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Biomedical subjects

P Fernandez-Salguero

Publications and source records attributed to P Fernandez-Salguero.

At least 19 recordsLinked to original sources
PubMed2000

Hepatic fibrosis and cytochrome P450: experimental models of fibrosis compared to AHR knockout mice.

Hepatic fibrosis is characterized by abnormal collagen deposition resulting from increased collagen synthesis and decreased collagen degradation. Cytochrome P450 mediates major drug metabolizing enzyme activity in the liver and this activity is reduced in hepatic fibrosis. In this study we assess cytochrome P450 and CYP 1A mRNA in livers of animals that have been induced to hepatic fibrosis using the heterologous serum induced model of fibrosis in rats compared to controls. Fibrosis was confirmed by assessing the collagen in liver sections as quantified by Sirius red/Fast green staining, a quantitative measure of fibrosis as well as by visualization of the hepatic fibrosis. Collagen levels in the liver sections of heterologous serum induced fibrotic rats was increased by 33% compared to controls and a typical fibrotic pattern was seen. Messenger RNA was prepared from heterologous serum induced fibrotic rats and compared to controls. CYP 1A2 was assessed using a specific probe and the CYP 1A2 level was significantly reduced in the heterologous serum induced fibrotic rats compared to controls. These results further suggest that cytochrome P450 is reduced in the presence of hepatic fibrosis. Thus, in three well established experimental models of hepatic fibrosis which had clearly developed hepatic fibrosis (as shown by Sirius red/Fast green staining), cytochrome P450 mediated enzyme activity, or specifically, CYP 1A messenger RNA is decreased. We then investigated a transgenic mouse, deficient in the arylhydrocarbon hydroxylase receptor (AHR), which has undetectable levels of CYP 1A messenger RNA. We quantitated the collagen in liver sections obtained from AHR knockout mice compared to controls, as an indication of the presence of hepatic fibrosis. Collagen concentration was significantly increased by 53% (P<0.0005) in sections from Ahr-/- (knockout) mice compared to wild-type controls. Collagen in livers of the Ahr+/- heterozygous mice was not different from wild-type controls. The increase in collagen concentration in liver sections is an indication of fibrosis in Ahr-/- mice. Collagen protein deposition was also elevated in liver sections from bile duct ligated rats (by 44%) compared to sham operated controls, was elevated in liver sections from heterologous serum induced fibrosis in rats (33%) compared to controls, and was elevated in liver sections from yellow phosphorous induced hepatic fibrosis (74%) compared to vehicle treated controls. In conclusion, these results indicate that cytochrome P450 and specific subtypes of P450, the CYP 1A subgroup, are significantly reduced in three experimental models of hepatic fibrosis when there is evidence of increased collagen deposition in the livers. These results also indicate that mice that are deficient in CYP 1A have elevated levels of hepatic collagen protein, an indication of hepatic fibrosis.

Journal Article↗
PubMed2000

Potassium-induced apoptosis in rat cerebellar granule cells involves cell-cycle blockade at the G1/S transition.

The role of regulators controlling the G1/S transition of the cell cycle was analyzed during neuronal apoptosis in post-mitotic cerebellar granule cells in an attempt to identify common mechanisms of control with transformed cells. Cyclin D1 and its associated kinase activity CDK4 (cyclin-dependent kinase 4) are major regulators of the G1/S transition. Whereas cyclin D1 is the regulatory subunit of the complex, CDK4 represents the catalytic domain that, once activated, will phosphorylate downstream targets such as the retinoblastoma protein, allowing cell-cycle progression. Apoptosis was induced in rat cerebellar granule cells by depleting potassium in presence of serum. Western-blot analyses were performed and protein kinase activities were measured. As apoptosis proceeded, loss in cell viability was coincident with a significant increase in cyclin D1 protein levels, whereas CDK4 expression remained essentially constant. Synchronized to cyclin D1 accumulation, cyclin-dependent kinase inhibitor p27Kip1 drastically dropped to 20% normal values. Cyclin D1/CDK4-dependent kinase activity increased early during apoptosis, reaching a maximum at 9-12 h and decreasing to very low levels by 48 h. Cyclin E, a major downstream target of cyclin D1, decreased concomitantly to the reduction in cyclin D1/CDK4-dependent kinase activity. We suggest that neuronal apoptosis takes place through functional alteration of proteins involved in the control of the G1/S transition of the cell cycle. Thus, apoptosis in post-mitotic neurons could result from a failed attempt to re-enter cell cycle in response to extracellular conditions affecting cell viability and it could involve mechanisms similar to those that promote proliferation in transformed cells.

