Ring hydroxylation of [o-3H]methoxychlor as a probe for liver microsomal CYP2B activity: potential for in vivo CYP2B assay

An in vitro radiometric assay selective for inducible CYP2B activity is described. The assay is based on the quantification of 3H2O release that occurs during o-ring hydroxylation of [o-3H]methoxychlor by liver microsomes in the presence of NADPH. 3H2O is isolated by removing > 99.9% of the parent compound and organic metabolites by facile charcoal extraction and filtration. There was no evidence for an NIH shift during ring hydroxylation, and there was little or no isotope effect. Selectivity for CYP2B was demonstrated using liver microsomes prepared from rats and mice treated with inducers of different CYP isoforms. Ring hydroxylation of [o-3H]methoxychlor was elevated 11.4-fold over control values in liver microsomes from male rats treated with phenobarbital. With mice, phenobarbital treatment elevated liver microsomal ring hydroxylation 7.1-fold. Clofibrate, 3-methylcholanthrene, or beta-naphthoflavone treatment of male rats or pyridine treatment of female rats did not elevate liver microsomal ring-hydroxylase activity, indicating that CYP4A, 1A, and 2E1 do not support this reaction. In female rats, dexamethasone and pregnenolone-16 alpha-carbonitrile treatment elevated ring hydroxylation up to 5.5- and 3.2-fold, respectively, an activity that may be attributed to CYP2B induction in those animals. Incubation of liver microsomes from phenobarbital-treated males with monospecific anti-CYP2B monoclonal antibodies (Mab) inhibited ring-hydroxylase activity up to 86%, demonstrating predominantly CYP2B-mediated catalysis. An 86% inhibition by these Mabs was also observed using liver microsomes from male mice treated with phenobarbital, indicating the assay is not limited to rats. The CYP2B mechanism-based inhibitor orphenadrine caused a 76% decline in activity, providing further evidence for CYP2B involvement. Unlike other CYP2B-selective assays, this method may be readily adapted to in vivo studies, by measuring urinary excretion of 3H2O as an indication of total body CYP2B activity.     

Multiple human cytochromes contribute to biotransformation of dextromethorphan in-vitro: role of CYP2C9, CYP2C19, CYP2D6, and CYP3A

Cytochromes mediating the biotransformation of dextromethorphan to dextrorphan and 3-methoxymorphinan, its principal metabolites in man, have been studied by use of liver microsomes and microsomes containing individual cytochromes expressed by cDNA-transfected human lymphoblastoid cells. In-vitro formation of dextrorphan from dextromethorphan by liver microsomes was mediated principally by a high-affinity enzyme (Km (substrate concentration producing maximum reaction velocity) 3-13 microM). Formation of dextrorphan from 25 microM dextromethorphan was strongly inhibited by quinidine (IC50 (concentration resulting in 50% inhibition) = 0.37 microM); inhibition by sulphaphenazole was approximately 18% and omeprazole and ketoconazole had minimal effect. Dextrorphan was formed from dextromethorphan by microsomes from cDNA-transfected lymphoblastoid cells expressing CYP2C9, -2C19, and -2D6 but not by those expressing CYP1A2, -2E1 or -3A4. Despite the low in-vivo abundance of CYP2D6, this cytochrome was identified as the dominant enzyme mediating dextrorphan formation at substrate concentrations below 10 microM. Formation of 3-methoxy-morphinan from dextromethorphan in liver microsomes proceeded with a mean Km of 259 microM. For formation of 3-methoxymorphinan from 25 microM dextromethorphan the IC50 for ketoconazole was 1.15 microM; sulphaphenazole, omeprazole and quinidine had little effect. 3-Methoxymorphinan was formed by microsomes from cDNA-transfected lymphoblastoid cells expressing CYP2C9, -2C19, -2D6, and -3A4, but not by those expressing CYP1A2 or -2E1. CYP2C19 had the highest affinity (Km = 49 microM) whereas CYP3A4 had the lowest (Km = 1155 microM). Relative abundances of the four cytochromes were determined in liver microsomes by use of the relative activity factor approach. After adjustment for relative abundance, CYP3A4 was identified as the dominant enzyme mediating 3-methoxymorphinan formation from dextromethorphan, although CYP2C9 and -2C19 were estimated to contribute to 3-methoxymorphinan formation, particularly at low substrate concentrations. Although formation of dextrorphan from dextromethorphan appears to be sufficiently specific to be used as an in-vitro or in-vivo index reaction for profiling of CYP2D6 activity, the findings raise questions about the specificity of 3-methoxymorphinan formation as an index of CYP3A activity.     

