Enzymes In Extrahepatic Tissues Research Articles

Brain Drug-Metabolizing Cytochrome P450 Enzymes are Active In Vivo, Demonstrated by Mechanism-Based Enzyme Inhibition

Individuals vary in their response to centrally acting drugs, and this is not always predicted by drug plasma levels. Central metabolism by brain cytochromes P450 (CYPs) may contribute to interindividual variation in response to drugs. Brain CYPs have unique regional and cell-type expression and induction patterns, and they are regulated independently of their hepatic isoforms. In vitro, these enzymes can metabolize endogenous and xenobiotic substrates including centrally acting drugs, but there is no evidence to date of their in vivo function. This has been difficult to demonstrate in the presence of hepatically derived metabolites that may cross the blood-brain barrier. In addition, because of the membrane location of brain CYPs and the rate limiting effect of endogenous heme levels on the activity and appropriate membrane insertion of some induced CYPs, it has been unclear whether sufficient cofactors and coenzymes are present for constitutive and induced CYP forms to be enzymatically active. We have developed a method using a radiolabeled mechanism-based inhibitor of CYP2B1, (3)H-8-methoxypsoralen, to demonstrate for the first time that both the constitutive and induced forms of this enzyme are active in situ in the living rat brain. This methodology provides a novel approach to assess the function of enzymes in extrahepatic tissues, where expression levels are often low. Selective induction of metabolically active drug metabolizing enzymes in the brain may also provide ways to control prodrug activation in specific brain regions as a novel therapeutic avenue.

Human extrahepatic cytochromes P450: function in xenobiotic metabolism and tissue-selective chemical toxicity in the respiratory and gastrointestinal tracts.

Cytochrome P450 (CYP) enzymes in extrahepatic tissues often play a dominant role in target tissue metabolic activation of xenobiotic compounds. They may also determine drug efficacy and influence the tissue burden of foreign chemicals or bioavailability of therapeutic agents. This review focuses on xenobiotic-metabolizing CYPs of the human respiratory and gastrointestinal tracts, including the lung, trachea, nasal respiratory and olfactory mucosa, esophagus, stomach, small intestine, and colon. Many CYPs are expressed in one or more of these organs, including CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2F1, CYP2J2, CYP2S1, CYP3A4, CYP3A5, and CYP4B1. Of particular interest are the preferential expression of certain CYPs in the respiratory tract and the regional differences in CYP expression profile in different parts of the gastrointestinal tract. Current research activities on the characterization of CYP expression, function, and regulation in these tissues, as well as future research needs, are discussed.

Expression profiling of sulfotransferases in human cell lines derived from extra-hepatic tissues.

To explore the physiological roles of sulfotransferases (SULTs) in extra-hepatic tissues, we examined the expression of eight SULT genes by reverse transcription (RT)-PCR in human cell lines that were established from various tissues. Expression levels of SULTs were low in neural cell lines such as NB-1 and GI-1, and high in epithelial cell lines, such as Caco-2 and BeWo. SULT1C2 expression was abundant in all cell types, whereas that of SULT1E1, SULTIBI or SULT2B1 was restricted to a specific cell type. SULT1C1, which can catalyze the sulfation of N-hydroxy-2-acetylaminofluorene, was expressed in Caco-2, BeWo and KB562. Induction of differentiation did not generally affect SULT expression, although that of SULT1C2 was reduced after differentiation of the neuroblastoma cell line, NB-1, was induced. The profile of SULT expression in the culture cells obtained here gives clues to understanding the physiological roles of SULT enzymes in extra-hepatic tissues or organs.

Characterization of UDP-glucuronosyltransferases active on steroid hormones

In recent years, the enzymes which are involved in the formation of DHT in steroid target tissues have been well investigated, however, enzymes responsible for the catabolism and elimination of steroids in these tissues, in particular the uridine diphospho-glucuronosyltransferase (UGT) family of enzymes, have received much less attention. We have recently demonstrated that human and monkey are unique in having high plasma levels of C19 steroid glucuronides. These circulating conjugates have been proposed to reflect the peripheral conversion of adrenal and gonadal C19 steroids to potent androgens, especially DHT. In humans, the presence of steroid UGT activities is found in the liver and several extrahepatic tissues including the prostate, mammary gland and ovary. In addition, UGT activities were observed in breast and prostate tumor cell lines such as MCF-7 and LNCaP, respectively. In agreement with the presence of steroid conjugating enzymes in extrahepatic tissues, UGT cDNA clones, which encode steroid conjugating proteins, have been isolated from libraries constructed from human and monkey prostate mRNA. The presence of UGT transcripts and proteins in extrahepatic tissues in both species, as determined by Northern blot, ribonuclease protection, specific RT–PCR, in situ hybridization, Western blot and immunocytochemistry analysis, indicate the relevance of steroid glucuronidation in tissues other than the liver. Knowing that both the human prostate and the human prostate cancer LNCaP cell line express steroid metabolizing proteins, including UGT enzymes, regulation of UGT mRNA and protein levels, as well as promoter activity was studied in these cells. The results demonstrate a differential regulation between the two highly related isoforms UGT2B15 and UGT2B17, where only the expression of UGT2B17 was affected following treatments of LNCaP cells with androgens, growth factors or cytokines. Steroid conjugation by UGT enzymes is potentially involved in hormone inactivation in steroid target tissues, thus modifications in UGT expression levels may influence hormonal responses.

