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H D Colby

Publications and source records attributed to H D Colby.

139 records · Page 8Linked to original sources
PubMedDate not supplied

Mechanisms responsible for the thermal sensitivity of adrenal microsomal monooxygenases.

Studies were done to determine the mechanism(s) responsible for the thermal lability of adrenal microsomal monooxygenases. Preincubation of guinea pig adrenal microsomal suspensions at 37 degrees C caused large time-dependent declines in benzo(a)pyrene (BP) hydroxylase and benzphetamine (BZ) demethylase activities. Similar preincubations with hepatic microsomes had little effect on enzyme activities. The decreases in adrenal enzyme activities were completely prevented by co-incubation of microsomes with cytosol, but were not diminished by reduced glutathione, ascorbic acid, or bovine serum albumin. Partial protection was afforded by EDTA, suggesting that lipid peroxidation might be involved, but malonaldehyde production was not demonstrable and MnCl2, a potent inhibitor of lipid peroxidation, did not affect the decline in enzyme activities. The decreases in the rates of BP and BZ metabolism were prevented by including NADPH or NADP+ in the preincubation medium. The preincubation conditions causing losses of adrenal enzyme activities did not affect cytochrome P-450 concentrations or substrate binding to cytochromes P-450, as indicated by type I difference spectra. NADH-cytochrome c reductase activity also was not affected, but there were decreases in NADPH-cytochrome c reductase activity that were proportionately similar to the declines in drug-metabolizing activities. Direct assessment of NADPH-cytochrome P-450 reductase revealed similarly large decreases in enzyme activity resulting from preincubation of adrenal microsomes. The results demonstrate a need for extra caution when doing preincubation experiments with adrenal microsomal preparations, and suggest that the thermal lability of adrenal monooxygenases is attributable to effects at the active site of NADPH-cytochrome P-450 reductase.

Adrenal Glands↗
PubMedDate not supplied

Spironolactone metabolism in target tissues. Characteristics of deacetylation in kidney, liver, adrenal cortex, and testes.

Prior investigations demonstrated that many of the actions of spironolactone (SL) required deacetylation of the parent compound as the first step in the formation of biologically active metabolites. Studies were done to characterize the process of deacetylation in several target tissues. The reaction was catalyzed by microsomal and cytosolic fractions of livers, kidneys, adrenal glands, and testes. Microsomal activity was greatest in liver and kidney and far exceeded cytosolic metabolism in those tissues. In adrenal glands and testes, by contrast, deacetylation was greater in cytosolic than microsomal fractions. The metabolism-mediated destruction of adrenal microsomal cytochromes P-450 by SL was enhanced by coincubation of microsomes with cytosol, illustrating the potential importance of combined microsomal and cytosolic metabolism in the actions of SL. The deacetylation of SL was decreased by various esterase inhibitors; the organophosphate compounds were the most potent inhibitors. The effectiveness of the esterase inhibitors varied from tissue to tissue, as well as from microsomes to cytosol within each tissue. The results indicate that SL deacetylation is catalyzed by microsomal and cytosolic esterases in various target tissues; several isozymes appear to be involved. These and prior observations suggest that tissue metabolism of SL is of major importance in the actions of the drug.

Adrenal Cortex↗
PubMedDate not supplied

The oxidative metabolism of hydralazine by rat liver microsomes.

Previous observations have demonstrated a relationship between the toxicity of the antihypertensive drug, hydralazine (HP), and acetylator phenotype, suggesting a role for metabolism in the adverse effects of HP. Experiments were done to characterize the metabolism of HP by rat liver microsomes using a newly developed HPLC assay. HP was metabolized by rat liver microsomes to products identified by HPLC and mass spectroscopy as s-triazolo[3,4-a]phthalazine (TP), 3-methyl-s-triazolo[3,4-a]phthalazine (MTP), phthalazine (P), and phthalazinone (PZ). An unknown metabolite was also formed. P, PZ, and the unknown metabolite were established as oxidation products of HP using the model oxidative systems, metal-catalyzed autooxidation and horseradish peroxidase. The results of incubations with [14C]HP indicated that P and the unknown metabolite were quantitatively the major metabolites and were produced in similar quantities by rat liver microsomes. The structure of the unknown metabolite based on mass spectral analyses is proposed to be a dimerization product consisting of P and 1-aminophthalazine. Production of all the microsomal metabolites, except TP, required NADPH and decreased when incubations were done with heat-treated microsomes or under an atmosphere of nitrogen or carbon monoxide. Pretreatment of rats with phenobarbital increased the rate of formation of all the metabolites except TP. In contrast, pretreatment with 3-methylcholanthrene or Arochlor 1254 had no effect on P, PZ, TP, or MTP formation and decreased formation of the dimer. Pretreatment with piperonyl butoxide had no effect on the formation of P, PZ, TP, or MTP but decreased formation of the dimer by approximately 50%.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

