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F J Gonzalez

Publications and source records attributed to F J Gonzalez.

At least 181 records · Page 10Linked to original sources
PubMed1996

Role of CYP2E1 in the hepatotoxicity of acetaminophen.

CYP2El, a cytochrome P-450 that is well conserved across mammalian species, metabolizes ethanol and many low molecular weight toxins and cancer suspect agents. The cyp2e1 gene was isolated, and a mouse line that lacks expression of CYP2E1 was generated by homologous recombination in embryonic stem cells. Animals deficient in expression of the enzyme were fertile, developed normally, and exhibited no obvious phenotypic abnormalities, thus indicating that CYP2E1 has no critical role in mammalian development and physiology in the absence of external stimuli. When cyp2el knockout mice were challenged with the common analgesic acetaminophen, they were found to be considerably less sensitive to its hepatotoxic effects than wild-type animals, indicating that this P-450 is the principal enzyme responsible for the metabolic conversion of the drug to its active hepatotoxic metabolite.

Acetaminophen↗
PubMed1996

Metabolic activation of the potent carcinogen dibenzo[a,h]anthracene by cDNA-expressed human cytochromes P450.

The metabolic activation of the potent carcinogen dibenzo[a,h]anthracene (DB[a,h]A) was investigated with recombinant human cytochrome P450 enzymes 1A2, 2B6, 2C8, 2C9, 2E1, 3A3, 3A4, and 3A5 expressed in hepatoma G2 cells and with 14 different human liver microsomes. Three dihydrodiols, three phenols, and one diphenol were formed and separated by high-performance liquid chromatography and identified by UV absorption and mass spectra. Of all P450s tested, 1A2 and 2C9 were the most active and 2B6 was moderately active in the rate of total DB[a,h]A metabolism (2.5- to 12-fold greater activity than that for other P450s). The trans-3,4-dihydrodiol, generally recognized as a precursor of the ultimate carcinogenic 3,4-diol-1,2-epoxides, was produced most actively by 2C9, then 1A2 and 2B6. The values of enzymatic kinetics (K(m) and V(max)) indicated that 2C9 had the highest catalytic efficiency (V(max)/K(m) = 9.7) in the formation of 3,4-dihydrodiol, in contrast to 1A2 (5.9) and 2B6 (4.4). 1A2 had the highest activity toward production of the 1,2-dihydrodiol, which is considered to be a weakly carcinogenic metabolite. Although specific activities of human liver microsomes in overall metabolism of DB[a,h]A markedly differed between individuals, metabolic patterns were observed similar to that generated from 1A2. Since human 1A1, a predominant enzyme for metabolism of polycyclic aromatic hydrocarbons, is not significantly expressed in the liver, hepatic microsomal 2C9, 1A2, and 2B6 all probably contribute to the metabolic activation of DB[a,h]A.

Benz(a)Anthracenes↗
PubMed1996

The T/ebp null mouse: thyroid-specific enhancer-binding protein is essential for the organogenesis of the thyroid, lung, ventral forebrain, and pituitary.

The thyroid-specific enhancer-binding protein (T/ebp) gene was disrupted by homologous recombination in embryonic stem cells to generate mice lacking T/EBP expression. Heterozygous animals developed normally, whereas mice homozygous for the disrupted gene were born dead and lacked the lung parenchyma. Instead, they had a rudimentary bronchial tree associated with an abnormal epithelium in their pleural cavities. Furthermore, the homozygous mice had no thyroid gland but had a normal parathyroid. In addition, extensive defects were found in the brain of the homozygous mice, especially in the ventral region of the forebrain. The entire pituitary, including the anterior, intermediate, and posterior pituitary, was also missing. In situ hybridization showed that the T/ebp gene is expressed in the normal thyroid, lung bronchial epithelium, and specific areas of the forebrain during early embryogenesis. These results establish that the expression of T/EBP, a transcription factor known to control thyroid-specific gene transcription, is also essential for organogenesis of the thyroid, lung, ventral forebrain, and pituitary.

Adrenal Cortex↗
PubMed1996

Specificity of cDNA-expressed human and rodent cytochrome P450s in the oxidative metabolism of the potent carcinogen 7,12-dimethylbenz[a]anthracene.