Types of carbamyl phosphate synthetases and subcellular localization of urea cycle and related enzymes in air-breathing walking catfish,Clarias batrachus

The types of carbamyl phosphate synthetase (CPS), the subcellular localization of urea cycle enzymes and glutamine synthetase (GS) in liver and kidney (potential ureogenic tissues) and also the possible involvement of some extra-hepatic tissues for urea synthesis via the urea cycle were studied in an Indian ureogenic amphibious air-breathing walking catfish, Clarias batrachus. Mitochondrial CPS III [glutamine- and N-acetyl-L-glutamate (NAG)-dependent] and cytosolic CPS II (glutamine-dependent) activities were found to be present in liver, analogous to that described for two other teleosts that have CPS III activity. The same activities and subcellular localization were found in kidney. Unexpectedly, a CPS I-like (ammonia- and NAG-dependent) activity was found to be present at levels higher than the CPS III activity in the mitochondrial fraction of both liver and kidney analogous to another Indian amphibious air-breathing teleost fish, Heteropneustes fossilis. The two other urea cycle enzymes, ornithine transcarbamylase (OTC) and arginase (ARG), and GS were found to be localized primarily in the mitochondria, and argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) in the cytosol analogous to H. Fossilis and elasmobranchs. Significant levels of urea cycle enzymes along with GS activity could also be detected in muscle, intestine and brain tissues, except ASL in muscle and ASS in brain. Our results also indicated that probably all three different types of CPS activities are also expressed in all three extra-hepatic tissues. The urea cycle–related CPS III found in invertebrates and fish is considered to be the evolutionary precursor of the urea cycle–related CPS I in ureotelic mammalian and amphibian species. Whether or not the CPS I-like activity reported here (1) is due to the presence of a separate CPS I gene in addition to a CPS III gene or (2) represents an adapted CPS III activity in C. batrachus, these results suggest that the presence of both CPS I-like and CPS III activities along with the presence of urea cycle enzymes in extra-hepatic tissues may play an important physiological adaptive role for survival of these walking catfish in higher ambient ammonia or living in air or while burrowing inside mud during habitat drying. J. Exp. Zool. 283:121–130, 1999. © 1999 Wiley-Liss, Inc.

Multiplicity and tissue specific expression of camel cytochrome P450(s)

The presence of multiple forms of the cytochrome P450 was demonstrated enzymatically in camel tissues using a variety of isoenzyme specific substrates and immunochemically using isoenzyme specific antibodies. The maximum catalytic activity using xenobiotics as substrate was observed in the liver followed by the kidney. However, lauric acid hydroxylation was found to be higher in the kidney than in any other tissues. Camel liver microsomal monooxygenase activity using aniline, aminopyrene, ethoxycoumarin, ethoxyresorufin and benzo(a)pyrene as substrates was comparable with those of rat and human livers. The activity of the enzymes in extrahepatic tissues of the camel was comparable with those of the rat extrahepatic tissues. The maximum expression of P450 protein was seen in the camel liver and kidney while the brain and intestine exhibited relatively low levels of expression. P450 expression in camel tissues appeared to be higher than in rat tissues. Immunohistochemical staining of P450 in the camel liver, kidney and brain confirmed the higher expression of P450 enzyme proteins in the liver and kidney as compared to other extrahepatic tissues. The maximum expression of P450 in the liver was observed in hepatocytes around the central vein and in the kidney it was observed in the proximal tubules. These results demonstrate that the multiple forms of P450s are differentially expressed in camel tissues and that the relative levels of expression are comparable with those of rat and human tissues. These observations may be important in understanding the differential susceptibility of camel tissues to the toxic/therapeutic effects of xenobiotics/drugs and environmental pollution.

Quantification of L-tryptophan and L-kynurenine by liquid chromatography/electron capture negative ion chemical ionization mass spectrometry.

