Biomedical subjects

F J Gonzalez

Publications and source records attributed to F J Gonzalez.

At least 19 recordsLinked to original sources

PubMed1998

A gender-related defect in lipid metabolism and glucose homeostasis in peroxisome proliferator- activated receptor alpha- deficient mice.

The peroxisome proliferator-activated receptor alpha (PPARalpha) is a nuclear receptor implicated in the control of cellular lipid utilization. To test the hypothesis that PPARalpha is activated as a component of the cellular lipid homeostatic response, the expression of PPARalpha target genes was characterized in response to a perturbation in cellular lipid oxidative flux caused by pharmacologic inhibition of mitochondrial fatty acid import. Inhibition of fatty acid oxidative flux caused a feedback induction of PPARalpha target genes encoding fatty acid oxidation enzymes in liver and heart. In mice lacking PPARalpha (PPARalpha-/-), inhibition of cellular fatty acid flux caused massive hepatic and cardiac lipid accumulation, hypoglycemia, and death in 100% of male, but only 25% of female PPARalpha-/- mice. The metabolic phenotype of male PPARalpha-/- mice was rescued by a 2-wk pretreatment with beta-estradiol. These results demonstrate a pivotal role for PPARalpha in lipid and glucose homeostasis in vivo and implicate estrogen signaling pathways in the regulation of cardiac and hepatic lipid metabolism.

Animals↗

PubMed1998

Characterization of the human dihydropyrimidine dehydrogenase gene.

Dihydropyrimidine dehydrogenase (DPD) catabolizes endogenous pyrimidines and pyrimidine-based antimetabolite drugs. A deficiency in human DPD is associated with congenital thymine-uraciluria in pediatric patients and severe 5-fluorouracil toxicity in cancer patients. The dihydropyrimidine dehydrogenase gene (DPYD) was isolated, and its physical map and exon-intron organization were determined by analysis of P1, PAC, BAC, and YAC clones. The DPYD gene was found to contain 23 exons ranging in size from 69 bp (exon 15) to 961 bp (exon 23). A physical map derived from a YAC clone indicated that DPYD is at least 950 kb in length with 3 kb of coding sequence and an average intron size of about 43 kb. The previously reported 5' donor splice site mutation present in pediatric thymine-uraciluria and cancer patients can now be assigned to exon 14. All 23 exons were sequenced from a series of human DNA samples, and three point mutations were identified in three racial groups as G1601A (exon 13, Ser534Asn), A1627G (exon 13, Ile543Val), and G2194A (exon 18, Val732Ile). These studies, which have established that the DPYD gene is unusually large, lay a framework for uncovering new mutations that are responsible for thymine-uraciluria and toxicity to fluoropyrimidine drugs.

Alleles↗

PubMed1998

Localization of CYP1A1 mRNA in human lung by in situ hybridization: comparison with immunohistochemical findings.

Cytochrome P4501A1 (CYP1A1) is involved in the bioactivation of polycyclic aromatic hydrocarbons into their reactive epoxide metabolites. CYP1A1 is considered to be important with regard to individual susceptibility to lung cancer since phenotypic and genotypic polymorphisms of CYP1A1 have been associated with an increased risk of lung cancer in a number of studies. We examined here the expression and localization of CYP1A1 mRNA in human lung tissue using in situ hybridization with a CYP1A1-specific RNA probe. A centrilobular expression of CYP1A1 mRNA was observed in the peripheral lung. The expression was intense in bronchiolar epithelium of peripheral lung, especially in terminal cuboidal epithelium. Type II alveolar epithelial cells were also intensely labelled. Type I alveolar epithelial cells and vascular epithelium exhibited binding but the hybridization signals were less intense. Our results are in good agreement with our previous work on immunohistochemical localization of CYP1A protein, in which we used the 1-7-1 MAb that recognizes both CYP1A1 and CYP1A2. In serial sections analyzed with in situ hybridization and immunohistochemistry, a similar distribution of CYP1A1 mRNA and CYP1A protein was observed. CYP1A1 mRNA is thus expressed in human lungs and the expression is particularly intense in the cell types involved in the development of peripheral lung cancers.

