Jeffrey Baron

A new spectral intermediate associated with cytochrome P-450 function in liver microsomes☆

The spectrophotometric examination of pigment reduction during the steady state of drug oxidative metabolism, as catalyzed by hepatic microsomes, has revealed the presence of a new spectral intermediate presumed associated with an oxygenated form of reduced cytochrome P-450. This new spectral intermediate has absorption maxima at about 440 and 590 mμ in the difference spectrum. The magnitude of this intermediate is dependent on the presence of a hydroxylatable substrate, the oxygen concentration, and the use of TPNH as the source of reducing equivalents. Further, changes in the extent of the TPNH-dependent reduction of cytochrome b5 during the steady state indicates that cytochrome b5 serves as a donor of electrons to the oxygenated form of reduced cytochrome P-450.

Immunohistochemical localizations of cytochromes P-450 in rat liver.

Abstract Antibodies produced against two forms of cytochrome P-450, PB-B and MC-B, which were purified to apparent homogeneity from hepatic microsomes of rats pretreated with phenobarbital and 3-methylcholanthrene, respectively, have been employed to localize these hemoproteins immunohistochemically at the light microscopic level in the livers of untreated rats. Using these antibodies in an unlabeled antibody peroxidase-antiperoxidase technique, immunohistochemical staining for the cytochromes P-450 was detected in parenchymal cells throughout the liver lobule. The patterns of immunohistochemical staining intensity observed with the two antibodies, however, were quite different. Exposure of liver sections to the antibody to cytochrome P-450 PB-B resulted in intense immunostaining within the centrilobular regions but produced staining of considerably weaker intensity in the peripheral regions of the lobule. In contrast to these observations, the antibody to cytochrome P-450 MC-B yielded a more uniform pattern of immunohistochemical staining, with the intensity of staining being only slightly greater in the centrilobular regions. The results of this immunohistochemical study thus demonstrate that different patterns of distribution exist for different forms of cytochrome P-450 within the liver lobule and that the greatest concentration of cytochrome P-450 occurs within the centrilobular regions of the liver.

Localization of a cytochrome P-450 isozyme (cytochrome P-450 PB-B) and NADPH-cytochrome P-450 reductase in rat nasal mucosa

Antibodies raised against cytochrome P-450 PB-B, the major phenobarbital-inducible isozyme of rat hepatic microsomal cytochrome P-450, and NADPH-cytochrome P-450 reductase (EC 1.6.2.4) were employed to determine the cellular localizations of these enzymes within the nasal mucosa of untreated rats. Immunohistochemical staining for each enzyme was detected at the light microscopic level within the respiratory and olfactory epithelia, duct and acinar cells of seromucous glands in the respiratory region, and duct and acinar cells of Bowmans glands in the olfactory region. These findings demonstrate that a number of different cell types in rat nasal mucosa contain enzymes which participate in the monooxygenations of chemical carcinogens and other xenobiotics.

Localization, distribution, and induction of xenobiotic-metabolizing enzymes and aryl hydrocarbon hydroxylase activity within lung.

The metabolism of xenobiotics within lung often leads to toxicity, although certain pulmonary cells are more readily damaged than others. This differential susceptibility can result from cell-specific differences in xenobiotic activation and detoxication. The localization and distribution of xenobiotic-metabolizing enzymes (cytochromes P-450, NADPH-cytochrome P-450 reductase, epoxide hydrolase, glutathione S-transferases, UDP-glucuronosyltransferases, and a sulfotransferase) and of aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity determined immunohistochemically and histochemically, respectively, within lung are discussed. Findings reveal that xenobiotics can be metabolized in situ, albeit to different extents, by bronchial epithelial cells, Clara and ciliated bronchiolar epithelial cells, and type II pneumocytes and other alveolar wall cells and that enzymes and activities are not necessarily induced uniformly among these cells.