7,12-Dimethylbenz[a]anthracene (DMBA), a potent carcinogen, requires metabolic activation by cytochrome P450s (P450s) to electrophilic metabolites that result in DNA modification, mutagenicity, and carcinogenicity. In this study, we used eight human forms, four rodent forms, and one rabbit form of P450 expressed from recombinant vaccinia or baculovirus vectors to define their specificity for metabolizing DMBA. Of the eight human P450s, 1A1 was the most active (specific activity = 14.7 nmol/min/nmol of P450) in total metabolism of DMBA and showed approximately 6- to 33-fold more activity than other P450s, 2B6, 2C9, and 1A2 were also capable of metabolizing DMBA (2.0-2.5 nmol/min/nmol of P450), whereas 2C8, 2E1, 3A4, and 3A5 exhibited relatively low activities. Among animal P450s, mouse 1A1 exhibited activity similar to that of human 1A1 and had 5.0- to 37-fold more activity than other rodent and rabbit P450s. In regard to enzyme regioselectivity, most human and rodent P450s predominantly formed the 8,9-diol, but human 2B6 and rat 2B1 preferentially formed the 5,6-diol. In the production of monohydroxymethyl metabolites, all the enzymes yielded more 7-hydroxymethyl-12-methylbenz[a]anthracene (7HOM12MBA) than 12-hydroxymethyl-7-methylbenz[a]anthracene (7M12HOMBA), except for human 1A1, which presented the reverse selectivity. Human liver microsomes from 10 organ donors were shown to metabolize DMBA and in most circumstances generated the metabolic profile DMBA trans-8,9-dihydrodiol > 7HOM12MBA > or = DMBA trans-5,6-dihydrodiol > or = 7,12-dihydroxymethylbenz[a]anthracene > 7M12HOMBA > DMBA trans-3,4-dihydrodiol. Thus, the combined activity of hepatic microsomal 2C9, 1A2, and 2B6 may contribute to the metabolic activation and the metabolism of DMBA in normal human liver.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1996

Aryl-hydrocarbon receptor-deficient mice are resistant to 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced toxicity.

Acute exposure of mammals to the environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) results in a diverse set of toxicologic and pathologic effects. The mechanism of some of these effects has been studied extensively in vitro and correlative studies have indicated the involvement of a transcription factor known as the aryl hydrocarbon receptor (AHR). However, a definitive association of the AHR with TCDD-mediated toxicity has been difficult to establish due to the diversity of effects and the ubiquitous expression of this receptor. In an effort to distinguish AHR-mediated TCDD toxicities from those resulting from alternative pathways, we have made use of the recently described AHR-deficient mouse that was generated by locus-specific homologous recombination in embryonic stem cells. Present studies demonstrate that AHR-deficient mice are relatively unaffected by doses of TCDD (2000 micrograms/kg) 10-fold higher than that found to induce severe toxic and pathologic effects in littermates expressing a functional AHR. Analyses of liver, thymus, heart, kidney, pancreas, spleen, lymph nodes, and uterus from AHR-deficient mice identified no significant TCDD-induced lesions. The resistance of AHR-deficient mice to TCDD-induced thymic atrophy appears restricted to processes involving AHR since the corticosteroid dexamethasone rapidly and efficiently induced cortical depletion in both AHR-deficient and normal littermate control mice. Taken together these results suggest that the pathological changes induced by TCDD in the liver and thymus are mediated entirely by the AHR. However, it is important to note that at high doses of TCDD, AHR-deficient mice displayed limited vasculitis and scattered single cell necrosis in their lungs and livers, respectively. The mechanism(s) responsible for these apparently receptor-independent processes remain unclear but may involve novel, alternative pathways for TCDD-induced toxicity.

Animals↗
PubMed1996

Reduction of benzene metabolism and toxicity in mice that lack CYP2E1 expression.