Adult↗

PubMed1998

Expression of putative fatty acid transporter genes are regulated by peroxisome proliferator-activated receptor alpha and gamma activators in a tissue- and inducer-specific manner.

Regulation of gene expression of three putative long-chain fatty acid transport proteins, fatty acid translocase (FAT), mitochondrial aspartate aminotransferase (mAspAT), and fatty acid transport protein (FATP), by drugs that activate peroxisome proliferator-activated receptor (PPAR) alpha and gamma were studied using normal and obese mice and rat hepatoma cells. FAT mRNA was induced in liver and intestine of normal mice and in hepatoma cells to various extents only by PPARalpha-activating drugs. FATP mRNA was similarly induced in liver, but to a lesser extent in intestine. The induction time course in the liver was slower for FAT and FATP mRNA than that of an mRNA encoding a peroxisomal enzyme. An obligatory role of PPARalpha in hepatic FAT and FATP induction was demonstrated, since an increase in these mRNAs was not observed in PPARalpha-null mice. Levels of mAspAT mRNA were higher in liver and intestine of mice treated with peroxisome proliferators, while levels in hepatoma cells were similar regardless of treatment. In white adipose tissue of KKAy obese mice, thiazolidinedione PPARgamma activators (pioglitazone and troglitazone) induced FAT and FATP more efficiently than the PPARalpha activator, clofibrate. This effect was absent in brown adipose tissue. Under the same conditions, levels of mAspAT mRNA did not change significantly in these tissues. In conclusion, tissue-specific expression of FAT and FATP genes involves both PPARalpha and -gamma. Our data suggest that among the three putative long-chain fatty acid transporters, FAT and FATP appear to have physiological roles. Thus, peroxisome proliferators not only influence the metabolism of intracellular fatty acids but also cellular uptake, which is likely to be an important regulatory step in lipid homeostasis.

Adipates↗

PubMed1998

Inhibitory monoclonal antibody to human cytochrome P450 2B6.

The human cytochrome P450 2B6 metabolizes, among numerous other substrates, diazepam, 7-ethoxycoumarin, testosterone, and phenanthrene. A recombinant baculovirus containing the human 2B6 cDNA was constructed and used to express 2B6 in Sf9 insect cells. The 2B6 was present at 1.8 +/- 0.4% of the total cellular protein and was purified to a specific content of 13.3 nmol/mg protein. Mice were immunized with the purified 2B6, and a total of 811 hybridomas were obtained from the fusion of NS-1 myeloma cells and spleen cells of the immunized mice. Monoclonal antibodies (MAbs) from 24 of the hybrids exhibited immunobinding to 2B6 as determined by ELISA. One of the MAbs, 49-10-20, showed a strong immunoblotting activity and was highly inhibitory to 2B6 enzyme activity. MAb 49-10-20 inhibited cDNA-expressed 2B6-catalyzed metabolism of diazepam, phenanthrene, 7-ethoxycoumarin, and testosterone by 90-91%. MAb 49-10-20 showed extremely high specificity for 2B6 and did not bind to 17 other human and rodent P450s or inhibit the metabolism of phenanthrene catalyzed by human 1A2, 2A6, 2C8, 2C9, 2D6, 2E1, 3A4, and 3A5. MAb 49-10-20 was used to determine the contribution of 2B6 to the metabolism of phenanthrene and diazepam in human liver. In ten liver samples, MAb 49-10-20 inhibited phenanthrene metabolism variably by a wide range of 8-42% and diazepam demethylation by 1-23%. The degree of inhibition by the 2B6 specific MAb 49-10-20 defines the contribution of 2B6 to phenanthrene and diazepam metabolism in each human liver. This technique using inhibitory MAb 49-10-20 determines the contribution of 2B6 to the metabolism of its substrates in a human tissue containing multiple P450s. This study is a prototype for the use of specific and highly inhibitory MAbs to determine individual P450 function.

Animals↗

PubMed1998

Establishment of a Salmonella tester strain highly sensitive to mutagenic heterocyclic amines.