Sites for xenobiotic activation and detoxication within the respiratory tract: Implications for chemically induced toxicity

Results of in situ immunohistochemical investigations on several enzymes which participate in the bioactivation and detoxication of xenobiotics and of histochemical studies on aryl hydrocarbon hydroxylase activity summarized in this report clearly demonstrate that there are numerous sites within the respiratory tract at which xenobiotics can be bioactivated and detoxicated. The data presented, however, also reveal that xenobiotic-metabolizing enzymes and benzo[a]pyrene hydroxylase activity may not be distributed uniformly within individual segments (e.g., the nasal mucosa) of this organ system. Thus, it should be apparent from these findings that one cannot generalize as to how a given xenobiotic-metabolizing enzyme or xenobiotic monooxygenase activity normally is distributed either within or among the different segments of the respiratory tract. Additionally, since enzymes catalyzing the bioactivation and detoxication of xenobiotics usually are present within the same respiratory tract cells, it obviously is difficult to predict from these results which cell types within individual segments of this organ system most likely would be damaged as a consequence of exposure to xenobiotics which are biotransformed into cytotoxic metabolites by cytochrome(s) P-450. Although the cellular localizations and intercellular distributions of cytochromes P-450 BNF-B and MC-B parallel those of benzo[a]pyrene hydroxylase activity within the different segments of the respiratory tract in untreated rats, immunohistochemical findings on the inductions of these cytochrome P-450 isozymes are not entirely consistent with histochemical observations on the enhancement of benzo[a]pyrene hydroxylase activity by Aroclor 1254 within the nasal mucosa and by both Aroclor 1254 and 3-methylcholanthrene within the lung. It must be appreciated, however, that other cytochrome P-450 isozymes undoubtedly are present and inducible in the nasal mucosa and lung and, further, that these hemeproteins, although being immunochemically unrelated to the cytochrome P-450 isozymes studied, also could catalyze aryl hydrocarbon hydroxylase activity. Nevertheless, these immunohistochemical and histochemical findings do demonstrate that one cannot generalize as to how chemicals which induce the same xenobiotic-metabolizing enzyme will affect that enzyme within different segments of the respiratory tract and, moreover, that inducers of cytochromes P-450 can alter differentially the extents to which different cells within a given segment of the respiratory tract (e.g., the nasal mucosa) participate in the oxidative metabolism of xenobiotics.

Immunochemical studies on electron transport chains involving cytochrome P-450. The role of the iron-sulfur protein, adrenodoxin, in mixed-function oxidation reactions

Abstract An antibody prepared to the homogeneous iron-sulfur protein (adrenodoxin) isolated from bovine adrenocortical mitochondria has been used to evaluate the role of adrenodoxin in cyt. P-450-mediated reactions in adrenal cortex and liver. As evidenced by the diminished magnitude of the g = 1.94 signal in electron paramagnetic resonance spectra of mitochondrial preparations at 100 °K, this antibody decreases the amount of adrenodoxin which can be reduced by either TPNH or sodium dithionite. The interaction between the antibody and adrenodoxin in mitochondrial preparations results in the concomitant inhibition of TPNH-cyt. c reductase and TPNH-cyt. P-450 reductase activities, as well as inhibition of the 11β-hydroxylation of 11-deoxycorticosterone. Inhibition by this antibody of TPNH-cyt. c reductase activity in a soluble fraction (fraction S 2 ) prepared from sonicated adrenocortical mitochondria was reversed by the addition of homogeneous adrenodoxin, demonstrating that the antibody is specific for adrenodoxin. The antibody did not inhibit DPNH-cyt. c reductase activity or the reduction of the dye, dichlorophenolindophenol, by either TPNH or DPNH in fraction S 2 of adrenocortical mitochondria. The antibody to adrenodoxin did not inhibit the TPNH-dependent reductions of either cyt. c or cyt. P-450 using microsomes prepared from bovine adrenal cortex or from the livers of untreated, phenobarbital-treated, or 3-methylcholanthrene-treated rats. No inhibition by this antibody was observed in substrate hydroxylations by these microsomal preparations. These observations indicate that adrenodoxin or an immunochemically similar iron-sulfur protein is not involved in cyt. P-450-mediated reactions in the microsomal fraction of liver or adrenal cortex.

