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H D Colby

Publications and source records attributed to H D Colby.

At least 55 records · Page 3Linked to original sources
PubMed1988

Adrenal mitochondrial metabolism of spironolactone. Absence of metabolic activation.

Previous investigations have established that spironolactone (SL) administration to guinea pigs decreases adrenal mitochondrial and microsomal cytochrome P-450 content, and that the latter requires microsomal activation of the drug. Studies were carried out to determine if adrenal mitochondrial metabolism (activation) of SL was similarly involved in the effects of the drug on mitochondrial cytochrome P-450 destruction. Incubation of guinea pig adrenal mitochondria with SL in the absence of NADPH resulted in the formation of 7 alpha-thio-SL as the only metabolite. In the presence of an NADPH-generating system, an unknown polar metabolite was also produced. The mass spectrum of the unknown compound suggested that it was a hydroxylated derivative of SL. Incubation of mitochondrial preparations with 7 alpha-thio-SL also resulted in the formation of a polar metabolite, but the latter had a different HPLC retention time than that of the SL metabolite. Formation of the polar SL metabolite was prevented by metyrapone, an 11 beta-hydroxylase inhibitor, and was greatest in mitochondria from the adrenal zone having the highest 11 beta-hydroxylase activity. Steroid substrates for 11 beta-hydroxylation inhibited the production of the SL metabolite. Mitochondrial incubations with SL or with 7 alpha-thio-SL in the presence or absence of an NADPH-generating system did not affect cytochrome P-450 concentrations. The results indicate that, unlike the microsomal effects of SL, local activation of SL is not responsible for the destruction of adrenal mitochondrial cytochromes P-450. The major adrenal mitochondrial metabolites of SL appear to be 11 beta-hydroxy-SL and 7 alpha-thio-SL.

Adrenal Glands↗
PubMed1988

Inhibition of hepatic microsomal drug metabolism by the steroid hydroxylase inhibitor SU-10'603.

SU-10'603 is a pyridine derivative that has been widely used as a steroid 17-hydroxylase inhibitor. Studies were done to compare the effects of SU-10'603 with those of the structurally related compound, metyrapone, on hepatic microsomal drug metabolism in vitro in rats and guinea pigs. In rat liver microsomes, SU-10'603 produced a concentration-dependent (0.01 to 1.0 mM) inhibition of ethylmorphine demethylation, aniline hydroxylation, and benzo[a]pyrene hydroxylation. A concentration of 0.1 to 0.2 mM decreased the metabolism of all three substrates by approximately 50%. SU-10'603 was a more potent inhibitor of ethylmorphine metabolism than metyrapone, and its relative potency was even greater with respect to aniline and benzo[a]pyrene metabolism. Similar results were obtained with guinea pig liver microsomes. SU-10'603 and metyrapone produced type II spectral changes in hepatic microsomes, but the apparent affinity of SU-10'603 for cytochrome(s) P-450 was greater than that of metyrapone. Both compounds inhibited the binding of type I substrates to microsomal cytochromes P-450; SU-10'603 was the more potent inhibitor. The results indicate that SU-10'603 is a potent inhibitor of hepatic microsomal monooxygenases whose mechanism of action is similar to that of metyrapone.

Aniline Hydroxylase↗
PubMed1988

Changes in adrenocortical monooxygenase activities in alloxan-diabetic rabbits.

Studies were carried out to determine if diabetes mellitus influenced the activities of adrenal steroidogenic enzymes. Adult male rabbits were made diabetic by an i.v. infusion of alloxan (100 mg/kg) and were killed 1 or 2 months later. Mitochondrial cytochrome P-450 concentrations were not affected by diabetes but steroid 11 beta-hydroxylase activity was greater in the diabetics than in controls after both 1 and 2 months. The type I spectral change produced by 11-deoxycorticosterone, the substrate for 11 beta-hydroxylation, was also greater in mitochondria from diabetics. By contrast, mitochondrial cholesterol side-chain cleavage activity was similar in controls and diabetics. Microsomal cytochrome P-450 concentrations were unaffected by diabetes but 21-hydroxylase activity was significantly lower in adrenal microsomes from diabetics than from controls. The results indicate that alloxan-induced diabetes alters adrenocortical steroid metabolism which may contribute to changes in the pattern of steroid secretion noted by other investigators.

