYP450 enzymes. The interest in extra-hepatic metabolism, e.g. due to metabolism in the gut wall, has increased during the last decade. Measurement of extra-hepatic enzyme activity using testosterone as a substrate requires a highly sensitive analytical method. A new liquid chromatography/electrospray tandem mass spectrometry (LC/MS/MS) method, using column switching for online cleaning and desalting of samples, was developed and validated for analysis of 2alpha-, 2beta-, 6alpha-, 6beta-, 7alpha-, 16alpha-, and 16beta-hydroxytestosterone and androstenedione. The samples were injected on a SB-CN column and detection was performed using MS/MS. The limits of quantification ranged from 0.3 to 3.33 nM for the different metabolites. The validated method was used to quantify the enzyme activity in rat intestine mucosa. The formation rates of 16alpha-, 16beta-hydroxytestosterone and androstenedione were quantified, and 2beta-and 6beta-hydroxytestosterone were formed above the limits of detection.

Androstenedione↗
PubMed1983

The effect of temperature and gonadotropin on testicular steroidogenesis in Sarotherodon (Tilapia) mossambicus in vitro.

Testes of sexually mature Sarotherodon mossambicus were incubated at 15, 22, 30, and 40 degrees with (a) tritiated testosterone and (b) salmon pituitary extract. Formation of 11-keto- and 11 beta-hydroxytestosterone from the tritiated precursor showed little change in yield between 15 and 30 degrees but yields of glucuronides rose dramatically between 22 and 30 degrees and a significant rise was observed for formation of 5 beta-androstane-3 alpha, 17 beta-diol between 15 and 40 degrees. Yields of 3 alpha, 17 beta-dihydroxy-5 beta-androstan-11-one followed a pattern similar to that of 11-ketotestosterone. With endogenous precursors under the stimulation of salmon pituitary extract, yields of testosterone, 11-ketotestosterone, and 11 beta-hydroxytestosterone were maximal at 22 degrees after which they declined to very low levels at 40 degrees. Yields of testosterone and 11-ketotestosterone glucuronides while showing a peak at 22 degrees declined much more slowly at higher temperatures than did those of the free steroids. In the absence of pituitary stimulation, levels of all steroids were below the limits of detection. Plasma levels of testosterone (15.3 +/- 1.5 ng/ml), 11-ketotestosterone (5.3 +/- 2.7 ng/ml), 11 beta-hydroxytestosterone (5.5 +/- 2.6 ng/ml), and their glucuromides (1.5 +/- 0.5, 0.14 +/- 0.1, and 1.5 +/- 0.5 ng/ml, respectively) were measured in fish held at 25 degrees. A rapid conchromatographic method for the assay of the three free steroids is described and the results are shown to be comparable to those obtained after chromatography.

Androgens↗
PubMed1989

Microbial transformation of steroids--II. Transformations of progesterone, testosterone and androstenedione by Phycomyces blakesleeanus.

Phycomyces blakesleeanus transformed progesterone, testosterone and androstenedione into mixtures of products. Five monohydroxylated metabolites were obtained in reasonable yields from the progesterone transformation. Only 7 alpha- and 15 beta-hydroxyprogesterone have been reported previously from this organism. We find that it gives these two metabolites and also 6 beta-, 14 alpha- and 15 alpha-hydroxyprogesterone as major products. Five compounds were also purified from testosterone transformation mixtures. Two of these were monohydroxylated, two were ring A dehydrogenation products, and two were oxidised at C-17. The products were identified as 6 beta-hydroxytestosterone, 7 alpha-hydroxytestosterone, androsta-1,4-diene-3,17-dione (1-dehydroandrostenedione), 17 beta-hydroxyandrosta-1,4-diene-3-one (1-dehydrotestosterone) and androstenedione. All five metabolites were produced in reasonable yields, although hydroxylation was the minor transformation in this case. Only two significant products were formed from androstenedione. Both were reduced at C-17; one was also monohydroxylated. They were testosterone and 14 alpha-hydroxytestosterone. The testosterone and androstenedione transformation products have not been reported previously for this organism. We also report for the first time the preparation of P. blakesleeanus cell-free extracts which transformed progesterone reasonably efficiently and faithfully in vitro, although the proportions of each product varied from one extract to another.

