Bufuralol metabolism by guinea pig adrenal and hepatic microsomes.

Previous investigations demonstrated that CYP2D16 was expressed at high levels in guinea pig adrenal microsomes. The studies presented here were done to determine whether adrenal metabolism of bufuralol (BUF), a model CYP2D substrate, was similar to that in the liver. Guinea pig adrenal microsomes converted BUF to 1'-hydroxybufuralol (1'-OH-BUF) as the major metabolite and smaller amounts of a compound identified as 6-hydroxybufuralol (6-OH-BUF). In contrast, 6-OH-BUF was the major product formed by hepatic microsomal preparations. The apparent Km values were similar for 1'-OH-BUF and 6-OH-BUF production in each tissue. Quinidine, a selective CYP2D inhibitor, decreased the production of both BUF metabolites equally in liver and adrenal microsomes. Cortisol also caused equivalent decreases in the rates of 1'-OH-BUF and 6-OH-BUF formation by adrenal microsomes, but had no effect on hepatic BUF metabolism. Although both BUF metabolites may be produced by CYP2D16, unknown factors appear to effect some differences in the catalytic characteristics of BUF metabolism in adrenal and liver. The large amount of 6-OH-BUF produced distinguishes BUF metabolism in guinea pigs from that in other species previously studied.

Changes in mitochondrial and microsomal lipid peroxidation and fatty acid profiles in adrenal glands, testes, and livers from alpha-tocopherol-deficient rats.

Studies were done to evaluate the effects of alpha-tocopherol deficiency in rats on the fatty acid composition and sensitivity to lipid peroxidation (LP) of mitochondria and microsomes from adrenal glands, testes, and livers. In control (alpha-tocopherol-sufficient) animals, adrenal concentrations of alpha-tocopherol were approximately 10 times greater than those in livers and testes. Dietary deficiency of alpha-tocopherol for 8 weeks decreased adrenal and hepatic concentrations by 80-90% and testicular concentrations by approximately 60-70%. Incubation of testicular or hepatic mitochondria and microsomes from control rats with FeSO(4) (1.0 mM) caused a time-dependent stimulation of LP as indicated by the formation of thiobarbituric acid reactive substances (TBARS); the rate of TBARS production increased in preparations from alpha-tocopherol-deficient animals. TBARS formation was not demonstrable in adrenal mitochondria or microsomes from alpha-tocopherol sufficient rats, but reached high levels in alpha-tocopherol-deficient preparations. The fatty acid composition of mitochondria and microsomes was tissue-dependent. In particular, arachidonic acid comprised approximately 40% of the total fatty acids in adrenal membranes, but only 20-25% in testes and livers. alpha-Tocopherol deficiency increased oleic acid concentrations in adrenal and hepatic mitochondria and microsomes but not in testes. In all three tissues, linoleic acid concentrations decreased by approximately 50%, but arachidonic acid levels were unaffected by alpha-tocopherol deficiency. The results indicate a close relationship between tissue sensitivity to LP in vitro and alpha-tocopherol concentrations. Nonetheless, any oxidative stress in vivo caused by alpha-tocopherol deficiency seems to spare arachidonic acid in mitochondria and microsomes but decreases linoleic acid concentrations. It is possible that because of the important physiological functions of arachidonic acid, metabolic adaptations serve to maintain membrane content during periods of oxidative stress.

Suppression of lipid peroxidation in adrenal microsomes following ACTH administration to guinea pigs.

Previous studies demonstrated high levels of lipid peroxidation (LP) in the guinea pig adrenal cortex. The present studies were done to determine if adrenal LP activity was influenced by ACTH, the major hormonal regulator of the gland. Guinea pigs were treated with ACTH for 1, 3 or 7 days. In addition, some guinea pigs received ACTH for 7 days and were killed 3 or 7 days later. After treatment, adrenal microsomal fractions were prepared and incubated in vitro with 1 mM ferrous sulfate to initiate LP. ACTH treatment caused a progressive decrease in adrenal LP; activity was almost totally inhibited within 3 days. The inhibitory effects of ACTH on LP were dose-dependent. Following cessation of ACTH treatment, adrenal LP gradually returned toward control levels. Microsomal concentrations of linoleic acid, a major substrate for adrenal LP, were increased by ACTH administration and then also returned to control levels after cessation of treatment. There were no significant changes in adrenal alpha-tocopherol or beta-carotene concentrations resulting from ACTH treatment. The results indicate that ACTH has a role in the regulation of adrenal LP. The actions of ACTH cannot be attributed to an increase in adrenal content of the antioxidants, alpha-tocopherol and beta-carotene, or to a decrease in LP substrate. The actions of ACTH to inhibit LP may contribute to an increase in adrenal hormone production by protecting steroidogenic enzymes from peroxidative degradation.