Adrenal Cortex↗
PubMed1988

Effects of tocopherol depletion on the regional differences in adrenal microsomal lipid peroxidation and steroid metabolism.

Studies were done to assess the contribution of alpha-tocopherol to the regional differences in microsomal lipid peroxidation (LP) and steroid metabolism in the guinea pig adrenal cortex. In normal guinea pigs, ferrous ion (Fe2+)- and ascorbic acid-induced LP are far greater in microsomal preparations from the inner adrenal zone (zona reticularis) than in those from the outer zones (zona fasciculata plus zona glomerulosa). The amounts of unsaturated fatty acids, substrates for LP, are similar in the two zones, but alpha-tocopherol concentrations are 4-5 times greater in outer than inner zone microsomes. Tocopherol depletion by dietary deprivation had little effect on LP in vitro in inner zone microsomes, but substantially increased LP in outer zone preparations. As a result, tocopherol deficiency eliminated the zonal differences in microsomal LP. Unsaturated FFA concentrations were lower in tocopherol-deficient microsomal preparations than in those from tocopherol-sufficient animals, suggesting peroxidative losses in vivo. Tocopherol deficiency decreased steroid C-17,20 lyase activity in outer zone microsomes, but had no effect on activity in inner zone preparations, eliminating the normal zonal difference in activity (outer greater than inner). The results indicate that alpha-tocopherol is a major determinant of adrenal LP and is responsible for the regional differences in microsomal LP in guinea pig adrenal cortex; the effects of ascorbic acid on LP in each zone are also affected by alpha-tocopherol. alpha-Tocopherol may influence the functional zonation of the adrenal cortex by selectively protecting outer zone steroidogenic enzymes from oxidative degradation.

Adrenal Glands↗
PubMed1987

Conversion of spironolactone to 7 alpha-thiomethylspironolactone by hepatic and renal microsomes.

Recent observations indicate that 7 alpha-thiomethylspironolactone is an important circulating metabolite of the mineralocorticoid antagonist spironolactone (SL). Studies were carried out to determine possible sites and pathways of 7 alpha-thiomethyl-SL formation and, in particular, to evaluate SL metabolism by guinea pig hepatic and renal microsomal preparations. In the absence of S-adenosylmethionine (SAM), liver and kidney microsomes rapidly converted SL to 7 alpha-thio-SL as the only metabolite. The rate of 7 alpha-thio-SL production was greater in liver than kidney. In the presence of SAM, 7 alpha-thio-SL was further converted to 7 alpha-thiomethyl-SL by liver and kidney microsomes. The rates of methylation with 7 alpha-thio-SL as substrate were three to four times greater for liver than for kidney, but the Km values were similar (approximately 30 microM) in the two issues. Maximal enzyme activity was obtained with SAM concentrations of 25-200 microM. NADPH had no effect on SL or 7 alpha-thio-SL metabolism by liver or kidney microsomes. To determine if a pathway involving the C-S lyase enzyme might contribute to circulating 7 alpha-thiomethyl-SL levels in vivo, guinea pigs were treated with SL or its dethioacetylated derivative, canrenone, and plasma metabolites were analyzed by HPLC. Both 7 alpha-thiomethyl-SL and canrenone were found to be circulating metabolites in SL-treated animals, but only canrenone was identified in the plasma of canrenone-treated guinea pigs. The results indicate that the liver and kidney are potential sites of 7 alpha-thiomethyl-SL production and that its formation probably does not involve the C-S lyase pathway.

