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F J Gonzalez

Publications and source records attributed to F J Gonzalez.

At least 217 records · Page 12Linked to original sources
PubMed1995

Human polymorphism in drug metabolism: mutation in the dihydropyrimidine dehydrogenase gene results in exon skipping and thymine uracilurea.

A condition called thymine uracilurea has been described that is due to a lack of dihydropyrimidine dehydrogenase (DPD) activity. Cancer patients experiencing acute 5-fluorouracil toxicity also have lower-than-normal DPD activities. However, to date, the molecular basis of this disorder has not been addressed. In this study, the phenotype and genotype of a family that presents a patient showing no DPD activity was determined. Fibroblast mRNAs from the patient and four family members were subjected to reverse transcriptase polymerase chain reaction (RT-PCR) using primers generated from the human DPD cDNA sequence. DPD mRNA from the patient was found to lack a segment of 165 nucleotides that results from exon skipping. DPD mRNA from the parents and a sibling were found to be heterozygous for the deleted and the normal mRNA, while a brother had two normal transcripts. DPD activities and levels of DPD protein correlated with genotype; the deficient patient had no detectable DPD protein. PCR analysis of the genomic DNA from this family revealed that the defective mRNA is not due to a deletion of a portion of the gene that contains the exon, thus implying that the mutation is the result of an as yet nonidentified point mutation that causes faulty splicing.

Base Sequence↗
PubMed1995

Role of the liver-enriched transcription factor HNF-1 alpha in expression of the CYP2E1 gene.

The role of the trans-acting factor HNF-1 alpha in activating CYP2E1 gene expression was confirmed by transient co-transfection of an HNF-1 alpha expression plasmid and the CYP2E1 promoter fused to the chloramphenicol acetyl transferase (CAT) reporter gene. Only HNF-1 alpha, and not HNF-1 beta, HNF-4, C/EBP alpha, C/EBP beta, or DBP, was able to activate the CYP2E1 promoter. The extent of activation was proportional to the number of copies of the HNF-1 binding sequence upstream of the promoter. Removal or mutation of the HNF-1 binding sequence led to inactivation of the promoter in response to HNF-1 alpha. Gel-shift Western blot analysis using a synthetic HNF-1 binding sequence derived from CYP2E1 and rat liver nuclear extract revealed that the protein-DNA complex obtained with adult rat liver nuclear extract consisted of both HNF-1 alpha and HNF-1 beta proteins. The shifted bands produced by nuclear extracts from adult, where the endogenous CYP2E1 gene is active, and fetal rat liver, where the gene is inactive, were found to migrate differently, suggesting that the population of factors, possibly including different ratios of HNF-1 alpha and HNF-1 beta proteins, may change during development. However, the co-transfection study did not show cooperativity between the two factors. Elements upstream of the HNF-1 binding site were found to affect the activity of the promoter negatively in the transfection assay. DNase I hypersensitive site mapping revealed a hypersensitive site in this inhibiting element in the adult rat liver sample but not in liver from newborn animals.

Aging↗
PubMed1995

Oxidative metabolism of bunitrolol by complementary DNA-expressed human cytochrome P450 isozymes in a human hepatoma cell line (Hep G2) using recombinant vaccinia virus.

We examined the oxidative metabolism of a beta-blocker bunitrolol (BTL) by 10 human cytochromes P450 (CYP) (1A2, 2A6, 2B6, 2C8, 2C9, 2D6, 2E1, 3A3, 3A4 or 3A5), which were individually expressed in Hep G2 cells with a vaccinia virus complementary DNA expression system. Among the 10 isozymes, only CYP2D6 and 1A2 at a substrate concentration of 5 microns, and CYP2C8 and 2C9 in addition to the two isozymes at a BTL concentration of 1 mM, exhibited detectable BTL 4-hydroxylase activities. The activities at 1 mM BTL were on the order of CYP2D6 (100% as relative activity) > CYP1A2 (86%) >> CYP2C8 and 2C9 (7-8%). Enzyme kinetic parameters of CYP2D6 were calculated to be 4.41 microns as a Km value and 0.442 nmol min-1 per nmol CYP as a Vmax value. Kinetic parameters of CYP1A2 were calculated as 295 microns and 0.411 nmol min-1 per nmol CYP for Km and Vm values, respectively. These results suggest that both CYP2D6 and 1A2 primarily catalyse BTL 4-hydroxylation, but that the former is a predominant isozyme responsible for the reaction at a low substrate concentration range of BTL in human liver.