In a number of infectious and inflammatory diseases, stimulation of the immune system can lead to increased accumulation of tryptophan metabolites via induction of kynurenine pathway enzymes in extrahepatic tissues. We developed a liquid chromatographic/mass spectrometric (LC/MS) method suitable for tracing the disposition of 13C isotopomers of L-tryptophan and L-kynurenine in various cultured cell, tissue slice, and whole animal model systems used to investigate tryptophan flux through the kynurenine pathway. The method employs extractive derivatization of the analytes and their 2H internal standards with pentafluorobenzyl bromide in order to enhance the negative ion chemical ionization (NICI) mass spectrometric response. Normal-phase liquid chromatographic separation of derivatized analytes was optimized using a silica column with organic solvents, followed by particle beam transfer and NICI-MS. Standard curves were linear over the range 1-250 ng per sample. Particle beam and mass spectrometric operating parameters were optimized with direct flow injections of 1-(methylamino) anthraquinone, which is an ideal test compound for the evaluation of LC/NICI-MS. The developed method was used to quantify the conversion of (13C6)L-tryptophan to (13C6)L-kynurenine by human monocytes (THP-1) stimulated with interferon-gamma, lung and brain tissue slices obtained from gerbils immune-stimulated with pokeweed mitogen. The effect of whole body immune stimulation on the plasma levels of endogenous L-kynurenine in mice stimulated with interferon-gamma was also quantified.

ROLE OF CYTOCHROME P-450 IN CHEMICAL TOXICITY AND CARCINOGENESIS

Most chemical carcinogens and toxicants formed in our environment require metabolic activation or detoxification to exert their harmful effects or be rendered harmless, respectively, in their target cells and tissues. The major activation pathway is oxidative metabolism, usually catalyzed by cytochrome P-450-dependent monooxygenase systems. Multiple forms of cytochrome P-450 have been isolated and characterized from certain species of experimental animals. In this review we attempt to present a discussion of 1) specificity of catalytic activity in cytochrome P-450 enzymes in chemical carcinogenesis, 2) distribution of cytochrome P-450 enzymes in different species of experimental animals, 3) specificity of cytochrome-P-450 enzymes in extrahepatic tissues, and 4) genetic regulation in the susoeptibility to cancer in experimental animals and human. One of the important problems is to evaluate the role of extrahepatic cytochrome P-450 in chemical carcinogenesis and toxicity. We know some forms of cytochrome P-450 are highly localized in particular regions or cells, as for example, in non-ciliated bronchiolar (Clara) cells of the lung. Whether or not cytochrome P-450 enzymes and other drug metabolizing enzymes play significant roles in the expression of chemical toxicity in target tissues, may depend on the stability of biologically reactive intermediates mediated in vivo by the liver, the major organ for these enzymes. It is of interest that there is an apparent tissue difference in the expression of the cytochrome P-450IAl gene and in the inducibility of benzo [a] pyrene hydroxylation by 3-methyl-cholanthrene between lung and liver in Syrian golden hamsters. Whenever we attempt to postulate a process explaining the primary prophylaxis leading to chemical carcinogenesis in human, we have to deal with the question of which similarities of cytochrome P-450 and the related drug-metabolizing enzymes encountered in the animal model situations may extend to those of interest in the human populations at potential risk.

Differential activation and inactivation of benzo[ a]pyrene in various hepatoma cell lines

Treatment of pregnant rats with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in a dose-dependent induction of a mixed-function oxidase system in fetal and maternal extra-hepatic tissues. At doses of 6 μmg/kg, aryl hydrocarbon hydroxylase (AHH) activity was increased 24-, 22- and 4-fold in fetal lung, kidney and skin, respectively, while maternal lung, kidney and adrenal AHH activity was increased 4-, 2- and 2-fold respectively. High-pressure liquid chromatographic (H.P.L.C.) analysis of benzo(a)pyrene (BP) metabolism after TCDD induction indicated that fetal lung, kidney and skin produced significant quantities of benzo(a)pyrene-7,8-dihydrodiol (BP-7,8-diol), benzo(a)pyrene-4,5-dihydrodiol (BP-4,5-diol) and 9- and 3-phenols of BP. The fetal liver produced benzo(a)pyrene-9,10-dihydrodiol (BP-9,10-diol), BP-4,5-diol, BP-7,8-diol and 9- and 3-phenols of BP. Maternal lung also produced BP-9,10-diol, while maternal adrenal gland yielded primarily the 9-phenol of BP. Epoxide hydratase activity was increased 2- to 3-fold in maternal lung, fetal lung and skin after TCDD pretreatment, but was not affected significantly in liver, kidney or placenta. Treatment of pregnant rats with TCDD increased the covalent binding of BP to DNA in preparations containing maternal liver, lung and placenta as well as fetal liver, lung and skin. Pretreatment with TCDD resulted in increased epoxide hydratase and AHH activities in extra-hepatic tissues but only AHH was increased in hepatic tissues, indicating that the inducing capabilities of TCDD differ from, but share some similarities with, both phenobarbital (PB) and 3-methylcholanthrene (MC). Thus, TCDD appears to provide an exceptionally potent and broad-spectrum transplacental induction of carcinogen-transforming enzymes in extra-hepatic tissues.