Maturational changes in CYP2D16 expression and xenobiotic metabolism in adrenal glands from male and female guinea pigs.

CYP2D16 is expressed at high levels in the zona reticularis (ZR) of guinea pig adrenal glands and contributes to adrenal metabolism of xenobiotics. Studies were done to evaluate the effects of age and gender on adrenal CYP2D16 expression and xenobiotic metabolism. In both male and female guinea pigs at 1, 7, 14, or 30 weeks of age, in situ hybridization and immunohistochemistry confirmed that CYP2D16 was highly localized to the ZR of the adrenal gland. The steroidogenic P450 isozyme, CYP17, by contrast, was expressed in both the zona fasciculata and ZR. The intensity of CYP2D16 staining was not age- or gender-dependent. However, the proportion of each adrenal gland comprised by ZR and thus expressing CYP2D16 increased with aging in both sexes and was greater in males than in females. The rates of metabolism of bufuralol, a CYP2D-selective substrate, by adrenal microsomal preparations generally correlated with the amount of ZR (and CYP2D16) in the gland. Thus, adrenal xenobiotic-metabolizing activities were greater in males than in females at all ages and increased with aging in males. However, the rates of bufuralol metabolism declined in sexually mature females (14 weeks) from the levels found in prepubertal females (7 weeks) and then increased markedly in retired breeders (30 weeks), suggesting an inhibitory effect of estrogens on enzyme activity. The results indicate that the age and gender differences in adrenal CYP2D16 content are largely determined by differences in the size of the ZR rather than the concentrations of CYP2D16 within cells of the ZR. However, adrenal xenobiotic-metabolizing activities in females seem to be further modulated by an inhibitory effect of estrogens.

Bimodal expression of CYP2D16 in the guinea pig adrenal cortex.

Prior studies revealed an apparent bimodal distribution of adrenal xenobiotic-metabolizing activities in outbred guinea pigs. High activities were characteristic of most animals but a minority had very low rates of metabolism. The present studies were done to determine the basis for this distribution. Among the enzymatic activities abundant in guinea pig adrenal microsomes is bufuralol 1'-hydroxylation, a marker for CYP2D isozymes. Western blot analyses revealed that adrenal expression of CYP2D16, the guinea pig CYP2D analogue, was highly correlated with xenobiotic-metabolizing activities. Of 12 animals studied, 4 had low levels of adrenal CYP2D16 expression and correspondingly low bufuralol 1'-hydroxylase and benzphetamine N-demethylase activities. In contrast, adrenal expression of steroidogenic P450 isozymes (CYP17 and CYP21) and the corresponding steroid hydroxylase activities were similar in all animals. There was also no evidence of polymorphism in hepatic xenobiotic metabolism. The results suggest that the bimodal expression of adrenal CYP2D16 is at least partly responsible for the variability in adrenal xenobiotic metabolism, and that this pattern of expression is not applicable to other adrenal P450 isozymes or to hepatic CYP2D16. Further studies are needed to determine if genetic polymorphism is responsible for the mode of adrenal CYP2D16 expression.

Species differences in adrenal lipid peroxidation: role of alpha-tocopherol.

