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F J Gonzalez

Publications and source records attributed to F J Gonzalez.

At least 163 records · Page 9Linked to original sources
PubMed1997

Role of PPAR alpha in the mechanism of action of the nongenotoxic carcinogen and peroxisome proliferator Wy-14,643.

Chronic administration of peroxisome proliferators to mice and rats results in hepatomegaly and ultimately carcinogenesis. The mechanism underlying the carcinogenic effect of nongenotoxic peroxisome proliferators is not well understood. To determine whether nongenotoxic carcinogenesis is receptor mediated, we evaluated the effect of the prototypical peroxisome proliferator Wy-14,643 on replicative DNA synthesis and carcinogenesis in the PPAR alpha-null mouse line. Male mice (F4, Sv/129 ter) of both genotypes (+/+) and (-/-) were fed either a control diet or one containing 0.1% Wy-14,643 for either 1 week, 5 weeks, or 11 months. Wild-type mice fed the Wy-14,643 diet for 1 or 5 weeks showed increased hepatic labeling by bromodeoxyuridine (BrDU) compared to untreated controls. In contrast, there was no increase in hepatic BrDU labeling index in (-/-) mice fed the Wy-14,643 diet for the same time periods compared to controls. After 11 months, 100% of the (+/+) mice fed the Wy-14,643 diet had multiple hepatocellular neoplasms, including adenomas and carcinomas, while the (-/-) mice fed the Wy-14,643 diet were unaffected. This work demonstrates that the effects of Wy-14,643 on replicative DNA synthesis and hepatocarcinogenesis are mediated by PPAR alpha.

Animals↗
PubMed1997

Aflatoxin B1-induced DNA adduct formation and p53 mutations in CYP450-expressing human liver cell lines.

Epidemiological evidence has been supporting a relationship between dietary aflatoxin B1 (AFB1) exposure, development of human primary hepatocellular carcinoma (HCC) and mutations in the p53 tumor suppressor gene. However, the correlation between the observed p53 mutations, the AFB1 DNA adducts and their activation pathways has not been elucidated. Development of relevant cellular in vitro models, taking into account species and tissue specificity, could significantly contribute to the knowledge of cytotoxicity and genotoxicity mechanisms of chemical procarcinogens, such as AFB1, in humans. For this purpose a non-tumorigenic SV40-immortalized human liver epithelial cell line (THLE cells) which retained most of the phase II enzymes, but had markedly reduced phase I activities was used for stable expression of the human CYP1A2, CYP2A6, CYP2B6 and CYP3A4 cDNA. The four genetically engineered cell lines (T5-1A2, T5-2A6, T5-2B6 and T5-3A4) produced high levels of the specific CYP450 proteins and showed comparable or higher catalytic activities related to the CYP450 expression when compared to human hepatocytes. The T5-1A2, T5-2A6, T5-2B6 and T5-3A4 cell lines exhibited a very high sensitivity to the cytotoxic effects of AFB1 and were approximately 125-, 2-, 2- and 15-fold, respectively, more sensitive than the control T5-neo cells, transfected with an expressing vector which does not contain CYP450 cDNA. In the CYP450-expressing cells, nanomolar doses of AFB1-induced DNA adduct formation including AFB1-N7-guanine, -pyrimidyl and -diol adducts. In addition, the T5-1A2 cells showed AFM1-DNA adducts. At similar levels of total DNA adducts, both the T5-1A2 and T5-3A4 cells showed, at codon 249 of the p53 gene, AGG to AGT transversions at a relative frequency of 15x10(-6). In contrast, only the T5-3A4 cells showed CCC to ACC transversion at codon 250 at a high frequency, whereas the second most frequent mutations found in the T5-1A2 cells were C to T transitions at the first and second position of the codon 250. No significant AFB1-induced p53 mutations could be detected in the T5-2A6 cells. Therefore, the differential expression of specific CYP450 genes in human hepatocytes can modulate the cytotoxicity, DNA adduct levels and frequency of p53 mutations produced by AFB1.

Adult↗
PubMed1997

Lack of correlation between phenotype and genotype for the polymorphically expressed dihydropyrimidine dehydrogenase in a family of Pakistani origin.

