Synergistic enhancement by 12-O-tetradecanoylphorbol-13-acetate and dibutyryl cAMP of 1alpha,25-dihydroxyvitamin D3 action in human promyelocytic leukemic HL-60 cells.
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We have reported that dibutyryl cAMP (dbcAMP), an activator of cAMP-dependent protein kinase (PKA), potentiated the effects of 1alpha,25-dihydroxyvitamin D3(1,25-(OH)2D3)-induced 24-hydroxylation activity in HL-60 cells by increasing 1,25-(OH)2D3 receptor (VDR). The present study demonstrated that 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent phorbol ester, also potentiated the effect of 1,25-(OH)2D3 on HL-60 cells and that TPA and dbcAMP acted in a synergistic manner to enhance the effect of 1,25-(OH)2D3. It is interesting that TPA induced 24-hydroxylation activity far more efficiently than dbcAMP, in addition to their effects in increasing VDR. TPA increased basal levels of c-fos mRNA to the maximum by 1 h after the treatment, whereas dbcAMP failed to affect c-fos gene expression. Together with the previous data indicating the presence of AP-1-like sequence in the promoter of 24-hydroxylase gene, it was suggested that TPA potentiated the effect of 1,25-(OH)2D3 through an activation of c-fos gene expression. This notion was further supported by the data showing that TPA and dbcAMP also acted in a synergistic manner to activate c-fos gene expression. Neither TPA nor dbcAMP affected c-jun early response gene in the HL-60 clone used in the present study. The present study suggested that the activation of early c-fos response gene by TPA might be another mechanism to enhance the effect of 1,25-(OH)2D3, besides up-regulation of VDR. (+info)
Enhancement of cytochrome P-450 3A4 catalytic activities by cytochrome b(5) in bacterial membranes.
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Activities of testosterone, nifedipine, and midazolam oxidation by recombinant cytochrome P-450 (P-450) 3A4 coexpressed with human NADPH-P-450 reductase (NPR) in bacterial membranes (CYP3A4/NPR membranes) were determined in comparison with those of other recombinant systems and of human liver microsomes with high contents of CYP3A4. Growth conditions for Escherichia coli transformed with the bicistronic construct affected expression levels of CYP3A4 and NPR; an excess of NPR over P-450 in membrane preparations enhanced CYP3A4-dependent testosterone 6beta-hydroxylation activities of the CYP3A4/NPR membranes. Cytochrome b(5) (b(5)) and apolipoprotein b(5) further enhanced the testosterone 6beta-hydroxylation activities of CYP3A4/NPR membranes after addition to either bacterial membranes or purified enzymes. NPR was observed to enhance catalytic activity when added to the CYP3A4/NPR membranes, either in the form of bacterial membranes or as purified NPR (in combination with cholate and b(5)). Apparent maximal activities of testosterone 6beta-hydroxylation in CYP3A4/NPR membranes were obtained when the molar ratio of CYP3A4/NPR/b(5) was adjusted to 1:2:1 by mixing membranes containing each protein. Testosterone 6beta-hydroxylation, nifedipine oxidation, and midazolam 4- and 1'-hydroxylation activities in CYP3A4/NPR membranes plus b(5) systems were similar to those measured with microsomes of insect cells coexpressing CYP3A4 with NPR and/or of human liver microsomes, based on equivalent CYP3A4 contents. These results suggest that CYP3A4/NPR membrane systems containing b(5) are very useful models for prediction of the rates for liver microsomal CYP3A4-dependent drug oxidations. (+info)
Studies on sulfation of synthesized metabolites from the local anesthetics ropivacaine and lidocaine using human cloned sulfotransferases.