Transgenic CYP2E1 knockout mice (cyp2e1-/-) were used to investigate the involvement of CYP2E1 in the in vivo metabolism of benzene and in the development of benzene-induced toxicity. After benzene exposure, absence of CYP2E1 protein was confirmed by Western blot analysis of mouse liver samples. For the metabolism studies, male cyp2e1-/- and wild-type control mice were exposed to 200 ppm benzene, along with a radiolabeled tracer dose of [14C]benzene (1.0 Ci/mol) by nose-only inhalation for 6 hr. Total urinary radioactivity and all radiolabeled individual metabolites were reduced in urine of cyp2e1-/- mice compared to wild-type controls during the 48-hr period after benzene exposure. In addition, a significantly greater percentage of total urinary radioactivity could be accounted for as phenylsulfate conjugates in cyp2e1-/- mice compared to wild-type mice, indicating the importance of CYP2E1 in oxidation of phenol following benzene exposure in normal mice. For the toxicity studies, male cyp2e1-/-, wild-type, and B6C3F1 mice were exposed by whole-body inhalation to 0 ppm (control) or 200 ppm benzene, 6 hr/day for 5 days. On Day 5, blood, bone marrow, thymus, and spleen were removed for evaluation of micronuclei frequencies and tissue cellularities. No benzene-induced cytotoxicity or genotoxicity was observed in cyp2e1-/- mice. In contrast, benzene exposure resulted in severe genotoxicity and cytotoxicity in both wild-type and B6C3F1 mice. These studies conclusively demonstrate that CYP2E1 is the major determinant of in vivo benzene metabolism and benzene-induced myelotoxicity in mice.

Animals↗
PubMed1996

Inhibitory and noninhibitory monoclonal antibodies to human cytochrome P450 2E1.

A panel of 17 hybridomas producing (MAbs) against human cytochrome P450 2E1 (h2E1) was generated by immunizing mice with baculovirus-expressed h2E1. All 17 hybridoma clones gave positive ELISA or immunoblots with either baculovirus-or vaccinia virus-expressed h2E1. Two of the latter were further developed due to their desirable characteristics. MAb 1-73-18 was found to be a powerful inhibitor of P450 h2E1; however, it did not yield a positive immunoblot. MAb 2-106-12 was found to be noninhibitory but formed a strong positive immunoblot with P450 h2E1. These MAbs to h2E1 were highly specific and did not recognize six other human P450s as tested with ELISA or immunoblot analyses. The MAbs to baculovirus-expressed h2E1 also reacted with h2E1 expressed from a vaccinia virus vector system as well as with microsomal fractions of human and acetone-treated rat liver. MAb 1-73-18 inhibited h2E1 enzyme activity catalyzing the metabolism of phenanthrene by 85%, p-nitroanisole by 90%, 4-methylanisole by 60-80%, toluene by 90%, and chlorzoxazone by 90%. The inhibitory MAb 1-73-18 is uniquely useful for determining the contribution of h2E1 to the metabolism of h2E1 substrates in human liver containing multiple P450s. The quantitatively determined contribution of h2E1 to the metabolism of the above substrates ranged from 25% to 75%. Thus, h2E1 was responsible for the following percentages of the total metabolism in human liver: p-nitroanisole (35%), phenanthrene (23%), methylanisole to cresol (25%), methylanisole to methoxybenzyl alcohol (12%), toluene (40%), and chlorzoxazone (72%). The MAb 2-106-12 forming a strong immunoblot is useful for determining the amount of h2E1 protein in a tissue. Thus the utility of the inhibitory and immunoblot positive MAbs is complementary and can determine both the contribution of h2E1 to the metabolism of specific substrates and the amount of h2E1 protein in human tissue. The analyses of metabolism with the inhibitory MAb 1-73-18 can be generalized and applicable to all h2E1 substrates.

Animals↗
PubMed1996

Metabolic activation of the potent carcinogen dibenzo[a,l]pyrene by human recombinant cytochromes P450, lung and liver microsomes.