Adrenergic beta-Antagonists↗
PubMed1995

Chlorzoxazone is metabolized by human CYP1A2 as well as by human CYP2E1.

Chlorzoxazone, a muscle-relaxing drug, is metabolized by carbon-hydroxylation at position 6. Chlorzoxazone has been suggested as an in vivo probe for CYP2E1. We studied the specificity of such a substrate using vaccinia virus expressed human P450 forms and the effect of inhibitors for chlorzoxazone metabolism by human liver microsomes. The 6-hydroxylation of chlorzoxazone was mediated by CYP1A2 as well as by CYP2E1. The Km value of CYP1A2 and CYP2E1 for the reaction was 5.69 microM and 232 microM, respectively. However, the Vmax value of CYP2E1 for the reaction was approximately 8.5-fold higher than that of CYP1A2. The CYP1A inhibitor, alpha-naphthoflavone, as well as the CYP2E1 inhibitor, diethyldithiocarbamate, decreased chlorzoxazone 6-hydroxylation at a low substrate concentration by human liver microsomes. Our results raise questions about the suitability of chlorzoxazone as an in vivo probe for hepatic CYP2E1 activity. In human liver microsomal samples, the Km = 40 microM was different from either the Km of CYP1A2 or CYP2E1. We think that this discrepancy is due to the co-expression of similar levels of CYP1A2 and CYP2E1 in human liver. Furthermore, it is suggested that the role of CYP2E1 in 6-hydroxychlorzoxazone formation at the physiological chlorzoxazone concentration of 30-60 microM is almost the same when compared to that of CYP1A2.

Benzoflavones↗
PubMed1995

Comparison of substrate metabolism by wild type CYP2D6 protein and a variant containing methionine, not valine, at position 374.

We have analysed kinetic parameters of cDNA-derived CYP2D6 proteins derived from the original CYP2D6 cDNA isolate (Gonzalez FJ et al. Nature 1988: 331, 442-446) which contains methionine at position 374 (CYP2D6-Met) and a modified cDNA which contains valine at position 374 (CYP2D6-Val). This latter protein is predicted from the CYP2D6 genomic sequence. Several quantitative differences, but no qualitative differences in metabolism were observed. CYP2D6-Met was found to have a two-fold lower Km and a three-fold lower turnover rate for (R)(+)-bufuralol 1'-hydroxylation as compared to CYP2D6-Val. In contrast, CYP2D6-Met and CYP2D6-Val had a similar Km for debrisoquine 4-hydroxylation while CYP2D6-Val had an 18-fold higher turnover rate. CYP2D6-Val and CYP2D6-Met had similar Kms for metoprolol but CYP2D6-Val showed a three-fold higher capacity for the O-demethylation reaction compared to alpha-hydroxylation which is more similar to that seen in human liver. In the case of sparteine, CYP2D6-Val and CYP2D6-Met showed similar capacities for formation of the 2-dehydrosparteine metabolite but the Km value for CYP2D6-Met was six-fold higher than that for CYP2D6-Val. Kinetic differences between CYP2D6-Met and CYP2D6-Val were further probed by examination of apparent Ki for inhibition of (R,S)(+/-)-bufuralol 1'-hydroxylation. Similar Ki values (within a factor of three) were observed for perhexiline and (R,S)-propranolol while quinidine and dextromethorphan were 8.5-fold and 21-fold more effective inhibitors of CYP2D6-Val relative to CYP2D6-Met. An allele specific polymerase chain reaction assay was developed for the CYP2D6-Met allele. The CYP2D6-Met allele was not found among 83 individuals. In the aggregate, these data indicated that the CYP2D6-Val allele is the more common allele in human populations. The quantitative kinetic differences between these two enzymes appears most pronounced for substrates/inhibitors with rigid structures. CYP2D6-Val more often has a substantially lower Km and/or a substantially higher capacity to metabolize those substrates.

