Ultrastructural effects of Nifurtimox on rat adrenal cortex related to reductive biotransformation

Nifurtimox (Nfx) (4(5-nitrofurfurylidene)amino)-3-methylthiomorpholine-1, 1-dioxide) is a drug used against Chagas' disease, a parasitic sickness afflicting several million Latin Americans. Nfx administration to Sprague-Dawley male rats (220-250 g) at a dose of 100 mg/kg caused pronounced alterations in the adrenal cortex involving the fasciculata and reticularis zones but which were not evident in the glomerulosa. Alterations observed involved mitochondria, nuclei, Golgi apparatus, and the endoplasmic reticulum but were more intense in the mitochondria. There is Nfx nitroreductase activity in the adrenal microsomal, mitochondrial, and cytosolic-rich fractions but most of it is in the mitochondrial-rich fraction. Activity in the first two fractions requires NADPH and that in the cytosol is only observed in the presence of hypoxanthine as substrate. Enzymatic activity in all fractions is inhibited by oxygen. CO does not inhibit mitochondrial Nfx nitroreductase and inhibits only 10% of the microsomal enzyme activity. Hypoxanthine-dependent cytosolic activity is inhibited by allopurinol. Present results suggest that Nfx is activated to damage-producing reactive metabolites by nitroreductive biotransformation in rat adrenal organelles. Mitochondrial and microsomal bioactivation would occur at the level of the flavoenzyme P-450 reductase rather than at P-450 itself, and cytosolic bioactivation would be mediated by xanthine oxidase. Epidemiological studies on adrenal function in patients undergoing Nfx treatment would be necessary to establish the potential toxicological relevance of these findings.     

Immunohistochemical localization of cytochrome P-450C21 in human adrenal cortex and its relation to endocrine function

Cytochrome P-450C21 was successfully demonstrated in the human adrenal glands by a peroxidase-antiperoxidase method. All three cortical layers were stained in the normal adrenal glands, particularly the zonae glomerulosa and reticularis. Well-stained and faintly stained cells were intermingled in the zona fasciculata, suggestive of intrazonal variations. The immunoreactivity was particularly intense at the site of ACTH action, i.e., cells in micronodules and cells around myelolipomatous lesions in adrenocortical hyperplasia of Cushing's disease and sites of regeneration in the normal adrenal glands. In adrenocortical adenomas with Cushing's syndrome and primary aldosteronism, cells with large nuclei were generally stained well. In the adrenocortical tissue adjacent to a functioning adenoma, the immunoreactivity was observed only in the zona glomerulosa, especially in cases of primary aldosteronism. This finding is consistent with morphologic observations.     

Influence of bile acids on stimulated lipid peroxidation and hydrogen peroxide production in rat liver microsomes.

Bile acids were found to be effective antioxidants in bile and intestine. The influence of different bile acids on the NADPH-Fe(++)-stimulated lipid peroxidation (LPO) and cytochrome P-450 dependent hydrogen peroxide production (H2O2) in rat liver microsomes was investigated in vitro. LPO was determined as production of thiobarbituric acid reactants (TBAR). Different tri-, di- and monohydroxylated bile acids and cholesterol were given to the incubation mixture in concentrations ranging from 10(-5) to 10(-3) M. Sodium salts of cholic, tauroglycocholic and deoxycholic acids as well as cheno-deoxycholic, ursodeoxycholic, lithocholic acids and cholesterol did not alter the microsomal production of TBAR. H2O2 formation was significantly decreased by sodium deoxycholate whereas cholesterol increased H2O2 production up to 4 times. These results show that bile acids were not able to protect microsomal membrane lipids against peroxidative damage. Cholesterol mediated H2O2 formation as a source of hydroxyl radicals had no toxic effect concerning LPO, TBAR were not enhanced significantly.     

Effects of glucocorticoids on differentiation of zona glomerulosa of fetal adrenal cortex of rats.