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in pyrimidine catabolism. DPD deficiency is associated with an increased risk of toxicity in cancer patients receiving 5-fluorouracil (5-PU) treatment. DPD deficiency causes an inborn error of metabolism called thymine-uraciluria that is in some instances associated with convulsive disorders and developmental delay in children. We have studied the molecular mechanism accounting for DPD deficiency in a Pakistani pedigree having 2-year-old child with thymine-uraciluria and exhibiting some degree of motor impairment and developmental delay. A common splice mutation was found in the patient's dihydropyrimidine dehydrogenase (DPYD) gene that produces a mutant mRNA resulting in the complete lack of DPD protein and activity in lymphocytes and primary fibroblast. This trait segregated in the family following a typical Mendelian distribution. Surprisingly, the patient's brother also had thymine-uraciluria and was homozygous for the splicing mutation but was clinically asymptomatic. Sequence tagged sites (STS) linkage analyses within 5 megabases of telomeric and centromeric DNA surrounding the DPYD gene revealed no allelic polymorphism between the two brothers. These results suggest that DPD deficiency might not be the only cause of the more severe clinical phenotypes observed in certain thymine-uraciluria patients and that an incomplete correlation between phenotype and genotype is present in the population.

Child, Preschool↗
PubMed1997

A monoclonal antibody inhibitory to human P450 2D6: a paradigm for use in combinatorial determination of individual P450 role in specific drug tissue metabolism.

Human cytochrome P450 2D6 metabolizes more than 50 common drugs and is polymorphically expressed, with 5-10% of the population lacking expression caused by mutant genes. This may result in a defective and toxic response in deficient individuals treated with 2D6 drug substrates. Baculovirus-expressed 2D6 was used to immunize mice for hybridoma production and two clones yielded monoclonal antibodies, that were positive against 2D6 by ELISA and inhibited 2D6 catalysed metabolism of bufuralol, dextromethorphan and phenanthrene by more than 90%. The inhibitory activity was highly specific to 2D6 and the monoclonal antibodies did not bind to 11 other P450s, nor inhibit seven human P450s tested. Analysis of eight human liver microsome samples showed that their basal bufuralol 1'-hydroxylase activity varied from 6.7-83.5 pmol min-1 nmol-1 P450. The monoclonal antibody 512-1-8 inhibited 2D6-dependent bufuralol 1'-hydroxylase in these samples by 10-70% indicating a widely variable role for 2D6 in human liver bufuralol 1'-hydroxylase activity and a role for other P450s in bufuralol metabolism. Independent analysis of several recombinant human P450s showed that 2D6, 2C8, 2C9, 2C19 and 1A2 exhibited bufuralol 1'-hydroxylase activity with 2D6 and 2C19 being the most active. Further analysis of three liver samples was made with individual inhibitory monoclonal antibodies. Inhibitory antibodies to 2D6, 2B6, 2E1, 2C8/9/19, 3A4 and 1A2 were added to the microsomes either singly or additively. Inhibitory activity of bufuralol 1'-hydroxylase was observed with antibodies to 2D6 (14-76%), 2C8/9/19 (24-69%) and 1A2 (2-25%) indicating a variable and different role for each of these P450s in the bufuralol 1'-hydroxylase of human liver. The monoclonal antibodies to 2B6, 2E1 and 3A4 were not inhibitory, indicating that these enzymes play no role in bufuralol 1'-hydroxylase metabolism. When the three antibodies to 2D6, 2C8/9/19 and 1A2, respectively, were all added, the total bufuralol 1'-hydroxylase of the liver samples was inhibited by more than 90%, indicating that the latter P450s catalyse all of liver bufuralol 1'-hydroxylase metabolism. These studies demonstrate that inhibitory monoclonal antibodies offer a simple and precise method for assessing the quantitative role of each P450 in the metabolism of a P450 substrate in a tissue, which include drugs, carcinogens, mutagens, toxic chemicals and endobiotics.

Animals↗
PubMed1997

Disruption of the c/ebp alpha gene in adult mouse liver.