Previous reports have noted high levels of lipid peroxidation (LP) in vitro in a variety of adrenocortical preparations. However, we have observed that susceptibility to adrenal LP seems to vary considerably from species to species. The current study was done to confirm these apparent species differences in adrenal LP in vitro and to determine if they were attributable to differences in alpha-tocopherol content. Incubation of mitochondrial or microsomal preparations from guinea pig or rabbit adrenal glands with ferrous ion (Fe2+) caused a time-dependent increase in the formation of thiobarbituric acid reactive substances (TBARS) accompanied by depletion of alpha-tocopherol. By contrast, incubation of adrenal mitochondria or microsomes from rats or monkeys with Fe2+ had little or no detectable effect on TBARS and basal adrenal alpha-tocopherol levels were five to ten-fold greater than those in guinea pigs or rabbits. In addition, there was little change in alpha-tocopherol concentrations during incubation of rat or monkey adrenal tissue. Dietary alpha-tocopherol deficiency in rats reduced adrenal alpha-tocopherol to concentrations approximating those in guinea pigs. Incubation with Fe2+ induced high levels of TBARS in adrenal mitochondria and microsomes from the alpha-tocopherol deficient rats. Conversely, dietary alpha-tocopherol supplementation in rabbits increased adrenal alpha-tocopherol levels and prevented Fe2+ induced TBARS formation in mitochondria and microsomes. The results indicate that there are large species differences in adrenal susceptibility to LP in vitro and that these differences are at least partly attributable to species differences in adrenal alpha-tocopherol concentrations.

Effects of adrenocorticotropic hormone and dexamethasone on adrenal and hepatic alpha-tocopherol concentrations.

Studies were done to determine the effects of ACTH treatment on adrenal alpha-tocopherol (alpha-T) concentrations in female rats. Administration of dexamethasone (DEX) to inhibit endogenous ACTH secretion increased whole adrenal alpha-T levels as well as the fractional amount in adrenal cytosol. Adrenal ascorbic acid (AA) concentrations were unaffected by DEX. DEX treatment also had no effect on hepatic AA content but decreased alpha-T concentrations in the liver. The subcellular distribution of alpha-T in the liver was not altered by DEX. Administration of ACTH to DEX-treated animals decreased adrenal alpha-T content and restored the pattern of subcellular distribution to that seen in controls. ACTH had no effect on hepatic alpha-T concentrations or subcellular distribution. ACTH treatment also had no effect on AA concentrations in adrenals or livers. The results demonstrate that ACTH has a role in the regulation of adrenal alpha-T but the mechanism(s) involved remain to be determined. The data also indicate that glucocorticoids such as DEX directly influence hepatic alpha-T levels independent of their effects on ACTH secretion.

Strain differences in adrenal microsomal steroid metabolism in guinea pigs.

We recently reported that CYP2D16, a xenobiotic-metabolizing P450 isozyme, was expressed at higher levels in adrenal microsomes from inbred Strain 13 guinea pigs than in those from outbred English Short Hair (ESH) animals. Studies were done to determine if there also were strain differences in adrenal microsomal steroid metabolism. In both inner (zona reticularis) and outer (zona fasciculata plus zona glomerulosa) zone preparations of the adrenal cortex, 21-hydroxylase activities were greater in microsomes from ESH than from Strain 13 guinea pigs. By contrast, 17alpha-hydroxylase activities were similar in the two strains. In both strains, 21-hydroxylase activities were greater in inner than outer zone microsomes, but the opposite was found for 17alpha-hydroxylase activities (outer>inner). Northern and Western analyses revealed higher levels of CYP21 mRNA and protein in adrenals from ESH than Strain 13 guinea pigs, but there were no strain differences in CYP17 mRNA or protein concentrations. Despite the zonal differences in adrenal 17alpha-hydroxylase and 21-hydroxylase activities, CYP17 and CYP21 mRNA and protein levels were similar in the inner and outer zones within each strain of guinea pig. The results demonstrate strain differences in microsomal steroid metabolism that are explained by differences in CYP21 expression. By contrast, the zonal differences in steroid hydroxylase activities may be attributable to post-translational mechanisms.

Inhibition of testicular steroid metabolism by administration of 1-aminobenzotriazole to rats.