B-Lymphocytes↗
PubMed1995

The CYP2A gene subfamily: species differences, regulation, catalytic activities and role in chemical carcinogenesis.

The CYP2A subfamily has been characterized in several mammalian species including mouse, rat, rabbit, hamster, cattle and human. Marked species differences have been demonstrated in the catalytic activities and regulation of this subfamily. In humans, the CYP2A genes are found as a cluster on the long arm of chromosome 19 with the CYP2B and CYP2F genes. Marked interindividual differences in expression of the CYP2A6 gene was found in livers analyzed in vitro and in humans phenotyped in vivo by using coumarin, a specific substrate for the enzyme. Efforts are underway to determine the existence of mutant and variant CYP2A6 alleles in the human population. Since CYP2A6 is able to metabolically-activate chemical carcinogens and is expressed in extrahepatic tissue, it will be of interest to determine whether genetic differences in expression of the gene is associated with cancer risk.

Animals↗
PubMed1995

Targeted disruption of the alpha isoform of the peroxisome proliferator-activated receptor gene in mice results in abolishment of the pleiotropic effects of peroxisome proliferators.

To gain insight into the function of peroxisome proliferator-activated receptor (PPAR) isoforms in rodents, we disrupted the ligand-binding domain of the alpha isoform of mouse PPAR (mPPAR alpha) by homologous recombination. Mice homozygous for the mutation lack expression of mPPAR alpha protein and yet are viable and fertile and exhibit no detectable gross phenotypic defects. Remarkably, these animals do not display the peroxisome proliferator pleiotropic response when challenged with the classical peroxisome proliferators, clofibrate and Wy-14,643. Following exposure to these chemicals, hepatomegaly, peroxisome proliferation, and transcriptional-activation of target genes were not observed. These results clearly demonstrate that mPPAR alpha is the major isoform required for mediating the pleiotropic response resulting from the actions of peroxisome proliferators. mPPAR alpha-deficient animals should prove useful to further investigate the role of this receptor in hepatocarcinogenesis, fatty acid metabolism, and cell cycle regulation.

Animals↗
PubMed1995

Characterisation of CYP3A gene subfamily expression in human gastrointestinal tissues.

The human CYP3A subfamily is of interest due to its multiplicity, activity toward known carcinogens, and extrahepatic expression. In situ hybridisation analysis of formalin fixed, routinely processed biopsy specimens was used to localise CYP3A mRNA in human gastrointestinal tissues from several individuals. CYP3A mRNA is abundant in human liver and in mucosal epithelial cells of all segments of the human small intestine. RNA blot analyses showed that the mRNA species observed in most livers and in human small intestine represent CYP3A3/3A4 transcripts. This was confirmed at the protein level by immunoblot comparison of small intestine microsomes to in vitro expressed CYP3A4 and CYP3A5 proteins. In liver and small intestine, CYP3A mRNA is not uniformly distributed, with grain density highest in cells within the respective non-proliferative compartments. CYP3A mRNA was also observed in human oesophagus and colon. RNA blot analysis of multiple colons showed heterogeneity in the CYP3A mRNAs present. Two CYP3A mRNAs (CYP3A3/3A4 and CYP3A5) were detected in colon samples from several individuals. In addition to those localisation studies, the capacity of expressed CYP3A4 and CYP3A5 to activate the dietary heterocyclic amine MeIQ in the presence of alpha-naphthoflavone was shown. These results show that there is considerable heterogeneity in the expression of the CYP3A subfamily in human gastrointestinal tissues.

Adult↗
PubMed1995

Cytochromes P450 expression systems.