The metabolic activation of dibenzo[a,l]pyrene (DB[a,l]P), recently considered the most potent carcinogen among all polycyclic aromatic hydrocarbons, to the 11,12-dihydrodiol, a precursor of the ultimate carcinogens, the 11,12-diol-13,14-epoxides, was investigated using eleven human recombinant cytochrome P450s, as well as human lung and liver microsomes. Of all human P450s, 1A1 was the most active in the metabolism of DB[a,l]P (310 pmol/min, nmol P450) and had 5-23-fold higher catalytic activity than other P450s examined. The order of activity in the formation of the 11,12-dihydrodiol was as follows: 1A1 (116 pmol/min, nmol P450) > 2C9 (29) > 1A2 (22) > 2B6 (18) > 3A4 (16) > others (< or = 5). The Km of 1A1 for DB[a,l]P and Vmax for the formation of 11,12-dihydrodiol were 3.9 microM and 0.13/min, respectively. Liver microsomes from 14 individuals were shown to metabolize DB[a,l]P and the rates for production of 11,12-dihydrodiol ranged from 4 to 71 pmol/min, nmol P450. Lung microsomes from six organ donors formed the 11,12-dihydrodiol at a rate from 0.1 to 1.3 pmol/min, mg of microsomal protein. These findings describe the potential of individual P450s present in liver and lung to contribute to the metabolic activation and the carcinogenicity of DB[a,l]P.

Adolescent↗
PubMed1996

Constitutive expression of hepatic cytochrome P450 genes.

Cytochromes P450 are a superfamily of heme proteins involved in oxidative metabolism of endogenous chemicals such as steroid hormones and human-made xenobiotics including drugs and environmental pollutants. Hundreds of P450s have been demonstrated by cDNA and gene cloning in animals, plants, fungi, and bacteria. Most of the mammalian xenobiotic-metabolizing P450s, found within the eight subfamilies comprising the CYP2 family, are constitutively expressed in the liver. Transcriptional activation of individual P450 genes in the liver commences at distinct stages of development. Some P450 genes are preferentially expressed in one sex. The mechanisms of liver-specific expression of the P450 genes are quite diverse. Recent studies have found that several different liver-enriched transcription factors including HNF-1 alpha, HNF-3, HNF-4, and C/EBP beta, and the more ubiquitously expressed factors Sp1, GABP alpha/beta, and NF2d9 are responsible for governing the transcription of P450 genes. In some cases, more than one factor can influence expression depending on the developmental stage of the animal, and ubiquitously expressed factors such as Sp1 have been found to cooperate with liver-enriched factors to maximally activate transcription of P450 genes. STAT protein- and phospholipase A2-mediated signal transduction have also been implicated in sex-dependent expression of certain P450 genes. These studies indicate that P450 genes, even within the same subfamily in the same mammalian species, can have unique regulatory circuits.-Gonzalez, F. J., Lee, Y.-H. Constitutive expression of hepatic cytochrome P450 genes.

Animals↗
PubMed1996

Nomenclature for human CYP2D6 alleles.

To standardize CYP2D6 allele nomenclature, and to conform with international human gene nomenclature guidelines, an alternative to the current arbitrary system is described. Based on recommendations for human genome nomenclature, we propose that alleles be designated by CYP2D6 followed by an asterisk and a combination of roman letters and arabic numerals distinct for each allele with the number specifying the key mutation and, where appropriate, a letter specifying additional mutations. Criteria for classification as a separate allele and protein nomenclature are also presented.

Alleles↗
PubMed1996

Role of CYP1A2 in caffeine pharmacokinetics and metabolism: studies using mice deficient in CYP1A2.

We investigated the involvement of CYP1A2 in the pharmacokinetics and metabolism of caffeine using mice lacking its expression (CYP1A2 -/-). The half-life of caffeine elimination from blood was seven times longer in the CYP1A2 -/- than wild-type mice. The clearance was concomitantly eight times slower. No parameter that could affect the pharmacokinetics differed between CYP1A2-/-and wild-type mice such as creatinine for kidney function; alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and bilirubin for liver function; or albumin for protein binding. Other P450s CYP2A, 2B, 2C, 2EI, and 3A were also unchanged in the knockout animals. Caffeine 3-demethylated metabolites thought previously to be characteristic of CYP1A2 (especially 1-methylxanthine and I-methylurate) were also found in the urines of the CYP1A2-/-animals, although at 40% of the level found in wild-type mice. These data indicate that the clearance of caffeine in wild-type mice is primarily determined by CYP1A2.

