Prevalence of human papillomavirus among oesophageal squamous cell carcinoma cases: systematic review and meta-analysis.

BACKGROUND: Oncogenic human papillomavirus (HPV) has been hypothesised as a risk factor for oesophageal squamous cell carcinoma (OSCC), but aetiological research has been limited by the varying methodology used for establishing HPV prevalence. The aims of this systematic review and meta-analysis were to estimate the prevalence of HPV DNA detected in OSCC tumours and the influence of study characteristics. METHODS: Study-level estimates of overall and type-specific HPV prevalence were meta-analysed to obtain random-effects summary estimates. RESULTS: This analysis included 124 studies with a total of 13 832 OSCC cases. The average HPV prevalence (95% confidence interval) among OSCC cases was 0.277 (0.234, 0.320) by polymerase chain reaction; 0.243 (0.159, 0.326) by in situ hybridisation; 0.304 (0.185, 0.423) by immunohistochemistry; 0.322 (0.154, 0.490) by L1 serology; and 0.176 (0.061, 0.292) by Southern/slot/dot blot. The highest HPV prevalence was found in Africa and Asia, notably among Chinese studies from provinces with high OSCC incidence rates. CONCLUSIONS: Future research should focus on quantifying HPV in OSCC cases using strict quality control measures, as well as determining the association between HPV and OSCC incidence by conducting large, population-based case-control studies. Such studies will provide a richer understanding of the role of HPV in OSCC aetiology.

Well-differentiated hepatocellular carcinoma in a ring-tailed lemur (Lemur catta).

A 16-year-old male ring-tailed lemur (Lemur catta) was presented with severe cachexia and an abdominal mass. The encapsulated, multilobular mass replaced the right medial lobe of the liver and compressed the adjacent gall bladder. Multiple haemorrhages and necrotic foci were found within the mass. Microscopically, neoplastic cells formed cords of moderately pleomorphic, polygonal cells with mild to moderate anaplasia. Immunohistochemical markers used for diagnosis of hepatocellular carcinomas in man were used to characterize the neoplastic cells, which expressed hepatocyte-specific antigen, but not glypican-3 or polyclonal carcinoembryonic antigen. Gross, microscopical and immunohistochemical features of the tumour were most consistent with a well-differentiated hepatocellular carcinoma. Although this tumour is common among prosimians, to the authors' knowledge this is the first documented case in a ring-tailed lemur. Hepatocellular carcinomas have been associated with hepatitis virus infections and excessive hepatic iron in man; however, no association was established between this tumour and viral infection or hepatic iron storage disease in the present case.

Granulomatous typhlocolitis, lymphangitis, and lymphadenitis in a horse infected with Listeria monocytogenes, Salmonella Typhimurium, and cyathostomes.

A 15-year-old American Quarter horse mare was euthanized because of poor response to therapy for severe diarrhea. Significant gross findings were limited to the large intestines. The walls of the cecum and colon were thickened with widely scattered nodules in the mucosa and submucosa that extended into the enlarged colic lymph nodes. Microscopically, there was severe granulomatous typhlocolitis, lymphangitis, and lymphadenitis, with many intralesional Gram-positive, non-acid-fast coccobacilli and few cyathostomes. Intralesional bacteria were immunohistochemically and polymerase chain reaction (PCR) assay positive for Listeria monocytogenes. Concurrent infection with Salmonella enterica serovar Typhimurium was detected by PCR and culture. Infection with L. monocytogenes in horses is rare, and coinfection with Salmonella and small strongyles probably contributed to the development of granulomatous typhlocolitis.

Common genetic variation near the connexin-43 gene is associated with resting heart rate in African Americans: a genome-wide association study of 13,372 participants.