Effects of Cr(VI) on the expression of the oxidative stress genes in human lung cells

Intracellular metabolism of chromium(VI) [Cr(VI)] may lead to oxidative stress and this may account for the ability of Cr(VI) to act as a complete carcinogen. Therefore, we examined the effects of Cr(VI) treatment on the expression of oxidative stress genes in normal human lung LL 24 cells and human lung adenocarcinoma A549 cells. RT-PCR and northern blot analyses were used to determine the steady-state mRNA levels of catalase, glutathione S-transferase, glutathione reductase, Cu/Zn- and Mn-superoxide dismutases, glutathione peroxidase, NAD(P)H:quinone oxidoreductase, heme oxygenase and interleukin 8 in control cells and cells treated with 5-200 microM of Cr(VI). We found that only expression of the heme oxygenase gene is strongly elevated under the treatment with Cr(VI), and only in normal human lung LL 24 cells. Our data showed that even in the absence of Cr(VI) treatment, the level of heme oxygenase gene expression is much higher in A549 cells than in LL 24 cells. As glutathione is believed to play a protective role in cells against different forms of oxidative stress, we studied the correlation between intracellular glutathione levels and the inducibility of the heme oxygenase gene after treatment of cells with Cr(VI). Our results demonstrate that glutathione levels are increased by 35 % of control values in LL 24 cells treated with Cr(VI). The data obtained indicate that heme oxygenase, known to be a stress-inducible gene, may be involved in cellular pathways critical to the carcinogenic activity of Cr(VI) in normal human lung cells. Intracellular glutathione levels and reactive oxygen species do not appear to be primarily responsible for the stress response, induced by Cr(VI) in the studied human cells.     

Human lymphocyte heme oxygenase 1 as a response biomarker to inorganic arsenic

We propose the use of human lymphocyte heme oxygenase 1 (HO1) as a biomarker of response to environmental arsenic exposure. We report the induction of HO1 in human lymphoblastoid cells (LBs) by arsenite in a dose-related manner. HO1 was identified by SDS-PAGE from its molecular weight and from its detection by Western blotting with anti-HO1. HO1 levels in LBs treated with arsenite increased by de novo synthesis as demonstrated by incorporation of 35S-methionine and by inhibition of HO1 synthesis by actinomycin D. The amount of HO1 in LBs was estimated by quantifying Western blots. HO1 was also induced by 10 microM cadmium or mercuric chloride. We suggest that circulating lymphocyte HO1 levels may be useful in assessing the biological activity of arsenic exposure in vivo under properly controlled conditions of simultaneous urinalysis for arsenic, cadmium, and mercury.     

Role of CYP3A4 in human hepatic diltiazem N-demethylation: inhibition of CYP3A4 activity by oxidized diltiazem metabolites