Animals↗
PubMed1987

Metabolic activation of hydralazine by rat liver microsomes.

There is evidence to suggest that the oxidative metabolism of hydralazine (HP), an antihypertensive drug, may represent a toxic pathway which could account for some of the adverse effects of the drug. Experiments were done to determine whether the hepatic oxidative metabolism of HP is associated with the formation of reactive metabolites. In the presence of NADPH, HP was metabolized by rat liver microsomes to three major oxidation products, phthalazine, phthalazinone (PZ), and a dimer compound. Under similar incubation conditions, radioactivity derived from [14C]HP was covalently bound to microsomal protein. Metabolite formation and covalent binding increased following pretreatment of rats with phenobarbital. In contrast, pretreatment with 3-methylcholanthrene or with the monooxygenase inhibitor, piperonyl butoxide, slightly decreased both metabolite formation and covalent binding. Electron spin resonance (ESR) analyses indicated that nitrogen-centered radicals were formed when rat liver microsomes were incubated with HP under conditions similar to those required for covalent binding and for the production of the oxidative metabolites. In addition, reduced glutathione (GSH) caused concentration-dependent decreases in the production of phthalazine, PZ, and the dimer, in the covalent binding of HP to microsomal protein, and in the formation of nitrogen-centered radicals. The results of these investigations indicate that the oxidative metabolism of HP by rat liver microsomes is highly correlated with the formation of nitrogen-centered radicals and the production of metabolites that become covalently bound to microsomal protein. These observations support the hypothesis that the oxidation of HP generates reactive metabolites which may contribute to the toxicity of the drug.

Animals↗
PubMed1987

Effects of cadmium in vitro on microsomal steroid metabolism in the inner and outer zones of the guinea pig adrenal cortex.

Studies were carried out to evaluate the effects of cadmium in vitro on microsomal steroid metabolism in the inner (zona reticularis) and outer (zona fasciculata and zona glomerulosa) zones of the guinea pig adrenal cortex. Microsomes from the inner zone have greater 21-hydroxylase than 17 alpha-hydroxylase activity, resulting in the conversion of progesterone primarily to 11-deoxycorticosterone and of 17 alpha-hydroxyprogesterone principally to its 21-hydroxylated metabolite, 11-deoxycortisol. Microsomes from the outer zones, by contrast, have far greater 17 alpha-hydroxylase and C17,20-lyase activities than 21-hydroxylase activity. As a result, progesterone is converted primarily to its 17-hydroxylated metabolite, 17 alpha-hydroxyprogesterone; and 17 alpha-hydroxyprogesterone is converted principally to delta 4-androstenedione, with only small amounts of 21-hydroxylated metabolites being produced. Addition of cadmium to incubations with inner zone microsomes causes concentration-dependent decreases in 21-hydroxylation and increases in 17 alpha-hydroxylase and C17,20-lyase activities, resulting in a pattern of steroid metabolism similar to that in normal outer zone microsomes. Cadmium similarly decreases 21-hydroxylation by outer zone microsomes but has no effect on the formation of 17-hydroxylated metabolites or on androgen (delta 4-androstenedione) production. In neither inner nor outer zone microsomes did cadmium affect cytochrome P-450 concentrations, steroid interactions with cytochrome(s) P-450, or NADPH-cytochrome P-450 reductase activities. The results indicate that cadmium produces both quantitative and qualitative changes in adrenal microsomal steroid metabolism and that the nature of the changes differs in the inner and outer adrenocortical zones. In inner zone microsomes, there appears to be a reciprocal relationship between 21-hydroxylase and 17 alpha-hydroxylase/C17,20-lyase activities which may influence the physiological function(s) of that zone.

Adrenal Cortex↗
PubMed1987

Regional differences in microsomal lipid peroxidation and antioxidant levels in the guinea pig adrenal cortex.