BACKGROUND: Genome-wide association studies have identified several genetic loci associated with variation in resting heart rate in European and Asian populations. No study has evaluated genetic variants associated with heart rate in African Americans. OBJECTIVE: To identify novel genetic variants associated with resting heart rate in African Americans. METHODS: Ten cohort studies participating in the Candidate-gene Association Resource and Continental Origins and Genetic Epidemiology Network consortia performed genome-wide genotyping of single nucleotide polymorphisms (SNPs) and imputed 2,954,965 SNPs using HapMap YRI and CEU panels in 13,372 participants of African ancestry. Each study measured the RR interval (ms) from 10-second resting 12-lead electrocardiograms and estimated RR-SNP associations using covariate-adjusted linear regression. Random-effects meta-analysis was used to combine cohort-specific measures of association and identify genome-wide significant loci (P≤2.5×10(-8)). RESULTS: Fourteen SNPs on chromosome 6q22 exceeded the genome-wide significance threshold. The most significant association was for rs9320841 (+13 ms per minor allele; P = 4.98×10(-15)). This SNP was approximately 350 kb downstream of GJA1, a locus previously identified as harboring SNPs associated with heart rate in Europeans. Adjustment for rs9320841 also attenuated the association between the remaining 13 SNPs in this region and heart rate. In addition, SNPs in MYH6, which have been identified in European genome-wide association study, were associated with similar changes in the resting heart rate as this population of African Americans. CONCLUSIONS: An intergenic region downstream of GJA1 (the gene encoding connexin 43, the major protein of the human myocardial gap junction) and an intragenic region within MYH6 are associated with variation in resting heart rate in African Americans as well as in populations of European and Asian origin.

Comparative inhibition of inducible and constitutive CYPs in rat hepatic microsomes by parathion.

1. In microsomal fractions, the phosphorothioate pesticide parathion inhibits cytochrome P450 (CYP) enzymes by reversible and irreversible mechanisms resulting in the long-term suppression of drug oxidation. The present study evaluated the relative susceptibilities of constitutive and inducible CYP2 and CYP3 steroid hydroxylases to inhibition by the pesticide. 2. Enzyme kinetic analysis indicated that constitutive and dexamethasone (DEX)-induced androst-4-ene-3,17-dione (AD) 6beta-hydroxylations were similarly susceptible to inhibition by parathion (Km/Ki ratios 1.5-1.6). However, preincubation of parathion with NADPH-fortified microsomes intensified the extent of inhibition of CYP3A-dependent 6beta-hydroxylation. Comparison of Km/Ki ratios indicated that 6beta-hydroxylation activity in fractions from DEX-pretreated rats was about twice as susceptible as the control activity to inactivation by parathion metabolites (Km/Ki ratio of 8.0 versus 4.0). 3. The time-dependent loss of AD 6beta-hydroxylation by parathion occurred more efficiently in fractions from DEX-induced liver than in control. Thus, half-times of 1.3 and 6.1 min, respectively, were determined for the inactivation of DEX-inducible and constitutive activities. Parathion concentrations required for half-maximal inactivation were 32 and 67 microM in microsomes from DEX-induced and control rats. 4. In phenobarbital (PB)-induced fractions CYP2B1-mediated AD 16beta-hydroxylation was inhibited potently in a reversible fashion by parathion (Ki = 0.37 microM; Km/Ki ratio about 73). Inhibition was not enhanced at parathion concentrations near the Ki by a preincubation step with NADPH. 5. In control microsomes parathion elicited a type I binding interaction with oxidized CYP (Ks=7.7 microM, deltaAmax=2.2 x 10(-2) a.u.nmol CYP-1; deltaAmax/Ks 2.86 x 10(3) a.u. nmol CYP(-1)/mM). Ligand binding was 13- and 1.6-fold more efficient in PB and DEX microsomes, respectively. 6. These findings indicate that pretreatment of rats with enzyme-inducing drugs like DEX and PB alters the profile of CYPs and their susceptibility to inhibition by parathion. Potent reversible inhibition of CYP2B1 occurred in PB-induced fractions and DEX-inducible CYPs 3A were more susceptible to mechanism-based inactivation than the corresponding constitutive CYPs from the same subfamily.

Differential regulation of endobiotic-oxidizing cytochromes P450 in vitamin A-deficient male rat liver.

