Howard D. Colby

Cytosolic factors which affect microsomal lipid peroxidation in lung and liver

Abstract Studies were carried out to determine the effects of lung and liver cytosol on pulmonary and hepatic mierosomal lipid peroxidation, to determine the cytosolic concentrations of various substances which affect lipid peroxidation, and to determine which of these substances is responsible for the effects of the cytosol on lipid peroxidation. Lung cytosol inhibits both enzymatic (NADPH-induced) and nonenzymatic (Fe2+-induced) lung microsomal lipid peroxidation. In contrast, liver cytosol stimulates lipid peroxidation in hepatic microsomes during incubation alone, enhances Fe2+-stimulated lipid peroxidation, and has no effect on the NADPH-induced response. Substances which are known to be involved in inhibition of lipid peroxidation, including glutathione, glutathione reductase, glutathione peroxidase, and superoxide dismutase, are found in greater concentrations in liver cytosol than in lung cytosol. However, ascorbate is found in approximately equal concentrations in pulmonary and hepatic cytosol. Most of the effects of the cytosol on lipid peroxidation seem to be due to ascorbate and glutathione. For example, ascorbate, in concentrations found in lung cytosol, inhibits lung microsomal lipid peroxidation to about the same extent as the cytosol. The effects of liver cytosol on hepatic microsomal lipid peroxidation can be duplicated by concentrations of ascorbate and glutathione normally found in the cytosol; i.e., ascorbate stimulates and glutathione inhibits lipid peroxidation with the net effect being similar to that of liver cytosol. The results indicate that ascorbate has opposite effects on pulmonary and hepatic microsomal lipid peroxidation and suggest that ascorbate plays a major role in protecting pulmonary tissue against the harmful effects of lipid peroxidation.

The relationship between chemiluminescence and lipid peroxidation in rat hepatic microsomes

Abstract Studies were carried out to determine the relationship between NADPH- and ascorbate-initiated chemiluminescence (CL) and lipid peroxidation (LP) in rat hepatic microsomes. NADPH-initiated CL and LP become maximal 15 min after addition of NADPH to the microsomes and ascorbate-initiated CL and LP become maximal 90 to 120 min following addition of ascorbate. There are four lines of evidence to indicate that both NADPH- and ascorbate-initiated chemiluminescence are related to lipid peroxidation. (i) The time courses for the increases in CL and in LP are identical. (ii) There is a linear relationship between total (integral) or maximal CL and LP. (iii) Drug substrates which inhibit LP also inhibit CL in a quantitatively similar manner. (iv) Inhibitors of lipid peroxidation, such as Co2+, Mn2+, Hg2+, para-chloromercuribenzenesulfonic acid, and EDTA, also inhibit chemiluminescence. The results of these experiments indicate that chemiluminescence initiated in hepatic microsomes by either NADPH or ascorbate is directly proportional to lipid peroxidation.

Dose-dependent actions of thyroxine on hepatic drug metabolism in male and female rats

Abstract Studies were carried out to compare the effects of various doses of thyroxine (T 4 ) on hepatic drug metabolism in male and female rats and to evaluate the role of the pituitary gland in the modulation of T 4 action. Administration of small amounts of T 4 (2.5 to 5 μg/100g body wt/day) to hypophysectomized rats of either sex increased hepatic ethylmorphine demethylase, benzo(a)pyrene hydroxylase and aniline hydroxylase activities. Larger amounts of T 4 (12.5 to 50 μg) reversed the stimulatory effects of the smaller doses. T 4 treatment produced dose-dependent decreases in hepatic cytochrome P-450 content and increases in NADPH-cytochrome c reductase activity in hypophysectomized rats of both sexes. Qualitatively similar effects were produced by T 4 administration to thyroidectomized male and female rats. However, larger doses of T 4 were required for maximum stimulation of drug metabolism in thyroidectomized than in hypophysectomized animals. The results indicate that physiological amounts of T 4 Uniformly stimulate hepatic drug metabolism in both male and female rats. Supraphysiological amounts, however, inhibit metabolism of some substrates and produce sex differences in T 4 actions. The effects of T 4 are demonstrable in the absence of the pituitary gland but pituitary-dependent factors appear to modulate the magnitude of the response to T 4 .