Lipid peroxidation (LP) and antioxidant levels were studied in the chromatically distinct inner (zona reticularis) and outer (zona fasciculata + zona glomerulosa) zones of the guinea pig adrenal cortex. Ferrous ion (Fe2+) produced a concentration-dependent (10(-5) to 10(-3) M) stimulation of microsomal LP in both zones, but LP, as estimated by malonaldehyde production, was far greater in the inner zone. Although cytosolic ascorbic acid content was similar in the two zones, microsomal tocopherol levels were approx 4 times greater in the outer than inner zone. Subphysiological concentrations of ascorbic acid, like Fe2+, initiated LP to a greater extent in inner than outer zone microsomes; optimal stimulation of LP by ascorbic acid occurred at concentrations of 100-200 microM in both zones. Physiological concentrations of ascorbic acid (1-5 mM), by contrast, did not initiate LP and, in fact, markedly inhibited Fe2+-induced LP in both inner and outer zone microsomal preparations. Outer zone microsomes were more sensitive to the antioxidant effects of ascorbic acid than were inner zone preparations. Addition of alpha-tocopherol to inner zone microsomal suspensions inhibited Fe2+-induced LP. The results indicate that there are regional differences in adrenocortical LP which may be caused by differences in tocopherol content. alpha-Tocopherol may serve important antioxidant functions within the adrenal cortex, thereby contributing to the functional zonation of the gland.

Adrenal Cortex↗
PubMed1986

Effects of organic solvent vehicles on benzo[a]pyrene metabolism in rabbit lung microsomes.

In order to study the metabolism of benzo[a]pyrene (BP), it must be dissolved in an organic solvent vehicle for delivery to the tissue. We studied the effects of five organic solvent vehicles, i.e. dimethyl sulfoxide (DMSO), acetone, methanol, ethanol, and ethyl acetate, on benzo[a]pyrene hydroxylase activity and the BP metabolite profile in rabbit lung microsomes. Fluorescence detection of 3- and 9-OH-BP was used to evaluate benzo[a]pyrene hydroxylase activity, and the BP metabolite profile was obtained by HPLC analysis. All solvent vehicles inhibited benzo[a]pyrene hydroxylase in a dose-dependent manner. When the smallest volume of each solvent (10 microliter/ml reaction mixture) was employed, the resulting enzyme activities as related to solvent type, from highest to lowest, were DMSO greater than or equal to methanol greater than ethanol greater than or equal to acetone greater than ethyl acetate. HPLC analysis of BP metabolites formed in the presence of the five solvent vehicles showed that production of all metabolites was greatest when DMSO was used and that linearity of product formation was retained longer with DMSO. The metabolites produced when DMSO was used as the solvent were BP-9,10-diol, BP-4,5-diol, BP-7,8-diol, BP-1,6-quinone, BP-3,6-quinone and 3-OH-BP. A similar metabolite profile was obtained when reactions were carried out with methanol as the solvent vehicle, although the magnitude of production was less than with DMSO. When acetone was used, there were greater amounts of BP-4,5-diol and BP quinone formation and lesser amounts of 3-OH-BP formed than with DMSO or methanol. When ethanol or ethyl acetate was used as a solvent, BP-9,10-diol and 3-OH-BP were the only metabolites produced. These results indicate that all solvent vehicles studied inhibit benzo[a]pyrene hydroxylase from rabbit lung microsomes in a dose-dependent manner and that the magnitudes and types of metabolites formed are highly dependent upon the specific solvent used as the vehicle. The study also indicates that DMSO is probably the solvent vehicle of choice for study of BP metabolism in rabbit lung microsomes.

Acetates↗
PubMed1986

Separation and quantitation of hydralazine metabolites by high-performance liquid chromatography.