Heterocyclic amines (HCAs) that are present in cooked foods require metabolic activation to exert their genotoxicity. They undergo activation via N-hydroxylation by cytochrome P450 1A2 (CYP1A2), followed by O-esterification by O-acetyltransferase (OAT). To develop a Salmonella tester strain that is highly sensitive to mutagenic HCAs, we introduced a coexpression plasmid (p1A2OR) carrying human CYP1A2 and NADPH-CYP reductase cDNAs and an expression plasmid (pOAT) carrying Salmonella OAT to Salmonella typhimurium TA1538 to yield a TA1538/ARO strain. The TA1538/ARO strain was proven to express the enzymes, as indicated by high activities of 7-ethoxyresorufin O-deethylase and isoniazid N-acetylase. The TA1538/ARO strain exhibited very high sensitivity to mutagenic HCAs 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline and a somewhat higher sensitivity to 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine compared with the parent Ames tester strain TA1538. The minimum concentrations of 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, IQ, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine giving positive results were defined by evidence that the number of colonies increased in a dose-dependent manner and reached a number two times higher than that obtained by vehicle alone as a control in the TA1538/ARO strain at concentrations of 0.3, 3, 30, and 1000 pM, respectively. When the membrane and cytosol fractions prepared from TA1538/ARO were added to a mixture containing the parental TA1538, the sensitivity of TA1538 to IQ was much lower than that seen with TA1538/ARO. These results indicate that the intracellular expression of drug-metabolizing enzymes makes the established strain of Salmonella highly sensitive to mutagenic HCAs.

Acetyltransferases↗

PubMed1998

Disruption of the DT diaphorase (NQO1) gene in mice leads to increased menadione toxicity.

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a flavoenzyme that catalyzes two-electron reductive metabolism and detoxification of quinones and their derivatives leading to protection of cells against redox cycling and oxidative stress. To examine the in vivo role of NQO1, a NQO1-null mouse was produced using targeted gene disruption. Mice lacking NQO1 gene expression showed no detectable phenotype and were indistinguishable from wild-type mice. However, NQO1-null mice exhibited increased toxicity when administered menadione compared with wild-type mice. These results establish a role for NQO1 in protection against quinone toxicity. The NQO1-null mice are a model for NQO1 deficiency in humans and can be used to determine the role of this enzyme in sensitivity to toxicity and carcinogenesis.

Alanine Transaminase↗

PubMed1998

Evaluation of atypical cytochrome P450 kinetics with two-substrate models: evidence that multiple substrates can simultaneously bind to cytochrome P450 active sites.

Some cytochrome P450 catalyzed reactions show atypical kinetics, and these kinetic processes can be grouped into five categories: activation, autoactivation, partial inhibition, substrate inhibition, and biphasic saturation curves. A two-site model in which the enzyme can bind two substrate molecules simultaneously is presented which can be used to describe all of these observed kinetic properties. Sigmoidal kinetic characteristics were observed for carbamazepine metabolism by CYP3A4 and naphthalene metabolism by CYPs 2B6, 2C8, 2C9, and 3A5 as well as dapsone metabolism by CYP2C9. Naphthalene metabolism by CYP3A4 and naproxen metabolism by CYP2C9 demonstrated nonhyperbolic enzyme kinetics suggestive of a low Km, low Vmax component for the first substrate molecule and a high Km, high Vmax component for the second substrate molecule. 7, 8-Benzoflavone activation of phenanthrene metabolism by CYP3A4 and dapsone activation of flurbiprofen and naproxen metabolism by CYP2C9 were also observed. Furthermore, partial inhibition of 7, 8-benzoflavone metabolism by phenanthrene was observed. These results demonstrate that various P450 isoforms may exhibit atypical enzyme kinetics depending on the substrate(s) employed and that these results may be explained by a model which includes simultaneous binding of two substrate molecules in the active site.

Aryl Hydrocarbon Hydroxylases↗

PubMed1998

Role of cDNA-expressed human cytochromes P450 in the metabolism of diazepam.