Inhibition of hepatic microsomal lipid peroxidation by drug substrates without drug metabolism

Experiments were performed to study the mechanism of action of drug substrates on lipid peroxidation in rat hepatic microsomes. Addition of the drug substrates, aniline, β-diethylaminoethyl diphenylpropylacetate (SKF-525A), aminopyrine, benzo[a]pyrene or ethylmorphine, to hepatic microsomes causes almost complete inhibition of NADPH-induced (enzymatic) lipid peroxidation. These substrates also produce similar inhibition of ascorbate-induced (non-enzymatic) lipid peroxidation in microsomes in which drug-metabolizing enzymes were inactivated by heat treatment. The substrate concentrations producing half-maximal inhibition (K12 are also similar for NADPH- and ascorbate-induced lipid peroxidation. Addition of metyrapone, an inhibitor of drug metabolism, has no effect on either the K12 values or on the maximal substrate inhibition of NADPH-induced lipid peroxidation. All five drug substrates also inhibit Fe2+-stimulated oxidation of linoleic acid. These results demonstrate that inhibition of lipid peroxidation in hepatic microsomes by drug substrates is independent of drug metabolism and is probably due to the antioxidant properties of the substrates.

Differential control of adrenal drug and steroid metabolism in the guinea pig.

Abstract Studies were carried out to compare the effects of several physiological variables on adrenal microsomal drug (ethylmorphine demethylation) and steroid (21-hydroxylation) metabolism in guinea pigs. The rate of adrenal ethylmorphine (EM) metabolism increased with maturation in males but not females, resulting in a sex difference (M > F) in adrenal enzyme activity in adult guinea pigs. Twenty-one hydroxylase activity, in contrast, was similar in adrenals from males and females. The concentration of adrenal microsomal cytochrome P-450 was unaffected by age or sex. ACTH administration decreased adrenal EM demethylase activity but did not affect 21-hydroxylation. Testosterone, when given to female guinea pigs, increased the rate of EM metabolism and decreased 21-hydroxylase activity. Various compounds known to interact with adrenal microsomal cytochrome P-450 had divergent effects on EM metabolism and 21-hydroxylation in vitro . Prostaglandins E1 and F2α, spironolactone, and canrenone inhibited EM demethylation but not 21-hydroxylation. Simple aromatic hydrocarbons (benzene, toluene), in contrast, inhibited 21-hydroxylation but did not affect EM metabolism. The results indicate that adrenal drug and steroid metabolism are independently regulated and that different terminal oxidases (cytochrome P-450) are probably involved in adrenal 21-hydroxylation and EM demethylation.

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.

Interaction of aromatic hydrocarbons and drugs with adrenal microsomal cytochrome P-450 in the guinea pig.

Abstract Addition of simple aromatic hydrocarbons (benzene, ethylbenzene, naphthalene) to guinea pig adrenal microsomes produced typical Type I difference spectra (ΔOD 385-420 ). Spectral dissociation constants ( K s ) for each indicated a far higher affinity for adrenal than hepatic cytochrome P-450. Hydrocarbon affinities for adrenal cytochrome P-450 were similar to that for progesterone, an endogenous steroid substrate. Ethylmorphine and aniline produced Type I and Type II spectral changes respectively in adrenal microsomes. The K s , and magnitude of spectrum for each in adrenals was similar to that in livers. Nonetheless, demethylation of ethylmorphine proceeded far more rapidly in adrenal than hepatic tissue. The Michaelis constants ( K m ) for ethylmorphine metabolism in both tissues were similar. Although the aniline-induced difference spectra in adrenal and hepatic microsomes did not differ substantially, aniline hydroxylase activity was far greater in liver. Pretreatment of guinea pigs with phenobarbital or 3-methylcholanthrene increased hepatic but not adrenal ethylmorphine metabolism. Spironolactone pretreatment, in contrast, did not affect hepatic metabolism, but significantly lowered adrenal demethylase activity. The results indicate a relative non-specificity of guinea pig adrenal microsomal cytochrome P-450 and suggest that the adrenal cortex may represent a significant site for the extra-hepatic metabolism of foreign compounds in the guinea pig.