The liver-enriched transcription factor C/EBP alpha has been implicated in the regulation of numerous liver-specific genes. It was previously reported that mice carrying a homozygous null mutation at the c/ebp alpha locus died as neonates due to the absence of hepatic glycogen and the resulting hypoglycemia. However, the lethal phenotype precluded further analysis of the role of C/EBP alpha in hepatic gene regulation in adult mice. To circumvent this problem, we constructed a conditional knockout allele of c/ebp alpha by using the Cre/loxP recombination system. Homozygous c/ebp-loxP mice, (c/ebp alpha(fl/fl);fl, flanked by loxP sites) were found to be indistinguishable from their wild-type counterparts. However, when Cre recombinase was delivered to hepatocytes of adult c/ebp alpha(fl/fl) mice by infusion of a recombinant adenovirus carrying the cre gene, more than 80% of the c/ebp alpha(fl/fl) genes were deleted specifically in liver and C/EBP alpha expression was reduced by 90%. This condition resulted in a reduced level of bilirubin UDP-glucuronosyltransferase expression in the liver. After several days, the knockout mice developed severe jaundice due to an increase in unconjugated serum bilirubin. The expression of genes encoding phosphoenolpyruvate carboxykinase, glycogen synthase, and factor IX was also strongly reduced in adult conditional-knockout animals, while the expression of transferrin, apolipoprotein B, and insulin-like growth factor I genes was not affected. These results establish C/EBP alpha as an essential transcriptional regulator of genes encoding enzymes involved in bilirubin detoxification and gluconeogenesis in adult mouse liver.

Adenoviruses, Human↗
PubMed1997

The ability of C/EBP beta but not C/EBP alpha to synergize with an Sp1 protein is specified by the leucine zipper and activation domain.

The rat CYP2D5 P-450 gene is activated in the liver during postnatal development. We previously showed that liver-specific transcription of the CYP2D5 gene is dictated by a proximal promoter element, termed 2D5, that is composed of a binding site for Sp1 or a related factor, and an adjacent cryptic C/EBP (CCAAT/enhancer-binding protein) site. Despite the fact that both C/EBP alpha and C/EBP beta are expressed abundantly in liver, only C/EBP beta is capable of stimulating the 2D5 promoter in HepG2 hepatocarcinoma cells. In addition, activation of the 2D5 promoter by C/EBP beta is completely dependent on the presence of the Sp1 site. Domain switch experiments reveal that C/EBP beta proteins containing either the leucine zipper or the activation domain of C/EBP alpha are unable to stimulate the 2D5 promoter yet are fully capable of transactivating an artificial promoter bearing a high-affinity C/EBP site. Thus, the leucine zipper and the activation domain of C/EBP beta are absolutely required to support transactivation of the 2D5 promoter. Using Drosophila cells that lack endogenous Sp1 activity, we show that the serine/threonine- and glutamine-rich activation domains A and B of Sp1 are required for efficient cooperatively with C/EBP beta. Furthermore, analysis of c/ebp beta-deficient mice shows that mutant animals are defective in expression of a murine CYP2D5 homolog in hepatic cells, confirming the selective ability of C/EBP beta to activate this liver-specific P-450 gene in vivo. Our findings illustrate that two members of a transcription factor family can achieve distinct target gene specificities through differential interactions with a cooperating Sp1 protein.

Animals↗
PubMed1997

Lesions of aryl-hydrocarbon receptor-deficient mice.

We have analyzed the possible role of the aryl-hydrocarbon receptor (AHR) in the aging process of mice using a homozygous null mouse (Ahr-/-) line as a model. We studied 52 male and female Ahr-/- mice aged from 6-13 months. Forty-six percent died or were ill by 13 months of age. Ahr-/- mice developed age-related lesions in several organs, some of which were apparent after only 9 months of age. Cardiovascular alterations included cardiomyopathy (100%) with hypertrophy and focal fibrosis. Vascular hypertrophy and mild fibrosis were found in the portal areas of the liver (81%), and vascular hypertrophy and mineralization were common in the uterus (70%). Gastric hyperplasia that progressed with age into polyps was evident in the pylorus of 71% of the mice over 9 months of age. Ahr-/- mice had T-cell deficiency in their spleens but not in other lymphoid organs. The immune system deficiency described previously could be the origin for the rectal prolapse found in 48% of the null mice, associated with Helicobacter hepaticus infection. In the dorsal skin (53% incidence), severe, localized, interfollicular and follicular epidermal hyperplasia, with hyperkeratosis and acanthosis, and marked dermal fibrosis, associated with the presence of anagenic hair follicles, were also evident. None of these lesions were found in 42 control (Ahr +/+ or +/-) mice of similar ages. These observations suggest that the AHR protein, in the absence of an apparent exogenous (xenobiotic) ligand, plays an important role in physiology and homeostasis in major organs in mice, and further supports an evolutionary conserved role for this transcription factor.