The metabolic conversion of diazepam (DZ) to temazepam (TMZ, a C3-hydroxylation product of DZ) and N-desmethyldiazepam (NDZ, an N1-demethylation product of DZ) was studied using cDNA-expressed human cytochrome P450 (CYP) isozymes 1A2, 2B6, 2C8, 2C9, 2C9R144C, 2E1, 3A4, and 3A5 and human liver microsomes from five organ donors. Of the CYPs examined, 3A5, 3A4, and 2B6 exhibited the highest enzymatic activities with turnovers ranging from 7.5 to 12.5 nmol of product formed/min/nmol for the total metabolism of DZ, while 2C8, 2C9, and 2C9R144C showed lesser and moderate activities. 1A2 and 2E1 produced insignificant amounts of metabolites of DZ. The regioselectivity of CYPs was determined, and 2B6 was found to catalyze exclusively and 2C8, 2C9, and 2C9R144C preferentially the N1-demethylation of DZ to form NDZ. 3A4 and 3A5 catalyzed primarily the C3-hydroxylation of DZ, which was more extensive than the N1-demethylation. The ratios of TMZ to NDZ formed in the metabolism of DZ by 3A4 and 3A5 were approximately 4:1. Enzyme kinetic studies indicated that 2B6- and 2C9-catalyzed DZ metabolism followed Michaelis-Menten kinetics, whereas 3A4 and 3A5 displayed atypical and non-linear curves in Lineweaver-Burk plots. Human liver microsomes converted DZ to both TMZ and NDZ at a ratio of 2:1. Our results suggest that hepatic CYP3A, 2C, and 2B6 enzymes have an important role in the metabolism of DZ by human liver.

Cytochrome P-450 Enzyme System↗

PubMed1998

Altered constitutive expression of fatty acid-metabolizing enzymes in mice lacking the peroxisome proliferator-activated receptor alpha (PPARalpha).

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a member of the steroid/nuclear receptor superfamily and mediates the biological and toxicological effects of peroxisome proliferators. To determine the physiological role of PPARalpha in fatty acid metabolism, levels of peroxisomal and mitochondrial fatty acid metabolizing enzymes were determined in the PPARalpha null mouse. Constitutive liver beta-oxidation of the long chain fatty acid, palmitic acid, was lower in the PPARalpha null mice as compared with wild type mice, indicating defective mitochondrial fatty acid catabolism. In contrast, constitutive oxidation of the very long chain fatty acid, lignoceric acid, was not different between wild type and PPARalpha null mice, suggesting that constitutive expression of enzymes involved in peroxisomal beta-oxidation is independent of PPARalpha. Indeed, the PPARalpha null mice had normal levels of the peroxisomal acyl-CoA oxidase, bifunctional protein (hydratase + 3-hydroxyacyl-CoA dehydrogenase), and thiolase but lower constitutive expression of the D-type bifunctional protein (hydratase + 3-hydroxyacyl-CoA dehydrogenase). Several mitochondrial fatty acid metabolizing enzymes including very long chain acyl-CoA dehydrogenase, long chain acyl-CoA dehydrogenase, short chain-specific 3-ketoacyl-CoA thiolase, and long chain acyl-CoA synthetase are also expressed at lower levels in the untreated PPARalpha null mice, whereas other fatty acid metabolizing enzymes were not different between the untreated null mice and wild type mice. A lower constitutive expression of mRNAs encoding these enzymes was also found, suggesting that the effect was due to altered gene expression. In wild type mice, both peroxisomal and mitochondrial enzymes were induced by the peroxisome proliferator Wy-14,643; induction was not observed in the PPARalpha null animals. These data indicate that PPARalpha modulates constitutive expression of genes encoding several mitochondrial fatty acid-catabolizing enzymes in addition to mediating inducible mitochondrial and peroxisomal fatty acid beta-oxidation, thus establishing a role for the receptor in fatty acid homeostasis.

17-Hydroxysteroid Dehydrogenases↗

PubMed1998

Effect of phenobarbital on hepatic CYP1A1 and CYP1A2 in the Ahr-null mouse.