Animals↗
PubMed1996

A new CYP2D6 allele with a nine base insertion in exon 9 in a Japanese population associated with poor metabolizer phenotype.

The CYP2D6 gene of a Japanese sparteine poor metabolizer (PM, proband) showing a urinary sparteine metabolic ratio of 31.6 was analysed, and a heterozygous CYP2D6(D), a deletional type, was found by restriction fragment length polymorphism analysis with Xba I enzyme. The PM did not have any other previously described mutations in the CYP2D6 gene causing the loss of catalytic activity of the CYP2D6 enzyme. Thus, a possible new allele(s) responsible for the PM phenotype was analysed. The results indicated that the PM possessed a new 9-base insertion in exon 9, designated CYP2D6(J9). The CYP2D6(J9) and CYP2D6(D) alleles were clarified to be inherited from the mother [2D6(W)/2D6(J9)] and the father [2D6(W)/2D6(D)], respectively. The 9-base insertion caused a large increase in the apparent K(m) value for bufuralol 1'-hydroxylation as examined by expression of the enzyme protein in yeast. Four of 300 Japanese carried a heterozygous CYP2D6(J9) allele (0.7%, 4/600 chromosomes) as determined by a polymerase chain reaction analysis.

Alleles↗
PubMed1996

Specificity of substrate and inhibitor probes for cytochrome P450s: evaluation of in vitro metabolism using cDNA-expressed human P450s and human liver microsomes.

1. We evaluated the specificity of 15 substrates and 14 inhibitors of the cytochrome P450s using nine human P450 forms expressed in HepG2 cells using a recombinant vaccinia virus and also in human liver microsomes. 2. Coumarin, 7-ethoxyresorufin, 7-benzyloxyresorufin, tolbutamide, aniline and diazepam were form-selective substrates towards CYP2A6, the CYP1A subfamily, CYP2B6, the CYP2C subfamily, CYP2E1 and the CYP3A subfamily respectively. However, a selective substrate for CYP2D6 was not found among the chemicals tested. 3. SKF-525A inhibited > 40% of the metabolic activity of all substrates tested, and the inhibitory effects differed among P450 forms. Sulphaphenazole, 7,8-benzoflavone, quinidine and troleandomycin were selective inhibitors of the CYP2C subfamily (except CYP2C19), the CYP1A subfamily, CYP2D6 and the CYP3A subfamily respectively. Methoxsalen (CYP2A6 inhibitor) inhibited the metabolic activity of CYP1A2 as well as that of CYP2A6. Diethyldithiocarbamate (CYP2E1 inhibitor) inhibited the metabolic activities of CYP2A6 and CYP2C19 in addition to that of CYP2E1. 4. Our results indicated that substrates and inhibitors reported as P450 selective probes are not necessarily specific for individual human P450 forms. These results may provide useful information regarding human P450 substrates and inhibitors in vitro using human liver microsomal samples.

Blotting, Western↗
PubMed1996

Human liver microsomal diazepam metabolism using cDNA-expressed cytochrome P450s: role of CYP2B6, 2C19 and the 3A subfamily.

1. We have examined the metabolism of diazepam by ten human cytochrome P450 forms (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4 and 3A5) expressed in HepG2 cells using a recombinant vaccinia virus system. 2. Among the P450 forms tested, diazepam was significantly demethylated by CYP2B6, 2C9, 2C19, 3A4 and 3A5, with 2C19 exhibiting the highest rate at concentrations < 0.1 mM, and hydroxylated only by the latter three enzymes, with 3A5 being the most active. The N-demethylation activity of diazepam by 2C19 at a concentration of 20 microM was six times of that by 3A4. However, that by 2C9 was detected at only a trace level. 3. CYP2C19, 3A4 and 3A5 of the ten human P450s catalysed the 3-hydroxylation of nordiazepam, and 2B6, the 2C subfamily and the 3A subfamily catalysed the N-demethylation of temazepam. CYP3A4 exhibited the highest activity of nordiazepam 3-hydroxylation and temazepam N-demethylation. 4. Diazepam N-demethylation by human liver microsomes correlated with diazepam 3-hydroxylation, but not S-mephenytoin 4'-hydroxylation. 5. Our results suggest that in the human liver, the metabolism of diazepam to nordiazepam is mediated by CYP3A4, which has been reported as the most abundant P450 form in human liver as well as 2C19, which has been reported as a polymorphic enzyme.