Animals↗
PubMed2000

Altered cell cycle control at the G(2)/M phases in aryl hydrocarbon receptor-null embryo fibroblast.

The aryl hydrocarbon receptor (AHR) is known to mediate the toxic and carcinogenic effects of polycyclic aromatic hydrocarbons and dioxins. High-affinity AHR ligands, such as 2,3,7, 8-tetrachlorodibenzeno-p-dioxin, have been shown to modify cell proliferation and differentiation. However, the mechanisms by which AHR affects cell proliferation and differentiation are not fully understood. To investigate the role of AHR in cell proliferation, mouse embryonic fibroblasts (MEFs) derived from AHR-null mice were obtained and characterized. Compared with wild-type MEFs, AHR-null cells exhibited a lower proliferation rate with an accumulation of 4N DNA content and increased apoptosis. The expression levels of Cdc2 and Plk, two kinases important for G(2)/M phase of cell cycle, were down-regulated in AHR-null MEFs. In contrast, transforming growth factor-beta (TGF-beta), a proliferation inhibitor in several cell lines, was present at high levels in conditioned medium from AHR-null MEFs. Concomitant with G(2)/M cell accumulation, treatment of wild-type MEFs with TGF-beta3 also resulted in down-regulation of both Cdc2 and Plk. Thus, overproduction of TGF-beta in AHR-deficient cells appears to be the primary factor that causes low proliferation rates and increased apoptosis. Taken together, these results suggest that AHR influences TGF-beta production, leading to an alteration in cell cycle control.

Animals↗
PubMed1999

Targeted disruption of the microsomal epoxide hydrolase gene. Microsomal epoxide hydrolase is required for the carcinogenic activity of 7,12-dimethylbenz[a]anthracene.

Microsomal epoxide hydrolase (mEH) is a conserved enzyme that is known to hydrolyze many drugs and carcinogens, and a few endogenous steroids and bile acids. mEH-null mice were produced and found to be fertile and have no phenotypic abnormalities thus indicating that mEH is not critical for reproduction and physiological homeostasis. mEH has also been implicated in participating in the metabolic activation of polycyclic aromatic hydrocarbon carcinogens. Embryonic fibroblast derived from the mEH-null mice were unable to produce the proximate carcinogenic metabolite of 7,12-dimethylbenz[a]anthracene (DMBA), a widely studied experimental prototype for the polycylic aromatic hydrocarbon class of chemical carcinogens. They were also resistant to DMBA-mediated toxicity. Using the two-stage initiation-promotion skin cancer bioassay, the mEH-null mice were found to be highly resistant to DMBA-induced carcinogenesis. In a complete carcinogenesis bioassay, the mEH mice were totally resistant to tumorigenesis. These data establish in an intact animal model that mEH is a key genetic determinant in DMBA carcinogenesis through its role in production of the ultimate carcinogenic metabolite of DMBA, the 3,4-diol-1,2-epoxide.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1999

Expression of CYP2A genes in human liver and extrahepatic tissues.

Members of the human cytochrome P450 2A (CYP2A) subfamily are known to metabolize several promutagens, procarcinogens, and pharmaceuticals. In this study, the expression of the three genes found in the human CYP2A gene cluster was investigated in the liver and several extrahepatic tissues by gene-specific reverse transcriptase-polymerase chain reaction (RT-PCR). All three transcripts (CYP2A6, CYP2A7, and CYP2A13) were found to be present in liver. Quantitative RT-PCR analysis showed that CYP2A6 and CYP2A7 mRNAs were present at roughly equal levels in the liver, while CYP2A13 was expressed at very low levels. Two putative splicing variants of CYP2A7 were found in the liver. Nasal mucosa contained a low level of CYP2A6 and a relatively high level of CYP2A13 transcripts. Kidney, duodenum, lung, alveolar macrophages, peripheral lymphocytes, placenta, and uterine endometrium were negative for all transcripts. This survey gives a comprehensive picture of the expression pattern of CYP2A genes in liver and extrahepatic tissues and constitutes a basis for a search for functional CYP2A forms and their roles in chemical toxicity in liver and nasal mucosa.

