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Biomedical subjects

H D Colby

Publications and source records attributed to H D Colby.

At least 73 records · Page 4Linked to original sources
PubMed1984

Sites of action of cadmium in vitro on hepatic, adrenal, and pulmonary microsomal monooxygenases in guinea pigs.

Preincubation of hepatic, adrenal, or pulmonary microsomal preparations with cadmium produced time-dependent decreases in monooxygenase (benzphetamine demethylase, benzo(a)pyrene hydroxylase) activities. Addition of cadmium after the preincubation period had little or no effect on microsomal metabolism. As a result of preincubation with cadmium, hepatic cytochrome P-450 levels declined and the magnitude of the benzphetamine-induced type I spectral change in hepatic microsomes decreased. Cadmium also decreased hepatic NADPH-cytochrome c and NADPH-cytochrome P-450 reductase activities but had no effect on NADH-cytochrome c reductase activity. Cadmium similarly decreased cytochrome P-450 concentrations and NADPH-cytochrome c reductase activity in lung microsomes without affecting NADH-cytochrome c reductase activity. Preincubation of adrenal microsomes with cadmium had no effects on cytochrome P-450 levels, on the benzphetamine-induced type I spectrum, or on NADH-cytochrome c reductase activity. However, decreases in adrenal NADPH-cytochrome c and NADPH-cytochrome P-450 reductase activities resulted which closely paralleled the decline in adrenal monooxygenase activities. EDTA extraction of hepatic, adrenal, or pulmonary microsomes after the preincubation exposure removed about 95% of the cadmium but did not diminish the effects of the metal on microsomal monooxygenases. The results indicate that cadmium has somewhat varying sites of action on hepatic, adrenal, and pulmonary monooxygenases, but in all three tissues electron transfer to cytochrome P-450 is compromised. In addition, the effects of cadmium on microsomal metabolism persist fully even after removal of approximately 95% of the metal.

Adrenal Glands↗
PubMed1984

Effects of carbon tetrachloride on adrenocortical structure and function in guinea pigs.

Studies were carried out to evaluate the effects of carbon tetrachloride (CCl4) on adrenocortical structure and function in guinea pigs. Treatment with CCl4 reduced adrenal microsomal cytochrome P-450 concentrations and markedly decreased adrenal benzo(a)pyrene (BP) hydroxylase and benzphetamine (BZ) demethylase activities. Adrenal microsomal 17 alpha- and 21-hydroxylase activities were relatively unaffected by CCl4. Similar changes in adrenal metabolism resulted from incubation of microsomal suspension with CCl4 plus NADPH in vitro. Morphologically, CCl4 treatment resulted in necrotic changes in the inner portions of the adrenal cortex. The zona reticularis and inner fasciculata contained numerous cells with pyknotic nuclei, fragmented nuclei, and vacuolated cytoplasm. Cells in the outer fasciculata and zona glomerulosa of the adrenals appeared normal. In adrenals obtained from normal guinea pigs, xenobiotic metabolism was highly localized to the inner portion of the cortex, the site of CCl4-induced necrosis. The CCl4-induced type I spectral change, a tentative measure of binding to cytochrome(s) P-450, was also greater in microsomes from the inner than from the outer zones. In addition, the initiation of lipid peroxidation by CCl4 plus NADPH, as well as the formation of covalently bound metabolites from 14CCl4, was far greater with inner than outer zone microsomes. The results indicate that the effects of CCl4 on the adrenal cortex are localized to the inner zone which probably represents the site of activation of the toxin. In addition, adrenal xenobiotic-metabolizing monooxygenases seem to be more vulnerable to the toxic effects of CCl4 than the microsomal steroid hydroxylases.

Adrenal Cortex↗
PubMed1984

Functional zonation of the guinea pig adrenal cortex: differences in mitochondrial steroid metabolism between the inner and outer zones.