Effects of 1-aminobenzotriazole (ABT) on testicular steroid metabolism were evaluated in rats. Administration of ABT to adult male rats caused dose-dependent decreases in testicular microsomal and mitochondrial cytochrome P450 concentrations. Significant losses of P450 occurred within 8 h of ABT treatment. Accompanying the declines in testicular P450 content were decreases in microsomal 17 alpha-hydroxylase and mitochondrial cholesterol sidechain cleavage activities. Incubation of testicular microsomes or mitochondria in vitro with ABT plus an NADPH-generating system had no effect on P450 concentrations or on rates of steroid metabolism. By contrast, incubation of hepatic microsomes with ABT under the same conditions decreased P450 levels and xenobiotic-metabolizing activity. The results indicate that ABT in vivo causes inactivation of steroidogenic P450 isozymes in the testis, but the mechanism of inactivation differs from that on xenobiotic-metabolizing isozymes.

Effects of ACTH administration on zonation of the guinea pig adrenal cortex.

Experiments were done to determine the actions of ACTH on the morphologic and functional characteristics of the zona fasciculata (ZF) and zona reticularis (ZR) in the guinea pig adrenal cortex. In control guinea pigs, a number of morphologic differences distinguished the ZF from the ZR, including the presence of far more lipid in the ZF than in the ZR. Treatment with ACTH decreased the lipid droplet content of the ZF cells, equalizing the amount of lipid in the two zones. Other morphologic differences between the ZF and ZR were also diminished by ACTH treatment. Immunohistochemical analyses indicated that CYP17 protein was found in both the ZF and ZR in control animals, but with greater immunostaining intensity in the ZF. The enzyme protein distribution corresponded with higher 17alpha-hydroxylase activity in the ZF than in the ZR. After ACTH treatment, the intensity of staining and enzyme activities in the two zones were similar, attributable largely to increases in the ZR. In situ hybridization-and immunohistochemistry showed that in control animals CYPD216 was highly expressed in the ZR but not in the ZF. ACTH treatment dramatically reduced the intensity of CYP2D16 mRNA and protein staining in the ZR. Bufuralol 1'-hydroxylase activity, a marker for CYP2D subfamily members, was also decreased significantly in the ZR by ACTH treatment. The data indicate that administration of ACTH to guinea pigs has opposite effects on the expression of CYP17 and CYP2D16 in the ZR, and diminishes or eliminates some of the structural and functional differences between the ZF and ZR. The results suggest a role for ACTH in establishing and maintaining adrenocortical zonation.

Localization of CYP2D16 in the guinea pig adrenal cortex by immunohistochemistry and in situ hybridization.

Recent reports indicate that the cytochrome P450 isozyme, CYP2D16, is expressed at high levels in the inner regions of the guinea pig adrenal cortex and may contribute to xenobiotic and/or steroid metabolism in the gland. In the present studies, immunohistochemical and in situ hybridization techniques were employed to definitively establish the localization of CYP2D16 within the adrenal cortex. In male guinea pigs of various ages, CYP2D16 protein and mRNA were highly localized to the zona reticularis (ZR); none was detectable in the zona fasciculata (ZF), zona glomerulosa (ZG) or the medulla. In contrast, the steroidogenic P450 isozyme, CYP17, was distributed throughout the ZF and ZR. From the earliest stages of development of the ZR, CYP2D16 staining was intense. As guinea pigs aged, the ZR progressively enlarged and comprised a proportionately greater amount of the cortex. At all ages, CYP2D16 was uniformly distributed throughout only the ZR. Coinciding with the age-related growth of the ZR and increase in adrenal CYP2D16 content was an increase in adrenal xenobiotic-metabolizing activity. The results establish that CYP2D16 has an intraadrenal localization that is unique among P450 isozymes, suggesting novel regulatory mechanisms and indicating that CYP2D16 may serve as a specific marker for ZR cells. The increase in CYP2D16 expression with age probably accounts for increasing levels of xenobiotic metabolism and may also contribute to an increase in intraadrenal cortisol degradation in older animals.

Strain differences in adrenal CYP2D16 expression in guinea pigs. Relationship to xenobiotic metabolism.