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The metabolism of the local anesthetics lidocaine and ropivacaine (ropi) involves several steps in humans. Lidocaine is mainly hydrolyzed and hydroxylated to 4-OH-2,6-xylidine (4-OH-xyl). The metabolism of ropi, involving dealkylation and hydroxylation, gives rise to 3-OH-ropi, 4-OH-ropi, 3-OH-2'6'-pipecoloxylidide (3-OH-PPX), and 2-OH-methyl-ropi. Because the metabolites are hydroxylated, they are particularly prone to subsequent Phase II conjugation reactions such as sulfation and glucuronidation. This study focused on the in vitro sulfation of these metabolites as well as another suspected metabolite of ropi, 2-carboxyl-ropi. All the metabolites were synthesized for the subsequent enzymatic studies. Five cloned human sulfotransferases (STs) were used in this study, namely, the phenol-sulfating form of ST (P-PST-1), the monoamine-sulfating form of ST (M-PST), estrogen-ST (EST), ST1B2, and dehydroepiandrosterone-ST (DHEA-ST), all of which are expressed in human liver. The results demonstrate that all of the metabolites except 2-OH-methyl-ropi and 2-carboxyl-ropi can be sulfated. It was also found that all of the STs can conjugate the remaining hydroxylated metabolites except DHEA-ST. However, there are large differences in the capacity of the individual human ST isoforms to conjugate the different metabolites. P-PST-1 sulfates 3-OH-PPX, 3-OH-ropi, and 4-OH-xyl; M-PST and EST conjugate 3-OH-PPX, 3-OH-ropi, and 4-OH-ropi whereas ST1B2 sulfates only 4-OH-xyl. The most extensively sulfated ropi metabolite is 3-OH-PPX. In conclusion, all of the hydroxylated metabolites of lidocaine and ropi can be sulfated if the hydroxyl group is attached to the aromatic ring in the metabolites. The human ST enzymes that are considered to be responsible for the sulfation of these metabolites in vivo are P-PST-1, M-PST, EST, and ST1B2. These enzymes are also found in the liver; this is the most important tissue for the metabolism of ropi in humans, demonstrated by. (+info)
Human and Escherichia coli beta-glucuronidase hydrolysis of glucuronide conjugates of benzidine and 4-aminobiphenyl, and their hydroxy metabolites.
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Individuals exposed to carcinogenic aromatic amines excrete arylamine N- and O-glucuronide metabolites. This study assessed the susceptibility of selected glucuronides to hydrolysis by human and Escherichia coli beta-glucuronidase. N- or O-glucuronides were prepared with the following aglycones: benzidine, N-acetylbenzidine, N'-hydroxy-N-acetylbenzidine, N-hydroxy-N-acetylbenzidine, N-hydroxy-N,N'-diacetylbenzidine, 3-hydroxy-N,N'-diacetylbenzidine, 3-hydroxy-benzidine, 4-aminobiphenyl, N-hydroxy-4-aminobiphenyl, and N-hydroxy-N-acetyl-4-aminobiphenyl. The (3)H- and (14)C-labeled glucuronides were prepared with human or rat liver microsomes using UDP-glucuronic acid as cosubstrate. Each of the 10 glucuronides (6-12 microM) was incubated at pH 5.5 or 7.0 with either human recombinant (pure) or E. coli (commercial preparation) beta-glucuronidase for 30 min at 37 degrees C. Hydrolysis was measured by HPLC. Reaction conditions were optimized, using the O-glucuronide of N-hydroxy-N,N'-diacetylbenzidine. Both enzymes preferentially hydrolyzed O-glucuronides over N-glucuronides and distinguished between structural isomers. With E. coli beta-glucuronidase at pH 7.0, selectivity was demonstrated by the complete hydrolysis of N-hydroxy-N-acetyl-4-aminobiphenyl O-glucuronide in the presence of N-acetylbenzidine N-glucuronide, which was not hydrolyzed. Metabolism by both enzymes was completely inhibited by the specific beta-glucuronidase inhibitor saccharic acid-1,4-lactone (0.5 mM). The concentration of human beta-glucuronidase necessary to achieve significant hydrolysis of glucuronides was substantially more than the amount of enzyme reported previously to be present in urine under either normal or pathological conditions. The bacterial enzyme may hydrolyze O-glucuronides, but not N-glucuronides, in urine at neutral pH. Thus, the nonenzymatic hydrolysis of N-glucuronides by acidic urine is likely a more important source of free amine than enzymatic hydrolysis. (+info)
New routes for lignin biosynthesis defined by biochemical characterization of recombinant ferulate 5-hydroxylase, a multifunctional cytochrome P450-dependent monooxygenase.