Aging↗
PubMed1997

Coexpression of cytochrome P4502A6 and human NADPH-P450 oxidoreductase in the baculovirus system.

Heterologous expression using baculovirus vectors has become a popular method for the production of catalytically active cytochrome P450s (CYPs). We have systematically optimized the multiplicity of infection (MOI) for a coinfection approach for the coexpression of CYP2A6 (viral vector designated v2A6) and NADPH-P450 oxidoreductase (OR; viral vector designated vOR) using Sf9 insect cells. A 3000-fold range of MOI was examined in stationary culture and stirred suspension culture. Surprisingly, our results indicate that the best CYP2A6 catalytic activity (850-1300 pmol/ min/mg total lysate protein as measured by coumarin 7-hydroxylase activity) was obtained only when using a low MOI of v2A6 (1.5-3 x 10(-2)) and a vOR of 10- to 20-fold less. This activity was approximately 7- to 11-fold higher than the best activity obtained when infecting cells with v2A6 alone. At this level of coinfection, the P450 content ranged from 180 to 250 pmol/mg total lysate protein, and the NADPH cytochrome c reductase activity ranged from 350 to 520 nmol/min/mg total lysate protein. Increasing the MOI of both viruses to 50-fold higher resulted in lower overall activity with the optimum (250 pmol/min/mg total lysate protein) being seen earlier postinfection (60 vs. 72 hr). Increasing the MOI of vOR to levels comparable with those of v2A6, decreased coumarin 7-hydroxylase activity 14-fold. These results suggest that the best CYP2A6 catalytic activity depends on properly posttranslationally modified proteins accumulating in a right ratio as a result of primary, secondary, and possibly tertiary infection of both viruses. These results also suggest that high OR expression results in degradation of P450.

Animals↗
PubMed1997

Human cytochrome P4502B6: interindividual hepatic expression, substrate specificity, and role in procarcinogen activation.

The level of expression and interindividual variation in human hepatic microsomal cytochrome P450 (CYP) 2B6 was characterized using a polyclonal antibody (WB-2B6) raised against rat CYP2B1. Immunoblot analysis using cDNA-expressed human CYPs revealed strong cross-reactivity of this antibody with CYP2B6 (limit of detection < 0.05 pmol) and only minor cross-reactivities with human CYP2A6, CYP2D6, and CYP2E1, all of which could be resolved from CYP2B6 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of human liver microsomes using this antibody revealed immunodetectable CYP2B6 protein in a majority of individual liver samples, with levels up to 74 pmol/mg protein in the CYP2B6-positive samples. Kinetic analysis of cDNA-expressed CYPs identified many of these enzymes as catalysts of 7-ethoxy-4-trifluoromethylcoumarin (7EFC) O-deethylation, but with significantly different apparent K(M) values (CYP1A2 < CYP2B6 approximately CYP1A1 < CYP2C19 < CYP2C9 < CYP2E1 < CYP2A6). By assaying liver microsomal 7EFC O-deethylase activity at a low 7EFC concentration (5 microM) and preincubating human liver microsomes with anti-CYP1A, anti-CYP2C, and anti-CYP2E1 antibodies, we were able to monitor CYP2B6-dependent 7EFC O-deethylase activity in a panel of 17 human liver microsomes and observe a significant correlation (r2 = 0.80) between this activity and CYP2B6 protein content. The ability of CYP2B6 to activate prodrugs and procarcinogens was examined using gene locus mutation assays in CYP2B6-expressing human lymphoblast cells. CYP2B6-expressing cells were found to be more sensitive than control cells to the cytotoxicity and mutagenicity of cyclophosphamide, aflatoxin B1, and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. CYP2B6 is thus a widely expressed human liver microsomal CYP that can contribute to a broad range of drug metabolism and procarcinogen activation reactions.