Androstadienes↗
PubMed2005

Characterization of testosterone 11 beta-hydroxylation catalyzed by human liver microsomal cytochromes P450.

A combination of accelerator mass spectrometry (AMS) and liquid chromatography-tandem mass spectrometry has been used to clarify some new aspects of testosterone metabolism. The main pathway of testosterone oxidative metabolism by human liver microsomes is the formation of 1beta-, 2alpha-/beta-, 6beta-, 15beta-, and 16beta-hydroxytestosterones, mainly catalyzed by cytochromes P450 2C9, 2C19, and 3A4. We now report the first determination that 11beta-hydroxytestosterone (11beta-OHT) can also be formed by human liver microsomal fractions. The structures of five hydroxylated metabolites of testosterone (2beta-, 6beta-, 11beta-, 15beta-, and 16beta-OHT) and the C-17 oxidative metabolite androstenedione were determined by liquid chromatography with UV detection at 240 nm and liquid chromatography-tandem mass spectrometry. Corresponding results were obtained by high-performance liquid chromatography-AMS analysis of incubations of [4-14C]testosterone with human liver microsomes. 6beta-Hydroxylation was always the dominant metabolic pathway, but 2beta-, 15beta-, and 16beta-OHT, and androstenedione were also formed. The previously undetected hydroxytestosterone, 11beta-OHT, was found to be a minor metabolite formed by human liver microsomal enzymes. It was formed more readily by CYP3A4 than by either CYP2C9 or CYP2C19. 11beta-Hydroxylation was inhibited by ketoconazole (IC50 = 30 nM) at concentrations similar to the IC50 (36 nM) for 6beta-hydroxylation Therefore, CYP3A4 could be mainly responsible for testosterone 11beta-hydroxylation in the human liver. These findings identify human hepatic biotransformation of testosterone to 11beta-OHT as a previously unrecognized extra-adrenal metabolic pathway.

Catalysis↗
PubMed1979

Effects of aromatizable androgens on aggressive behaviour among rats (rattus norvegicus).

Three experiments were used to tests the applicability of the aromatization hypothesis of androgen action to aggressive behaviour among Norway rats. In Expt 1, administration of testosterone propionate was highly effective in restoring aggressive behaviour to castrated rats while 17 beta-hydroxy-5 alpha-androstan-3-one was of intermediate effectiveness. Of the steriods tested in Expt 2, androstenedione and testosterone were highly effective, 17 beta, 19-dihydroxyandrost-4-en-3-one was of intermediate effectiveness and cholesterol was ineffective. The results of Expt 3 indicated that treatment with testosterone or oestradiol both resulted in increased aggression while treatment with (5 alpha,17 beta)-17,19-bis(acetyloxy)-andostan-3-one diacetate (5 alpha-19-hydroxytestosterone) was without effect. Androgens which were aromatizable and could be 5 alpha reduced, i.e. testosterone, testosterone propionate and androstenedione, were highly effective in restoring aggressive behaviour; however, two other steroids, 5 alpha,19-hydroxytestosterone which is 5 alpha reduced, and 19-hydroxytestosterone, which can be aromatized, were respectively of low or medium effectiveness on behaviour. However, oestradiol, which did not maintain sexual development of accessory glands, was highly effective in the restoration of aggressive behaviour. Since the behaviourally active steroids in the present experiments were not only those predicted by the aromatization hypothesis, it is proposed that several steroids are capable of activating aggressive behaviour and that the aromatization hypothesis does not adequately explain the hormonal basis of aggressive behaviour among Norway rats.