Experiments were done to determine the mechanisms responsible for differences in adrenal microsomal xenobiotic metabolism between Strain 13 and English Short-Hair (ESH) guinea pigs. The rates of adrenal xenobiotic metabolism (bufuralol 1'-hydroxylase, benzo[a]pyrene hydroxylase, benzphetamine N-demethylase) were 2-3 times greater in microsomes from the Strain 13 animals. In both strains, xenobiotic-metabolizing activities were far greater in the inner zone (zona reticularis) than in the outer zones (zona fasciculata and zona glomerulosa) of the adrenal cortex. Northern blot analyses of total adrenal RNA with a CYP2D16 cDNA as the probe revealed significantly greater amounts of CYP2D16 mRNA in the Strain 13 guinea pigs. In addition, SDS-PAGE and Western blotting of adrenal microsomes demonstrated higher concentrations of CYP2D16 protein in Strain 13 than in ESH animals. Expression of CYP2D16 was predominantly in the inner zone of the adrenal, coinciding with the major site of xenobiotic metabolism. The results demonstrated higher levels of expression of CYP2D16 in adrenal glands from Strain 13 than from ESH guinea pigs, which may account for the strain differences in adrenal xenobiotic metabolism. Strain 13 guinea pigs should serve as a good experimental model for further studies on the regulation of adrenal CYP2D16.

Differential effects of adrenocorticotropin in vivo on cytochromes P4502D16 and P450c17 in the guinea pig adrenal cortex.

Studies were performed to compare the effects of ACTH treatment in vivo on cytochromes P4502D16 and P450c17 in the guinea pig adrenal cortex. In untreated animals, CYP2D16 protein and messenger RNA (mRNA) expression as well as xenobiotic-metabolizing activities (bufuralol 1'-hydroxylase, benzphetamine N-demethylase, and benzo(a)pyrene hydroxylase) were far greater in the inner (zona reticularis) than the outer (zona fasciculata plus zona glomerulosa) zones of the cortex. ACTH treatment for 3 or 7 days significantly decreased the rates of xenobiotic metabolism in both the inner and outer adrenal zones. Western and Northern blot analyses revealed that adrenal CYP2D16 protein and mRNA concentrations were significantly decreased by ACTH. In contrast to its inhibitory effects on CYP2D16, ACTH treatment increased steroid 17 alpha-hydroxylase activity in the adrenal inner zone, but did not affect outer zone activity. Microsomal CYP17 protein concentrations were not affected by ACTH despite increases in CYP17 mRNA levels in both zones. The results indicate that ACTH causes down-regulation of adrenal CYP2D16, probably at the transcriptional level. Thus, modulation of CYP2D16 by ACTH is opposite that for the steroidogenic P450 isozymes, suggesting unique regulatory mechanisms. In addition, the data suggest that posttranscriptional mechanisms contribute to ACTH regulation of 17 alpha-hydroxylase activity in the guinea pig adrenal cortex.

Expression and zonal distribution of CYP2D16 in the guinea pig adrenal cortex: relationship to xenobiotic metabolism.

We recently cloned a CYP2D subfamily member (CYP2D16) from a guinea pig adrenal cDNA library and investigated the expression of CYP2D16 in the guinea pig adrenal cortex and its relationship to adrenal xenobiotic metabolism. A modified sodium dodecyl sulfate-polyacrylamide gel electrophoresis technique revealed three major bands in the molecular mass range of cytochromes P450 in guinea pig adrenal microsomes. Two of the bands were immunoreactive with anti-CYP17 (54 kDa) or anti-CYP21 (52 kDa) antibody. The third band (50 kDa) was immunoreactive with antibody raised against CYP2D1 and with anti-CYP1A1/1A2 antibody. Microsequencing of the 50-kDa band yielded an amino-terminal sequence of 38 amino acids identical to that deducted from the CYP2D16 cDNA. In addition, Northern blot analyses indicated the CYP1A1 was not expressed in the adrenal gland, suggesting that only CYP2D16 composed the microsomal 50-kDa band. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analyses demonstrated greater expression of CYP2D16 in microsomes from the inner zone (zona reticularis) of the adrenal cortex than from the outer zones, coinciding with the major site of adrenal xenobiotic metabolism. Bufuralol-1'-hydroxylase activity, a marker for CYP2D isozymes, was also greater in inner- than in outer-zone microsomal preparations and was highly correlated with CYP2D16concentrations. Northern blot analysis with a full-length CYP2D16 cDNA as the probe gave strong bands with adrenal inner zone RNA preparations and relatively weak bands with outer zone RNA. CYP2D16 mRNA was also detectable in liver and kidney RNA preparations, but at lower levels than in the adrenal inner zone, and it was not detectable in testes, lung, intestines, or heart. Overall, the results demonstrate that CYP2D16 is expressed at highest levels in the inner zone of the guinea pig adrenal cortex and suggest a major role for this isozyme in adrenal xenobiotic metabolism.