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The enzymes and genes of the lignin biosynthetic pathway have been studied for several decades, but the gene encoding ferulate 5-hydroxylase (F5H) was cloned only 3 years ago by T-DNA tagging in Arabidopsis. To characterize the enzyme in detail, we have expressed F5H in yeast. According to current models of the phenylpropanoid pathway, F5H catalyzes the hydroxylation of ferulate to 5-hydroxyferulate; however, our studies indicate that the enzyme also uses coniferaldehyde and coniferyl alcohol as substrates. Unexpectedly, the K(m) values measured for the latter two substrates are three orders of magnitude lower than that measured for ferulic acid, suggesting that in lignifying tissues, syringyl monomers may be derived from their guaiacyl counterparts by hydroxylation and subsequent methylation. Thus, F5H may function later in the lignin biosynthetic pathway than was originally proposed. To further test this model, recombinant F5H was incubated together with ferulic acid, coniferaldehyde, or coniferyl alcohol in the presence of native or recombinant Arabidopsis caffeic acid/5-hydroxyferulic acid O-methyltransferase and [(14)C]S-adenosylmethionine. In all cases, the corresponding radiolabeled sinapyl derivatives were synthesized, indicating that the necessary enzymes required for this pathway are present in Arabidopsis. Taken together, these data suggest that the previously accepted pathway for lignin biosynthesis is likely to be incorrect. (+info)
Benzylic hydroxylation of 1-methylpyrene and 1-ethylpyrene by human and rat cytochromes P450 individually expressed in V79 Chinese hamster cells.
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Alkyl-substituted polycyclic aromatic hydrocarbons may be metabolized to highly reactive benzylic sulfuric acid esters via benzylic hydroxylation and subsequent sulfonation. We have studied the benzylic hydroxylation of 1-methylpyrene (MP), a hepatocarcinogen in rodents, and 1-ethylpyrene (EP), whose benzylic hydroxylation would produce a secondary alcohol (alpha-HEP), in contrast to the primary alcohol (alpha-HMP) formed from MP. The hydrocarbons were incubated with hepatic microsomal preparations from humans and rats, as well as with V79-derived cell lines engineered for the expression of individual cytochrome P450 (CYP) forms from human (1A1, 1A2, 1B1, 2A6, 2E1, 3A4) and rat (1A1, 1A2, 2B1). All microsomal systems and CYP-expressing cell lines used, but not CYP-deficient V79 cells, showed biotransformation of both hydrocarbons. Formation of the benzylic alcohol was detected in each case. alpha-HMP and its oxidation product, 1-pyrenylcarboxylic acid (COOH-P), accounted for a major part of the total amount of the metabolites formed from MP in the presence of human liver microsomes (38-64%) and cells expressing human 3A4, 2E1 or 1B1 (80-85%). Likewise, cells expressing human 1A1 showed a higher contribution of alpha-HMP and COOH-P to the total metabolites (45%) than cells expressing the orthologous enzyme of the rat (3%). EP was metabolized at a higher rate and with modified regioselectivity compared with MP, although omega-hydroxylation of the side chain was not detected with the cell lines and only accounted for a small percent of the biotransformation by the microsomal preparations. The highest contributions of alpha-HEP to the total metabolites from EP were detected with the cells expressing human 1A1, 1B1 and 3A4 (38-51%). alpha-HEP accounted for 16% of the metabolites formed in the presence of human hepatic microsomes. Thus, benzylic hydroxylation is a major initial step in the metabolism of MP and EP. This pathway appears to be even more important in humans than in rats. Previously, we had shown that the second step of the activation, the sulfonation of alpha-HMP and alpha-HEP, is also efficiently catalysed by various forms of human sulfotransferases. (+info)
Biotransformation of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in freshly isolated human lung cells.
