RESUMO
Involvement of cytochrome P450 genes (CYPs) in breast cancer (BCa) may differ between populations, with expression patterns affected by tumorigenesis. This may have an important role in the metabolism of anticancer drugs and in the progression of cancer. The aim of this study was to determine the mRNA expression patterns of four cytochrome P450 genes (CYP2W1, 3A5, 4F11 and 8A1) in Mexican women with breast cancer. Real- time PCR analyses were conducted on 32 sets of human breast tumors and adjacent non-tumor tissues, as well as 20 normal breast tissues. Expression levels were tested for association with clinical and pathological data of patients. We found higher gene expression of CYP2W1, CYP3A5, CYP4F11 in BCa than in adjacent tissues and only low in normal mammary glands in our Mexican population while CYP8A1 was only expressed in BCa and adjacent tissues. We found that Ki67 protein expression was associated with clinicopathological features as well as with CYP2W1, CYP4F11 and CYP8A1 but not with CYP3A5. The results indicated that breast cancer tissues may be better able to metabolize carcinogens and other xenobiotics to active species than normal or adjacent non-tumor tissues.
Assuntos
Neoplasias da Mama/enzimologia , Citocromo P-450 CYP3A/biossíntese , Sistema Enzimático do Citocromo P-450/biossíntese , Neoplasias da Mama/genética , Citocromo P-450 CYP3A/genética , Sistema Enzimático do Citocromo P-450/genética , Família 2 do Citocromo P450 , Família 4 do Citocromo P450 , Feminino , Humanos , Antígeno Ki-67/biossíntese , México , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Primary hepatocytes are widely used in investigating drug metabolism and its toxicological effects. N-Nitrosodiethylamine (NDEA)-induced genotoxicity and cytotoxicity was used in primary cultures of female rat hepatocytes in the presence of phenobarbital (PB). PB pre-treatment (1mM) increased the number of necrotic (2-fold) and apoptotic cells (4-fold) after NDEA treatment (0.21-105 µg/mL). The mitotic indices and the number of micronucleated cells decreased, thus suggesting cytotoxicity. An increased number of chromosomal aberrations were observed after pre-treatment with PB. NDEA-treatment (0.21-21 µg/mL) induced expression of the CYP2B1 and CYP2B2 mRNA and PB treatment alone induced ~6-fold and ~2-fold increases of CYP2B1 and CYP2B2 mRNA, respectively. NDEA treatment following PB exposure increased CYP2B1 mRNA expression under all tested concentrations and also increased CYP2B2 expression at 21 and 105 µg/mL. Our data suggest that the alteration of CYP2B1/2 expression by PB increased the cytotoxicity and genotoxicity of NDEA leading to the final genotoxic metabolite.
Assuntos
Carcinógenos/toxicidade , Sistema Enzimático do Citocromo P-450/biossíntese , Dietilnitrosamina/toxicidade , Hepatócitos/efeitos dos fármacos , Mutagênicos/toxicidade , Fenobarbital/toxicidade , Regulação para Cima/efeitos dos fármacos , Animais , Hidrocarboneto de Aril Hidroxilases/biossíntese , Hidrocarboneto de Aril Hidroxilases/genética , Morte Celular/efeitos dos fármacos , Células Cultivadas , Aberrações Cromossômicas/efeitos dos fármacos , Cocarcinogênese , Citocromo P-450 CYP2B1/biossíntese , Citocromo P-450 CYP2B1/genética , Sistema Enzimático do Citocromo P-450/genética , Indução Enzimática/efeitos dos fármacos , Feminino , Hepatócitos/patologia , Micronúcleos com Defeito Cromossômico/efeitos dos fármacos , Índice Mitótico , RNA Mensageiro/metabolismo , Ratos , Esteroide Hidroxilases/biossíntese , Esteroide Hidroxilases/genéticaRESUMO
Constitutive overexpression of Cyp6g1 and Cyp6a2 genes in DDT-resistant line Oregon-flare of the Drosophila melanogaster wing spot test (SMART) has been reported. Cyp6g1 and Cyp6a2 expression levels were compared against the ß-actin gene in the standard (ST) and high bioactivation (HB) crosses of the Somatic Mutation and Recombination test (SMART) treated with sulforaphane or phenobarbital as the control inductor. The CYP450s' enzymatic activity was determined by overall NADH consumption. The expression levels of both genes and the CYP450s activity was higher in the HB cross. The Cyp6g1 levels were higher than those of Cyp6a2 in both crosses, but lower than the expression of ß-actin. Sulforaphane decreased Cyp6g1 in the HB cross and increased it in the ST cross; Cyp6a2 expression was inhibited in the ST cross. Sulforaphane resulted mutagenic in the ST cross, which could be related to the inhibition of Cyp6a2. Phenobarbital did not modify the Cyp6g1 levels but increased the Cyp6a2 and CYP450s basal activity. Although the transcript levels were always higher in the HB cross than in the ST, the expression of Cyp6a2 and Cyp6g1 was not constitutive and was independent one from the other. Sulforaphane modulated both genes in a differential way in each cross and, in contrast to its putative protective effect, it resulted to be mutagenic.