The tissue differentiation of the zona glomerulosa of the fetal adrenal cortex of rats was studied by giving experimental treatments to the fetus in vivo. A low-glucocorticoid-condition was given to the fetus by bilateral adrenalectomy of pregnant rats for removing exogenous glucocorticoids from the fetus, and by brain aspiration of the fetuses for removing the fetal pituitary gland (ACTH) and endogenous glucocorticoids. When the fetus was placed under a low-glucocorticoid-condition for the last couple of days of gestation, poor differentiation of the zona glomerulosa occurred specifically in the fetal adrenal cortex. The degree of the poor differentiation seemed to be proportional to the duration of the low-glucocorticoid-condition. Supplemental administration of glucocorticoids could prevent this poor differentiation of the zona glomerulosa. These results indicate that the tissue differentiation of the zona glomerulosa of the fetal adrenal cortex depends much on glucocorticoids.     

The adrenal cortex in spontaneously hypertensive rats. A quantitative ultrastructural study.

The adrenal cortex of spontaneously hypertensive rats (SHR) has been examined by quantitative morphologic techniques for electron microscopy. The volume and surface area of smooth endoplasmic reticulum and the volume of Golgi apparatuses in zona glomerulosa cells of SHR was significantly greater than those of Wistar-Kyoto strain (W/KY) normotensive controls; the volume of lipid droplets and nucleus was significantly less in SHR than in W/KY animals. A stimulation of the zona glomerulosa in SHR may well be attributable to the elevation in systolic blood pressure. A distinct lipid-free subglomerulosa was observed in the adrenal gland of W/KY rats; the cell volume was similar to that of the zona glomerulosa although the cells showed a significantly greater volume of mitochondria and surface area of mitochondrial membranes and greater volume of smooth endoplasmic reticulum and lysosomes. In the zona fasciculata, cell volume, volumes and surface area of mitochondria and smooth endoplasmic reticulum, and volume of lipid droplets were significantly lower in SHR than in W/KY rats. The volume of the Golgi apparatus was greater in SHR than in W/KY rats. Glycogen particles were observed in focal areas of some zona fasciculata cells. The adrenal cortex of another strain of normotensive Wistar rat (W/CFN) was compared with that of the W/KY and SHR. Although the relative adrenal weights of SHR and W/KY animals were identical, the weight of that in W/CFN was significantly smaller. The volume of the zona glomerulosa of SHR was significantly greater than that of W/KY although the volume of the zona glomerulosa in W/CFN was significantly greater than the other two groups. The volume of nucleus and lipid droplets of zona glomerulosa in W/KY was significantly greater than that in the S/CFN; the volume of the cell, mitochondria, smooth endoplasmic reticulum, lipid droplets, and lysosomes, and the surface area of smooth endoplasmic reticulum and mitochondrial membranes of W/KY animals was significantly greater than those of W/CFN animals. It is concluded that the W/CFN rat is not an appropriate control for spontaneously hypertensive rats.     

Quantitative ultrastructural study of the rat adrenal cortex in renal encapsulation-induced hypertension.

Hypertension was induced in young rats by latex encapsulation of both kidneys. By the fourth week, 85% of the renal-encapsulated (RE) rats became hypertensive. Varying degrees of cardiovascular involvement were evident in the moderately to severely hypertensive rats. The level of systolic blood pressure was directly correlated with the width and the volume of zona glomerulosa of the adrenal cortex. Electron microscopy combined with morphometric-stereologic techniques was employed to quantitate change in the adrenal cortex. The cells of both zona glomerulosa and zona fasciculata of RE rats showed significant increases in the volume of the cell, nucleus, smooth endoplasmic reticulum, and lipid droplets; only in the zona glomerulosa cells was the increase in surface area of the smooth endoplasmic reticulum statistically significant. It is suggested that these structural changes associated with renal-encapsulation hypertension are related at least in part to stress of the hypertensive cardiovascular disease.     

The innervation of the mouse adrenal cortex.

