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F J Gonzalez

Publications and source records attributed to F J Gonzalez.

At least 199 records · Page 11Linked to original sources
PubMed1996

Peroxisome proliferator-activated receptor alpha required for gene induction by dehydroepiandrosterone-3 beta-sulfate.

Peroxisome proliferator-activated receptor alpha (PPAR alpha) mediates the effects of foreign chemical peroxisome proliferators on liver and kidney, including the induction of peroxisomal, mitochondrial, and microsomal enzymes involved in beta-oxidation of fatty acids. However, the role of this receptor in the peroxisome proliferative effects of the endogenous steroid dehydroepiandrosterone (DHEA) is not known. DHEA-3 beta-sulfate fd(DHEA-S) is shown to induce a liver peroxisome proliferative response in rats in vivo at a dose at which DHEA is much less active, which is consistent with cultured hepatocyte studies indicating a requirement for sulfation for the activation of DHEA. Transient transfection experiments demonstrated that in contrast to the prototypic foreign chemical peroxisome proliferator pirinixic acid, DHEA-S and its 17 beta-reduced metabolite, 5-androstene-3 beta, 17 beta-diol-3 beta-sulfate, are inactive in mediating trans-activation by PPAR alpha in COS-1 cells. Two other mammalian PPAR isoforms, gamma and delta/Nucl, were also inactive with respect to DHEA-S trans-activation. To test whether PPAR alpha mediates peroxisomal gene induction by DHEA-S in intact animals, we administered DHEA-S or clofibrate to mice lacking a functional PPAR alpha gene. Both peroxisome proliferators markedly increased hepatic expression of two microsomal cytochrome P450 4A proteins as well as six mRNAs known to be associated with the peroxisomal proliferative response in wild-type mice. In contrast, neither DHEA-S nor clofibrate induced these hepatic proteins and mRNAs in PPAR alpha-deficient mice. Clofibrate-induced expression of kidney CYP4A mRNAs was also blocked in the PPAR alpha gene knockout mice. Thus, despite its unresponsiveness to steroidal peroxisome proliferators in transfection assays, PPAR alpha is obligatory for DHEA-S-stimulated hepatic peroxisomal gene induction. DHEA-S, or one of its metabolites, may thus serve as an important endogenous regulator of liver peroxisomal enzyme expression via a PPAR alpha-mediated pathway.

Acetophenones↗
PubMed1996

Piperonyl butoxide and acenaphthylene induce cytochrome P450 1A2 and 1B1 mRNA in aromatic hydrocarbon-responsive receptor knock-out mouse liver.

It has been suggested that acenaphthylene (ACN), piperonyl butoxide (PBO) and other methylenedioxyphenyl (benzodioxole) compounds can function as aromatic hydrocarbon-responsive receptor (AHR)-independent inducers of the cytochrome P450 (CYP) 1A2 in mouse liver. Although much indirect evidence has supported this hypothesis, direct proof was lacking until the present study. PBO and ACN were used to examine the expression of CYP1A1, CYP1A2 and CYP1B1 in mouse liver. These three CYP isozymes are included in the AHR battery of proteins. In this study, AHR knock-out mice were dosed intraperitoneally with PBO (200 mg/kg) or ACN (100 mg/kg). Induction of hepatic CYP1A1 by PBO or ACN was not detected by northern blots. In contrast, both CYP1A2 and CYP1B1 mRNA, constitutively expressed at low levels in this tissue, were induced by each compound in the livers of AHR knock-out mice. In addition, the use of heterogenous nuclear RNA reverse transcription-polymerase chain reaction procedures revealed that the transcriptional activities of CYP1A2 were increased by PBO and ACN treatments. These results show that AHR-independent pathway(s) can be involved in induction of CYP1A2 and CYP1B1.

Acenaphthenes↗
PubMed1996

Baculovirus-mediated expression and characterization of rat CYP2A3 and human CYP2a6: role in metabolic activation of nasal toxicants.