1. The hepatic CYP4A-dependent omega-hydroxylation of arachidonic acid and CYP2C11-dependent 2alpha-/16alpha-hydroxylations of testosterone were decreased to 74 and 60% of respective control in microsomal fractions from vitamin A-deficient rats. Decreases in the rates of arachidonic acid omega-1-hydroxylation and testosterone 6beta-, 7alpha- and 17alpha-hydroxylations were less pronounced. 2. Corresponding decreases in microsomal CYP4A and CYP2C11 immunoreactive protein expression to 64 and 68% of respective control were observed in vitamin A-deficient rat liver. Expression of CYP3A proteins was unchanged from vitamin A-adequate control. 3. Northern analysis revealed a selective decrease in CYP4A2 mRNA expression in vitamin A-deficient rat liver to approximately 5% of control; expression of the related CYP4A1/4A3 mRNAs was not decreased. CYP2C11 mRNA expression was also decreased in vitamin A-deficient male rat liver to 39% of control levels. 4. Intake of the deficient diet containing all-trans-retinoic acid (ATRA) during the final week of the experiment restored CYP4A2 mRNA and CYP4A protein. Administration of exogenous androgen or episodic growth hormone was ineffective. In contrast, CYP2C11 expression was restored by ATRA and androgen, but not by growth hormone. 5. From these studies it emerges that CYP4A2, a fatty acid omega-hydroxylase in rat liver, is highly dependent on vitamin A for optimal expression, whereas CYP2C11 is indirectly down regulated by androgen deficiency resulting from vitamin A-deficiency. Altered CYP expression in vitamin A-deficiency provides insights into the relationship between dietary constituents and the intracellular formation of vasoactive eicosanoids as well as the clearance of androgenic steroids.

Characterization of a novel heterodimeric cathepsin L-like protease and cDNA encoding the catalytic subunit of the protease in embryos of Artemia franciscana.

Embryos and larvae of the brine shrimp, Artemia franciscana, contain a novel cathepsin L-like cysteine protease (ACP) composed of 28.5- and 31.5-kDa subunits. Both subunits of the ACP are glycosylated, and seven isoforms of the protease were identified by isoelectric focusing with pI values ranging from 4.6 to 6.2. Several clones containing sequences coding for the 28.5-kDa subunit of the ACP were isolated from an Artemia embryo cDNA library in lambda ZAP II. One clone of 1229 bp, with an open reading frame of 1014 bp, was sequenced and found to contain 50-65% amino acid sequence identity with several members of the cathepsin L subfamily of cysteine proteases. The mature protein predicted from this sequence consisted of 217 amino acids with a mass of 23.5 kDa prior to post-translational modifications. The mature protein showed 68.6% amino acid sequence identity with human cathepsin L and 73.9% identity with cathepsin L-like proteases from Sarcophaga. peregrina and Drosophila melanogaster. The full-length cDNA clone analyzed in this study (pCP-3b) was renamed AFCATL1 (A. franciscana Cathepsin L1) and the sequence has been deposited in the Genbank database, accession number AF147207. Northern blot analyses identified a single transcript of about 1.4 kb in both embryos and young larvae of Artemia. Southern blot analyses of Artemia genomic DNA treated with various restriction endonucleases indicated a single gene for the ACP. The catalytic subunit of the ACP was tightly associated with a 31.5-kDa protein, which may localize the protease to nonlysosomal sites in embryos and larvae.

Pretranslational up-regulation of the hepatic microsomal delta4-3-oxosteroid 5alpha-oxidoreductase in male rat liver by all-trans-retinoic acid.

Administration of all-trans-retinoic acid (ATRA; 60 mg/kg daily for 3 days) to male rats increased the rate of 5alpha-dihydrotestosterone (5alpha-DHT) formation from testosterone in microsomal fractions in vitro. The formation of androstane-3alpha,17beta-diol from testosterone was also increased because of the higher concentration of 5alpha-DHT produced in microsomal incubations. Northern analysis confirmed that the increased rate of 5alpha-DHT formation was due to the pretranslational up-regulation in delta4-3-oxosteroid 5alpha-oxidoreductase (EC 1.3.99.5) mRNA expression in ATRA-treated male rat liver. Thus, ATRA elicited in male rat liver a partial feminization of the expression of this enzyme, which normally exhibits a female-selective distribution in the rat. Subsequent experiments evaluated whether the administration of human chorionic gonadotropin or thyroxine to ATRA-treated male rats decreases 5alpha-reductase activity to that observed in untreated male rat liver. Although these treatments did not decrease 5alpha-reductase to untreated male levels, it was found that administration of ATRA to gonadectomized male rats produced complete feminization of the enzyme. Again, up-regulation was confirmed at the mRNA level. The activity of the male-specific cytochrome P450 2C11 (as reflected by microsomal testosterone 16alpha-hydroxylation activity) was correspondingly decreased by treatments that increased steroid 5alpha-reductase activity. Thus, gonadectomy in combination with ATRA administration effected a more pronounced decrease in 16alpha-hydroxylation activity than either treatment alone. These findings suggest that ATRA is a novel positive regulator of the 5alpha-reductase that in combination with the removal of circulating androgen, which normally suppresses 5alpha-reductase levels, feminizes the expression of this enzyme in rat liver.