Comparison of the activities of some peroxisomal and extraperoxisomal lipid-metabolizing enzymes in liver and extrahepatic tissues of the rat.

Peroxisomal (acyl-CoA oxidase and peroxisomal dihydroxyacetone-phosphate acyltransferase) and extraperoxisomal (mitochondrial fatty acid oxidation, extraperoxisomal dihydroxyacetone-phosphate acyltransferase, mitochondrial and microsomal glycerophosphate acyltransferases) lipid-metabolizing enzymes were measured in homogenates from rat liver and from seven extrahepatic tissues. Except for jejunal mucosa and kidney, extrahepatic tissues contained very little acyl-CoA oxidase activity. Peroxisomal dihydroxyacetone-phosphate acyltransferase, taken as the activity that was not inhibited by 5 mM-glycerol 3-phosphate, was present in all tissues examined, and its specific activity in liver and extrahepatic tissues was roughly of the same order of magnitude. Clofibrate treatment increased the activity of acyl-CoA oxidase in liver, and to a smaller extent also in kidney, but did not influence the activity of peroxisomal dihydroxyacetone-phosphate acyltransferase. Comparison of the activities of peroxisomal and extraperoxisomal lipid-metabolizing enzymes in extrahepatic tissues and in liver, an organ in which the contribution of peroxisomes to fatty acid oxidation and to glycerolipid synthesis has been estimated previously, suggests that, as in liver, peroxisomal long-chain fatty acid oxidation is of minor quantitative importance in extrahepatic tissues, but that in these tissues (micro)-peroxisomes are responsible for most of the dihydroxyacetone phosphate acylation and, consequently, for initiating ether glycerolipid synthesis.

Transplacental induction of mixed-function oxygenases in extra-hepatic tissues by 2,3,7,8-tetrachlorodibenzo- p-dioxin

Treatment of pregnant rats with 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) resulted in a dose-dependent induction of a mixed-function oxidase system in fetal and maternal extra-hepatic tissues. At doses of 6 μmg/kg, aryl hydrocarbon hydroxylase (AHH) activity was increased 24-, 22- and 4-fold in fetal lung, kidney and skin, respectively, while maternal lung, kidney and adrenal AHH activity was increased 4-, 2- and 2-fold respectively. High-pressure liquid chromatographic (H.P.L.C.) analysis of benzo(a)pyrene (BP) metabolism after TCDD induction indicated that fetal lung, kidney and skin produced significant quantities of benzo(a)pyrene-7,8-dihydrodiol (BP-7,8-diol), benzo(a)pyrene-4,5-dihydrodiol (BP-4,5-diol) and 9- and 3-phenols of BP. The fetal liver produced benzo(a)pyrene-9,10-dihydrodiol (BP-9,10-diol), BP-4,5-diol, BP-7,8-diol and 9- and 3-phenols of BP. Maternal lung also produced BP-9,10-diol, while maternal adrenal gland yielded primarily the 9-phenol of BP. Epoxide hydratase activity was increased 2- to 3-fold in maternal lung, fetal lung and skin after TCDD pretreatment, but was not affected significantly in liver, kidney or placenta. Treatment of pregnant rats with TCDD increased the covalent binding of BP to DNA in preparations containing maternal liver, lung and placenta as well as fetal liver, lung and skin. Pretreatment with TCDD resulted in increased epoxide hydratase and AHH activities in extra-hepatic tissues but only AHH was increased in hepatic tissues, indicating that the inducing capabilities of TCDD differ from, but share some similarities with, both phenobarbital (PB) and 3-methylcholanthrene (MC). Thus, TCDD appears to provide an exceptionally potent and broad-spectrum transplacental induction of carcinogen-transforming enzymes in extra-hepatic tissues.

Failure of various drugs to induce drug-metabolizing enzymes in extrahepatic tissues of the rat

The effect of a tranquilizer (chlorpromazine HCl), a diuretic (hydrochlorthiazide), an antihydroxylation agent (metyrapone), and known inducers of hepatic microsomal drug-metabolizing enzymes (phenobarbital, 3-methylcholanthrene, 4-methylcoumarin) on drug metabolism in various organs of the rat has been studied. In all organs tested (liver, brain, kidneys, intestines, adrenals) various drug-metabolizing enzymes (aminopyrine demethylase, aniline hydroxylase, coumarin 3-hydroxylase, hexobarbital oxidase) could be demonstrated at various levels. However, the liver was the only organ in which drug metabolizing enzymes were induced by these compounds in various degrees. Even the hydroxylase inhibitor, metyrapone, increased hepatic hydroxylation. There was no change in the activity of these enzymes in any other organs. It appears, therefore, that the specific pharmacologic action of various drugs on target organs may not be connected with induction of drug metabolism.