Cytochrome P450 2A3 (CYP2A3) was previously identified in rat lung by cDNA cloning and recently found to be expressed at a high level in the olfactory mucosa. In the current study, CYP2A3 was expressed in insect cells lacking endogenous cytochrome P450 (P450) activity, and the substrate specificity of the recombinant cytochrome was characterized and compared with that of CYP2A6, a human ortholog of rat CYP2A3, which has been detected in human olfactory mucosa as well as in liver. The CYP2A3 and CYP2A6 cDNAs were cloned into baculovirus, and recombinant viruses were used to produce active enzymes in Spodoptera frugiperta (SF9) cells. The metabolic activities of S. frugiperta cell microsomal fractions containing CYP2A3 or CYP2A6 were studied in a reconstituted system with purified rabbit NADPH-P450 reductase. CYP2A3 was found to be active toward testosterone, producing 15 alpha-hydroxytestosterone and several other metabolites, but it had only low activity toward coumarin. On the other hand, CYP2A6 was active toward coumarin but not toward testosterone. However, both enzymes were active in the metabolic activation of hexamethylphosphoramide, a nasal procarcinogen, and 2,6-dichlorobenzonitrile (DCBN), a herbicide known to cause tissue-specific toxicity in the olfactory mucosa of rodents at very low doses. In addition, both enzymes were active toward 4-nitrophenol, a preferred substrate for CYP2E1. Consistent with CYP2A3 being a major catalyst in microsomal metabolism of DCBN, the activities of both CYP2A3 and rat olfactory microsomes in DCBN metabolism were inhibited strongly by metyrapone and methoxsalen (ID50 < 1 microM, with DCBN at 30 microM), but only marginally by 4-methylpyrazole, an inhibitor of CYP2E1. In contrast, the activity of CYP2A6 was only weakly inhibited by metyrapone or methoxsalen (ID50 > 50 microM). Thus, rat CYP2A3 and human CYP2A6 have differences in substrate specificity as well as tissue distributor. These findings should be taken into account when assessing the risk of exposure to potential nasal toxicants in humans.

Animals↗
PubMed1995

Inhibitory and non-inhibitory monoclonal antibodies to human cytochrome P450 3A3/4.

Cytochromes P450 3A3/4 are inordinately important P450 enzymes catalyzing the metabolism of a large variety of clinically useful drugs, steroids, and carcinogens. Two monoclonal antibodies, MAb 3-29-9 and MAb 275-1-2, were prepared to human P450 3A4 from mice immunized with baculovirus-expressed human P450 3A4. MAb 3-29-9 was a powerful inhibitor of the enzymatic activity of P450 3A3/4/5. MAb 3-29-9 inhibited the P450 3A3, 3A4, and 3A5 catalyzed metabolism of substrates of divergent molecular weights, e.g., p-nitroanisole, phenanthrene, diazepam, testosterone, taxol, and cyclosporin. However, MAb 3-29-9 did not give a western blot with P450 3A3 or 3A4. MAb 275-1-2 was non-inhibitory but yielded a strong western blot with P450 3A3 and 3A4 but not with 3A5, and thus distinguished between 3A3/4 and 3A5. The two MAbs did not cross-react with human 2E1, 1A2, 2B6, 2C8, and 2C9; rat 2A1, 3A1/2, 4A1, 4A3, and 2B1; and mouse 1A1 and 1A2. MAb 3-29-9 has been used successfully to measure the quantitative contribution of P450 3A3 and 3A4 to the metabolism of the above-designated substrates in human adult liver. MAb 3-29-9 and MAb 275-1-2 are precise and sensitive reagents for P450 3A studies.

Anisoles↗
PubMed1995

Human liver oxidative metabolism of O6-benzylguanine.

The oxidation of O6-benzylguanine, an inactivator of O6-alkylguanine-DNA alkyltransferase, was examined using human liver cytosol, microsomes, and several P450 isoforms. Incubation of O6-benzylguanine with human liver cytosol resulted in the formation of O6-benzyl-8-oxoguanine, which was inhibited by menadione, a potent inhibitor of aldehyde oxidase. Inhibition by allopurinol, a xanthine oxidase inhibitor, was less dramatic. Oxidation of O6-benzylguanine also occurred with pooled human liver microsomes and was inhibited by both furafylline and troleandomycin, selective inhibitors of CYP1A2 and CYP3A4, respectively. Human P450s CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2E1, and CYP3A4 expressed in Hep G2 hepatoma cells using vaccinia virus vectors were incubated with 10 or 200 microM O6-benzylguanine. At 10 microM, O6-benzylguanine was oxidized primarily by CYP1A2 and to a lesser extent by CYP3A4. However, an appreciable increase in CYP3A4 contribution was noted at 200 microM. CYP1A2 exhibited a more than 200-fold higher relative catalytic activity (Vmax/Km) compared with CYP3A4. Therefore, at therapeutically relevant concentrations of O6-benzylguanine, CYP1A2 could be primarily involved in its oxidation since it shows a much lower Km value (1.3 microM) than CYP3A4 (52.2 microM) and cytosol (81.5 microM). However, one would expect interindividual variation in the extent of oxidation of O6-benzylguanine depending on the levels of aldehyde oxidase, CYP1A2, and CYP3A4.