Studies have suggested that phenobarbital (PB) induces members of the CYP1A subfamily by both transcriptional and post-transcriptional mechanisms. Using the Ahr -/- mice, we examined the induction of CYP1A1 and CYP1A2 by PB. CYP1A2 mRNA and protein were induced by PB in the null mice, suggesting that CYP1A2 is regulated by PB by a mechanism independent of the aryl hydrocarbon receptor (AHR). In contrast, the regulation of CYP1A1 is highly dependent on the AHR.

Animals↗

PubMed1998

Protection against acetaminophen toxicity in CYP1A2 and CYP2E1 double-null mice.

Acetaminophen (APAP) hepatotoxicity is due to its biotransformation to a reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI), that is capable of binding to cellular macromolecules. At least two forms of cytochrome P450, CYP2E1 and CYP1A2, have been implicated in this reaction in mice. To test the combined roles of CYP1A2 and CYP2E1 in an intact animal model, a double-null mouse line lacking functional expression of CYP1A2 and CYP2E1 was produced by cross-breeding Cyp1a2-/- mice with Cyp2e1-/- mice. Animals deficient in the expression of both P450s developed normally and exhibited no obvious phenotypic abnormalities. Comparison of the dose-response to APAP (200-1200 mg/kg) indicated that double-null animals were highly resistant to APAP-induced toxicity whereas the wild-type animals were sensitive. Administration of 600 to 800 mg/kg of this drug to male wild-type animals resulted in increased plasma concentrations of liver enzymes (alanine aminotransferase, sorbitol dehydrogenase), lipidosis, hepatic necrosis, and renal tubular necrosis. In contrast, when APAP of equivalent or higher dose was administered to the double-null mice, plasma levels of liver enzymes and liver histopathology were normal. However, administration of 1200 mg of APAP/kg to the double-null mice resulted in infrequent liver lipidosis and mild kidney lesions. Consistent with the protection from hepatotoxicity, the expected depletion of hepatic glutathione (GSH) content was significantly retarded and APAP covalent binding to hepatic cytosolic proteins was not detectable in the double-null mice. Likewise, in vitro activation of APAP by liver microsomes from the double-null mice was approximately one tenth of that in microsomes from wild-type mice. Thus, the protection against APAP toxicity afforded by deletion of both CYP2E1 and CYP1A2 likely reflects greatly diminished production of the toxic electrophile, NAPQI.

Acetaminophen↗

PubMed1998

Dihydropyrimidine dehydrogenase pharmacogenetics in patients with colorectal cancer.

Individuals with a deficiency in the enzyme dihydropyrimidine dehydrogenase (DPD) may experience severe life-threatening toxicity when treated with 5-fluorouracil (5-FU). As routine measurement of enzyme activity is not practical in many clinical centres, we have investigated the use of DNA mutation analysis to identify cancer patients with low enzyme levels. We have identified two new mutations at codons 534 and 543 in the DPD cDNA of a patient with low enzyme activity and screened the DNA from 75 colorectal cancer patients for these mutations and the previously reported splice site mutation (Vreken et al, 1996; Wei et al, 1996). In all cases, DPD enzyme activity was also measured. The splice site mutation was detected in a patient (1 out of 72) with low enzyme activity whereas mutations at codons 534 (2 out of 75) and 543 (11 out of 23) were not associated with low enzyme activity. These studies highlight the need to combine DPD genotype and phenotype analysis to identify mutations that result in reduced enzyme activity.

Adult↗

PubMed1998

Dihydropyrimidine dehydrogenase pharmacogenetics in Caucasian subjects.