Catalytically active cytochrome P450 enzymes have been successfully expressed in bacterial, yeast, and mammalian cells. A variety of expression vectors have been used, resulting in both transient and stable expression. The system of choice depends on the goals of a particular project. Factors such as expense, ease of use, and yields required should govern the decision whether to use bacterial, yeast, insect, or mammalian cDNA expression. High-level expression of mammalian P450s in bacteria usually requires modifications of the amino-terminal region of the enzyme. The Escherichia coli P450-OR fusion proteins may also come of age for use in fermentation-production processes for the chemical industry. Many cytochromes P450 have been expressed in yeast, with variable levels of expression. Baculovirus, albeit somewhat tedious in having to individualize expression conditions, can produce high levels of enzyme. The standard mammalian cell expression systems, both transient and stable, have been of tremendous value to drug metabolism and carcinogenesis research and will continue to play a role in these areas.

Animals↗
PubMed1995

cDNA-directed expression of human cytochrome P450 CYP1A1 using baculovirus. Purification, dependency on NADPH-P450 oxidoreductase, and reconstitution of catalytic properties without purification.

A recombinant baculovirus containing the human cytochrome P450 (CYP) 1A1 cDNA was constructed and used to express CYP1A1 in Spodoptera frugiperda (SF9) insect cells (0.14 +/- 0.04 nmol/mg protein, 53 +/- 14 nmol/liter, N = 30). The enzyme represented approximately 1% of total cellular protein and was partially purified by a three-column procedure to a specific content of 5.0 nmol/mg protein. Catalytic activity was reconstituted with both the purified enzyme using lipid and NADPH-P450 oxidoreductase, and the SF9 insect cell membrane fraction without purification using NADPH-P450 oxidoreductase and small amounts of detergent. Catalytic activity of the enzyme after reconstitution was optimum using molar ratios of CYP1A1 to NADPH-P450 oxidoreductase of 1:8. Cytochrome b5 had no additional stimulating effect. The enzyme metabolized substrates characteristic for CYP1A1:benzo[a]pyrene (4.0 +/- 0.3 nmol/min/nmol CYP), 7-ethoxy-4-trifluoromethyl- coumarin (36 +/- 2), ethoxyresorufin (37 +/- 1), but not pentoxyresorufin (0.77 +/- 0.02). Recombinant baculovirus expresses the highest amounts of all expression systems published to date of catalytically active CYP1A1. Because human CYP1A1 has never been isolated in a catalytically active state from human tissue, nor has recombinant unmodified human CYP1A1, this system is an excellent alternative for the isolation and characterization of this CYP.

Animals↗
PubMed1995

Modulation of cardiac intermediate filament proteins in the chick heart by fibric acid derivatives.

The effects of different fibrates (bezafibrate, fenofibrate and gemfibrozil) on intermediate filaments were studied in cultured chick embryo heart cells after treatment for 6 or 24 h. Treatment led to alterations in total protein levels, as well as changes in protein levels, in the cytoplasmic and cytoskeletal fractions of cultured cells. Desmin was increased in the cytoskeletal fraction of all cultures after 6 h of treatment regardless of the drug tested, whereas vimentin was decreased in the cytoskeletal fraction only in cells treated with fenofibrate. These findings suggest that the alterations caused by fibrates in desmin and vimentin protein content may be related with the secondary effects that these drugs have on the cardiovascular system in patients treated with fibrates.

Animals↗
PubMed1994

Protection against SR 4233 (Tirapazamine) aerobic cytotoxicity by the metal chelators desferrioxamine and tiron.