Aryl Hydrocarbon Hydroxylases↗
PubMed1999

CYP1A2 is not the primary enzyme responsible for 4-aminobiphenyl-induced hepatocarcinogenesis in mice.

4-Aminobiphenyl (4-ABP), a potent carcinogen in rodents (liver cancer) and human (bladder cancer), is found as an environmental contaminant and in tobacco smoke. Hemoglobin adducts and lung DNA adducts of 4-ABP are found in tobacco smokers. In vitro metabolism studies with human and rat liver microsomes have shown that CYP1A2 is primarily responsible for catalyzing N-hydroxylation, the initial step in the metabolic activation of 4-ABP. To determine whether this P450 is a rate limiting pathway for hepatocarcinogenesis, CYP1A2-null mice were analyzed at 16 months of age and were compared with wild-type mice in their response to 4-ABP using the neonatal mouse bioassay and two different doses of the carcinogen. Overall differences in incidences of hepatocellular adenoma, carcinoma and preneoplastic foci were not significant between either genotypes or 4-ABP doses used, whereas small, but significant, differences were found for specific types of foci. These results suggest that while CYP1A2 levels may not be rate limiting for 4-ABP metabolism to produce tumors and foci, it may modulate the induction process of some types of liver foci in either a positive or negative manner. In vitro studies using CYP1A2-null and wild-type mouse liver microsomes revealed that CYP1A2 is not the sole P450 required for 4-ABP N-hydroxylation and that another, yet to be identified, P450 is likely to be involved.

Adenoma↗
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

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

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

Nomenclature for human DPYD alleles.

To standardize DPYD 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 each distinct allele be designed by DPYD followed by an asterisk and an Arabic numeral. The number specifies the key mutation and, where appropriate, a letter following the number indicates an additional mutation on the mutant allele. Criteria for classification as a distinct allele are also presented.

Alleles↗
PubMed1998

Aryl-hydrocarbon receptor is an inhibitory regulator of lipid synthesis and of commitment to adipogenesis.

The aryl-hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates the biological effects of 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD). In mouse embryo fibroblasts, TCDD activates expression of multiple genes, including CYP1B1, the predominant cytochrome P450 expressed in these cells. Here, we analyze constitutive functions of the AhR in primary mouse embryo fibroblasts (MEFs) and spontaneously immortalized MEF cell lines derived from wild-type (WT) C57BL/6 mice and also from congenic mice with a targeted disruption of the AhR gene (AhR-/-). After multiple passages, primary MEFs exhibit spontaneous differentiation, growth cessation and senescence. Eventually, colonies of immortalized MEFs arise to provide clonal lines. The senescent phase occurs much earlier for AhR-/- MEFs, while immortalization is substantially delayed. Comparison of AhR-/- and WT MEFs also indicates that constitutive AhR activity is required for basal expression of CYP1B1 and suppresses lipogenesis in subconfluent cultures. Primary WT and AhR-/- MEFs and the corresponding lines undergo adipogenesis when treated at confluence with the appropriate hormonal inducers. Addition of TCDD before or concurrent with hormonal induction suppressed PPAR gamma mRNA and adipogenesis, as measured by lipid accumulation, glycerol phosphate dehydrogenase activity and stearoyl CoA desaturase type 1 mRNA expression. This effect of TCDD treatment was absent in AhR-/- MEFs, establishing the role of AhR in hormone-induced adipogenesis. Such hormonal activation of confluent MEFs and preadipocytes results in a limited proliferative expansion followed by irreversible growth arrest. TCDD-treated MEFs undergo the mitotic expansion but fail to exit the cell cycle. In AhR-/- MEFs, there is no such effect of TCDD. These findings implicate the AhR as a constitutive inhibitor of triglyceride synthesis, and as an early regulator of adipocyte differentiation. AhR interference with cell-cycle arrest in differentiation may be linked to the increased rate of senescence.