Maturational changes in adrenal xenobiotic metabolism in male and female guinea pigs.

In young (25-day-old) guinea pigs, adrenal and hepatic benzo[a]pyrene (BP) hydroxylase activities were similar but the rates of ethylmorphine (EM) demethylation were greater in adrenals than liver. Between 25 and 50 days of age no sex differences in adrenal or hepatic enzyme activities were demonstrable. The rates of adrenal BP and EM metabolism increased with age in guinea pigs of both sexes; activities reached significantly higher levels in males than females. In contrast, hepatic metabolism of BP and EM declined with maturation and activities were similar in males and females at all ages. Neither microsomal cytochrome P-450 concentrations no NADPH-cytochrome c reductase activities correlated with the maturational changes or sex differences in xenobiotic metabolism. Adrenal microsomal steroid 21-hydroxylase activity did not change significantly with aging and was not sex-dependent. The results indicate that opposite changes occur in adrenal and hepatic xenobiotic metabolism as a function of aging, resulting in substantially greater adrenal than hepatic activity in sexually mature animals. The data also suggest that adrenal microsomal drug and steroid metabolism are independently regulated.

Adrenal Glands↗
PubMedDate not supplied

Strain differences in adrenal xenobiotic metabolism in guinea pigs.

Studies were carried out to compare adrenal and hepatic xenobiotic metabolism in various strains of guinea pigs. In all strains studied (Hartley, English Short Hair, NIH, Strain 2, Strain 13), microsomal protein and cytochrome P-450 levels and NADPH-cytochrome c reductase activities were greater in adrenals than livers. Neither adrenal values nor hepatic values for these parameters differed across strains. Ethylmorphine (EM) demethylase and benzo[a]pyrene (BP) hydroxylase activities were also greater in adrenals than livers in all strains. However, the rates of adrenal xenobiotic metabolism were far greater in the highly inbred Strain 2 and Strain 13 guinea pigs than in other strains. In contrast, hepatic metabolism of EM and BP was not strain-dependent. Adrenal steroid 21-hydroxylase activity was also similar in all strains. The results indicate that strain is an important determinant of adrenal but not hepatic xenobiotic metabolism in the guinea pig. In addition, genetic control of adrenal microsomal drug and steroid metabolism appear to be independent of one another.

Adrenal Glands↗
PubMedDate not supplied

Differential effects of adrenocorticotropic hormone on adrenal microsomal xenobiotic and steroid metabolism in guinea pigs.

The effects of adrenocorticotropic hormone (ACTH) administration to guinea pigs on the activities of adrenal microsomal monooxygenases were studied. ACTH treatment decreased the rates of adrenal benzphetamine (BZ) demethylation and benzo[a]pyrene (BP) hydroxylation but had no effect on the same reactions in hepatic microsomes. Adrenal microsomal steroid hydroxylation reactions were unaffected (21-hydroxylation) or stimulated (17 alpha-hydroxylation) by ACTH. Although ACTH treatment decreased adrenal BP hydroxylase activity, the relative quantity of the various BP metabolites, as determined by HPLC, did not change. Adrenal microsomal cytochrome P-450 concentrations were decreased by ACTH but proportionately less than the decreases in adrenal xenobiotic metabolism. The maximal type I spectral changes produced by xenobiotics in adrenal microsomes were decreased in size by ACTH treatment, but steroid-induced difference spectra were unaffected. The results indicate that ACTH selectively decreases the rates of adrenal xenobiotic metabolism, perhaps by producing a selective decline in the concentration(s) of those cytochromes P-450 involved in the metabolism of foreign compounds.