PURPOSE: Metal chelating agents and antioxidants were evaluated as potential protectors against aerobic SR 4233 cytotoxicity in Chinese hamster V79 cells. The differential protection of aerobic and hypoxic cells by two metal chelators, desferrioxamine and Tiron, is discussed in the context of their potential use in the on-going clinical trials with SR 4233. METHODS AND MATERIALS: Cytotoxicity was evaluated using clonogenic assay. SR 4233 exposure was done in glass flasks as a function of time either alone or in the presence of the following agents: superoxide dismutase, catalase, 5,5-dimethyl-1-pyrroline, Trolox, ICRF-187, desferrioxamine, Tiron (1,2-dihydroxybenzene-3,5-disulfonate), and ascorbic acid. Experiments done under hypoxic conditions were carried out in specially designed glass flasks that were gassed with humidified nitrogen/carbon dioxide mixture and with a side-arm reservoir from which SR 4233 was added to cell media after hypoxia was obtained. Electron paramagnetic resonance studies were also performed. RESULTS: Electron paramagnetic resonance and spectrophotometry experiments suggest that under aerobic conditions SR 4233 undergoes futile redox cycling to produce superoxide. Treatment of cells during aerobic exposure to SR 4233 with the enzymes superoxide dismutase and catalase, the spin trapping agent DMPO, the water-soluble vitamin E analog Trolox, and the metal chelator ICRF-187 provided little or no protection against aerobic SR 4233 cytotoxicity. However, two other metal chelators, desferrioxamine and Tiron, afforded significant protection against aerobic SR 4233 cytotoxicity (protection factors at 50% survival were 3.8 and 3.1, respectively), while exhibiting minimal protection to hypoxic cells treated with SR 4233. CONCLUSIONS: One potential mechanism of aerobic cytotoxicity is redox cycling of SR 4233 with molecular oxygen resulting in several potentially toxic oxidative species that overburden the intrinsic intracellular detoxification systems such as superoxide dismutase, catalase, and glutathione peroxidase. This study identifies two metal chelating agents, desferrioxamine and Tiron, that were able to protect against aerobic but not hypoxic SR 4233 cytotoxicity.

1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium ↗
PubMed1994

Selective biotransformation of taxol to 6 alpha-hydroxytaxol by human cytochrome P450 2C8.

The principal taxol biotransformation reaction of humans and of human hepatic in vitro preparations is 6 alpha-hydroxylation of the taxane ring, but a separate, minor hydroxylation pathway (metabolite B formation) also exists. Taxol metabolism was studied using membrane fractions from Hep G2 cells infected with recombinant vaccinia viruses that contain complementary DNAs encoding several human cytochrome P450 enzymes. Only P450 2C8 formed detectable 6 alpha-hydroxytaxol. Metabolite B formation was catalyzed by complementary DNA-expressed 3A3 and 3A4, but not by 3A5. Each P450 3A preparation catalyzed felodipine oxidation. The apparent Km and Vmax values for taxol 6 alpha-hydroxylation were 5.4 +/- 1.0 microM and 30 +/- 1.5 nmol/min/nmol P450, respectively, for complementary DNA-expressed P450 2C8; the values were 4.0 +/- 1.0 microM and 0.87 +/- 0.06 nmol/min/mg protein, respectively, for human hepatic microsomes. The inhibition of 6 alpha-hydroxytaxol formation by quercetin was competitive with an apparent Ki of 1.3 or 1.1 microM with 2C8 or hepatic microsomes, respectively; retinoic acid was inhibitory, showing an apparent Ki of 27 microM with hepatic microsomes; inhibition by tolbutamide was complex, weak, and unlikely to be clinically relevant. The correlation between hepatic 2C8 protein content and 6 alpha-hydroxytaxol formation was high (r2 = 0.82), while the correlation with 2C9 content was low (r2 = 0.38). These data show that human biotransformation routes of taxol result from catalysis by specific enzymes of two P450 families and that taxol 6 alpha-hydroxylation is a useful indicator of P450 2C8 activity in human hepatic microsomes.

Biotransformation↗
PubMed1994

Evidence for the transient expression of P-450 during the neonatal period in rat.