The fine structure of the mouse adrenal cortex was examined with the electron microscope for the presence of neural elements. Several axon terminals containing mostly clear vesicles (60 nm) were noted in the vicinity (250 nm) of the capsular fibroblasts. In the subcapsular region, myelinated as well as unmyelinated fibers were commonly found. Preterminal and terminal axons were also found in close relationship to the parenchymal cells in the zona glomerulosa. Nerve bundles were the most common neural elements in the zona fasciculata. In the zona reticularis axon terminals containing both clear (60 nm) and dense core (120 nm) vesicles were seen in close proximity (30 nm) to parenchymal cells. Although this study did not delineate the type of fibers involved, the axon terminals resemble those of autonomic nerves. This study demonstrates innervation of the mouse adrenal cortex, thus corroborating similar reports by others in different species.     

Evidence for and characterization of cytochrome P-450 in Neurospora crassa

Cytochrome P-450 was detected in microsomes and presumably in cytosol of Neurospora crassa, and was found to be inducible by progesterone. In the microsomal fraction cytochrome b5 and NADPH-cytochrome c reductase activities were measurable, too. Cytochrome P-450 of Neurospora crassa is inhibited by SKF-525 A and by inhibitors of ergosterol biosynthesis. After induction of cytochrome P-450 with progesterone 11α-hydroxyprogesterone as one metabolite of progesterone was detected in the culture media as well as in the mycelia. After 42 hours about 70% of progesterone were metabolized.     

The effect of IFN-alpha-Con1 on hepatic cytochrome P-450 and protein synthesis and degradation in hepatic microsomes.

Interferon and its inducers are well known to depress drug biotransformation in the liver by decreasing the levels of cytochrome P-450 in that organ. We now report that IFN-alpha-Con1, which was constructed from the most frequently observed amino acid sequences in human alpha-interferon subtypes, causes a loss in cytochrome P-450 which could be prevented by pretreating animals with either puromycin or actinomycin D. This suggests that the loss in drug biotransformation is mediated via the production of an intermediate protein. When the turnover of microsomal protein was examined this interferon appeared to depress the synthesis of proteins with molecular weights 46-60 kd and had little effect on the synthesis of other proteins. The in vitro translation of proteins of molecular weights 45-60 kd was also depressed in an in vitro translation system using mRNA isolated from the livers of interferon treated hamsters. Interferon had no effect on the degradation of microsomal proteins of all molecular weights. It is concluded that interferon probably depresses the levels of cytochrome P-450 in the liver by decreasing the synthesis of the apoprotein and that interferon has little effect on the degradation of the hemoprotein.     

Resistance of different forms of cytochrome P-450 of rat liver to factors destabilizing the microsomal membrane

The effect of factors destabilizing the membrane of the liver microsomes on the spectral properties of cytochrome P-450 (P-448) was investigated in intact rats and rats receiving phenobarbital (PB) or 3-methylcholanthrene (MC). Considerable resistance of microsomes induced by PB and MC to enzymic and nonenzymic peroxidation of polyunsaturated fatty acids of membrane phospholipids was discovered. A clear difference was shown in the sensitivity of cytochrome P-448 and cytochrome P-450 of intact rats and rats receiving PB to in vitro treatment with sodium deoxycholate. The results indicate structural changes in the microsomal membrane during induction by PB and MC, which are two different types of inducers of the monooxygenases of the liver.Institute of Clinical and Experimental Medicine, Siberian Branch, Academy of Medical Sciences of the USSR, Novosibirsk. (Presented by Academician of the Academy of Medical Sciences of the USSR V. P. Kaznacheev.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 5, pp. 553–555, May, 1977.     

Effects of aging and caloric restriction on hepatic drug metabolizing enzymes in the Fischer 344 rat. I: The cytochrome P-450 dependent monooxygenase system

The effects of long-term caloric restriction on the hepatic cytochrome P-450 dependent monooxygenase system were investigated in the 22-month-old Fischer 344 rat. Caloric restriction decreased the age-related changes in hepatic testosterone metabolism, which are associated with demasculinization of the liver. Caloric restriction also increased hepatic microsomal testosterone 6 beta-hydroxylase, lauric acid 12-hydroxylase and 4-nitrophenol hydroxylase activities over corresponding values in both ad libitum fed 22-month and 60-day-old control male rats. This suggests that cytochrome P-450 isozymes, P-450 pcn1&2, P-452 and P450j may be induced by caloric restriction. Such changes in cytochrome P-450 isozyme profiles could result in altered carcinogen activation, radical formation or drug detoxication in the calorically restricted rat.     