Comparative induction of CYP3A and CYP2B in rat liver by 3-benzoylpyridine and metyrapone.

3-Benzoylpyridine (3BP) is a major metabolite of HGG-12, and oxime that has been synthesized as a potential antidote to the toxic effects of soman and other anticholinesterases. Structural similarities exist between 3BP, the cytochrome P450 (CYP)-inducer metyrapone (MET) and other 3-substituted pyridines that interact with CYPs. The present study evaluated the regulatory effects of 3BP on CYP expression in rat liver. Both 3BP and MET (100 mg/kg) increased total hepatic microsomal holo-CYP content significantly 24 h after administration to male rats. Pronounced increases in activities mediated by CYP2B (androstenedione 16 beta-hydroxylation and 7-pentylresorufin O-depentylation) were produced by 3BP and MET, which correlated with respective 9- and 14-fold increases in CYP2B immunoreactive protein. In addition, both agents slightly increased rates of microsomal CYP3A-dependent steroid 6 beta-hydroxylation, troleandomycin metabolite complex formation and total CYP3A immunoreactive protein. Induction of the dexamethasone-inducible CYP3A23 mRNA to 4.5- and 2.5-fold of control was detected in liver of MET- and 3BP-induced rats; CYP3A2 mRNA levels were unchanged. Analogous in vitro studies revealed that MET was a preferential inhibitor of CYP3A-mediated steroid 6 beta-hydroxylation activity, but 3BP was inactive against constitutive steroid hydroxylase CYPs. These findings indicate that the structurally related 3BP and MET elicit similar induction effects on CYPs 2B and 3A23 in rat liver after in vivo administration, but differential inhibitory effects of the chemicals on CYP activity in vitro. Recent reports have implicated a microsomal binding site in the induction of CYP3A1/3A23 in rat liver. In light of the present findings, substituted pyridines like 3BP may be useful tools in structure-activity studies to evaluate the physicochemical requirements for binding to this protein.

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.

Biotransformation of parathion in human liver: participation of CYP3A4 and its inactivation during microsomal parathion oxidation.

Studies in rat liver have shown that cytochrome P450 (CYP) enzymes mediate the oxidative biotransformation of the phosphorothioate pesticide parathion to paraoxon and 4-nitrophenol. Transfer of the phosphorothioate thionosulfur atom to the CYP apoprotein results in amino acid modification and enzyme inactivation. Our study investigated the role of human hepatic CYP in parathion oxidation and their relative susceptibilities to inhibition and inactivation. Rates of parathion oxidation varied about 10-fold in microsomes from 23 individual livers (1.72-18.33 nmol total metabolites/mg protein/min). Linear regression of rates of parathion oxidation with those of other microsomal CYP reactions implicated CYP3A4 in the reaction. Thus, parathion oxidation was correlated strongly with testosterone 6beta-hydroxylation (r2 = 0.95, n = 11), but not with activities mediated by CYP 1A2, 2C9 or 2E1. CYP 3A4 expressed in lymphoblastoid cell lines was an efficient catalyst of parathion oxidation, although CYP 1A2 and 2B6 also catalyzed the activity. The CYP3A4 inhibitors ketoconazole and triacetyloleandomycin decreased the observed rate of microsomal parathion oxidation, but chemicals known to interact preferentially with other human CYP were essentially noninhibitory. P450 was lost during parathion biotransformation in human hepatic microsomes. Thus, incubation (10 min) of parathion (25 microM) with NADPH-supplemented microsomes led to an apparent 19 +/- 4% decrease in holo-P450 content. Several CYP-specific oxidation reactions were inhibited and inactivated by parathion. Testosterone 6beta-hydroxylation (mediated by CYP3A4), 7-ethylresorufin O-deethylation (CYP1A2) and tolbutamide methyl hydroxylation (CYP2C9/10), but not aniline 4-hydroxylation (CYP2E1), were inhibited effectively by parathion. Preincubation of microsomes with parathion and NADPH intensified the extent of inhibition (i.e., elicited inactivation) of reactions mediated by 3A4 and 1A2 and, to a lesser extent, 2C9. In summary, these findings strongly implicate CYP 3A4 as the principal catalyst of parathion oxidation in human liver, although other CYP may play a lesser role. During parathion oxidation CYP3A4 undergoes significant inactivation. In view of the role of this enzyme in the oxidation of many therapeutic agents, exposure to phosphorothioate pesticides may adversely affect drug elimination in humans.