A cytochrome P-450 N (N for Neonatal) was resolved and partially purified from 15-day-old newborn rat livers. Antibody raised in rabbit against P-450 N detects two bands in liver microsomes, the major one comigrating with the partially purified P-450 N. This protein is not expressed in lung and kidney and exhibits an unusual ontogenic profile in the liver with maximal levels in 2-week-old animals. Expression of P-450 N is not modified in animals given beta-naphthoflavone or pregnenolone 16 alpha-carbonitrile, but is markedly increased by phenobarbital. Its amino-acid composition differs from that of other P-450s and it is not recognized by antibodies directed against CYP1A, CYP2A1, CYP2B, CYP2C11, CYP3A or epoxide hydrolase. It could thus be considered as an unique P-450 expressed transiently before puberty.

Amino Acid Sequence↗
PubMed1994

cDNA cloning and chromosome mapping of human dihydropyrimidine dehydrogenase, an enzyme associated with 5-fluorouracil toxicity and congenital thymine uraciluria.

The pig and human dihydropyrimidine dehydrogenase (DPD) cDNAs were cloned and sequenced. The pig enzyme, expressed in Escherichia coli, catalyzed the reduction of uracil, thymine, and 5-fluorouracil with kinetics approximating those published for the enzyme purified from mammalian liver. DPD could be expressed in significant quantities only when uracil was added to the bacterial growth medium. The pig and human enzymes contained 1025 amino acids and calculated M(r) = 111,416 and 111,398, respectively. Conserved domains corresponding to a possible NADPH binding site and FAD binding site were found in the NH2-terminal half of the proteins and two motifs of putative [4Fe-4S] binding sites were found near to the carboxyl terminus of the enzyme. The latter corresponds to the labile COOH-terminal fragment previously shown to contain the iron sulfur centers. A sequence encompassing a peptide corresponding to the uracil binding site was found between the NADPH/FAD-containing NH2-terminal portion of the protein and the iron-sulfur binding sites near to the COOH terminus. Thus, the DPD appears to be derived from at least three distinct domains. The DPYD gene was localized to the centromeric region of human chromosome 1 between 1p22 and q21.

Amino Acid Sequence↗
PubMed1994

CYP2C11 and CYP2B1 are major cytochrome P450 forms involved in styrene oxidation in liver and lung microsomes from untreated rats, respectively.

The contribution of cytochrome P450s (P450s) to the formation of styrene glycol from styrene in rat liver microsomes was investigated using monoclonal antibodies to P450s. Anti-CYP2E1 inhibited the formation to a similar extent in ethanol-treated microsomes and in control microsomes in terms of percentage inhibition, whereas to a greater extent in the former than the latter in terms of net inhibition, and only at low substrate concentration. Anti-CYP2C11/6 also inhibited the formation in control and in ethanol-treated microsomes at both low and high concentrations of styrene, and the net degree of inhibition was greater than that obtained with anti-CYP2E1, even in ethanol-treated microsomes where CYP2E1 was induced. Anti-CYP2B1/2 and anti-CYP1A1/2 inhibited the formation only in phenobarbital (PB)- and 3-methylcholanthrene (MC)-induced microsomes, respectively. These results suggest that (1) at least four P450s, CYP2C11/6, CYP2E1, CYP2B1/2 and CYP1A1/2, contribute to the metabolism of styrene, (2) CYP2C11/6, which probably corresponds to CYP2C11, is the major form of P450 responsible for the metabolism in untreated rat liver microsomes, and also in those treated with ethanol. Anti-CYP2E1 inhibited styrene oxidation more prominently in microsomes from styrene-treated rats than in those from control rats at a low substrate concentration. Although styrene treatment did not influence the total metabolism of styrene in liver microsomes at a high substrate concentration, inhibition of the metabolism by anti-CYP2C11/6 decreased with increasing styrene dose, whereas that by anti-CYP2B1/2 increased, suggesting that styrene treatment increases CYP2B1/2 but decreases CYP2C11/6 in rat liver, and the major form of P450 which mediates styrene oxidation is CYP2B1/2 after the treatment. Only anti-CYP2B1/2, which probably corresponds to CYP2B1, inhibited styrene oxidation in lung microsomes from untreated and even styrene-treated rats. Thus, the major form of P450 responsible for the metabolism of styrene is different in each tissue.

Animals↗