Carbon tetrachloride-induced changes in adrenal microsomal mixed-function oxidases and lipid peroxidation

Abstract Studies were carried out to compare the effects of carbon tetrachloride (CCl 4 ) in vivo and in vitro on adrenal and hepatic microsomal metabolism in guinea pigs. CCl 4 administration in vivo decreased adrenal and hepatic microsomal cytochrome P -450 concentrations and lowered benzphetamine (BZ) demethylase and benzo[ a ]pyrene (BP) hydroxylase activities in both tissues. NADPH-cytochrome c reductase activity was decreased in hepatic but not in adrenal microsomes. Addition of CCl 4 to adrenal or hepatic microsomes in vitro produced a type I difference spectrum suggesting binding of CCl 4 to cytochrome(s) P -450; its magnitude was far greater in adrenal than in liver. Incubation of adrenal or hepatic microsomes in vitro with CCl 4 alone had little or no effect on mixed-function oxidase activity or on lipid peroxidation. However, when microsomes were incubated with CCl 4 + NADPH, the rates of BZ and BP metabolism were decreased, cytochrome P -450 concentrations were decreased, and lipid peroxidation was increased. The effects of CCl 4 + NADPH on enzyme activities were greater in adrenal than in hepatic microsomes. Addition of 1.0 m M EDTA or 0.1 m M MnCl 2 to the incubation medium blocked the effects of CCl 4 + NADPH on lipid peroxidation in adrenal and liver but had no effect on the decreases in mixed-function oxidase activities. The results indicate the following: (1) the adrenal cortex in the guinea pig is an active site of CCl 4 metabolism; (2) CCl 4 metabolism results in a loss of microsomal enzyme activities in the adrenal as well as liver; and (3) lipid peroxidation is not obligatory for the CCl 4 -mediated destruction of microsomal enzymes.

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.

Lipid peroxidation in guinea pig lung microsomes

The effects of substances known to influence lipid peroxidation were studied in guinea pig lung microsomes by measuring the formation of malonaldehyde in vitro. Incubation of lung microsomes at 37 degrees C results in lipid peroxidation which appears to be an enzymatic process but is not dependent upon iron. Lipid peroxidation can be initiated non-enzymatically in lung microsomes by Fe2+, but ascorbate and Fe3+ have very little effect on malonaldehyde formation. The effects of NADPH on lipid peroxidation are dependent upon the concentration of Fe2+ in the incubation medium. At concentrations of Fe2+ between 0.05 mM and 1 mM, addition of NADPH causes an increase in lipid peroxidation over that produced by Fe2+ alone. This stimulation by NADPH is an enzymatic process and phosphate is required for the maximal effect. Addition of NADPH to lung microsomes in the presence of Fe3+ does not increase malonaldehyde formation over that produced by Fe3+ alone, suggesting that NADPH does not influence lipid peroxidation by maintaining iron in the reduced form. At concentrations of Fe2+ greater than 1 mM, NADPH inhibits Fe2+-induced lipid peroxidation in normal microsomes and in microsomes in which enzymes have been inactivated with heat. This latter result suggests that the inhibition by NADPH is at least partially non-enzymatic. The result suggests that the inhibition by NADPH is at least partially non-enzymatic. The results of all of these experiments are discussed and compared with those obtained during lipid peroxidation in liver microsomes. We conclude that the processes involved in pulmonary microsomal lipid peroxidation differ significantly from those in hepatic microsomes.