Cytology and clinical spectrum of sexually transmitted infections in Lebanese women as revealed by Pap smear: a cross-sectional study from 2002-2006.

To study the cytology profile of cervical smears and the respective prevalence and incidence of certain cervico-vaginal infections detectable by routine Pap smear cytology in Lebanese women from 2002 to 2006. Pap smear cytology results were compiled from the archives of the Institut National de Pathologie for the period extending from 2002 until 2006. This study covered 118,230 cervical specimens obtained from Lebanese women attending clinics and hospitals in all the five districts of Lebanon; prevalence and incidence rates for infections detectable by routine Pap smear examination were determined. A rise in prevalence of these infections by 2.1 percent (2,555) from 2002 to 2006 was revealed. A doubling of Pap smears showing HPV-associated changes was detected (1.4 percent in 2002 to 2.9 percent in 2006), and a simultaneous almost 7-fold increase of Atypical Squamous Cells of Undetermined Significance (ASCUS) cytology was detected during this period; the rise in ASCUS cytology was age-dependent. Moreover, a 60 percent increase in prevalence of bacterial vaginosis (2.3 percent in 2002 to 3.7 percent in 2006) and a more than 3-fold decline in Trichomonas vaginalis infection (1 percent in 2002 to 0.3 percent in 2006) were also noted in this population during this period. An increase in the prevalence and incidence of cervico-vaginal infections detectable by Pap smear cytology in Lebanese women was revealed from 2002 to 2006. Such changes could point to recent modifications of sexual and health behaviours in the Lebanese community.

Regulation of peroxisome proliferator-activated receptor alpha-induced transactivation by the nuclear orphan receptor TAK1/TR4.

Recently, we reported the cloning of the nuclear orphan receptor TAK1. In this study, we characterized the sequence requirements for optimal TAK1 binding and analyzed the repression of the peroxisome proliferator-activated receptor alpha (PPARalpha) signaling pathway by TAK1. Site selection analysis showed that TAK1 has the greatest affinity for direct repeat-1 response elements (RE) containing AGGTCAAAGGTCA (TAK1-RE) to which it binds as a homodimer. TAK1 is a very weak inducer of TAK1-RE-dependent transcriptional activation. We observed that TAK1, as PPARalpha, is expressed within rat hepatocytes and is able to bind the peroxisome proliferator response elements (PPREs) present in the promoter of the PPARalpha target genes rat enoyl-CoA hydratase (HD) and peroxisomal fatty acyl-CoA oxidase (ACOX). TAK1 is unable to induce PPRE-dependent transcriptional activation and represses PPARalpha-mediated transactivation through these elements in a dose-dependent manner. Two-hybrid analysis showed that TAK1 does not form heterodimers with either PPARalpha or retinoid X receptor (RXRalpha), indicating that this repression does not involve a mechanism by which TAK1 titrates out PPARalpha or RXRalpha from PPAR.RXR complexes. Further studies demonstrated that the PPARalpha ligand 8(S)-hydroxyeicosatetraenoic acid strongly promotes the interaction of PPARalpha with the co-activator RIP-140 but decreases the interaction of PPARalpha with the co-repressor SMRT. In contrast, TAK1 interacts with RIP-140 but not with SMRT and competes with PPARalpha for RIP-140 binding. These observations indicated that the antagonistic effects of TAK1 on PPARalpha.RXRalpha transactivation act at least at two levels in the PPARalpha signaling pathway: competition of TAK1 with PPARalpha.RXR for binding to PPREs as well as to common co-activators, such as RIP-140. Our results suggest an important role for TAK1 in modulating PPARalpha-controlled gene expression in hepatocytes.

Induction of replicative DNA synthesis and PPAR alpha-dependent gene transcription by Wy-14 643 in primary rat hepatocyte and non-parenchymal cell co-cultures.