The antihypertensive agent diltiazem (DTZ) impairs hepatic drug metabolism by inhibition of cytochrome P450 (CYP). The accumulation of DTZ metabolites in serum occurs during prolonged therapy and leads to decreased DTZ elimination. Thus, DTZ metabolites may contribute to CYP inhibition. This study assessed the role of human CYPs in microsomal DTZ oxidation and the capacity of DTZ metabolites to inhibit specific CYP activities. DTZ N-demethylation varied 10-fold in microsomal fractions from 17 livers (0.33-3.31 nmol/mg of protein/min). DTZ oxidation was correlated with testosterone 6beta-hydroxylation (r = 0.82) and, to a lesser extent, tolbutamide hydroxylation (r = 0.59) but not with activities mediated by CYP1A2 or CYP2E1. CYP3A4 in lymphoblastoid cell microsomes catalyzed DTZ N-demethylation but CYP2C8 and CYP2C9 were also active (approximately 20% and 10% of the activity supported by CYP3A4); seven other CYPs produced little or no N-desmethyl DTZ from DTZ. The CYP3A4 inhibitors ketoconazole and troleandomycin decreased microsomal DTZ oxidation, but inhibitors or substrates of CYP2C, CYP2D and CYP2E1 produced no inhibition. Some inhibition was produced by alpha-naphthoflavone, a chemical that inhibits CYP1As and also interacts with CYP3A4. In further experiments, the capacities of DTZ and three metabolites to modulate human CYP 1A2, 2E1, 2C9 and 3A4 activities were evaluated in vitro. DTZ and its N-desmethyl and N,N-didesmethyl metabolites selectively inhibited CYP3A4 activity, whereas O-desmethyl DTZ was not inhibitory. The IC50 value of DTZ against CYP3A4-mediated testosterone 6beta-hydroxylation (substrate concentration, 50 microM) was 120 microM. The N-desmethyl (IC50 = 11 microM) and N,N-didesmethyl (IC50 = 0.6 microM) metabolites were 11 and 200 times, respectively, more potent. From kinetic studies, N-desmethyl DTZ and N,N-didesmethyl DTZ were potent competitive inhibitors of CYP3A4 (Ki = approximately 2 and 0.1 microM, respectively). CYP3A4 inhibition was enhanced when DTZ and N-desmethyl DTZ underwent biotransformation in NADPH-supplemented hepatic microsomes in vitro, supporting the contention that inhibitory metabolites may be generated in situ. These findings suggest that N-demethylated metabolites of DTZ may contribute to CYP3A4 inhibition in vivo, especially under conditions in which N-desmethyl DTZ accumulates, such as during prolonged DTZ therapy.     

Modulation of corneal heme oxygenase expression by oxidative stress agents

Heme oxygenase, the rate-limiting enzyme in the degradation of heme to bile-pigments and carbon monoxide, is induced in response to increased oxidative stress and is believed to provide a cytoprotective effect. We investigated the role of heme oxygenase in cultured rabbit corneal epithelial cells (RCE), and its potential to alleviate oxidative stress-induced cell damage. Heme oxygenase in RCE was effectively and potently induced by most metals tested, including tin, silver, and gold, and cytokines such as IL-6, and TGF beta. Stannous chloride and heme-induced heme oxygenase mRNA by 40 and 100 fold within 1-3 hours and increased enzyme activity by 9.2- and 10-fold, respectively, over a 24 hour period. IL-6, TGF beta and H2O2 induced heme oxygenase by 2-3 fold. Zinc protoporphyrins were effective inhibitors of heme oxygenase activity in vitro. However, when incubated with cells for 24 h they induced heme oxygenase mRNA but decreased or had no effect on its activity. Administration of heme, SnCl2, and H2O2 resulted in some degree of glutathione perturbation (GSH/GSSG). However, in all cases, depletion of glutathione was exacerbated if heme oxygenase was simultaneously inhibited. Conversely, perturbation of glutathione levels was minimized if heme oxygenase was induced by heme or stannous chloride. These results demonstrate that RCE cells exhibit functional heme oxygenase activity which is inducible in response to inflammatory cytokines and oxidative stress agents and suggest a cytoprotective role for heme oxygenase against cell injury.     

Inhibition of CYP1A1-dependent activity by the polynuclear aromatic hydrocarbon (PAH) fluoranthene

Polynuclear aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants, and recently bioassay-based induction studies have been used to determine exposures to complex mixtures of PAHs. Induction of CYP1A1-dependent activity in H4IIE rat hepatoma cells has been used extensively as a bioassay for halogenated aromatic hydrocarbons and more recently for PAHs. Fluoranthene (FL) is a prevalent PAH contaminant in diverse environmental samples, and FL did not induce CYP1A1-dependent ethoxyresorufin O-deethylase (EROD) activity significantly in H4IIE cells. However, in cells cotreated with 2 x 10(-5) M FL plus the potent inducers 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or benzo[k]fluoranthene (BkF) (2 x 10(-8) M), there was a significant decrease in EROD activities. Furthermore, treatment of TCDD-induced rat microsomes with FL caused an 80% decrease in EROD activity. Studies showed that FL did not affect induction of CYP1A1 protein or mRNA levels in H4IIE cells, and analysis of enzyme inhibition data using microsomal CYP1A1 indicated that FL noncompetitively inhibited CYP1A1-dependent activity. 32P-Postlabeling revealed no significant FL-DNA adduct formation in H4IIE cells treated with FL. However, in cells cotreated with FL plus BkF or benzo[a]pyrene (BaP), certain PAH-DNA adducts were induced 2-fold. This study demonstrated that FL is an inhibitor of CYP1A1-dependent enzyme activity in rat hepatoma H4IIE cells and that the genotoxic potency of some carcinogenic PAHs may be modulated by FL in mixtures containing relatively high levels of this compound.     