Anti-Anxiety Agents↗
PubMed1996

Metabolism of 3-methylindole by vaccinia-expressed P450 enzymes: correlation of 3-methyleneindolenine formation and protein-binding.

The toxicity of 3-methylindole (3 MI), a selective pneumotoxin, is dependent upon cytochrome P450-mediated bioactivation 3. Using vaccinia-expressed P450 enzymes, the metabolites of radiolabeled 3 MI produced by 14 individual P450s were identified and quantified by high performance liquid chromatography. Indole-3-carbinol was produced from incubations of 3 MI with only four enzymes. Although 3-methyloxindole was a product of several P450s, human 1A2 was most efficient in producing this metabolite. The toxic intermediate of 3 MI is believed to be a reactive methylene imine, 3-methyleneindolenine. In this study, this intermediate was detected as its mercapturate adduct, when N-acetylcysteine was added to the incubations. 3-Methyleneindolenine was produced by CYP2A6 at a rate of 50.9 +/- 8.9 pmol/mg protein/hr and by CYP2F1 at a rate of 205.7 +/- 12.5 pmol/mg/hr. The mouse 1a-2 and rabbit 4B1 enzymes produced the reactive intermediate in amounts that exceeded that of the human 2F1 enzyme by 1.4-fold and 1.9-fold, respectively. The toxicity of 3 MI is believed to be due to covalent binding of a P450-generated intermediate to critical pulmonary proteins. Comparison of covalent binding studies to the formation of the metabolites revealed a strong correlation between the amount of the 3 MI adduct detected and covalent binding. This study showed that the methylene imine electrophile is produced by only a few P450 enzymes and is the metabolite responsible for the covalent binding and presumably, the toxicity of 3 MI. Remarkable product preferences between the desaturation pathway to form the methyleneindolenine by CYP2F1 and the ring epoxidation pathway to form the oxindole by CYP1A2, were observed.

Animals↗
PubMed1996

Transcriptional induction of the cytochrome P4501A1 gene by a thiazolium compound, YH439.

The molecular mechanism of induction of cytochromes P4501A1/2 (CYP1A1/2) by a synthetic compound YH439 was studied in rodents as well as in cultured hepatoma cells. CYP1A1-mediated ethoxyresorufin-O-deethylase activity and amounts of its immunoreactive protein were increased in a time- and concentration-dependent manner after a single dose of YH439 (150 mg/kg). Northern blot analyses revealed that YH439 rapidly increased (< or = 2 hr) the levels of CYP1A1/2 mRNAs, resulting in an increase in CYP1A protein level by > 6-fold at 8 hr after injection. After YH439 administration, the levels of CYP1A1 and CYP1A2 mRNAs peaked at 8 hr and 16 hr, respectively, before returning to control levels at 16 and 24 hr. The CYP1A protein level, on the other hand, reached a maximum at 24 hr after YH439 treatment and returned to near-control levels at 72 hr. Nuclear run-on analyses revealed that YH439 induces CYP1A1/2 gene transcription as early as 2 hr after YH439 treatment. Cytosolic electrophoretic mobility shift assays suggested that YH439 activates the CYP1A1/2 genes through the aryl hydrocarbon (Ah) receptor and the xenobiotic response elements. The dependency on the Ah receptor for the induction of CYP1A1/2 by YH439 was confirmed by the lack of CYP1A1/2 induction in the Ah receptor knock-out mice (Ahr-1-) as well as in murine hepatoma cells without a functional Ah receptor. Molecular structural analysis of YH439 and several other compounds indicated that the planarity and size of a molecule are important in its interaction with the Ah receptor and subsequent CYP1A1/2 induction. YH439 is a thiazolium compound with little aromaticity and with a two-dimensional structure different from that of the Ahs. Therefore, it represents a new class of Ah receptor ligand and CYP1A inducer.

Animals↗