Adolescent↗
PubMed1997

Human CYP2C9 and CYP2A6 mediate formation of the hepatotoxin 4-ene-valproic acid.

Cytochrome P450-dependent desaturation of the anticonvulsant drug valproic acid (VPA) results in formation of the hepatotoxin, 4-ene-VPA. Polytherapy with other anticonvulsants which are known P450 inducers increases the flux through this bioactivation pathway. The aim of the present study was to identify specific, inducible forms of human liver P450 which catalyze terminal desaturation of VPA. Oxidized VPA metabolites formed in an NADPH-dependent manner by human liver microsomes were quantified by gas-chromatography/mass spectrometry. In vitro reaction conditions were established which reflected the product profile found in vivo. Production of 4-ene-VPA by microsomal P450s could be inhibited significantly by coumarin, sulfaphenazole and diethyldithiocarbamate, but not by triacetyloleandomycin, quinidine or furafylline. Recombinant human CYP3A4 did not form detectable levels of 4-ene-VPA and, of nine additional isoforms expressed in either HepG2 or lymphoblastoid cells which were screened for VPA desaturase activity, only CYP2C9 and CYP2A6 formed detectable levels of metabolite. Consequently, CYP3A4, the isoform usually associated with induction by anticonvulsants cannot be responsible for the enhanced 4-ene-VPA formation that occurs during polytherapy. Instead, enhanced activity in vivo likely results from induction of CYP2A6 and/or CYP2C9.

Adolescent↗
PubMed1996

Stereoselective epoxidation and hydration at the K-region of polycyclic aromatic hydrocarbons by cDNA-expressed cytochromes P450 1A1, 1A2, and epoxide hydrolase.

Stereoselective epoxidation by cytochrome P450s (P450s) and regioselective hydration by epoxide hydrolase determine the carcinogenic potency of some polycyclic aromatic hydrocarbons (PAHs). In this report, cDNA-expressed human and mouse P450s 1A1 and 1A2 and epoxide hydrolase were used to characterize the stereoselective epoxidation and regioselective hydration at the K-region of benz[a]-anthracene (BA), 7,12-dimethylbenz[a]anthracene (DMBA), chrysene (CR), benzo[a]pyrene (B[a]P), dibenz[a,h]anthracene (DB[a,h]A), and benzo[c]phenanthrene (B[c]Ph) by direct chiral stationary-phase HPLC (CSP-HPLC) analyses. Our results indicated that all P450 isoforms preferentially produced major K-region, S,R-epoxides of BA (95-98%), DMBA (94-97%), B[a]P (91-96%), DB[a,h]A (94-98%), and B[c]Ph (87-92%), and major R,S-epoxide of CR (74-85%) in the presence of 3,3,3-trichloropropylene 1,2-oxide (TCPO), an inhibitor of epoxide hydrolase, suggesting that P450 enzymes exhibited the high stereoselectivity toward one of two stereoheterotopic faces of K-region double bond of the PAHs. Epoxide hydrolase either expressed from recombinant vaccinia virus or contained in human hepatoma G2 cells (HepG2) hydrated the C-O bond of epoxy-ring at the S-carbon of major metabolically-formed K-region epoxide enantiomers of BA, CR, DMBA, B[a]P, and DB[a,h]A to yield 80-98% dihydrodiols enriched in R,R-form and that at the R-carbon of B[c]Ph epoxide to yield 77-92% dihydrodiol enriched in S,S-form, suggesting that epoxide hydrolase was highly regioselective. The various enantiomeric components of dihydrodiol products in the metabolism of PAHs were apparently due to the combined effect of stereoselectivity of the P450s and regioselectivity of epoxide hydrolase. Our results provide a clear understanding of how these enzymes catalyze overall stereoselective metabolism at the K-region of the PAHs.

Animals↗
PubMed1996

The PPARalpha-leukotriene B4 pathway to inflammation control.

Inflammation is a local immune response to 'foreign' molecules, infection and injury. Leukotriene B4, a potent chemotactic agent that initiates, coordinates, sustains and amplifies the inflammatory response, is shown to be an activating ligand for the transcription factor PPARalpha. Because PPARalpha regulates the oxidative degradation of fatty acids and their derivatives, like this lipid mediator, a feedback mechanism is proposed that controls the duration of an inflammatory response and the clearance of leukotriene B4 in the liver. Thus PPARalpha offers a new route to the development of anti- or pro-inflammatory reagents.