Imprinted overinduction of hepatic CYP2B1 and 2B2 in adult rats neonatally exposed to phenobarbital

Neonatally administered phenobarbital has been shown to cause a permanent, but delayed overexpression of hepatic drug-metabolizing enzymes, occurring at the time of sexual maturity. The present studies indicate that these above-normal levels of hepatic monooxygenases are not a result of a persistent overexpression of CYP2B1 and 2B2 proteins, the major phenobarbital-inducible isoforms of cytochrome P450. However, early exposure to the barbiturate permanently alters (i.e., imprints) the inductive responsiveness of CYP2B1 and 2B2 to subsequent phenobarbital challenge in adulthood. That is, neonatal administration of therapeutic-like levels of phenobarbital causes an overinduction (approximately 30-40%) of CYP2B1 and 2B2 mRNAs, proteins and specific catalytic activity (androstenedione 16 beta-hydroxylase) levels when the rats are rechallenged as adults with as little as 1 mg or 10 mg/kg b.wt. of the barbiturate. This "latent" defect in the inductive mechanism is associated in both sexes with an abnormality in the circulating growth hormone profiles characterized by subnormal secretory pulses. Because endogenous growth hormone normally inhibits phenobarbital induction of CYP2B1 and 2B2, and the level of inhibition is directly related to the height of the secretory pulse, we have proposed that the overinduction of CYP2B1 and 2B2 in adult rats neonatally exposed to phenobarbital results, at least in part, from a "deinhibition" by the subnormal pulse amplitudes in the plasma growth hormone profiles.     

3,3'-Diindolylmethane induces CYP1A2 in cultured precision-cut human liver slices

1. The effect of 3,3'-diindolylmethane (DIM), an indole derivative derived from cruciferous vegetables, on cytochrome P450 (CYP) isoforms in the CYP1A and CYP3A subfamilies has been studied in 72-h cultured human liver slices. 2. In cultured human liver slices 50 microM DIM induced 7-ethoxyresorufin O-deethylase and to a lesser extent 7-methoxyresorufin O-demethylase activities. 3. Western immunoblotting of liver slice microsomes was performed with antibodies to rat CYP1A2 and human CYP3A4. Compared with control liver slice microsomes (dimethyl sulphoxide-only treated), DIM induced levels of CYP1A2 but had little effect on levels of CYP3A4. The treatment of human liver slices with 2 microg/ml of the polycholorinated biphenyl mixture Aroclor 1254 also resulted in an induction of levels of CYP1A2, but had no effect on CYP3A4. 4. These results demonstrate that DIM induces CYP1A isoforms in cultured human liver slices. Some variability in the magnitude of induction of enzyme activities by DIM was observed in four human liver samples examined. For 7-ethoxyresorufin O-deethylase, the magnitude of induction by 50 microM DIM ranged from 2.3- to 19.3-fold. 5. These results demonstrate that cultured human liver slices can be used to evaluate the effect of chemicals derived from cruciferous and other vegetables on CYP isoforms.     

Shimming a high-resolution MAS probe

The gene expression and interrelation of the constitutive type nitric oxide (NO) synthase-III as a NO-forming enzyme and heme oxygenase-2 as a carbon monoxide-forming enzyme were studied in cultured rat aortic endothelial cells. Both NO synthase-III and heme oxygenase-2 mRNAs were demonstrated in the endothelial cells by RNAase protection analysis. NO synthase-III mRNA was upregulated in the presence of the heme oxygenase inhibitor, zinc protoporphyrin IX, but not in the presence of the NO synthase inhibitor, N(G)-nitro-L-arginine. Although heme oxygenase-2 mRNA was significantly upregulated in the presence of both NO synthase inhibitor and heme oxygenase inhibitor, the increase was greater with the NO synthase inhibitor. These results provide the first evidence for the concomitant gene expression of NO synthase-III and heme oxygenase-2, and their compensatory interrelation in endothelial cells.     