Distribution of prothymosin alpha in rat and human adrenal cortex

Background: Prothymosin alpha (ProT) is a polypeptide widely distributed in the organism and expressed by cell types with a high proliferative capacity. The aim of the current work was to investigate if ProT was localized in the progenitor compartment of the adrenal cortex which, following the cell migration theory, corresponds to the zona glomerulosa. Methods: We studied by immunohistochemical and in situ hybridization methods the distribution of ProT in rat and human adrenal cortex. Immunohistochemical techniques for the study of the proliferating cell nuclear antigen (PCNA) and incorporation of 5-bromo-2′-deoxyuridin during DNA synthesis were also done. Immunoelectron microscopic procedures were performed to determine the exact subcellular localization of ProT. Results: ProT was found in the zona glomerulosa cells, but not in the cells of the remaining cortical layers (zonae fasciculata and reticularis). Glomerulosa cells showed immunostaining for ProT only in the nuclei, excluding the nucleoli. Variability in immunostaining intensity was found between different glomerulosa cells. In situ hybridization of ProT mRNA confirmed that ProT synthesis in adrenal cortex occurs only in the zona glomerulosa. The results obtained for proliferating cell nuclear antigen and incorporation of 5-bromo-2′-deoxyuridin confirmed that adrenocortical proliferation occurs in the zona glomerculosa. Ultrastructural immunocytochemistry showed labelling for ProT over the euchromatin, but not on the heterochromatin aggregations nor the nucleoli. Conclusions: The results presented here: (1) support the migration theory for the adrenocortical cell renewal, (2) demonstrate that ProT is present in the nuclei of proliferating cells (being associated with euchromatin), and (3) suggest that the study of ProT expression would be useful in distinguishing cycling from resting cells. © 1994 Wiley-Liss, Inc.     

Subcellular organization of alkane oxidation in the yeast Candida maltosa

After spheroplast lysis and differential centrifugation the alkane monooxygenase system consisting of cytochrome P-450 and the NADPH-cytochrome P-450 reductase of alkane-grown Candida maltosa cells is enriched in the microsomal fraction. This membrane fraction is nearly free of intact mitochondria (cytochrome oxidase) and peroxisomes (catalase), but contains considerable amounts of plasma membrane fractures (azide insensitive, vanadate-sensitive Mg²⁺-ATPase) as demonstrated by biochemical an electron microscopic examinations. By means of sucrose density gradient centrifugation it was possible to separate the cytochrome P-450 containing membranes ( = 1,11 g/cm³) from the plasma membranes ( = 1,18 g/cm³). Therefore the cytochrome P-450 alkane monooxygenase system is most likely localized in the endoplasmic reticulum of the yeast cells. For the following enzymatic steps of terminal alkane oxidation to the corresponding fatty acid a quite different subcellular distribution was observed. The fatty alcohol oxidase and aldehyde dehydrogenase activities are mainly localized in the mitochondrial peroxisomal membrane fraction. During the oxidation of n-alkanes by yeast cells the fatty alcohol should be regarded as an intracellular transport from between the cytochrome P-450 containing endoplasmic reticulum and the sites of its further oxidation in peroxisomes and mitochondria.     

Aldehydic products of lipid peroxidation inactivate cytochrome P-450.