Aggression↗
PubMed1984

Assay of hepatic microsomal testosterone hydroxylases by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) method for the assay of the hepatic microsomal polysubstrate monooxygenase catalyzed hydroxylation of testosterone is described. The metabolites are extracted from the incubation mixture with dichloromethane and the extract is washed with dilute alkali and water, dried over anhydrous sodium sulfate, and evaporated to dryness. The residue is dissolved in methanol and an aliquot analyzed. The products are separated by reverse-phase chromatography with a methanol/water/tetrahydrofuran gradient and quantitated at 240 nm by the internal standard technique. The assay does not use radioactively labeled testosterone and can measure hydroxylase activity in microsomal samples containing less than 1.0 mg protein. At least seven products, 2 alpha-, 2 beta-, 6 beta-, 7 alpha-, 16 alpha-, and 16 beta-hydroxytestosterone and androstenedione, are resolved by HPLC. The major products formed by microsomes from untreated adult male rats are 2 alpha- (not 2 beta-) and 16 alpha-hydroxytestosterone and androstenedione which constituted 60% of the total products, followed by 6 beta-, 7 alpha-, and smaller quantities of 2 beta- and 16 beta-hydroxytestosterone. The carrier of the substrate in the incubation mixture was found to affect significantly the metabolite pattern and total activity, and of the several solvents studied methanol yielded the highest total activity. Since the 6 beta-, 7 alpha-, and 16 alpha-hydroxylation of testosterone is catalyzed by distinct forms of cytochrome P-450, this assay which measures seven products may serve as a useful qualitative probe of the cytochrome P-450 population of the monooxygenase.

Animals↗
PubMed2004

Characterization of the rates of testosterone metabolism to various products and of glutathione transferase and sulfotransferase activities in rat intestine and comparison to the corresponding hepatic and renal drug-metabolizing enzymes.

Metabolism of testosterone to various products (catalyzed by several different CYP isozymes) and the activities of phenol sulfotransferase (pST) and glutathione transferase (GST) in S9 fractions prepared from the mucosa of the duodenum, jejunum, ileum, caecum and upper and lower colon of male Sprague-Dawley rats were determined and compared to the corresponding hepatic and renal activities. Incubation of the S9 fraction prepared from the jejunum with testosterone and NADPH resulted in the formation of 2alpha-, 6alpha-, 6beta- and 16alpha-hydroxytestosterone and androstenedione at rates that were 1.6, 24, 1.3, 0.6 and 1.3%, respectively, of the corresponding hepatic values. The production of 2alpha-hydroxytestosterone was catalyzed only by the preparations from the duodenum and jejunum; whereas 6alpha-, 6beta- and 16alpha-hydroxytestosterone and androstenedione were produced in all regions of the intestine. In the case of the rat kidney, the rates of formation of the different testosterone metabolites were between 0.6 and 35% of the corresponding liver activity. The activity of glutathione transferase was approximately 12-26% of the corresponding hepatic activity throughout the intestine. The highest activity of phenol sulfotransferase was observed in the lower colon (almost 6% of the liver activity) and the lowest activity in the duodenum (1%). The renal activities of GST and pST were 70 and 1%, respectively, of the corresponding liver values. In summary, the metabolism of testosterone and the activities of GST and pST in rat intestine are generally low to very low in comparison to the corresponding activities in rat liver. In most cases, these activities are present throughout the entire intestine and not restricted to a particular portion(s) of this organ.

Animals↗
PubMed2005

Effects of oral androstenedione on steroid metabolism in liver of pregnant and non-pregnant female rats.

It is unknown whether androstenedione, a steroidal dietary supplement taken to enhance athletic performance, can affect physiological hormone levels by altering liver enzyme activities that metabolize steroid hormones. Altered hormone levels could be especially devastating during pregnancy. Mature female rats were gavaged with 0, 5, 30 or 60 mg/kg/day androstenedione beginning two weeks prior to mating and continuing through gestation day 19. Non-pregnant female rats were gavaged over the same time frame with 0 or 60 mg/kg/day androstenedione. Livers were removed from dams on gestation day 20 and from non-pregnant rats after five weeks' treatment. Liver microsomes were incubated with 200 microM testosterone, and the reaction products were isolated and analyzed by HPLC. In pregnant rats, formation of 6alpha-, 15beta-, 7alpha-, 16beta-, and 2beta-hydroxytestosterone was increased significantly vs. control at the highest dose level only. Formation of 6beta-hydroxytestosterone increased significantly at both the 30 and 60 mg/kg/day dose levels. In non-pregnant rats, 60 mg/kg/day androstenedione significantly increased formation of 15beta-, 6beta-, 16beta-, and 2beta-hydroxytestosterone. The data suggest that high oral doses of androstenedione can induce some female rat liver cytochromes P450 that metabolize steroid hormones and that the response to androstenedione does not differ between pregnant and non-pregnant female rats.