A selective high-performance liquid chromatographic assay for the separation and quantitation of the proposed hepatic microsomal metabolites of hydralazine (HP), phthalazine, phthalazinone, s-triazolo[3,4-a]phthalazine, 3-methyl-s-triazolo[3,4-a]phthalazine and 3-hydroxymethyl-s-triazolo [3,4-a]phthalazine, is described. An extraction technique was developed for the removal of HP from hepatic microsomal samples in order to minimize the measurement of products resulting from the chemical degradation of HP. The effects of pH and composition of the mobile phase on the retention times and resolution of the five compounds were examined. The methods presented are accurate and reliable, permitting the baseline separation of five HP metabolites with reasonable analysis time and sensitivity.

Animals↗
PubMed1986

Metabolism of spironolactone by adrenocortical and hepatic microsomes: relationship to cytochrome P-450 destruction.

Previous investigations have established that spironolactone (SL) is converted to a reactive metabolite by adrenocortical NADPH-dependent enzymes, resulting in the destruction of microsomal cytochrome(s) P-450 and decreases in steroid hydroxylase activities. Hepatic microsomes, by contrast, do not activate SL. Studies were done to characterize the activation pathway by comparing adrenal with hepatic metabolism of SL in guinea pigs. In the absence of NADPH, both adrenal and hepatic microsomal preparations converted SL to its deacetylated metabolite, 7 alpha-thio-SL. NADPH had no effect on hepatic SL metabolism but stimulated adrenal metabolism of SL. In the presence of NADPH, very little 7 alpha-thio-SL was recovered from the adrenal incubations, suggesting that the 7 alpha-thio-SL was further metabolized by NADPH-dependent enzymes. The latter hypothesis was confirmed by incubating microsomal preparations with 7 alpha-thio-SL as the substrate. In the presence of NADPH, 7 alpha-thio-SL was rapidly metabolized by adrenal microsomes but was not metabolized by hepatic preparations. Under the same incubation conditions, 7 alpha-thio-SL promoted the destruction of adrenal cytochrome(s) P-450 but had no effect on hepatic monooxygenases. 7 alpha-Thio-SL was far more potent than SL in promoting the destruction of cytochrome(s) P-450, suggesting that the metabolite might be an intermediate in the actions of the parent compound. Indeed, inhibition of SL conversion to 7 alpha-thio-SL by the esterase inhibitor, diethyl p-nitrophenyl phosphate blocked the effects of SL on adrenal cytochrome(s) P-450. Diethyl p-nitrophenyl phosphate did not affect the actions of 7 alpha-thio-SL on cytochrome(s) P-450.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Cortex↗
PubMed1985

Mitochondrial steroid metabolism in the inner and outer zones of the guinea-pig adrenal cortex.

Previous investigations have demonstrated that cells isolated from the outer zone (zona fasciculata + zona glomerulosa) of the guinea-pig adrenal cortex produce far more cortisol than those from the inner zone (zona reticularis). Studies were carried out to compare mitochondrial steroid metabolism in the two zones. Protein and cytochrome P-450 concentrations were similar in outer and inner zone mitochondria. However, the rate of 11 beta-hydroxylation was significantly greater in the outer zone despite the fact that substrates for 11 beta-hydroxylation (11-deoxycortisol, 11-deoxycorticosterone) produced larger type I spectral changes in inner zone mitochondria. The apparent affinities of 11-deoxycortisol and 11-deoxycorticosterone for mitochondrial cytochrome(s) P-450 were similar in the two zones. In both inner and outer zone mitochondria, 11 beta-hydroxylation was inhibited by metyrapone but unaffected by aminoglutethimide. Cholesterol sidechain cleavage activity, measured as the rate of conversion of endogenous cholesterol to pregnenolone, was far greater in outer than inner zone mitochondria. Addition of exogenous cholesterol or 25-hydroxycholesterol to the mitochondrial preparations did not affect pregnenolone production in either zone. Addition of pregnenolone to outer zone mitochondria produced a reverse type I spectral change (delta A 420-390 nm), suggesting displacement of endogenous cholesterol from cytochrome P-450. In inner zone mitochondria, pregnenolone induced a difference spectrum (delta A 425-410 nm) similar to the reduced vs oxidized cytochrome b5 spectrum. A b5-like cytochrome was found to be present in the mitochondrial preparations. Prior reduction of the cytochrome with NADH eliminated the pregnenolone-induced spectral change in inner zone mitochondria but had no effect in outer zone preparations. The results suggest that differences in mitochondrial steroid metabolism between the inner and outer adrenocortical zones account in part for the differences in cortisol production by cells in each zone.