Involvement of human CYP1A isoenzymes in the metabolism and drug interactions of riluzole in vitro

Cytochrome P450 (CYP) and uridine diphosphate glucuronosyltransferase (UGT) isoenzymes involved in riluzole oxidation and glucuronidation were characterized in (1) kinetic studies with human hepatic microsomes and isoenzyme-selective probes and (2) metabolic studies with genetically expressed human CYP isoenzymes from transfected B-lymphoblastoid and yeast cells. In vitro incubation of [14C]riluzole (15 microM) with human hepatic microsomes and NADPH or UDPGA cofactors resulted in formation of N-hydroxyriluzole (K(m) = 30 microM) or an unidentified glucuroconjugate (K(m) = 118 microM). Human microsomal riluzole N-hydroxylation was most strongly inhibited by the CYP1A2 inhibitor alpha-naphthoflavone (IC50 = 0.42 microM). Human CYP1A2-expressing yeast microsomes generated N-hydroxyriluzole, whereas human CYP1A1-expressing yeast microsomes generated N-hydroxyriluzole, two additional hydroxylated derivatives and an O-dealkylated derivative. CYP1A2 was the only genetically expressed human P450 isoenzyme in B-lymphoblastoid microsomes to metabolize riluzole. Riluzole glucuronidation was inhibited most potently by propofol, a substrate for the human hepatic UGT HP4 (UGT1.8/9) isoenzyme. In vitro, human hepatic microsomal hydroxylation of riluzole (15 microM) was weakly inhibited by amitriptyline, diclofenac, diazepam, nicergoline, clomipramine, imipramine, quinine and enoxacin (IC50 approximately 200-500 microM) and cimetidine (IC50 = 940 microM). Riluzole (1 and 10 microM) produced a weak, concentration-dependent inhibition of CYP1A2 activity and showed competitive inhibition of methoxyresorufin O-demethylase. Thus, riluzole is predominantly metabolized by CYP1A2 in human hepatic microsomes to N-hydroxyriluzole; extrahepatic CYP1A1 can also be responsible for the formation of several other metabolites. Direct glucuronidation is a relatively minor metabolic route. In vivo, riluzole is unlikely to exhibit significant pharmacokinetic drug interaction with coadministered drugs that undergo phase I metabolism.     

Interaction of nonylphenol and hepatic CYP1A in rats

Both estradiol and nonylphenol (NP) inhibited hepatic microsomal 7-ethoxyresorufin O-deethylase (EROD) activity of beta-naphthoflavone-treated rats. Enzyme kinetic analyses (Lineweaver-Burk plots) using different estradiol and NP concentrations with graded increases in the concentrations of the substrate, ethoxyresorufin, showed that the inhibition was of a competitive nature at all concentrations of estradiol or NP used. Thus, the mechanism by which NP inhibits EROD activity is similar to that of estradiol. NP, however, was much less potent than estradiol. Young rats treated in vivo with 80 mg/kg body weight of NP demonstrated a slight but significant decrease in their hepatic microsomal EROD activity and CYP1A protein as measured by western blot analysis. In addition, treatment with NP led to a decrease in the steady-state levels of hepatic CYP1A mRNA in rats, suggesting that NP acted at the pre-translational level. The competitive nature of inhibition by NP on hepatic microsomal EROD activity indirectly suggests that this compound is a possible substrate of the CYP1A enzyme. Furthermore, NP had a moderate modulating effect on the expression of CYP1A in rat liver.     