3T3 Cells↗
PubMed1998

The aryl hydrocarbon receptor: studies using the AHR-null mice.

The aryl hydrocarbon receptor (AHR) is believed to mediate the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polychlorinated biphenyls, and polycyclic aromatic hydrocarbons. AHR is a member of the Per, ARNT, Sim/basic-helix-loop-helix superfamily of ligand-activated transcription factors that also harbors the transcription factors involved in the hypoxia response, development of the central nervous system, and day-night adaptations. To investigate the role of AHR in chemical toxicity and carcinogenesis and to determine any possible function in mammalian development and physiological homeostasis, AHR-null mice were developed. The AHR-null mice were resistant to the acute toxicity of TCDD and had an altered teratogenic response to this compound. These mice were found to have a number of abnormal phenotypes, thus confirming that AHR plays an important developmental and physiological role. Among the most consistent phenotypes was an altered liver pathology that was associated with accelerated rates of apoptosis. Evidence suggests that this may be related to an abnormal accumulation of levels of hepatic retinoic acid that cause an activation of transforming growth factor beta, resulting in stimulation of apoptosis. AHR may directly or indirectly control levels of a cytochrome P450 that is responsible for catabolizing retinoic acid.

Animals↗
PubMed1997

Selenocysteine tRNA[Ser]Sec levels and selenium-dependent glutathione peroxidase activity in mouse embryonic stem cells heterozygous for a targeted mutation in the tRNA[Ser]Sec gene.

To investigate the effect of a reduced level of selenocysteine (Sec) tRNA[Ser]Sec in selenoprotein biosynthesis, two mouse embryonic stem (ES) cell lines heterozygous for the corresponding gene were generated by homologous recombination of the host genome with targeting vectors encoding a deleted or a disrupted tRNA[Ser]Sec gene. The presence of a single functional gene in ES cells afforded us an opportunity to determine directly in the cell line the effect of reduced gene dosage on (1) the levels of the Sec tRNA[Ser]Sec population, (2) the distributions of the isoacceptors within the Sec tRNA population, and (3) selenoprotein biosynthesis. We therefore determined the amounts and distributions of the two major tRNA[Ser]Sec isoacceptors, designated mcm5U and mcm5Um, within the Sec tRNA population and determined the activity of the anti-oxidant, selenium-containing glutathione peroxidase (GPx) in the heterozygotes and in wild type cells grown in media with and without added selenium. The level of the Sec tRNA[Ser]Sec population in the heterozygotes was approximately 60% of that of wild type cells grown in media under normal conditions, while the ratio of the mcmU isoacceptor in wild type vs mutant cells was approximately 2:1 and of the mcmUm isoacceptor approximately 1:1. In the presence of media supplemented with selenium, the Sec tRNA[Ser]Sec population increased about 20% in wild type cells and virtually not all in heterozygous cells, and the level of the Sec tRNA[Ser]Sec population was, therefore, approximately 50% of that of wild type cells. GPx activity was indistinguishable among these cell lines in either selenium-supplemented or unsupplemented media, indicating that the resultant changes in tRNA[Ser]Sec levels did not have a measurable effect on GPx biosynthesis.

Animals↗
PubMed1997

Mutations at codon 974 of the DPYD gene are a rare event.

A mutation at codon 974 of the dihydropyrimidine dehydrogenase (DPD) gene was previously described in a cancer patient with undetectable DPD enzyme activity who experienced severe toxicity when treated with 5-fluorouracil. We have studied the frequency of this mutation in 29 Scottish subjects with low DPD enzyme activity and in 274 American subjects. We detected no mutations in the 606 alleles studied and conclude that mutations at codon 974 are a rare event.

Dihydrouracil Dehydrogenase (NADP)↗
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

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↗
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↗