Adrenal Glands↗
PubMedDate not supplied

The duration of exposure of microsomal preparations to cadmium or zinc in vitro influences the inhibition of mono-oxygenases.

Preincubation of guinea pig hepatic, pulmonary, or adrenal microsomes with cadmium or zinc decreased mono-oxygenase [benzo(a)pyrene hydroxylase, benzphetamine demethylase] activities. Addition of the same concentrations of the metals to the microsomal suspensions after the preincubation period had little or no effect on enzyme activities. The decline in mono-oxygenase activities produced by cadmium or zinc was dependent on the length of the preincubation period as well as the concentration of metal present during the preincubation. In addition, the preincubation effects of both metals were temperature dependent; at temperatures between 4 and 37 degrees C, loss of enzyme activity increased with increasing temperature. Cadmium and zinc produced greater decreases in mono-oxygenase activities in pulmonary and adrenal microsomes than in hepatic microsomes. The results indicate that the duration of exposure of hepatic and extrahepatic microsomal preparations to cadmium or zinc in vitro is an important determinant of effects on mono-oxygenases.

Adrenal Glands↗
PubMedDate not supplied

Hepatic metabolism of spironolactone. Production of 3-hydroxy-thiomethyl metabolites.

Spironolactone (SP) is used clinically as a renal aldosterone antagonist and as an antiandrogen. It is known that the drug is extensively metabolized and that metabolites mediate its therapeutic actions, but hepatic metabolism of SP has not been comprehensively investigated. Hepatic disposition may also be important in the toxicity of SP, because the parent compound prevents the hepatocarcinogenic effects of its metabolite, canrenone (CAN). Using a combination of in vivo and in vitro approaches, we studied the metabolism of SP by guinea pig livers. The major compounds detected in livers in vivo following SP treatment were the known metabolites, 7 alpha-thiomethyl-spirolactone (TM) and CAN, and a previously uncharacterized compound whose mass spectral and UV absorption characteristics suggested that it was an A-ring-reduced derivative of TM. In vitro incubation of liver homogenates with SP also resulted in the formation of the unknown metabolite. A combination of MS and NMR spectroscopy was used to identify unequivocally the unknown metabolites as 3 alpha-hydroxy-TM. Another metabolite produced in vitro was identified as 3 beta-hydroxy-TM. It is possible that these two new metabolites of SP contribute to the pharmacological actions of the drug. In addition, production of 3 alpha-hydroxy-TM suggests a mechanism to account for the prevention of CAN-induced carcinogenicity by SP. TM may block the conversion of CAN to mutagenic 3-hydroxy-CAN metabolites by serving as a competitive substrate for hepatic 3-keto reductases.

Animals↗
PubMedDate not supplied

Identification of spironolactone metabolites in plasma and target organs of guinea pigs.

Spironolactone (SL) is a renal aldosterone antagonist that is used clinically in the treatment of hypertension and congestive heart failure. Among the side effects of the drug are degradation of cytochrome P-450 and inhibition of steroidogenesis in the testes. It has long been recognized that the effects of SL are mediated by metabolites of the drug, but questions remain about the identities of the active metabolites. Because tissue metabolites of SL had not previously been investigated, experiments were done to determine the identities of metabolites in target organs after SL administration to guinea pigs. Metabolites were identified by HPLC and MS. The major plasma metabolite was 7 alpha-thiomethyl-SL (TM) with smaller amounts of canrenone (CAN) and 7 alpha-thio-SL (TH) also present. In kidneys, TM also was the principal metabolite, but CAN was the only other compound consistently found. By contrast, in testes, substantial amounts of SL and TH were present in addition to TM and CAN. It is possible that local metabolism of SL contributes to the differences in metabolite profiles between plasma and target organs. Data also suggest that TM is principally responsible for the renal antimineralocorticoid effects of SL and support the purported role of TH in the degradation of testicular cytochrome P-450.

Animals↗