Carcinoma, Hepatocellular↗
PubMed1995

Cytochrome P450 2A1, 2E1, and 2C9 cDNA-expression by insect cells and partial purification using hydrophobic chromatography.

High-level expression of three cloned cytochrome P450 enzymes was accomplished using the baculovirus-insect cell expression system. The amount of enzyme expression was enhanced by cell infections in the presence of medium-supplements containing hemin and by growth in suspension cultures. Human cytochromes P450 2E1 and 2C9 and rat cytochrome P450 2A1 were partially purified from cell extracts using hydrophobic interaction and hydroxyapatite chromatography. The resulting enzymes were of estimated molecular masses similar to those reported previously and analyzed by PAGE. Reconstitution of enzyme activity resulted when the enzymes were incubated together with NADPH-cytochrome P450 reductase, phospholipid, NADPH, and appropriate substrates. The cytochrome P450 activity of the partially purified enzymes was comparable to that of the corresponding enzymes expressed in the vaccinia virus-Hep G2 system. These results provide evidence for a general means of obtaining cytochrome P450 enzymes for mechanistic, immunochemical, and biophysical investigations.

Animals↗
PubMed1995

Analysis of cis-acting regions upstream of the rat Na+/K(+)-ATPase alpha 1 subunit gene by in vivo footprinting.

By means of in vivo footprinting, we examined the putative cis-acting DNA elements located between -50 and -122 of rat Na+/K(+)-ATPase alpha 1 subunit gene ATP1A1. Proximal and distal GC box sequences and a consensus sequence for the active transcription factor (ATF) were protected for all the tissues examined (kidney, brain and liver). Putative cooperation between two binding factors on the ATF site and the proximal GC box was observed. The overall in vivo footprinting profiles of the three tissues did not exhibit any marked differences that could account for the variation in the extent of tissue-specific transcription. The alpha 1 regulatory element (ARE) found by Suzuki-Yagawa et al. does not appear to be an element responsible for tissue-specific regulation of the gene.

Animals↗
PubMed1995

Correlation between catalytic activity and protein content for the polymorphically expressed dihydropyrimidine dehydrogenase in human lymphocytes.

A TLC procedure was developed to determine dihydropyrimidine dehydrogenase (DPD) activity in human peripheral lymphocytes. The assay, which used radiolabeled uracil as a substrate, was validated using recombinant pig DPD in which it was demonstrated to yield kinetic constants similar to those found by methods that rely on either spectroscopic determination of NADPH oxidation or HPLC. DPD activity was measured in a group of human lymphocyte extracts, including an extract from a subject that actually presented toxicity to 5-fluorouracil treatment. Measurements of DPD protein content using western immunoblots revealed a significant correlation with activity levels in human lymphocytes. Thus, this correlation could be used to determine not only the levels of expression of this enzyme, which is the cause of an inherited genetic deficiency in pyrimidine catabolism, but also to estimate the degree of sensitivity to pyrimidine-based cancer drugs such as 5-fluorouracil.

Animals↗
PubMed1995

CYP4 isozyme specificity and the relationship between omega-hydroxylation and terminal desaturation of valproic acid.

The cytochrome P450-dependent terminal desaturation of valproic acid (VPA) is of both toxicological and mechanistic interest because the product, 4-ene-VPA, is a more potent hepatotoxin than the parent compound and its generation represents a rather novel metabolic reaction for the cytochrome P450 system. In the present study, lung microsomes from rabbits were identified as a rich source of VPA desaturase activity. Monospecific polyclonal antibodies directed against CYP4B1 (anti-4B) inhibited 82% of 4-ene-VPA formation, whereas monospecific polyclonal antibodies directed against CYP2B4 (anti-2B) inhibited only 15% of 4-ene-VPA formation. Anti-4B also inhibited 95% of the 5-hydroxy-VPA formation, but only 42% of 4-hydroxy-VPA formation. These data suggest that CYP4B1 accounts for more than 80% of the 4-ene- and 5-hydroxy-VPA metabolites generated by rabbit lung microsomes. CYP4B1 expressed in HepG2 cells metabolized VPA with a turnover number of 35 min-1 and formed the 5-hydroxy-, 4-hydroxy-, and 4-ene-VPA metabolites in a ratio of 110:2:1, respectively. In contrast, the lauric acid omega-hydroxylases, CYP4A1 and CYP4A3, did not give rise to detectable levels of any of these VPA metabolites. Therefore, these studies demonstrate a new functional role for CYP4B1 in the terminal desaturation and omega-hydroxylation of this short, branched-chain fatty acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Neonatal lethality associated with respiratory distress in mice lacking cytochrome P450 1A2.