Previous studies established that cells isolated from the chromatically distinct inner (primarily zona reticularis) and outer (zona fasciculata + zona glomerulosa) zones of the guinea pig adrenal cortex had vastly different steroidogenic capabilities; the outer zone produced far more cortisol than the inner zone. The mechanism(s) responsible for those differences were investigated by comparing mitochondrial steroid metabolism in the inner and outer zones. Cytochrome P-450 concentrations were similar in the two zones, but 11 beta-hydroxylase activity was approximately twice as great in the outer zone. More importantly, cholesterol sidechain cleavage, the rate-limiting step in steroidogenesis, was nearly 10 times greater in outer than inner zone mitochondria. Free cholesterol concentrations were also far higher in outer zone mitochondria. The results suggest that the relatively low level of steroid secretion by cells of the zona reticularis is attributable, at least in part, to deficiencies in mitochondrial cholesterol content and/or metabolism.

Adrenal Cortex↗
PubMed1983

Effects of lipid peroxidation on adrenal microsomal monooxygenases.

Incubation of guinea pig adrenal microsomes with 10(-6) M ferrous (Fe2+) ion and adrenal cytosol initiated high levels of lipid peroxidation as measured by the production of malonaldehyde. Cytosol or Fe2+ alone had little effect on microsomal malonaldehyde formation. When microsomes were incubated in the presence of Fe2+ and cytosol, malonaldehyde levels continued to increase for at least 60 min. Accompanying the lipid peroxidation was a decline in adrenal microsomal monooxygenase activities. The rates of metabolism of xenobiotics (benzphetamine demethylase, benzo[a]pyrene hydroxylase) as well as steroids (21-hydroxylation) decreased as malonaldehyde levels increased. In addition, cytochrome P-450 levels, NADPH- and NADH-cytochrome c reductase activities, and substrate interactions with cytochrome(s) P-450 decreased as lipid peroxidation progressed. Inhibition of lipid peroxidation by increasing microsomal protein concentrations during the incubation period prevented the changes in microsomal metabolism. Malonaldehyde had no direct effects on adrenal microsomal enzyme activities. The results indicate that lipid peroxidation may have significant effects on adrenocortical function, diminishing the capacity for both xenobiotic and steroid metabolism.

Adrenal Glands↗
PubMed1983

Regional distribution of microsomal drug and steroid metabolism in the guinea pig adrenal cortex.

The regional distribution of steroid and drug metabolism was studied in intact cells and microsomal fractions obtained from the chromatically distinct inner (zona reticularis) and outer (zona fasciculata plus zona glomerulosa) zones of the guinea pig adrenal cortex. Cells isolated from the outer cortical zone produced far more cortisol than cells from the inner zone and cortisol production was stimulated by adrenocorticotropic hormone only in cells from the outer zone. Among the factors which may contribute to the greater cortisol production by the outer zone are a higher rate of 17 alpha-hydroxylation and ratio of 17 alpha- to 21-hydroxylase activities in that zone, both of which favor cortisol synthesis. In contrast, steroid 21-hydroxylase activity was far greater than 17 alpha-hydroxylase activity in microsomes obtained from the inner zone of the adrenal cortex. Microsomal metabolism of various xenobiotics such as benzo(a)pyrene and ethylmorphine proceeded far more rapidly in the inner than outer cortical zone. The zonal differences in metabolism appeared to result in part from differences in the ability of xenobiotics to interact with microsomal cytochromes P-450 in the two zones. The results indicate that the inner zone has a minor role in cortisol production by the adrenal cortex, but its involvement in the production of other steroids cannot be excluded. In contrast, the inner zone appears to have the major role in the metabolism of at least some xenobiotics which may account for its greater vulnerability to the toxic effects of chemicals requiring metabolic activation.

Adrenal Cortex↗
PubMed1983

Ascorbate uptake by isolated rat alveolar macrophages and type II cells.

Studies were conducted to measure intracellular ascorbate content and to characterize ascorbate uptake in three fractions of isolated rat pneumocytes (i.e., alveolar macrophages, alveolar type II epithelial cells, and another fraction of small pneumocytes that contains neither macrophages nor type II cells). When cells are incubated in medium containing 0.1 mM ascorbate (i.e., the concentration normally found in plasma), intracellular ascorbate concentrations are 3.2 mM in alveolar macrophages and type II cells and 0.9 mM in other lung cells; ascorbate influx is 1.5 nmol . 10(7) cells-1 . h-1 for alveolar macrophages, 0.24 nmol . 10(7) cells-1 . h-1 for type II cells, and very slow in other pneumocytes. Ascorbate influx displays saturation kinetics in both alveolar macrophages (K1/2 = 2 mM; Vmax = 32.2 nmol . 10(7) cells-1 . h-1) and type II cells (K1/2 = 5 mM; Vmax = 14.2 nmol . 10(7) cells-1 . h-1). After correction for differences in the membrane surface areas of these two types of lung cells, the rates for maximum ascorbate influx (Vmax) are similar in alveolar macrophages and type II cells. In addition, ascorbate uptake by alveolar macrophages and type II cells is dependent on metabolic activity and extracellular sodium. In contrast, ascorbate uptake in other lung cells does not exhibit saturation kinetics and is not dependent on metabolism or sodium. Thus alveolar macrophages and type II cells possess an energy-dependent cotransport system for ascorbate and sodium influx. The high ascorbate content and the existence of a specialized transport mechanism for ascorbate uptake may explain the relative resistance of alveolar macrophages and type II cells to oxidant injury.