The effects of aldehydic products of lipid peroxidation, malondialdehyde (MDA) and 4-hydroxynonenal (HNE), on the structure of rat liver microsomal membrane and cytochrome P-450 was studied. MDA (15-30 microM) similarly to p-chlormercuribenzoate decreased the cytochrome P-450 content by 50 % and lowered microviscosity of lipid surrounding of the spin label OTMB bound to SH-groups of membrane proteins. OTMB was effectively reduced by K3Fe(CN)6 in microsomes preincubated with MDA (20 (M), but not in native microsomes. HNE (10 microM) decreased the cytochrome P-450 content by 90 %. Reduced glutathione and cysteine (5 mM) prevented the decrease of cytochrome P-450 under influence of both MDA or HNE, whereas cytochrome P-420 formation remains unchanged. MDA and HNE decreased activities of NADPH oxidase and NADPH cytochrome c reductase. HNE increased microviscosity of the OTMB lipid environment. The further increase of HNE concentration did not affect this parameter. Both MDA and HNE increased the absorbance at 420 nm, which indicated inactivation of cytochrome P-450 by changes in hydrophobicity of lipid surrounding. We suggest that HNE and aliphatic aldehydes at low concentrations can enter into hydrophobic environment of cytochrome P-450 binding to its SH-groups, which led to inactivation of cytochrome P-450. At the same time, the modification of membrane surface layer and subsequent decrease of hydrophobicity of cytochrome P-450 environment preceded the binding of MDA to SH-groups of cytochrome P-450 to develop its inactivating effect.     

Immunohistochemical localization of cytochrome P-450 in rat brain

The presence of cytochrome P-450 in rat brain was studied by immunohistochemistry, using antibodies to cytochrome P-450 purified from livers of phenobarbital- or 3-methylcholanthrene-treated rats. Immunoreactive nerves were observed only in brain sections incubated with immunoglobulin-G to 3-methylcholanthrene-induced cytochrome P-450. This immunoreactivity was abolished by preabsorption of the antibody with highly purified rat liver cytochrome P-450c, the major cytochrome P-450 isozyme induced by 3-methylcholanthrene, but was not affected by other cytochrome P-450 isozymes induced by phenobarbital, isosafrole or pregnenolone-16-carbonitrile.

The most abundant concentration of nerve fibers with cytochrome P-450 immunoreactivity was observed in the globus pallidus. Immunoreactive fibers were also observed in the caudate putamen, amygdala, septum, ventromedial nucleus of the hypothalamus, medial forebrain bundle, ansa lenticularis, and ventromedial portion of the internal capsule and crus cerebri. Cell bodies with cytochrome P-450 immunoreactivity were observed in the caudate putamen and in the perifornical area of the hypothalamus. The cytochrome P-450 immunoreactive fibers in the globus pallidus and caudate putamen do not appear to emanate from cell bodies in the substantia nigra, since there was no reduction in the density of these fibers after unilateral stereotaxic electrolytic destruction of the substantia nigra (zona compacta and reticulata). Our data suggest that these striatal nerve processes are derived from cell bodies within the caudate putamen itself.

The present results indicate that rat brain contains a form of cytochrome P-450 with antigenic relatedness to the hepatic 3-methylcholanthrene-inducible cytochrome P-450c. This cytochrome P-450 isozyme was detected in brain areas which metabolize morphine and convert estradiol and estrone into catecholestrogens, which suggests an important role for this enzyme in the metabolism of both ex´ogenous and endogenous compounds in brain.     

Immunohistochemical study of cytochrome P-45017 alpha in human adrenocortical disorders

Cytochrome P-450 specific for steroid 17 alpha-hydroxylation (P-45017 alpha) was immunolocalized in normal and hyperfunctioning adrenal glands of pigs, bovines, and humans, using a specific IgG fraction raised against the enzyme. P-45017 alpha was present in the zona fasciculata (ZF) and zona reticularis (ZR), but not in the zona glomerulosa (ZG), in pig, bovine, and human adrenal glands. In the adrenal glands of patients with Cushing's disease, the positive immunoreactivity to P-45017 alpha was intense in ZF and ZR, particularly in cortical micronodules, corresponding to the sites of active steroidogenesis. Cells of hyperplastic ZG and outer ZF in the adrenal glands of idiopathic hyperaldosteronism were negative for P-45017 alpha. In aldosteronoma, positive immunoreactivity was observed in some tumor cells, which is consistent with cortisol production and its responsiveness to ACTH in aldosteronoma. In the attached adrenal glands of aldosteronoma, the immunoreactive P-45017 alpha was clearly present in the inner ZF and ZR, suggesting persistent androgen production. In Cushing's adenoma, the positive immunoreactivity was intense in tumor cells, and the ZR of the attached adrenal glands was weakly immunoreactive.     