The nitric oxide donor SIN-1 protects endothelial cells from tumor necrosis factor-alpha-mediated cytotoxicity: possible role for cyclic GMP and heme oxygenase

In cultured endothelial cells, incubation with TNF-alpha (50 ng/ml) for 72 h markedly reduced viability of endothelial cells. A 6-h pre-incubation with the nitric oxide (NO) donor linsidomine (SIN-1, 10-150 microM) protected endothelial cells in a concentration-dependent manner and increased viability by up to 59% of control. The unmetabolized parent compound molsidomine and the NO-free metabolite of SIN-1 3-morpholinoiminoacetonitrile (SIN-1C) were without cytoprotective effect. Cytoprotection by SIN-1 was completely abolished by the NO scavenger 2-phenyl-4,4,5,5, -tetramethylimidazoline-1-oxyl-3-oxide (PTIO, 30 microM). A cytoprotective effect comparable to SIN-1 was observed when preincubating the cells with dibutyryl cyclic GMP (10-100 microM). Moreover, no protection by SIN-1 occurred in the presence of cycloheximide (1 microM) or 1H--1,2,4-oxadiazole-4, 3-a-quinoxalin-1-one (ODQ, 0.1 microM), a selective inhibitor of soluble guanylyl cyclase. Tin protoporphyrin-IX (SnPP, 25 microM), an inhibitor of heme oxygenase, was found to attenuate SIN-1-induced cytoprotection. Our results demonstrate that SIN-1 produces a long-term endothelial protection against cellular injury by TNF-alpha, presumably via a cyclic GMP-dependent pathway leading to up-regulation of protective proteins such as heme oxygenase.     

Human liver CYP2B6-catalyzed hydroxylation of RP 73401

RP 73401 is a potent inhibitor of cyclic nucleotide phosphodiesterase type IV. RP 73401 is metabolized by human liver microsomes almost exclusively by transhydroxylation of the cyclopentyl group to RPR 113406. Liquid chromatography/mass spectrometry/mass spectrometry analysis of plasma from patients given RP 73401 also revealed a molecular ion and fragmentation consistent with RPR 113406. Thus, the objective was to determine the oxidative enzyme(s) responsible for RP 73401 hydroxylation. Kinetic constants of RP 113406 formation ranged from 8 to 26 MM and 0.83 to 5.99 nmol/min/mg protein for K(m) and V(max), respectively (n = 3). Enzyme activity varied 23-fold among 15 human liver microsome samples and correlated with CYP2A6-catalyzed coumarin hydroxylase (r2 = 0.85, P < .01) and CYP2B6-catalyzed 7-ethoxytrifluoromethylcoumarin O-deethylase (r2 = 0.82, P < .01) activities. Chemical inhibition studies showed a 63% decrease in RP 73401 hydroxylation by 500 microM orphenadrine. Coumarin (10 microM), however, did not inhibit RP 73401 hydroxylation. Also, anti-CYP2B1 IgG produced 85% inhibition of RP 73401 hydroxylation, but only a negligible decline in coumarin hydroxylase activity. Of the 10 expressed P450 forms studied, only CYP2B6 catalyzed RP 73401 hydroxylation. Finally, expressed CYP2B6 showed a high affinity (K(m) = 22.5 microM) for RP 73401 hydroxylation, similar to the human liver microsome studies.     

Tissue and sex-dependent differences in CYP2A activities in hamsters

Three enzymatic activities of the CYP2A subfamily, coumarin 7-hydroxylase (COH), testosterone 15 alpha-hydroxylase (T15 alpha OH) and testosterone 7 alpha-hydroxylase (T7 alpha OH), were characterized in liver, kidney and lung microsomes from control, pyrazole (PYR), 3-methylcholanthrene (MC) and phenobarbital (PB) treated female and male Syrian golden hamsters. Sex-dependent changes in the enzymatic activities were found. Among control animals COH and T15 alpha OH activities were higher in males. T7 alpha OH activity was five times higher in female kidneys than in males. Inducers changed this metabolic profile. MC and PB were potent CYP2A inducers in extrahepatic tissues: significant increases were found in COH (5-fold) and T15 alpha OH (12-fold) activities in female MC lung microsomes and T7 alpha OH (7-fold) in MC male kidney microsomes. PB increased significantly activities of COH (5-fold), T15 alpha OH (3-fold) and T7 alpha OH (10-fold) in male kidney microsomes. All inducers significantly increased T7 alpha OH activity in male kidney microsomes but decreased hepatic T7 alpha OH activity in both sexes. PYR treatment decreased hepatic CYP2A activities. Anti-mouse CYP2A4/5 antibody inhibited COH activity by a variable extent depending on the tissue and pretreatment and recognised three 52-, 49-, 48-kDa bands in liver and two major bands in kidney (48 and 49 kDa) and lung (49 and 52 kDa) microsomes. COH and T15 alpha OH activities correlated well with 49 kDa protein (r = 0.95 and r = 0.99, respectively) in lung microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selenium regulation of hepatic heme metabolism: induction of delta-aminolevulinate synthase and heme oxygenase