Cytochrome P450 1A2 (CYP1A2) is a constitutively expressed hepatic enzyme that is highly conserved among mammals. This protein is primarily involved in oxidative metabolism of xenobiotics and is capable of metabolically activating numerous procarcinogens including aflatoxin B1, arylamines, heterocyclic amine food mutagens, and polycylic aromatic hydrocarbons. Expression of CYP1A2 is induced after exposure to certain aromatic hydrocarbons (i.e., 2,3,7,8-tetrachlorodibenzo-p-dioxin). Direct evidence for a role of CYP1A2 in any physiological or developmental pathway has not been documented. We now demonstrate that mice homozygous for a targeted mutation in the Cyp1a-2 gene are nonviable. Lethality occurs shortly after birth with symptoms of severe respiratory distress. Mutant neonates display impaired respiratory function associated with histological signs of lung immaturity, lack of air in alveoli at birth, and changes in expression of surfactant apoprotein in alveolar type II cells. The penetrance of the phenotype is not complete (19 mutants survived to adulthood out of 599 mice). Surviving animals, although lacking expression of CYP1A2, appear to be normal and are able to reproduce. These findings establish that CYP1A2 is critical for neonatal survival by influencing the physiology of respiration in neonates, thus offering etiological insights for neonatal respiratory distress syndrome.

Animals↗
PubMed1995

Role of cytochrome P450 2C9 and an allelic variant in the 4'-hydroxylation of (R)- and (S)-flurbiprofen.

Flurbiprofen is a chiral non-steroidal anti-inflammatory drug used in the treatment of pain or inflammation. The primary routes of biotransformation for (R)- and (S)-flurbiprofen are oxidation (presumably cytochrome P450) and conjugation. To date, the specific cytochrome P450 (P450) involved in the oxidative metabolism of this compound (specifically 4'-hydroxylation) has not been elucidated. Experiments were conducted to characterize the kinetic parameters (Km and Vmax) for the 4'-hydroxylation of (R)- and (S)-flurbiprofen in human liver microsomes, to determine if enantiomeric interactions occur when both enantiomers are present, and to identify the specific P450 form(s) involved in this reaction. In human liver microsomes, the Km and Vmax (mean +/- SD) for (R)-4'-hydroxy-flurbiprofen formation were 3.1 +/- 0.8 microM and 305 +/- 168 pmol.min-1.mg protein)-1, respectively. In comparison, the Km and Vmax (mean +/- SD) for (S)-4'-hydroxy-flurbiprofen formation were 1.9 +/- 0.4 microM and 343 +/- 196 pmol.min-1.mg protein-1, respectively. Enantiomeric interaction studies revealed a decrease in Km and Vmax for both enantiomers and an apparent loss of stereoselectivity. Racemic-warfarin, tolbutamide, alpha-naphthoflavone and erythromycin were studied as potential inhibitors of this process. The estimated Ki values for the inhibition of (R)- and (S)-4'-hydroxy-flurbiprofen formation by racemic-warfarin were 2.2 and 4.7 microM. This reaction was also inhibited by tolbutamide. In contrast, erythromycin and alpha-naphthoflavone had no appreciable effect on 4'-hydroxy-flurbiprofen formation. cDNA-expression of individual forms was used to determine which P450 was involved in 4'-hydroxy-flurbiprofen formation. P450 2C9 and an allelic variant (R144C) readily catalyzed the formation of 4'-hydroxy-flurbiprofen. P450 1A2 was also active albeit with a turnover rate 1/140th that of P450 2C9R144C (P450s 2C8, 2E1 and 3A4 were not active toward either enantiomer). The results of these studies indicate that the enantiomers of flurbiprofen may exhibit stereoselectivity with respect to enzyme affinity but have roughly equal maximum formation velocities. Additionally, these two enantiomers may compete for the enzyme resulting in lower maximum velocities for both enantiomers. Finally, of those P450 forms examined, only P450 2C9 and an allelic variant catalyzed the 4'-hydroxylation of both (R)- and (S)-flurbiprofen.

Aryl Hydrocarbon Hydroxylases↗
PubMed1995

Immune system impairment and hepatic fibrosis in mice lacking the dioxin-binding Ah receptor.