Stimulation of microsomal spironolactone metabolism by reduced glutathione.

The first step in the conversion of spironolactone (SP) to its biologically active metabolites is deacetylation to 7 alpha-thiospirolactone (7 alpha-thio-SL). Studies were done to evaluate the effects of reduced glutathione (GSH) on SP deacetylation by adrenal microsomal preparations. In the absence of GSH, adrenal microsomes catalyzed the conversion of SP to 7 alpha-thio-SL at low rates. Addition of GSH to the incubation medium caused a concentration-dependent stimulation of SP deacetylation. At a concentration of 10 mM, GSH caused a 4- to 5-fold increase in the rate of 7 alpha-thio-SL production. The results suggest that GSH may have an important role in the overall disposition of SP, including the formation of active metabolites.

Inhibition of adrenal steroid metabolism by administration of 1-aminobenzotriazole to guinea pigs.

Prior in vitro investigations demonstrated that the P450 suicide substrate, 1-aminobenzotriazole (ABT), was a potent inhibitor of xenobiotic metabolism but had no effect on steroidogenic enzymes in the guinea pig adrenal cortex. Studies were done to determine if ABT administration of guinea pigs in vivo also selectively inhibited adrenal xenobiotic metabolism. At single doses of 25 or 50 mg/kg, ABT effected rapid decreases in spectrally detectable adrenal P450 concentrations. The higher dose caused approx. 75% decreases in microsomal and mitochondrial P450 levels within 2 h. The decreases in P450 were sustained for 24 h but concentrations returned to control levels within 72 h. Accompanying the ABT-induced decreases in adrenal P450 content were proportionately similar decreases in P450-mediated xenobiotic and steroid metabolism. Microsomal benzo(a)pyrene hydroxylase, benzphetamine N-demethylase, 17 alpha-hydroxylase and 21-hydroxylase activities were decreased to 20-25% of control values by the higher dose of ABT. Mitochondrial 11 beta-hydroxylase and cholesterol sidechain cleavage activities were similarly diminished by ABT treatment. Adrenal 3 beta-hydroxysteroid dehydrogenase activity, by contrast, was not affected by ABT, indicating specificity for P450-catalyzed reactions. The results demonstrate that ABT in vivo is a non-selective inhibitor of adrenal steroid- and xenobiotic-metabolizing P450 isozymes. The absence of ABT effects on steroid metabolism in vitro suggests that an extra-adrenal metabolite may mediate the in vivo inhibition of steroidogenesis.

Inactivation of adrenal cytochromes P450 by 1-aminobenzotriazole. Divergence of in vivo and in vitro actions.

Recent investigations demonstrated that administration of 1-aminobenzotriazole (ABT) to rats caused adrenal gland enlargement. Studies were done to pursue the mechanism(s) involved. Preliminary experiments revealed that the adrenal enlargement caused by ABT was associated with a decline in plasma corticosterone concentrations, suggesting inhibition of adrenal steroidogenesis. Indeed, a single injection of ABT (25 or 50 mg/kg body weight) to rats caused concentration-dependent declines (60-80%) in adrenal mitochondrial and microsomal cytochrome P450 (P450) concentrations. The decreases in adrenal P450 levels exceeded those in hepatic microsomes. Accompanying the declines in adrenal P450 concentrations were decreases in steroid hydroxylase activities. Mitochondrial 11 beta-hydroxylase and cholesterol side-chain cleavage activities and microsomal 21-hydroxylase activity were diminished markedly (60-90%) by ABT treatment. In contrast, activity of adrenal 3 beta-hydroxysteroid dehydrogenase-isomerase was not affected by ABT, indicating specificity for P450-dependent reactions. Incubation of adrenal microsomes or mitochondria in vitro with ABT plus an NADPH-generating system had no effect on P450 concentrations or on steroid hydroxylase activities. Similar incubations with hepatic microsomes caused declines in P450 levels and in the rates of P450-mediated xenobiotic metabolism. The results demonstrate that ABT is a potent inhibitor of adrenal steroid hydroxylases in vivo, but the in vitro studies indicate that the mechanism of action differs from that on other P450 isozymes. The absence of inhibitor effects in vitro suggests that an extra-adrenal metabolite of ABT is responsible for the in vivo inactivation of steroidogenic enzymes.