Adaptation, Physiological↗
PubMed1996

Studies of flurbiprofen 4'-hydroxylation. Additional evidence suggesting the sole involvement of cytochrome P450 2C9.

Flurbiprofen, a non-steroidal anti-inflammatory drug (NSAID), is metabolized by both oxidation via the cytochrome P450 system and by glucuronidation. The major oxidative pathway in flurbiprofen metabolism is to a 4'-hydroxy metabolite, and recently we demonstrated that cytochrome P450 2C9 and its R144C variant were involved in this process (Tracy et al., Biochem Pharmacol 49: 1269-1275, 1995). Using complementary DNA (cDNA)-expressed cell systems, it has been demonstrated that at physiological concentrations of flurbiprofen there is a lack of involvement of P450s 1A2, 2C8, 2E1, and 3A4. In evaluating flurbiprofen as a potential probe for cytochrome P450 2C9, it is important to assess the involvement of additional P450s in this process. To this end, further studies were undertaken using specific inhibitors of P450 2C9 and P450 cDNA-expressed microsomes for P450 1A1, 2A6, 2B6, 2C19, and 2D6 to assess their potential involvement. We observed the inhibition of (R)- and (S)-flurbiprofen 4'-hydroxylation by an inhibitor of P450 2C9, sulfaphenazole (Ki = 0.07 and 0.06 microM, respectively), and the NSAID piroxicam (Ki = 10 and 7 microM, respectively). Furthermore, using microsomes from a lymphoblastoid cell line, we found that P450s 1A1, 2A6, 2B6, 2C19, and 2D6 were not involved in flurbiprofen hydroxylation at physiological concentrations of flurbiprofen. This finding is particularly important due to the sequence homology and potential substrate overlap of P450 2C9 and 2C19. These studies then provide additional evidence to suggest that P450 2C9 may be the only isoform involved to any substantial degree in flurbiprofen 4'-hydroxylation, and thus this reaction is useful as an in vitro probe for this particularly cytochrome P450 isoform and may be useful as an in vivo probe.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1996

Differential regulation of mouse Ah receptor gene expression in cell lines of different tissue origins.

The dioxin-binding Ah receptor (AHR) is a ligand-activated transcription factor that regulates the expression of several drug-metabolizing enzymes and has been implicated in immunosuppression, teratogenesis, cell-specific hyperplasia, and certain types of malignancies and toxicities. In order to examine tissue-specific regulation of the mouse Ah receptor gene (Ahr), we studied chimeric deletion constructs, containing the Ahr 5' flanking region and the firefly luciferase reporter gene (Luc). Transient transfection assays were performed in five established mouse cell lines: Hepa-1c1c7 (derived from hepatoma), JB6-C1 41-5a (epidermis), MLE-12 (lung epithelium), F9 (embryonal carcinoma), and NIH/3T3 (fibroblasts). Treatment of the cell lines included: dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin), retinoic acid (RA), cyclic adenosine 3':5'-monophosphate (cAMP), or 12-O-tetradecanoylphorbol 13-acetate (TPA). Expression levels of Luc varied widely from one untreated cell line to another, this finding was also confirmed by measurements of AHR mRNA steady-state levels. In all cell lines except F9 cells, maximal constitutive expression was observed with constructs containing 78 bp of Ahr promoter sequences, which include several putative binding sites for the transcription factor Sp1. In contrast, in F9 cells, inclusion of sequences between -174 and -78 resulted in a fourfold stimulation of constitutive expression, suggesting that other transcription factors are important in Ahr gene expression in these cells. In MLE-12 and 41-5a cells, expression was significantly decreased by treatment with dioxin, RA, cAMP, or TPA. A similar inhibitory effect was observed in cAMP-treated MLE-12 and F9 cells; this result was confirmed by RT-PCR measurements of AHR mRNA steady-state levels. These results indicate that both up- and down-regulation of the Ahr gene occur and exhibit tissue-and cell-type specificity.

3T3 Cells↗
PubMed1996

Activation of a human peroxisome proliferator-activated receptor by the antitumor agent phenylacetate and its analogs.