AIMS: Dihydropyrimidine dehydrogenase (DPD) catalyses the reduction of pyrimidines, including the anticancer agent 5-fluorouracil (5FU). Impaired 5FU degradation, through low DPD activity, has led to severe, life-threatening or fatal toxicity after administration of 5FU. Complete DPD deficiency is associated with the inherited metabolic disease thymine uraciluria. Several mutations in the gene encoding DPD have recently been identified, but the phenotype-genotype concordance of these alterations in the general population has not been reported. METHODS: Mononuclear cells were isolated from whole blood and DPD activity was determined after ex vivo incubation with 14C-5FU followed by h.p.1.c. analysis of 5FU metabolites. Analysis of mutations in the DPD gene at an exon splice site, codons 534, 543, and 732, and a deletion at base 1897 (deltaC1897) were performed in 30 subjects with the lowest and 30 subjects with the highest enzyme activity using PCR-RFLP. RESULTS: DPD activity was measured in 226 Caucasian subjects and was highly variable (range 19.1-401.4 pmol min(-1)mg(-1) protein). Mutations were frequently observed at codons 543 (allele frequency 28%), 732 (allele frequency 5.8%), and 534 (allele frequency 0.8%), but were not associated with low DPD activity. There were no splice site or deltaC1897 mutations found in this population. CONCLUSIONS: The five mutations analysed in this study are insufficient for identification of patients at risk for 5FU toxicity or thymine uraciluria. Both the splice site mutation and deltaC1897 are relatively rare in the general Caucasian population. Therefore, identification of further molecular alterations is required to facilitate the use of DPD analysis in genetic diagnosis and cancer therapeutics.

Alleles↗

PubMed1998

2,3-epoxy-4-hydroxynonanal, a potential lipid peroxidation product for etheno adduct formation, is not a substrate of human epoxide hydrolase.

Our previous studies have shown that 2,3-epoxy-4-hydroxynonanal, a reactive epoxy aldehyde capable of forming etheno adducts with DNA bases, is mutagenic and tumorigenic (Carcinogenesis, 14, 2073). The epoxy aldehyde can be generated from trans-4-hydroxy-2-nonenal, a lipid peroxidation product of omega-6 polyunsaturated fatty acids, by autoxidation or by incubation with fatty acid hydroperoxides or hydrogen peroxides (Chem. Res. Toxicol., 9, 306). These are plausible in vivo pathways for the formation of 2,3-epoxy-4-hydroxynonanal. The possibility that 2,3-epoxy-4-hydroxynonanal is a tumorigen of endogenous origin is suggested by recent observations that etheno bases are detected as background DNA lesions in untreated rodents and humans. A metabolic pathway critical for detoxification of 2,3-epoxy-4-hydroxynonanal involves the ring-opening by epoxide hydrolase, which abolishes its ability to form cyclic etheno DNA adducts. In this study, we examined whether 2,3-epoxy-4-hydroxynonanal is a substrate of cDNA expressed human epoxide hydrolase. Human epoxide hydrolase was expressed in TK- 143 cells (thymidine kinase-deficient human embryoblast) infected with recombinant vaccinia virus encoding human epoxide hydrolase cDNA. Controls consisted of the cells infected with vaccinia virus in the absence of human epoxide hydrolase cDNA. No hydrolysis occurred when [2,3-(3)H]2,3-epoxy-4-hydroxynonanal was incubated at 37 degrees C for 30 min at pH 7.4 with cells expressing human epoxide hydrolase, as indicated by the presence of a pair of radioactive peaks in reversed-phase HPLC chromatography, which comigrated with the UV standards of the two diastereomers of the epoxy aldehyde. The identity of these compounds as the intact epoxy aldehyde was further supported by derivatization to the 2,4-dinitrophenylhydrazones followed by reversed phase HPLC analysis. Similar results were observed with the control cells or with the heat deactivated human epoxide hydrolase. The epoxide hydrolase activity in the expressed cells was demonstrated by their ability to convert benzo[a]pyrene-4,5-dihydroepoxide to benzo[a]pyrene-trans-4,5-dihydrodiol under the same conditions. These results clearly indicate that 2,3-epoxy-4-hydroxynonanal is not a substrate of human epoxide hydrolase, and, thus strengthen its possible endogenous role in the formation of promutagenic exocyclic etheno adducts in vivo.

Aldehydes↗

PubMed1998

Molecular basis of the Dark Agouti rat drug oxidation polymorphism: importance of CYP2D1 and CYP2D2.