Identification of intratissue sites for xenobiotic activation and detoxication.

Results of immunohistochemical and histochemical investigations on xenobiotic-metabolizing enzymes and aryl hydrocarbon hydroxylase activity have demonstrated that xenobiotic activation and detoxication do not occur uniformly throughout the liver, skin, respiratory tract, and pancreas, four tissues that are targets for the toxic actions of xenobiotics that are biotransformed into reactive metabolites. It has been shown that there can be significant differences in the levels and activities of xenobiotic-metabolizing enzymes among even morphologically similar cells, that an inducer can affect a specific xenobiotic-metabolizing enzyme to significantly different extents within different cells in a tissue, and that inducers of xenobiotic-metabolizing enzymes can alter differentially the extents to which different cells within a tissue participate in xenobiotic metabolism. These studies also have revealed that the route of administration of an inducer can affect significantly the induction of xenobiotic-metabolizing enzymes and aryl hydrocarbon hydroxylase activity within an organ such as the pancreas. Some of the immunohistochemical findings reported for the cellular localizations of xenobiotic-metabolizing enzymes within specific tissues, e.g., the nasal mucosa, may not appear to be entirely consistent with the intratissue distribution of benzo[a]pyrene hydroxylase activity, especially after induction. However, it must be appreciated that other cytochrome P-450 isozymes undoubtedly are present within these tissues which, although not studied, also are capable of catalyzing aryl hydrocarbon hydroxylase activity.

Biotransformation and Zonal Toxicity

Hepatotoxins are ubiquitous in nature. Chemical injury to the liver is dependent on the nature of the hepatotoxic agent and the circumstances of exposure (for a comprehensive review, see Zimmerman1). Products of plant, fungal, and bacterial metabolism, minerals,2–4 chemicals and pharmaceuticals, industrial byproducts, and waste materials can damage the liver.5 The types of hepatic injury that result from exposure to hepatotoxins are quite diverse. Some agents cause necrosis, fat accumulation, cirrhosis, or carcinoma,2 while others interfere with bile secretion, cause jaundice, and produce little or no injury to hepatocytes.2,5

Studies on adrenal δ-aminolevulinic acid synthetase

Abstract The presence of δ-aminolevulinic acid synthetase (EC 2.3.1.37) in rat and bovine adrenals has been demonstrated. When untreated animals are employed, the activity of δ-aminolevulinic acid synthetase in rat and bovine adrenal homogenates is comparable to the activity found in hepatic homogenates. Adrenal δ-aminolevulinic acid synthetase is localized in the mitochondrial fraction and appears to be refractory to induction by agents that induce the hepatic enzyme. Starvation of rats increased adrenal δ-aminolevulinic acid synthetase activity without altering the activity of the hepatic enzyme. Treatment of rats with adrenocorticotropin also dramatically increased adrenal δ-aminolevulinic acid synthetase activity. These results suggest that the adrenal enzyme may be controlled by factors that differ from those which regulate the activity of the hepatic enzyme.

Immunohistochemical localization of NADPH-cytochrome c reductase in rat liver.

Abstract NADPH-cytochrome c reductase (NADPH-cytochrome reductase, EC 1.6.2.4), the flavoprotein which is responsible for the NADPH-dependent reduction of cytochromes P-450 in hepatic microsomes, has been localized immunohistochemically at the light microscopic level in rat liver. Localization was achieved through the use of sheep antiserum to rat hepatic microsomal NADPH-cytochrome c reductase in an unlabeled antibody peroxidase-antiperoxidase technique. Parenchymal cells throughout the liver lobule were found to be stained positively for NADPH-cytochrome c reductase, although the intensity of immunostaining was slightly greater in the centrilobular regions. Immunostaining for NADPH-cytochrome c reductase was not detected in Kupffer cells, connective tissue cells, or in cells of the hepatic vasculature.