Induction of cytochromes P450 2B and 2E1 in rat liver by isomeric picoline N-oxides.

Pyridine derivatives are widely used solvents and precursors for the synthesis of chemicals of industrial importance. Oxidized metabolites have been implicated in the observed toxicity of pyridines and are known to induce drug-metabolizing enzymes in rat liver. In this study the three isomeric picoline (methylpyridine) N-oxides, as major oxidized metabolites of 2-, 3- and 4-picoline, were evaluated as inducers of cytochrome P450 (CYP) enzymes in rat liver. After a single dose of 100 mg/kg 24 h before sacrifice the 3- and 4-isomers were effective inducers of microsomal substrate oxidations associated with the phenobarbital-inducible CYPs 2B; upregulation of CYP2B protein was confirmed by immunoblotting. In contrast, the 2-isomer did not increase CYP2B protein or activity in rat liver but CYP2E1 protein expression was upregulated by the isomers to 160-200% of control. The three chemicals increased aniline 4-hydroxylation activity in rat liver, which is consistent with induction of CYPs 2B or 2E1 and 4-nitrophenol 2-hydroxylation activity was increased in microsomal fractions from 3- and 4-picoline N-oxide-treated rats. The activities of several other CYPs were also determined and CYP1A-dependent 7-ethylresorufin O-deethylation was increased (to approximately 6- and 2-fold of control) by the 3- and 4-isomer, respectively, whereas the activity of CYP3A-mediated androstenedione 6beta-hydroxylation was decreased by the agents--most notably by the 2-isomer. During NADPH-supported oxidation of CCl4, lipid peroxidation was increased in microsomes from 3- and 4-picoline N-oxide-pretreated rats and was modulated in vitro by the CYP2B inhibitor orphenadrine, but not by the CYP2E1 inhibitor 4-methylpyrazole. These findings establish that particular isomers of picoline N-oxide rapidly upregulate CYP2B or, to a lesser extent, CYP2E1 and implicate CYP2B in the enhanced lipid peroxidation observed in microsomes from rats treated with 3- and 4-picoline N-oxides. Such induction process may contribute to the hepatotoxicity of pyridines by enhancing the capacity for microsomal lipid peroxidation.

Enhanced inhibition of microsomal cytochrome P450 3A2 in rat liver during diltiazem biotransformation.

Pharmacokinetic drug interactions involving the calcium channel blocker diltiazem (DTZ) have been attributed to inhibition of microsomal cytochrome P450 (P450)-mediated drug oxidation. Accumulation of certain DTZ metabolites during dosage with the drug, as well as dose-related differences in DTZ pharmacokinetics, suggests that DTZ metabolites may also participate in P450 inhibition. The present study evaluated a series of putative DTZ metabolites as inhibitors of major constitutive P450s in rat liver in vitro, in relation to DTZ biotransformation. The principal finding to emerge was that the N-demethylated metabolite of DTZ was a more potent competitive inhibitor than DTZ of CYP3A2-dependent testosterone 6 beta-hydroxylation. This P450 appeared to be the preferred target for inhibition, because the observed K/K(m) ratio for inhibition of CYP3A2-dependent steroid hydroxylation was approximately 4- and 100-fold lower than those for CYP2C11 and CYP2A1-dependent pathways, respectively. It was also established that N-desmethyl-DTZ was a major metabolite formed during microsomal DTZ biotransformation in rat liver in vitro. The other primary metabolites, desacetyl-DTZ and O-desmethyl-DTZ, were ineffective inhibitors of any pathways of steroid oxidation by P450s, but several other potential metabolites, which were not detected in microsomal incubations, also inhibited P450 activity. Consistent with previous reports, there was no evidence of P450 inactivation or complexation by DTZ, but the drug and its N-desmethyl metabolite generated binding interactions with ferric P450 in rat hepatic microsomes. Considered together, the findings of the present study establish that N-desmethyl-DTZ is a preferential inhibitor of CYP3A2 in rat hepatic microsomes, with greater potency than the parent drug. This is consistent with clinical reports in which this metabolite accumulates during multiple-dose therapy with DTZ. The competitive nature of the inhibitory interaction suggests that the eventual elimination of N-desmethyl-DTZ should restore normal hepatic oxidation capacity.