Molecular cloning and sequencing of a guinea pig cytochrome P4502D (CYP2D16): high level expression in adrenal microsomes.

Studies were done to characterize a guinea pig adrenal microsomal P450 that had been linked with xenobiotic metabolism in the inner zone of the gland. N-terminal amino acid sequencing of the isolated protein revealed homology with members of the CYP2D subfamily. A human CYPD2D6 cDNA probe was used to screen a guinea pig adrenal cDNA library and a full-length clone was obtained having an open reading frame encoding a 500 amino acid protein. The sequence was found to be highly homologous with all members of the CYP2D subfamily and was designated CYP2D16. The N-terminal sequence of 38 amino acids obtained from the protein microsequencing was identical to that deduced from the nucleotide sequence of the cloned CYP2D16. Northern blot analysis confirmed that CYP2D16 is expressed at high levels in the inner zone of the guinea pig adrenal cortex. The results suggest that CYP2D16 may account, at least in part, for the high rates of xenobiotic metabolism in the guinea pig adrenal.

Zonal differences in adrenocortical lipid peroxidation: role of alpha-tocopherol.

Studies were done to evaluate the relationship between alpha-tocopherol (alpha-T) concentrations and lipid peroxidation (LP) in vitro in microsomal preparations from the inner (zona reticularis) and outer (zona fasciculata plus zona glomerulosa) zones of the guinea pig adrenal cortex. Microsomes were incubated with ferrous ion (Fe2+) to promote free radical production, and alpha-T levels and LP were monitored after various incubation times. alpha-T concentrations were far lower in inner than outer zone preparations and were rapidly depleted from inner zone microsomes by incubation with Fe2+. Coinciding with alpha-T depletion was a large and rapid increase in LP. With outer zone microsomes, alpha-T depletion required more than 30 min, and very little LP was demonstrable during this period. However, once alpha-T depletion occurred, LP was rapidly initiated and reached levels similar to those obtained with inner zone preparations. Inhibition of LP by MnCl2 prevented the Fe(2+)-induced declines in alpha-T in both zones. The results demonstrate the importance of alpha-T as a modulator of adrenal LP and indicate that the zonal differences in LP are largely attributable to the differences in alpha-T concentrations.

Adrenal activation of carbon tetrachloride: role of microsomal P450 isozymes.

Previous investigations demonstrated that carbon tetrachloride (CCl4) was activated by adrenal microsomes, resulting in various functional changes and ultimately in necrosis of the zona reticularis of the gland. Experiments were done to identify the adrenal P450 isozyme(s) involved in the bioactivation of CCl4. Incubation of microsomes from the zona reticularis (ZR) of the guinea pig adrenal cortex with CCl4 plus NADPH caused initiation of lipid peroxidation, covalent binding of CCl4-derived radioactivity to protein, and degradation of cytochrome(s) P450. Preincubation of the microsomal preparations with inhibitory antibodies to P450(17 alpha) or P450C21 decreased the corresponding enzyme activities (17 alpha-hydroxylation and 21-hydroxylation), but did not affect the activation of CCl4. 1-Aminobenzotriazole (ABT), a suicide inhibitor of some P450 isozymes, decreased the enzyme activities catalysed by an adrenal 52,000 Da (52 kDa) isozyme, but had no effect on the function of P450(17 alpha) or P450C21. However, ABT completely inhibited the CCl4-induced LP and covalent binding in adrenal microsomes. The results indicate that adrenal CCl4 activation is catalysed by the 52 kDa P450 isozyme and not by the steroid hydroxylases. Localization of the 52 kDa isozyme to the ZR probably accounts for the selective necrosis of this region of the gland by CCl4.