Peroxisome proliferators (PP) are known hepatocarcinogens in rats and mice. We have investigated the ability of Wyeth-14 643 (Wy), a PP and potent rodent carcinogen, to induce replicative DNA synthesis and to modulate the levels of peroxisome proliferator activated receptor-alpha (PPAR alpha) transcriptionally-dependent genes in primary rat hepatocyte (HPC) cultures and hepatocyte/nonparenchymal cell (HPC/NPC) co-cultures maintained on Matrigel. Four days after plating, cells were treated with Wy and replicative DNA synthesis was quantitated using [3H]thymidine incorporation and specific mRNA transcript levels were determined by reverse-transcriptase polymerase chain reaction (RT-PCR). An increase in HPC replicative DNA synthesis was detected at 48 h in both Wy-treated HPC and HPC/NPC co-cultures relative to controls. This increase was approximately 3- and 6-fold in HPC and HPC/NPC cultures respectively, and was Wy concentration-dependent. The levels of PPAR alpha-transcriptionally dependent genes [cytochrome P4504A1, acyl-CoA oxidase (AOxase), and liver-fatty acid binding protein (L-FABP)] transcripts were determined as indicators of PPAR alpha activation. These transcripts increased dose-dependently at 48 h in HPC/NPC cultures up to 10 microM Wy. Similarly, RT-PCR product levels were also increased in HPC cultures with 10 microM Wy at 48 h. In conclusion, we have investigated the transcription of PPAR alpha-dependent genes and HPC replicative DNA synthesis by Wy in HPC/NPC co-cultures. Results of this work are clearly more reflective of the known in vivo effects of PP and suggest that HPC/NPC co-cultures are more appropriate than HPC cultures for such studies. The effect of PP on human HPC/NPC co-cultures is currently being investigated in our laboratory in an attempt to assess human risks to these chemicals more directly.

Human CYP2C19 is a major omeprazole 5-hydroxylase, as demonstrated with recombinant cytochrome P450 enzymes.

Omeprazole (OP) is a potent antiulcer drug that is metabolized by liver cytochrome P450 (P450) enzymes. However, the identities of the P450 isoforms responsible for its metabolism have been controversial. 5-Hydroxyomeprazole (5OH-OP) formation cosegregates with the polymorphism of (S)-mephenytoin 4'-hydroxylation in humans, which is now known to be mediated by CYP2C19. Previous in vitro studies have indicated that liver microsomal 50H-OP formation correlates with both (S)-mephenytoin 4'-hydroxylase and CYP3A content. Inhibitor and CYP2C antibody studies also suggested that both enzymes may be involved in the 5-hydroxylation of OP, whereas CYP3A appears to be the predominant enzyme involved in OP sulfone (OP-S) formation. The present studies assessed the contribution of various CYP2C and CYP3A4 enzymes to OP metabolism by using recombinant human enzymes. CYP2C19, CYP2C8, CYP2C18, and CYP2C9 formed a single metabolite with an HPLC retention time identical to that of 5OH-OP. The turnover number for CYP2C19 was 13.4 +/- 1.4 nmol/min/nmol of P450, whereas those for CYP2C8, CYP2C18, and CYP2C9 were 2.2 +/- 0.1, 1.5 +/- 0.1, and approximately equal to 0.5 nmol/min/nmol of P450, respectively. Recombinant human CYP3A4 formed 5OH-OP and OP-S with turnover numbers of 5.7 +/- 1.1 and 7.4 +/- 0.9 nmol/min/nmol of P450, respectively, and formed a minor unidentified metabolite. CYP2C19 had a substantially lower KM for 5OH-OP formation than did CYP3A4, CYP2C8, or CYP2C18. Antibody to CYP2C proteins inhibited approximately equal to 70% of OP 5-hydroxylation at low substrate concentrations, comparable to those that may be encountered at therapeutically relevant doses, whereas antibody to CYP3A4 inhibited approximately equal to 30% of the activity. At high substrate concentrations, the contributions of the two enzymes to OP hydroxylation were roughly comparable (40-50%). In contrast, OP-S formation was completely inhibited by antibody to CYP3A4 proteins. The present study provides the first direct confirmation, using human recombinant P450 enzymes and selective antibody inhibition, that CYP2C19 is a major high affinity OP 5-hydroxylase and CYP3A4 is a low affinity OP-hydroxylating enzyme. The current work also shows, for the first time, that other CYP2C enzymes (CYP2C8, CYP2C9, and CYP2C18) may contribute to OP hydroxylation at high substrate concentrations. In contrast, OP-S was formed principally by CYP3A4.

Expression and purification of human cholesterol 7 alpha-hydroxylase in Escherichia coli.