Restoration of cytochrome P450 2C11 in vitamin A-deficient rat liver by exogenous androgen.

Down-regulation of microsomal androgen-dependent CYP2C11 is produced in male rat liver by dietary vitamin A deficiency. Decreased circulating androgen concentrations also occur in vitamin A-deficient male rats. Both effects are prevented by addition of all-trans-retinoic acid to the diet. The present study evaluated directly whether androgen deficiency may be responsible for the down-regulation of 2C11 in vitamin A-deficient male rats. The major finding was that subcutaneous administration of the androgen methyltrienolone (MT) during the final week of the study restored CYP2C11 protein and its associated steroid 16alpha-hydroxylation activities to control levels; CYP2C11 mRNA was also restored. Despite the efficient restoration of CYP2C11 at a pretranslational level, no alteration in vitamin A status was apparent and animals remained vitamin A deficient after MT treatment. The possibility was assessed that vitamin A can maintain the microsomal content of CYP2C11 in normal liver. However, in contrast to MT, administration of ATRA to gonadectomized male rats did not restore 2C11 in liver. These findings establish that the major effect of vitamin A deficiency on CYP2C11 in male rat liver is mediated indirectly by androgen deficiency.

Pretranslational down-regulation of cytochromes P450 2C11 and 3A2 in male rat liver by tumor necrosis factor alpha.

BACKGROUND & AIMS: The cytokine tumor necrosis factor alpha (TNF-alpha) is a primary inflammatory mediator after liver injury. Several cytokines impair the regulation of cytochrome P450 (CYP) genes in liver, but the specificity of these effects remains unclear. This study investigated the effects of recombinant murine TNF-alpha on the expression of specific constitutive CYPs in male rat liver. METHODS: Microsomal steroid hydroxylation was used to indicate the activities of specific CYPs after TNF-alpha treatment and immunoblotting to correlate CYP activities with protein contents. CYP messenger RNA levels were measured by solution hybridization. RESULTS: Testosterone 2 alpha/16 alpha- and 6 beta-hydroxylations, mediated respectively by CYPs 2C11 and 3A2, were decreased after TNF-alpha treatment, whereas 7 alpha-hydroxylation (CYP 2A1) was unchanged. Similarly, progesterone 2 alpha/16 alpha- (CYP 2C11) and 6 beta-hydroxylations (CYP 3A2), but not 21-hydroxylation (CYP 2C6), were decreased after TNF-alpha treatment. 2C11 and 3A2 apoproteins and messenger RNAs, but not 2A1 apoprotein, were decreased after TNF-alpha treatment; changes in messenger RNAs were evident 4 hours after treatment. CONCLUSIONS: TNF-alpha down-regulates CYPs 2C11 and 3A2 in male rat liver at a pretranslational level, whereas two other constitutive CYPs, 2A1 and 2C6, seem refractory to TNF-alpha. Thus, impaired CYP regulation by TNF-alpha resembles the combined effects of autologous interferons (on 3A2) and interleukins (on 2C11).

Pretranslational down regulation of cytochrome P450 2C11 in vitamin A-deficient male rat liver: prevention by dietary inclusion of retinoic acid.