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Metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was characterized in human lung cells isolated from peripheral lung specimens obtained from 12 subjects during clinically indicated lobectomy. NNK biotransformation was assessed in preparations of isolated unseparated cells (cell digest), as well as in preparations enriched in alveolar type II cells, and alveolar macrophages. Metabolite formation was expressed as a percentage of the total recovered radioactivity from [5-(3)H]NNK and its metabolites per 10(6) cells per 24 h. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) was the major metabolite formed in all lung cell preparations examined, and its formation ranged from 0.50 to 13%/10(6) cells/24 h. Formation of alpha-carbon hydroxylation end-point metabolites (bioactivation) and pyridine N-oxidation metabolites (detoxification), ranged from non-detectable to 0.60% and from non-detectable to 1.5%/10(6) cells/24 h, respectively, reflecting a large degree of intercellular and inter-individual variability in NNK metabolism. Formation of the alpha-hydroxylation end-point metabolite 4-hydroxy-1-(3-pyridyl)-1-butanol (diol) was consistently higher in alveolar type II cells than in cell digest or alveolar macrophages (0.0146 +/- 0.0152, 0.0027 +/- 0.0037 and 0.0047 +/- 0.0063%/10(6) cells/24 h, respectively; n = 12; P < 0.05). SKF-525A was used to examine cytochrome P450 contributions to the biotransformation of NNK. SKF-525A inhibited keto reduction of NNK to NNAL by 85, 86 and 74% in cell digest, type II cells, and macrophages, respectively (means of 11 subjects, P < 0.05). Type II cell incubates treated with SKF-525A formed significantly lower amounts of total alpha-hydroxylation metabolites compared with type II cells without SKF-525A (0.0776 +/- 0.0841 versus 0.1694 +/- 0. 2148%/10(6) cells/24 h, respectively; n = 11; P < 0.05). The results of this first study examining NNK biotransformation in freshly isolated human lung cells indicate that NNK metabolism is subject to a large degree of inter-individual and intercellular variability, and suggest a role for P450s in human lung cell NNK metabolism. Both alveolar type II cells and alveolar macrophages may be potential target cells for NNK toxicity based on their alpha-carbon hydroxylation capabilities. In addition, carbonyl reduction of NNK to NNAL is SKF-525A sensitive in human lung cells. (+info)
CYP1A2 is not the primary enzyme responsible for 4-aminobiphenyl-induced hepatocarcinogenesis in mice.
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4-Aminobiphenyl (4-ABP), a potent carcinogen in rodents (liver cancer) and human (bladder cancer), is found as an environmental contaminant and in tobacco smoke. Hemoglobin adducts and lung DNA adducts of 4-ABP are found in tobacco smokers. In vitro metabolism studies with human and rat liver microsomes have shown that CYP1A2 is primarily responsible for catalyzing N-hydroxylation, the initial step in the metabolic activation of 4-ABP. To determine whether this P450 is a rate limiting pathway for hepatocarcinogenesis, CYP1A2-null mice were analyzed at 16 months of age and were compared with wild-type mice in their response to 4-ABP using the neonatal mouse bioassay and two different doses of the carcinogen. Overall differences in incidences of hepatocellular adenoma, carcinoma and preneoplastic foci were not significant between either genotypes or 4-ABP doses used, whereas small, but significant, differences were found for specific types of foci. These results suggest that while CYP1A2 levels may not be rate limiting for 4-ABP metabolism to produce tumors and foci, it may modulate the induction process of some types of liver foci in either a positive or negative manner. In vitro studies using CYP1A2-null and wild-type mouse liver microsomes revealed that CYP1A2 is not the sole P450 required for 4-ABP N-hydroxylation and that another, yet to be identified, P450 is likely to be involved. (+info)