The aromatic fatty acid phenylacetate and its analogs induce tumor cytostasis and differentiation in experimental models. Although the underlying mechanisms of action are not clear, effects on lipid metabolism are evident. We have now examined whether these compounds, structurally similar to the peroxisome proliferator clofibrate, affect the human peroxisome proliferator-activated receptor (hPPAR), a homolog of the rodent PPAR alpha, a transcriptional factor regulating lipid metabolism and cell growth. Gene transfer experiments showed activation of hPPAR, evident by the increased expression of the reporter gene chloramphenicol acetyltransferase linked to PPAR-response element from either the rat acyl-CoA oxidase or rabbit CYP4A6 genes. The relative potency of tested drugs in the co-transfection assay was: 4-iodophenylbutyrate > 4-chlorophenylbutyrate > clofibrate > phenylbutyrate > naphthylacetate > 2,4-D > 4-chlorophenylacetate > phenylacetate >> indoleacetate. Phenylacetylglutamine, in which the carboxylic acid is blocked, was inactive. The ability of the aromatic fatty acids to activate PPAR was confirmed in vivo, as CYP4A mRNA levels increased in hepatocytes of treated rats. Further studies using human prostate carcinoma, melanoma, and glioblastoma cell lines showed a tight correlation between drug-induced cytostasis, increased expression of the endogenous hPPAR, and receptor activation documented in the gene-transfer model. These results identify phenylacetate and its analogs as a new class of aromatic fatty acids capable of activating hPPAR, and suggest that this nuclear receptor may mediate tumor cytostasis induced by these drugs.

Animals↗
PubMed1996

Stable expression of human CYP2E1 in Chinese hamster cells: high sensitivity to N,N-dimethylnitrosamine in cytotoxicity testing.

Involvement of human CYP2E1 expressed in genetically engineered cells in the metabolic activation of promutagens and procarcinogens was studied. An expression plasmid containing an insert of CYP2E1 cDNA and SR alpha promoter was constructed and transfected into the cultured cell line CR-119 which had previously been established by introducing a cDNA coding for NADPH-cytochrome P450 reductase. Among newly established cell lines, ER-181 showed the highest expression of CYP2E1 mRNA. Production of the CYP2E1 protein was confirmed by Western blot analysis using anti-rat CYP2E1 antibodies. Assay of 7-ethoxycoumarin O-deethylase activity demonstrated that ER-181 cells acquired the catalytic function of CYP2E1. ER-181 cells showed higher sensitivity to N,N-dimethylnitorosamine (DMN) in cytotoxicity assays as compared to parental CR-119 cells. Hypersensitivity to DMN of ER-181 cells was completely suppressed by 3-amino-1,2,4-triazole, a known inhibitor of CYP2E1. These results indicate that ER-181 cells which express human CYP2E1 are a useful tool to investigate toxicological functions of the cytochrome.

Animals↗
PubMed1996

Molecular basis of the human dihydropyrimidine dehydrogenase deficiency and 5-fluorouracil toxicity.

Dihydropyrimidine dehydrogenase (DPD) deficiency constitutes an inborn error in pyrimidine metabolism associated with thymine-uraciluria in pediatric patients and an increased risk of toxicity in cancer patients receiving 5-fluorouracil (5-FU) treatment. The molecular basis for DPD deficiency in a British family having a cancer patient that exhibited grade IV toxicity 10 d after 5-FU treatment was analyzed. A 165-bp deletion spanning a complete exon of the DPYD gene was found in some members of the pedigree having low DPD catalytic activity. Direct sequencing of lymphocyte DNA from these subjects revealed the presence of a G to A point mutation at the 5'-splicing site consensus sequence (GT to AT) that leads to skipping of the entire exon preceding the mutation during pre-RNA transcription and processing. A PCR-based diagnostic method was developed to determine that the mutation is found in Caucasian and Asian populations. This mutation was also detected in a Dutch patient with thymine-uraciluria and completely lacking DPD activity. A genotyping test for the G to A splicing point mutation could be useful in predicting cancer patients prone to toxicity upon administration of potentially toxic 5-FU and for genetic screening of heterozygous carriers and homozygous deficient subjects.

Alleles↗