Manipulation of vitamin A intake has been associated with altered rates of cytochrome P450 (P450)-mediated microsomal drug oxidation. Dietary vitamin A deficiency reportedly results in decreased rates of P450-dependent substrate oxidation, but the mechanisms underlying these changes remain unclear. In this study, the effects of dietary vitamin A modulation, as well as dietary inclusion of all-trans-retinoic acid (ATRA), on major constitutive P450s were defined. Total microsomal P450 in deficient male rats was decreased to 72% of control (0.63 +/- 0.07 vs. 0.88 +/- 0.08 nmol/mg of protein; P < .05); this was prevented by inclusion of ATRA (12 micrograms/g) in the deficient diet. Dietary vitamin A deficiency decreased rates of P450 2C11-mediated testosterone 2 alpha- and 16 alpha-hydroxylation in rat liver to 44 and 47% of respective adequate control, whereas rates of 6 beta- and 7 alpha-hydroxylation of the steroid were unaltered; inclusion of ATRA into the deficient diet prevented the loss of 2C11 activities. Immunoblot and RNA analysis revealed decreases in P450 2C11 apoprotein and its corresponding mRNA in liver from deficient rats that was prevented by inclusion of ATRA in the deficient diet. Serum testosterone concentrations were reduced in deficient rats and this also was prevented by dietary ATRA. To discern whether this was a direct effect of vitamin A on P450 2C11 regulation, further experiments evaluated the effect of ATRA administration to male rats maintained on standard rat chow (vitamin A-adequate). Dose- and time-dependent decreases in P450 2C11 activity were observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of a reversible component in the in vitro inhibition of rat hepatic cytochrome P450 2B1 by parathion.

Cytochrome P450 (P450) enzymes are inactivated in suicidal fashion during microsomal parathion oxidation. In the present study, two distinct components of the inhibition of the phenobarbital (PB)-inducible P450 2B1 by parathion were characterized. Here we report for the first time that low concentrations of parathion potently and reversibly inhibited, but did not inactivate, 2B1. In contrast, the previously described inactivation process occurred only at considerably higher parathion concentrations, at which concentrations enzyme activity was already extensively inhibited. At low concentration, parathion was a competitive inhibitor of 2B1-mediated androstenedione 16 beta-hydroxylation (Ki = 0.44 +/- 0.07 microM) and of 7-pentylresorufin O-depentylation (Ki = 0.40 +/- 0.03 microM) in microsomes from PB-pretreated rats and was similarly effective against androstenedione 16 alpha- and 16 beta-hydroxylation catalyzed by purified 2B1. Although preincubation of higher concentrations of parathion (> 5 microM) with NADPH-supplemented microsomes from PB-pretreated rat liver decreased holo-P450, heme loss was not observed near the Ki values. Instead, half-maximal loss of P450 occurred at 6 microM and at 9 microM parathion in PB-pretreated microsomes and in the reconstituted system, respectively. Parathion metabolism was efficient in PB-microsomes (Km values for 4-nitrophenol and paraoxon formation were 13 microM and 10 microM, respectively) and in the reconstituted system (corresponding Km values were 19 microM and 14 microM). Thus the constants for P450 inactivation and for parathion metabolism were similar and were at least 15-fold greater than the Ki values for the reversible process.(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatic biotransformation of parathion: role of cytochrome P450 in NADPH- and NADH-mediated microsomal oxidation in vitro.

In vitro studies have established that cytochrome P450 (P450) is deactivated by the electrophilic sulfur atom released during the enzymic oxidation of parathion to paraoxon. However, in vivo studies in rats have been unable to demonstrate significant P450 loss. This study evaluated the possibility that there may be alternate pathways of parathion biotransformation in liver, other than those mediated by P450 and supported by NADPH. Initial experiments confirmed that parathion administration did not decrease microsomal P450 or testosterone hydroxylation activities. Subsequent in vitro experiments identified an NADH-dependent pathway of parathion biotransformation, and MS was used to confirm that paraoxon and 4-nitrophenol were the products of both the NADH- and NADPH-dependent reactions. The Michaelis constants of the NADH-dependent formation of paraoxon and 4-nitrophenol (26 +/- 6 microM and 53 +/- 10 microM, respectively) were approximately 3-fold greater than those for the NADPH-supported reactions (9 +/- 1 microM and 18 +/- 3 microM, respectively). Induction of male rats with phenobarbital and dexamethasone, but not beta-naphthoflavone, produced similar increases in the rates of NADH- and NADPH-mediated parathion metabolism. Rates of NADH- and NADPH-dependent metabolism were highly correlated in linear relationships. An anti-NADPH-cytochrome P450 reductase (NADPH-P450 reductase) antibody partially inhibited microsomal parathion oxidation mediated by either cofactor, and the P450 inhibitor clotrimazole was similarly effective against the NADH- and NADPH-supported oxidation of parathion. Finally, a reconstituted system containing P450 2B1, NADPH-P450 reductase, and phospholipid supported parathion oxidation mediated by NADH.(ABSTRACT TRUNCATED AT 250 WORDS)