Animals↗
PubMed1983

Transport properties of isolated type II alveolar epithelial cells.

Type II cells are granular cells located in the alveolar epithelium. In addition to the synthesis and secretion of surfactant, these pneumocytes exhibit several other interesting properties. Although type II cells possess a high permeability to sodium, they maintain a low free intracellular sodium concentration by the presence of a Na-K pump. The activity of the Na-K pump is high and can result in substantial net movement of solute and water. Therefore, type II cells may employ this pumping capacity to play a significant role in the transepithelial transport of water. Type II cells are also relatively resistant to oxidant damage and play a role in the regeneration of the alveolar epithelium after oxidant injury. Ascorbate is a known antioxidant that is accumulated by type II cells via a specialized transport system for the uptake of ascorbate and sodium. The presence of this specialized transport system in type II cells and alveolar macrophages may explain in part why these cells are more resistant to oxidant injury than other pneumocytes.

Animals↗
PubMed1982

Metabolism of benzo [a] pyrene by guinea pig adrenal and hepatic microsomes.

Studies were carried out to compare the metabolism of benzo [a] pyrene (BP) by adrenal and hepatic microsomes obtained from adult male guinea pigs. Adrenal microsomes produced fluorescent metabolites (primarily phenols) approximately three to four times more rapidly than hepatic microsomes, but the differences in the rates were considerably smaller when total BP metabolism was assessed using an isotopic assay. The apparent discrepancy between the two assays is attributable to differences in the profiles of BP metabolites produced by adrenal and liver. Separation of metabolites by high pressure liquid chromatography revealed that adrenal microsomes converted BP to primarily a phenolic metabolite with a retention time identical to that of 3-hydroxy-BP. Liver microsomes, by contrast, produced approximately equal amounts of compounds co-chromatographing with 3-hydroxy-BP and BP-4,5-dihydrodiol. Small amounts of other metabolites were also produced by adrenal and hepatic microsomes. Liver microsomes catalyzed the conversion of BP to metabolites that became covalently bound to exogenous DNA. The amount of binding was dependent upon the duration of incubation and concentration of microsomal protein. Adrenal microsomes, by contrast, did not promote BP binding to DNA. Inhibition of microsomal epoxide hydratase activity with trichloropropene oxide (TCPO) blocked the formation of dihydrodiol metabolites of BP by adrenal and liver microsomes. In the presence of TCPO, liver microsomes produced large amounts of a BP metabolite co-chromatographing with BP-4,5-oxide. TCPO also increased the rate of production of DNA-binding metabolites by liver microsomes but had no effect on the formation of DNA-binding metabolites by adrenal microsomes. The results demonstrate major differences in the pathways of BP metabolism by guinea pig adrenal and hepatic microsomes. Although adrenal microsomes metabolize BP more rapidly than hepatic microsomes, far greater amounts of reactive metabolites are produced by the liver. Thus, adrenal metabolism of BP may be of little toxicological significance.

Adrenal Glands↗
PubMed1981

Ascorbate uptake by isolated rat lung cells.