The Dark Agouti rat has been proposed as a model for the human debrisoquine 4-hydroxylase polymorphism. Earlier studies suggested that the poor metabolizer phenotype in the Dark Agouti rat is caused by the absence of the expression of CYP2D1 mRNA. Although CYP2D1 is the major enzyme catalyzing debrisoquine 4-hydroxylation, other reports have indicated the involvement of a CYP2D, purified from rat hepatic microsomes and presumed to be CYP2D2, which also exhibits this activity. The levels of CYP2D1 and CYP2D2 mRNAs were markedly lower in Dark Agouti as compared to Sprague Dawley rats. Using a baculovirus expression system, recombinant CYP2D1 and CYP2D2 from Spodoptera frugiperda insect cells were examined and were found to both forms catalize debrisoquine 4-hydroxylase activity. These results suggest that reduced debrisoquine 4-hydroxylase activity in the Dark Agouti rat is caused by the low level expression not only of CYP2D1, but also of CYP2D2. Interestingly, bunitrolol 4-hydroxylation was catalyzed by recombinant CYP2D2, while CYP2D1 was inactive toward this substrate. Thus, the low bunitrolol 4-hydroxylation in Dark Agouti rats was caused by the low level of CYP2D2 expression in this rat strain.

Adrenergic beta-Antagonists↗

PubMed1998

The involvement of aryl hydrocarbon receptor in the activation of transforming growth factor-beta and apoptosis.

The aryl hydrocarbon receptor (AHR) is believed to mediate many of the toxic, carcinogenic, and teratogenic effects of environmental contaminants such as dioxins, polycyclic aromatic hydrocarbons, and polyhalogenated biphenyls. Ligands for the AHR have been shown to influence cell proliferation, differentiation, and apoptosis, but the mechanism by which the AHR affects the cell cycle is not known. Increased levels of mature transforming growth factor-beta (TGFbeta) has been correlated with reduced cell proliferation and increased rates of apoptosis and fibrosis. Based on the increase in portal fibrosis and small liver size observed in AHR-null (Ahr-/-) mice, the relationship between TGFbeta expression and apoptosis in this mouse line was analyzed. Livers from Ahr-/- mice had marked increase in active TGFbeta1 and TGFbeta3 proteins and elevated numbers of hepatocytes undergoing apoptosis compared with wild-type mice. Furthermore, increases in TGFbeta and apoptotic cells were found in the portal areas of the liver, where fibrosis is found in the Ahr-/- mice. In vitro, primary hepatocyte cultures from Ahr-/- mice exhibited a high number of cells in later stages of apoptosis and an elevated secretion of active TGFbeta into the media compared with cultures from wild-type mice, which have previously been shown to secrete only latent forms of the molecule. Conditioned media from Ahr-/- hepatocytes stimulated apoptosis in cultured hepatocytes from wild-type mice. Taken together, these findings suggest that the phenotypic abnormalities in Ahr-/- mice could be mediated in part by abnormal levels of active TGFbeta and altered cell cycle control.

Animals↗

PubMed1998

Laron dwarfism and non-insulin-dependent diabetes mellitus in the Hnf-1alpha knockout mouse.

Mice deficient in hepatocyte nuclear factor 1 alpha (HNF-1alpha) were produced by use of the Cre-loxP recombination system. HNF-1alpha-null mice are viable but sterile and exhibit a phenotype reminiscent of both Laron-type dwarfism and non-insulin-dependent diabetes mellitus (NIDDM). In contrast to an earlier HNF-1alpha-null mouse line that had been produced by use of standard gene disruption methodology (M. Pontoglio, J. Barra, M. Hadchouel, A. Doyen, C. Kress, J. P. Bach, C. Babinet, and M. Yaniv, Cell 84:575-585, 1996), these mice exhibited no increased mortality and only minimal renal dysfunction during the first 6 months of development. Both dwarfism and NIDDM are most likely due to the loss of expression of insulin-like growth factor I (IGF-I) and lower levels of insulin, resulting in stunted growth and elevated serum glucose levels, respectively. These results confirm the functional significance of the HNF-1alpha regulatory elements that had previously been shown to reside in the promoter regions of both the IGF-I and the insulin genes.

Animals↗