Competitive inhibition of human liver microsomal cytochrome P450 3A-dependent steroid 6 beta-hydroxylation activity by cyclophosphamide and ifosfamide in vitro.

The prodrugs cyclophosphamide (CP) and ifosfamide (IF) are oxidized by hepatic cytochrome P450 (P450) to the active cytotoxic species, phosphoramide mustard. Acrolein (prop-2-enal) is also formed during CP and IF activation in rat liver and has been associated with P450 destruction. Analogous inactivation of human liver P450s by CP or IF could lead to pharmacokinetic interactions with coadministered drugs. The present study investigated the susceptibilities of human hepatic P450s to inhibition and inactivation by CP and IF in vitro. Unlike the situation in rat liver microsomes, total P450 was not decreased after incubation of CP or IF with NADPH and human fractions. However, CP and IF inhibited testosterone 6 beta-hydroxylation mediated by P450s 3A but not P450 1A2-dependent 7-ethylresorufin O-deethylation, P450 2C-dependent tolbutamide methyl hydroxylation or P450 2E1-mediated N-nitrosodimethylamine N-demethylation. Kinetic analysis indicated that the drugs were reversible (competitive) inhibitors of testosterone 6 beta-hydroxylation (Km, 94 +/- 8 microM) in human liver microsomes (KiS, 510 +/- 20 microM and 490 +/- 40 microM for CP and IF, respectively). Time-dependent intensification of the inhibition of the activity by CP or IF did not occur; this supports the observation that P450 was refractory to inactivation. The rates of acrolein formation from CP and IF in human hepatic microsomes (0.76 +/- 0.23 and 0.19 +/- 0.07 nmol min-1 mg-1 of protein, respectively) were only 18% and 10% of the rates estimated in fractions from untreated rat liver (4.20 +/- 0.04 and 1.96 +/- 0.12 nmol min-1 mg-1 of protein, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of microsomal 17 beta-hydroxysteroid oxidoreduction activities in rat liver by all-trans-, 9-cis- and 13-cis-retinoic acid.

Retinoids and steroid hormones mediate their biological effects through nuclear receptors. However, retinoids may alter the intracellular availability of ligands for steroid receptor activation by modulating the activity of biotransformation enzymes. This study investigated the modulation of NAD(H)-linked steroid oxidoreductases in rat hepatic microsomes by retinoids. 13-cis-Retinoic acid inhibited testosterone 17 beta-dehydrogenation (Ki 2.4 +/- 0.5 microM; Km/Ki ratio 0.34 +/- 0.06) but androstenedione reduction was less susceptible to inhibition (Ki 27 +/- 13 microM; Km/Ki ratio 0.045 +/- 0.12). All-trans-retinoic acid was less potent than the 13-cis-isomer and 9-cis-retinoic acid was of intermediate potency. In vivo administration of all-trans-retinoic acid (60 mg/kg i.p. for 7 days) decreased hepatic microsomal oxidoreduction activity, but exposure over shorter periods and 13-cis-retinoic acid were without effect. Thus, all-trans-retinoic acid elicits direct inhibition and may also alter the normal regulation of the oxidoreductase under certain conditions. Three geometric isomers of retinal were potent inhibitors of testosterone dehydrogenation (IC50s approximately 6 microM), but were ineffective against androstenedione reduction. These findings suggest that certain anti-hormonal effects of retinoids may be attributable in part to modulation of endobiotic biotransformation prior to receptor binding and activation.