Immunohistochemical demonstration of cholesterol side-chain cleavage cytochrome P-450 in bovine and human adrenals

Cytochrome P-450 specific for cholesterol side-chain cleavage (P-450SCC) was purified from the bovine adrenal and a specific antibody was raised in rabbits. The antiserum was applied for immunohistochemical visualization of the P-450SCC in the bovine and human adrenal cortex. The immunoreactivity was intense in the zona fasciculata (ZF) and reticularis (ZR) while weak in the zona glomerulosa (ZG) in the normal adrenals. In adrenocortical hyperplasia, a marked immunoreactivity was observed in the ZG in idiopathic hyperaldosteronism and the inner ZF and reticularis particularly in cells of micronodules in Cushing's disease, corresponding to cells with active steroidogenesis. In aldosteronoma and adenoma with Cushing's syndrome, P-450SCC was generally present in compact cells of adenomas.     

The innervation of the mouse adrenal cortex

The fine structure of the mouse adrenal cortex was examined with the electron microscope for the presence of neural elements. Several axon terminals containing mostly clear vesicles (60 nm) were noted in the vicinity (250 nm) of the capsular fibroblasts. In the subcapsular region, myelinated as well as unmyelinated fibers were commonly found. Preterminal and terminal axons were also found in close relationship to the parenchymal cells in the zona glomerulosa. Nerve bundles were the most common neural elements in the zona fasciculata. In the zona reticularis axon terminals containing both clear (60 nm) and dense core (120 nm) vesicles were seen in close proximity (30 nm) to parenchymal cells. Although this study did not delineate the type of fibers involved, the axon terminals resemble those of autonomic nerves. This study demonstrates innervation of the mouse adrenal cortex, thus corroborating similar reports by others in different species.     

Turnover of hepatic cytochrome P-450 in experimental cholestasis

In mechanical experimental chllestasis, hypertrophy of smooth microsomal membranes was observed. In contrast to typical induction, the membranes were deficient in cytochrome P-450. The total cytochrome P-450 content of the liver, however, as determined in the liver homogenate remained unchanged. To clarify the mechanism of the development of cytochrome P-450 deficient membranes in cholestasis, the half life of the heme portion of cytochrome P-450, and the initial rate of synthesis of cytochrome P-450 and b₅ hemes were compared in bile duct ligated rats and in control animals after labeling the heme by injection of the precursor δ-[4-¹⁴C]aminolevulinic acid. The half lives were not significantly different, which eliminates the possibility that selective destruction of cytochrome P-450 has occurred. Depression of cytochromal heme synthesis was not observed. During mechanical cholestasis, the relative cytochrome P-450 deficiency is probably caused by proliferation of components of the endoplasmic reticulum other than the hemoprotein.     

Immunohistochemical study of cytochrome P-45011β -hydroxylase in human adrenal cortex with mineralo- and glucocorticoid excess

Summary Cytochrome P-450 specific for steroid 11-hydroxylation (P-450₁₁ ) was immunohistochemically demonstrated in the adrenal glands of human, pig and bovine and of mineralo- and glucocorticoid excess using a specific monoclonal antibody against P-450₁₁ of bovine adrenocortical mitochondria. P-450₁₁ was present in all three cortical zones of the histologically normal adrenal glands of bovine, pig and human, particularly in the zona fasciculata (ZF) and reticularis (ZR). The P-450₁₁ immunoreactivity was intensive in cortical micronodules and inner ZF and ZR in Cushing's disease, and relatively intensive in the zona glomerulosa (ZG) and outer ZF in idiopathic hyperaldosteronism (IHA), corresponding to the sites of active steroidogenesis. In adenomas with Cushing's syndrome and primary aldosteronism, compact cells were generally stained well. In the adrenal glands attached to the adenomas, immunoreactivity was observed only focally in ZG cells but not in ZF and ZR cells.This study was in part supported by a Grant from the Ministry of Health and Welfare Disorders of adrenal hormone Research Committee, Japan