Experiments were done to determine the intracellular concentration of ascorbate in isolated rat lung cells and the concentration in plasma and to study ascorbate influx in these cells. The intracellular ascorbate concentration was 2.25 mM and the plasma level was about 0.14 mM; i.e., the lung cell ascorbate concentration was about 16 times greater than the plasma level. When the cells were incubated in medium containing physiological levels of ascorbate (0.1 mM), influx increased linearly up to 60 min of incubation and was 0.54 +/- 0.04 nmol.10(7) cells-1.h-1. Influx was dependent on the extracellular ascorbate concentration. At concentrations ranging from 0.025 to 1 mM, uptake appeared to exhibit saturation kinetics with an apparent Km of 0.16 mM. At physiological levels of extracellular ascorbate (0.1 mM) at least 90% of the uptake appeared to be carrier mediated, and this influx was inhibited by various metabolic inhibitors. In addition, ascorbate influx was inhibited by ouabain and removal of extracellular sodium. These results suggest that lung cells contain a transport mechanism for ascorbate that is energy-dependent and that may be coupled to Na+ influx.

Animals↗
PubMed1981

Lipid peroxidation in adrenal and testicular microsomes.

Studies were carried out to determine the actions of and interactions between ascorbate, NADPH, Fe2+, and Fe3+ on lipid peroxidation in adrenal and testicular microsomes. Ascorbate-induced malonaldehyde production was maximal in adrenal and testicular microsomes at an ascorbate concentration of 1 X 10(-4)M. Fe2+, at levels between 10(-6) and 10(-3)M, produced concentration-dependent increases in lipid peroxidation in adrenal and testicular microsomes; Fe2+ had a far greater effect than Fe3+ in both tissues. In liver microsomes, by contrast, Fe2+ and Fe3+ had quantitatively similar effects on lipid peroxidation. NADPH alone had no effect on malonaldehyde production in adrenal or testicular microsomes. However, in the presence of low Fe2+ concentrations (10(-6)M), NADPH stimulated adrenal malonaldehyde production. The stimulation of lipid peroxidation by NADPH plus low Fe2+ was not demonstrable in testicular microsomes nor in adrenal microsomes which had been heat-treated to inactivate microsomal enzymes. Testicular malonaldehyde production was stimulated by NADPH if Fe3+ (5 X 10(-5) to 1 X 10(-3)M) was added to the incubation medium; the stimulation was not demonstrable in heat-treated microsomes. Fe3+ plus NADPH had little effect on adrenal lipid peroxidation. In the presence of high Fe2+ levels (10(-3)M), NADPH produced a concentration-dependent inhibition of adrenal lipid peroxidation; the inhibition was fully demonstrable in heat-treated microsomes. NADPH similarly inhibited ascorbate-induced lipid peroxidation in adrenal microsomes. In testicular microsomes, NADPH did not inhibit ascorbate or Fe2+-induced lipid peroxidation. The results indicate that various endogenous substances may be important in the control of adrenal and testicular lipid peroxidation and that the nature of the regulation differs from tissue to tissue.

Adrenal Glands↗
PubMed1981

Chemical suppression of steroidogenesis.

A large number of chemicals are known to interfere with steroidogenesis in the adrenal cortex and other tissues. Many xenobiotics inhibit steroid hormone production as a result of interactions with cytochrome P-450-containing hydroxylases in adrenal mitochondria or microsomes. For example, metyrapone, a compound used clinically in the evaluation of pituitary-adrenocortical function, binds to various cytochromes P-450 in the adrenal, preventing the interactions of steroid substrates with the enzymes and inhibiting steroidogenesis. The mineralocorticoid antagonist, spironolactone, and its major circulating metabolite, canrenone, also competitively interact with adrenal steroid hydroxylases. In addition, spironolactone is converted by adrenal microsomes to an unknown metabolite which promotes the destruction of cytochromes P-450, decreasing the activities of steroid hydroxylases. Carbon tetrachloride is similarly "activated" by adrenal microsomal mixed function oxidases resulting in a decline in steroidogenic enzyme activity. Carbon tetrachloride (in the presence of NADPH) initiates lipid peroxidation in adrenal microsomes but its toxic effects on steroid hydroxylases are fully demonstrable when lipid peroxidation is inhibited by EDTA. A number of heavy metals, including cadmium, also inhibit adrenal steroid hydroxylases. When incubated with adrenal microsomes, cadmium does not affect cytochrome P-450 levels but decreases basal and substrate stimulated NADPH-cytochrome P-450 reductase activity. Although inhibitory effects of many chemicals on steroidogenesis have been described, the toxicological significance as well as definitive mechanisms of action have in most cases yet to be determined.

Adrenal Cortex↗