The aryl hydrocarbon (Ah) receptor (AHR) mediates many carcinogenic and teratogenic effects of environmentally toxic chemicals such as dioxin. An AHR-deficient (Ahr-/-) mouse line was constructed by homologous recombination in embryonic stem cells. Almost half of the mice died shortly after birth, whereas survivors reached maturity and were fertile. The Ahr-/- mice showed decreased accumulation of lymphocytes in the spleen and lymph nodes, but not in the thymus. The livers of Ahr-/- mice were reduced in size by 50 percent and showed bile duct fibrosis Ahr-/- mice were also nonresponsive with regard to dioxin-mediated induction of genes encoding enzymes that catalyze the metabolism of foreign compounds. Thus, the AHR plays an important role in the development of the liver and the immune system.

Animals↗
PubMed1995

Metabolism of polycyclic aza-aromatic carcinogens catalyzed by four expressed human cytochromes P450.

The role of human cytochromes P4501A1, -1A2, -3A4, and -3A5 in the metabolism of the polycyclic aza-aromatic hydrocarbons 7-methylbenz(c)acridine and dibenz(aj)acridine was investigated. The regioselectivity of the reactions was determined, as well as the associated stereoselectivity in the production of dihydrodiol metabolites and K-region oxides. Metabolite distributions were also examined in the presence of the epoxide hydrolase inhibitor 1,1,1-trichloropropylene-2,3-oxide and the P450 modulator alpha-naphthoflavone. P4501A2 was most regioselective for the production of the proximate carcinogen; the 3,4-dihydrodiol of 7-methylbenz(c)acridine and P4503A4 showed the highest regioselectivity for K-region oxidation. In contrast, the analogous putative proximate carcinogen of dibenz(aj)acridine was formed with the highest relative abundance by P4503A4, while P4501A2 was most regioselective for K-region oxidation. For both compounds the proximate carcinogens possessed predominantly the 3R,4R-absolute configuration, independent of the P450 catalyzing the reaction. The K-region dihydrodiols of 7-methylbenz(c)acridine were formed with no stereoselectivity, except with P4501A2 which favored production of the S,S isomer. In contrast the K-region dihydrodiol of dibenz(aj)acridine was formed by P4501A1 and P4501A2 as the R,R isomer with almost 100% optical purity. P4501A2 and 3A4 showed no stereoselectivity in the formation of the K-region oxide of 7-methylbenz(c)acridine, while P4501A1 produced the 5R,6S-oxide with low optical purity. For dibenz(aj)acridine 5,6-oxide, P4501A1 predominantly formed 5S,6R-oxide (80% pure). These results emphasize the importance of the composition and levels of expressed P450s of an individual in relation to the activation and detoxification of toxicants.

Acridines↗
PubMed1995

Organization and evolution of the cytochrome P450 CYP2A-2B-2F subfamily gene cluster on human chromosome 19.

Cytochrome P450 genes from the CYP2A, CYP2B, and CYP2F subfamilies form a tight cluster which we have localized on the detailed physical map of human chromosome 19. The corresponding three gene subfamilies are also clustered in the mouse genome, on the region of chromosome 7 known to be syntenic to human chromosome 19. One hundred eight cosmid clones from the human P450 region were assembled into a single contig of 350 kb, restriction mapped, and probed with cDNAs from the three gene subfamilies. A total of 11 genes were identified in humans, including five from the 2A subfamily, three from the 2B subfamily, and three from the 2F subfamily; at least six of the 11 are pseudogenes. The organization of the genes, with members of the three subfamilies intermixed, indicates that the evolution of this gene cluster has been complex. The modern gene arrangement in humans is probably the result of a series of tandem duplications, plus at least one inverted duplication. The identification of all genes and pseudogenes in this cluster also makes it possible to determine the origins of some previously known variant P450 transcripts.

Animals↗
PubMed1995

Xenobiotic receptor knockout mice.

Administration of certain foreign chemicals to animals elicits responses that are due to receptor-mediated activation of gene expression. Among the most well studied receptors are the Ah receptor (AHR) that binds 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds and the peroxisome proliferator-activated receptors, PPARs, that mediate gene activation by the diverse group of peroxisome proliferators. These receptors may also have critical roles in development or physiological homeostasis in addition to their abilities to allow animals to interact with exogenous chemicals or xenobiotics. To explore the function of AHR and PPAR alpha and to determine whether they participate in the adverse effects of dioxins and peroxisome proliferators, gene knockout mice were developed.

Animals↗