Administration, Oral↗
PubMed2004

Liquid chromatographic-mass spectrometric method to assess cytochrome P450-mediated metabolism of testosterone by rat everted gut sacs.

A rapid, sensitive and specific method was developed for the simultaneous assay of testosterone, androstenedione and 6beta-hydroxytestosterone (6beta-OHT) in the TC199 tissue culture medium used in intestinal drug metabolism studies with the rat everted gut sac model. An electrospray LC-MS method was validated in the concentration range of 0.025-9.5 microM (7.2 ng-2.7 microg/mL) for testosterone and androstenedione and 0.01-4 microM (3 ng-1.2 microg/mL) for 6beta-hydroxytestosterone. The limits of quantification (LOQ) with an injection volume of 10 microL were 0.0005 microM (4.9 fmol, 1.4 pg injected), 0.004 microM (0.04 pmol, 11.4 pg injected) and 0.03 microM (0.3 pmol, 91 pg injected), respectively. The method also detected the other testosterone metabolites, the 16alpha-, 16beta-, 2beta- and 2alpha-hydroxytestosterones and was then used to study the metabolism of testosterone during its absorption by rat intestine in vitro, using everted gut sacs.

Animals↗
PubMed1994

Deuterium isotope effects on A-ring and D-ring metabolism of testosterone by CYP2C11: evidence for dissociation of activated enzyme-substrate complexes.

Cytochrome P450 systems are unusual in that many of them can convert a substrate to a number of different metabolites by several possible kinetic mechanisms. Steady-state equations describing the deuterium isotope effects for mechanisms in which different orientations of the substrate relative to the perferryl oxygen in the active site of the enzyme are achieved before a hydrogen (or possibly an electron) is abstracted have been derived and solved (Gillette et al., 1994). These equations have been used to elucidate the kinetic mechanisms by which CYP2C11 converts testosterone to 2 alpha-hydroxytestosterone on the one hand and 16 alpha-hydroxytestosterone and androstenedione on the other. We have synthesized testosterone-2,2,4,6,6-2H5 and compared its metabolism by CYP2C11 with that of nondeuterated testosterone. In this system, deuterated 2 alpha-hydroxytestosterone would be formed by a deuterium abstraction pathway via the active oxygen intermediate (EOSw) and the D-ring metabolites would be formed by non-deuterium abstraction pathways from active oxygen intermediates represented by (EOSx). The results revealed that testosterone in the activated enzyme-substrate complexes, (EOSw) and (EOSx), does not change orientations while it is in the active site of CYP2C11. Instead, two of the noncompetitive experiments indicated that testosterone is able to dissociate from the (EOS) complexes and reassociate in either the same or different orientations. A third noncompetitive experiment suggested that testosterone in the (EOS) complexes does not change orientations while it is in the active site of CYP2C11, nor does it dissociate from the (EOS) complexes; instead, the pattern of metabolite formation is governed almost solely by the orientation of testosterone in the (ESw) and ESx) complexes.

Aryl Hydrocarbon Hydroxylases↗
PubMed1975

Hydroxylation of testosterone at carbons 1, 2, 6, 7, 15 and 16 by the hepatic microsomal fraction from adult female C57BL/6J mice.

The metabolism of a mixture of [4-14C]- and [7 beta-2H]testosterone by the hepatic microsomal fraction from adult femal C57BL/6J mice has been investigated. The following metabolites were identified by their mass spectra and by their retention times on gas chromatography on one or two phases: 1epsilon-, 2beta-, 6alpha-, 6beta-, 7alpha-, 15alpha-, 15beta-, 16alpha- and 16beta-hydroxytestosterone; 6alpha-, 6beta- and 7alpha-hydroxy-4-androstene-3,17-dione; and 4-androstene-3,17-dione. A compound tentatively identified as 6- or 7-oxotestosterone was also isolated. 17beta-Hydroxy-4,6-androstadien-3-one, 17beta-hydroxy-1,4-androstadien-3-one and 4,6-androstadiene-3,17-dione were identified but are considered to arise non-enzymatically from 7alpha-hydroxytestosterone, 1epsilon-hydroxytestosterone and 7alpha-hydroxy-4-androstene-3,17-dione, respectively.