Adrenal Cortex↗
PubMed1985

Differences in microsomal steroid metabolism between the inner and outer zones of the guinea pig adrenal cortex.

Previous investigations established that cells isolated from the outer zone (zona fasciculata + zona glomerulosa) of the guinea pig adrenal cortex produced far more cortisol and androstenedione than those from the inner zone (zona reticularis). Studies were done to determine whether differences in microsomal metabolism might contribute to the zonal differences in steroid secretion. Cytochromes P-450 and b5 concentrations were greater in inner zone microsomes as were the magnitudes of the type I difference spectra produced by progesterone and 17 alpha-hydroxyprogesterone. Basal NADPH-cytochrome P-450 reductase activity was greater in the outer zone, but steroid substrates (progesterone, 17 alpha-hydroxyprogesterone) increased reductase activity in the inner zone and decreased activity in the outer zone. 21-Hydroxylase activity was far greater in inner than outer zone microsomes, but 17 alpha-hydroxylase activity was greater in the outer zone. As a result, progesterone was converted primarily to 17 alpha-hydroxyprogesterone by outer zone microsomes, but 11-deoxycorticosterone was the major metabolite produced by inner zone preparations. In addition, with 17 alpha-hydroxyprogesterone as substrate, the major product produced by outer zone microsomes was androstenedione, indicating relatively high C17-20-lyase activity. Inner zone microsomes by contrast, converted 17 alpha-hydroxyprogesterone primarily to the 21-hydroxylated metabolite, 11-deoxycortisol, with little production of androstenedione. The rate of conversion of pregnenolone to progesterone was also greater with outer than inner zone microsomes. The results suggest that differences in the patterns of microsomal steroid metabolism contribute to the greater secretion of cortisol and androstenedione by adrenocortical outer zone cells than by inner zone cells.

Adrenal Cortex↗
PubMed1984

Metabolism of 4-chlorobiphenyl by guinea pig adrenocortical and hepatic microsomes.

Studies were carried out to determine if 4-chlorobiphenyl (4-CB) was a substrate for adrenal monooxygenases and to compare its interactions with adrenal and hepatic microsomal enzymes. Addition of 4-CB to guinea pig adrenal microsomes produced a typical type I spectral change, indicative of binding to cytochrome(s) P-450 and similar to that seen in hepatic microsomal preparations. The activities of several adrenal and hepatic microsomal monooxygenases were decreased by 4-CB in vitro. High pressure liquid chromatographic analyses revealed that both adrenal and hepatic microsomes, in the presence of NADPH, converted 4-CB to a major metabolite which eluted with a retention time identical to that of 4-chloro-4'-biphenylol (4'-OH-4-CB). The identity of 4'-OH-4-CB was confirmed by mass spectrometry. The maximal rate of 4-CB metabolism was greater in adrenal, compared with liver microsomes, but 4-CB had a higher affinity for hepatic than for adrenal enzymes. The rate of adrenal 4-CB metabolism was four to five times greater in microsomes derived from the inner cortical zone (zona reticularis) than those from the outer zones (zona fasciculata and zona glomerulosa). Hepatic microsomes also converted 4-CB to a minor metabolite whose production was blocked by epoxide hydrolase inhibitors, suggesting it might be a diol. 4-CB metabolism was not demonstrable in adrenal mitochondrial preparations. The results indicate that chlorinated biphenyls can serve as substrates for adrenal microsomal monooxygenases, suggesting that local activation may contribute to their adrenocortical toxicity.

Adrenal Cortex↗