Selenium was found to be a novel regulator of cellular heme methabolism in that the element induced both the mitochondrial enzyme delta-aminolevulinate synthase [succinyl-CoA:glycine C-succinyltransferase (decarboxylating); EC 2-3-1-37] and the microsomal enzyme heme oxygenase [heme, hydrogen-donor:oxygen oxidoreductase(alpha-methene-oxidizing, hydroxylating); EC 1-14-99-3] in liver. The effect of selenium on these enzyme activities was prompt, reaching a maximum within 2 hr after a single injection. Other changes in parameters of hepatic heme metabolism occurred after administration of the element. Thirty minutes after injection the cellular content of heme was significantly increased; however, this value slightly decreased below control values within 2 hr, coinciding with the period of rapid induction of heme oxygenase. At later peroids heme content returned to normal values. Selenium treatment caused only a slight decrease in microsomal cytochrome P-450 content. However, drug-metabolizing activity was severely inhibited by higher doses of the element. Unlike other inducers of delta-aminolevulinate synthase, which as a rule are also porphyrinogenic agents, selenium induction of this enzyme was not accompanied by an increase in the cellular content of prophyrins. When rats were pretreated with selenium 90 min before administration of heme, a potent inhibitor of delta-aminolevulinate synthase production, the inhibitory effect of heme of formation of this mitochondrial enzyme was completely blocked. Selenium, at high concentrations in vitro, was inhibitory to delta-aminolevulinate synthase activity. It is postulated that selenium may not be a direct inducer of heme oxygenase as is the case with trace metals such as cobalt, but may mediate an increase in heme oxygenase through increased production and cellular availability of "free" heme, which results from the increased heme synthetic activity of hematocytes. Subsequently, the increased heme oxygenase activity is in turn responsible for the lack of increase in the microsomal heme content, thus maintaining heme levels at normal values despite the highly increased activities of both heme oxygenase and delta-aminolevulinate synthase. It is further suggested that the increase in delta-aminolevulinate synthase activity is not due to a decreased rate of enzyme degradation or an activation of preformed enzyme, but to increased rate of synthesis of enzyme protein. Although selenium in trace amounts has been postulated to be involved in microsomal electron transfer process, the data from this study indicate that excess selenium can substantially inhibit microsomal drug metabolism.     

Differential induction of cytochrome P450 gene expression by 4n-alkyl-methylenedioxybenzenes in primary rat hepatocyte cultures

A well-characterized primary rat hepatocyte culture system was used to examine induction patterns of cytochrome 450 gene expression by a series of 4-n-alkyl-methylenedioxybenzene (MDBs) derivatives. Hepatocytes were treated for 24, 48, or 72 hours with 0-500 microM of the MDB compounds, and total cellular RNA and protein from each treatment was evaluated by hybridization and immunochemical techniques. Exposure to MDB congeners possessing increasing 4-n-alkyl side-chain length (C0-C8) resulted in dose- and structure-dependent activation of CYP2B1, 2B2, 3A1, 1A1, and 1A2 gene expression. At equivalent 100 microM concentrations, the C6 and C8 MDB congeners were more effective than the prototypical inducer phenobarbital (PB) with respect to induction potency of CYP2B1, CYP2B2, and CYP3A1 gene expression. In contrast to PB, longer side-chain-substituted MDBs effectively induced CYP1A1 and CYP1A2 gene expression, in addition to the CYP2B and CYP3A genes. At equivalent molar concentrations, the catechol derivative of C6-MDB was ineffective in its ability to induce CYP gene expression, indicating the importance of the intact methylenedioxy bridge in the induction mechanism. Levels of MDB-inducible CYP2B1 and CYP2B2 mRNA were highly correlated with CYP2B1/2 apoprotein levels, ascertained by immunoblot analysis of cultured hepatocyte S9 fractions. Compared with results from previous in vivo analysis (12), the current data indicate that pharmacodynamic factors may influence MDB induction profiles and that differences in MDB effects on CYP gene expression result depending on distinct structure-activity relationships.     