Acclimatization↗
PubMed2003

Inhibition and activation of the human liver microsomal and human cytochrome P450 3A4 metabolism of testosterone by deployment-related chemicals.

Cytochrome P450 (P450) enzymes are major catalysts involved in the metabolism of xenobiotics and endogenous substrates such as testosterone (TST). Major TST metabolites formed by human liver microsomes include 6beta-hydroxytestosterone (6beta-OHTST), 2beta-hydroxytestosterone (2beta-OHTST), and 15beta-hydroxytestosterone (15beta-OHTST). A screen of 16 cDNA-expressed human P450 isoforms demonstrated that 94% of all TST metabolites are produced by members of the CYP3A subfamily with 6beta-OHTST accounting for 86% of all TST metabolites. Similar K(m) values were observed for production of 6beta-, 2beta-, and 15beta-OHTST with human liver microsomes (HLM) and CYP3A4. However, V(max) and CL(int) were significantly higher for 6beta-OHTST than 2beta-OHTST (approximately 18-fold) and 15beta-OHTST (approximately 40-fold). Preincubation of HLM with a variety of ligands, including chemicals used in military deployments, resulted in varying levels of inhibition or activation of TST metabolism. The greatest inhibition of TST metabolism in HLM was following preincubation with organophosphorus compounds, including chlorpyrifos, phorate, and fonofos, with up to 80% inhibition noticed for several metabolites including 6beta-OHTST. Preincubation of CYP3A4 with chlorpyrifos, but not chlorpyrifos-oxon, resulted in 98% inhibition of TST metabolism. Phorate and fonofos also inhibited the production of most primary metabolites of CYP3A4. Kinetic analysis indicated that chlorpyrifos was one of the most potent inhibitors of major TST metabolites followed by fonofos and phorate. Chlorpyrifos, fonofos, and phorate inhibited major TST metabolites noncompetitively and irreversibly. Conversely, preincubation of CYP3A4 with pyridostigmine bromide increased metabolite levels of 6beta-OHTST and 2beta-OHTST. Preincubation of human aromatase (CYP19) with the test chemicals had no effect on the production of the endogenous estrogen, 17beta-estradiol.

Chlorpyrifos↗
PubMed1985

Purification and characterization of a new form (RLM2) of liver microsomal cytochrome P-450 from untreated rat.

A new cytochrome P-450 isozyme (RLM2) has been purified to electrophoretic homogeneity from liver microsomes of the untreated rat. It has an apparent minimum molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 49,000. Absolute spectrum of the oxidized form indicates that this isozyme is essentially all in the low spin state. The maximum of the reduced CO complex is at 449 nm. Amino-terminal partial amino acid sequence and amino acid composition are different from those of RLM3 and RLM5, two other native forms of cytochrome P-450 previously reported from this laboratory as well as other forms reported in the literature. RLM2 is capable of oxidizing a variety of drug substrates, like benzphetamine and aminopyrine, and to a lesser extent ethoxycoumarin. With the steroid substrate multiple isomeric products are formed differentially. Progesterone is preferentially hydroxylated at the 15-position (15 beta-hydroxylation (34%) and 15 alpha-hydroxylation (13%) of the total) and at the 6 beta-position (21%). The major metabolite when testosterone was the substrate, 15 alpha-hydroxytestosterone, comprised 43% of the total, while a modest amount of 6 beta-hydroxytestosterone (12%) is formed. Another major metabolite (31%) has yet to be unequivocally identified, but is suggested to be 7 beta-hydroxytestosterone. Examination of the substrate dependence of major and minor isomeric metabolites provides evidence for a single substrate-binding site on RLM2. Regardless of the position hydroxylated, a common Km value was obtained. It is suggested that differences in formation of the isomeric and epimeric products relate to differences in distance from the active oxygen center and the position of attack.

Amino Acid Sequence↗