Cholesterol 7 alpha-hydroxylase (P450c7) is the first and rate-limiting enzyme in bile acid biosynthesis and is the product of a cytochrome P450 gene, CYP7. We have previously reported the cloning of a full-length human cholesterol 7 alpha-hydroxylase cDNA (Karam, W. G., and J. Y. L. Chiang. 1992. Biochem. Biophys. Res. Commun. 185: 588-595). Using this clone in a polymerase chain reaction, we have generated a cDNA (H7 alpha 1.5) in which the codons for the N-terminal 24 amino acid residues were deleted. The translational product of this cDNA would be a truncated protein, P450c7(delta 2-24) with a hydrophilic NH2-terminal sequence, Met-Ala-Arg-Arg-Arg-Gln... This cDNA was cloned into the expression vector pJL and the construct pJL/H7 alpha 1.5 was transformed into E. coli strain TOPP3. We have also ligated a truncated rat cholesterol 7 alpha-hydroxylase cDNA obtained previously (Li, Y. C., and J. Y. L. Chiang. 1991. J. Biol. Chem. 266: 19186-19191) into the pJL vector and have transformed this construct (pJL/R7 alpha 1.5) into E. coli strain MV1304. Both of these systems expressed functional cholesterol 7 alpha-hydroxylase in E. coli. A fivefold improvement in the expression of rat enzyme over the previous expression system was obtained. About 70-80% of the truncated human P450 in the clear lysate was localized in the cytosol. The truncated human and rat P450c7(delta 2-24) were purified to homogeneity. Reconstitution of cholesterol 7 alpha-hydroxylase activity using purified rat or human P450c7(delta 2-24) showed a similar Km of 6 and 7 microM for cholesterol, a Vmax of 0.13 and 0.14 nmol/min, and a turnover number of 1.3 and 1.5 per min, respectively. Immunoblotting experiment revealed that a polyclonal antibody raised against rat microsomal cholesterol 7 alpha-hydroxylase recognized both rat and human P450c7(delta 2-24). This expression system provides a method for isolation of a large quantity of purified and catalytically active cholesterol 7 alpha-hydroxylase for the study of structure and function of this important enzyme in bile acid synthesis and cholesterol homeostasis.

Effects of bile acids and steroid/thyroid hormones on the expression of cholesterol 7 alpha-hydroxylase mRNA and the CYP7 gene in HepG2 cells.

The expression of cholesterol 7 alpha-hydroxylase mRNA levels in confluent HepG2 cultures was reduced by tauro- or glyco-conjugates of deoxycholate and chenodeoxycholate, but not by cholate. Ursodeoxycholates, on the other hand, stimulated the mRNA level. The 5'-upstream regions of rat cholesterol 7 alpha-hydroxylase gene (CYP7) were fused to luciferase reporter gene and the constructs, p-3616/Luc, p-224/Luc and p-160/Luc, were transiently transfected into HepG2 cells. Tauro-conjugates of deoxycholate and chenodeoxycholate inhibited the transcriptional activities of the gene constructs in the confluent cells, but not in subconfluent cells. These results reveal that bile acid responsive elements are located in the -160 fragment and also between nt -3616 and -224. Thyroid and steroid hormones stimulated transcriptional activity expressed in the confluent cells and their responsive elements are located upstream of nt -224. It appears that adult phenotypes are responsible for bile acid feedback and hormone response in HepG2 cells.

Polymorphisms of human cholesterol 7 alpha-hydroxylase.

Human liver cholesterol 7 alpha-hydroxylase (CYP7) cDNAs were isolated from a human liver cDNA library. A full-length cDNA has 2901 nucleotides which encode a typical P450 polypeptide of 504 amino acid residues. Two different sequences of codon 100, TTT (Phe) and TCT (Ser), were identified in cDNA clones. In addition, codons 347 and 385 are GAT (Asp) and GAC (Asp) in all cDNA clones, whereas those reported previously (FEBS Lett. 268, 137-140, 1990) are AAT (Asn) and AGC (Ser), respectively. Since there is only one 7 alpha-hydroxylase gene in the human genome, it is likely that polymorphisms at the codon 100 of cDNA clones arise from two different alleles in the 7 alpha-hydroxylase gene of this human liver.