Dimensional changes related to ordering in an AuCu-3wt%Ga alloy at intraoral temperature

In this study, the overfed rat was employed as a model for examining the influence of obesity on the regulation of hepatic cytochromes P450 3A and 2C11 (CYP3A and CYP2C11, respectively). These proteins represent the predominant constitutive hepatic P450 enzymes of male rats. Sprague-Dawley rats were chronically fed a standard pelleted diet or an energy-dense diet which typically results in significant increases in body weight, serum triglyceride levels and liver lipid content. Obesity did not influence baseline levels of spectral cytochrome P450 content. Similar baseline activities of CYP3A (testosterone 6 beta-hydroxylation), comparative CYP3A protein levels (Western blot) and steady-state CYP3A mRNA (slot blot), were found in rats fed either diet. Likewise, obesity did not appear to influence CYP2C11 at the enzyme activity (testosterone 2 alpha-hydroxylation) or mRNA levels. Half of the animals in each group received 20 mg phenobarbital (intraperitoneal injection) per animal every 12 hours for three consecutive days. This resulted in similar phenobarbital plasma concentrations in both groups. Phenobarbital treatment increased the concentrations of total cytochrome P450 in both lean and obese rats to the same extent. CYP3A activity, protein and mRNA levels were induced to a similar magnitude in rats fed either diet. Furthermore, obesity did not influence CYP2C11 activity or mRNA levels following administration of phenobarbital. A lack of an effect of obesity and the altered lipid environment on the regulation of CYP3A and CYP2C11 is in contrast to other enzymes studied previously. It is apparent that the consequences of obesity on hepatic cytochrome P450 may be enzyme-specific.     

Induction of CYP1A1 by GABA receptor ligands

The induction of CYP1A1 is mediated via the aromatic hydrocarbon (Ah) receptor. Studies from our laboratory show CYP1A1 induction by picrotoxin and phenobarbital which prompted us to examine if other ligands of the gamma-aminobutyric acid (GABA) receptor could also induce CYP1A1. Here we report the nuclear translocation of the Ah receptor and its DNA binding activity to radiolabeled double-stranded synthetic xenobiotic response elements (XREs) in nuclear extracts, increased accumulation of CYP1A1 mRNA, and alterations in intracellular calcium concentrations in cells exposed to GABA receptor ligands.     

Intracellular signaling by reactive oxygen species during hypoxia in cardiomyocytes

Cardiomyocytes suppress contraction and O2 consumption during hypoxia. Cytochrome oxidase undergoes a decrease in Vmax during hypoxia, which could alter mitochondrial redox and increase generation of reactive oxygen species (ROS). We therefore tested whether ROS generated by mitochondria act as second messengers in the signaling pathway linking the detection of O2 with the functional response. Contracting cardiomyocytes were superfused under controlled O2 conditions while fluorescence imaging of 2, 7-dichlorofluorescein (DCF) was used to assess ROS generation. Compared with normoxia (PO2 approximately 107 torr, 15% O2), graded increases in DCF fluorescence were seen during hypoxia, with responses at PO2 = 7 torr > 20 torr > 35 torr. The antioxidants 2-mercaptopropionyl glycine and 1,10-phenanthroline attenuated these increases and abolished the inhibition of contraction. Superfusion of normoxic cells with H2O2 (25 microM) for >60 min mimicked the effects of hypoxia by eliciting decreases in contraction that were reversible after washout of H2O2. To test the role of cytochrome oxidase, sodium azide (0.75-2 microM) was added during normoxia to reduce the Vmax of the enzyme. Azide produced graded increases in ROS signaling, accompanied by graded decreases in contraction that were reversible. These results demonstrate that mitochondria respond to graded hypoxia by increasing the generation of ROS and suggest that cytochrome oxidase may contribute to this O2 sensing.