Assuntos
Anticarcinógenos/farmacologia , Sistema Enzimático do Citocromo P-450/biossíntese , Proteínas de Drosophila/biossíntese , Mutagênicos , Tiocianatos/farmacologia , Asas de Animais/anatomia & histologia , Actinas/biossíntese , Actinas/genética , Animais , Anticarcinógenos/toxicidade , Cruzamentos Genéticos , Sistema Enzimático do Citocromo P-450/genética , Família 6 do Citocromo P450 , Proteínas de Drosophila/genética , Drosophila melanogaster , Vetores Genéticos , Hipnóticos e Sedativos/toxicidade , Isotiocianatos , Larva/metabolismo , Testes de Mutagenicidade , NAD/metabolismo , Oligonucleotídeos/síntese química , Oligonucleotídeos/genética , Fenobarbital/toxicidade , Recombinação Genética/genética , Sulfóxidos , Tiocianatos/toxicidadeRESUMO
Monocrotaline (MCT) is a pyrrolizidine alkaloid present in the plants of the Crotalaria species that causes cytotoxicity and genotoxicity in animals and humans, and it is hepatically metabolized to the alkylating agent dehydromonocrotaline by cytochrome P-450. The exact cellular and molecular mechanisms of MCT-induced tissue injury remain unclear. We previously demonstrated that dehydromonocrotaline, but not monocrotaline, inhibits the activity of NADH-dehydrogenase at micromolar concentrations in isolated liver mitochondria, an effect associated with significantly reduced ATP synthesis. Impairment of energy metabolism is expected to lead to several alterations in cell metabolism. In this work, the action of different concentrations of monocrotaline (250, 500, and 750microM) on energy metabolism-linked parameters was investigated in isolated perfused rat livers. In the fed state, monocrotaline increased glycogenolysis and glycolysis, whereas in the livers of fasted rats, it decreased gluconeogenesis and urea synthesis from l-alanine. These metabolic alterations were only found in livers of phenobarbital-treated rats, indicating that active metabolites including dehydromonocrotaline were responsible for the observed activity. Our findings indicate that hepatic metabolic changes may be implicated, partly at least, in the hepatotoxicity of monocrotaline in animals and humans.
Assuntos
Carcinógenos/farmacologia , Metabolismo Energético/efeitos dos fármacos , Fígado/metabolismo , Monocrotalina/farmacologia , Alanina/metabolismo , Animais , Sistema Enzimático do Citocromo P-450/biossíntese , Interpretação Estatística de Dados , Gluconeogênese/efeitos dos fármacos , Glicogênio/metabolismo , Glicólise/efeitos dos fármacos , Fígado/efeitos dos fármacos , Circulação Hepática/efeitos dos fármacos , Masculino , Consumo de Oxigênio/efeitos dos fármacos , Fenobarbital/farmacologia , Ácido Pirúvico/metabolismo , Ratos , Ratos Wistar , Ureia/metabolismoRESUMO
We describe the structure, organization, and transcriptional impact of repetitive elements within the lignin-degrading basidiomycete, Phanerochaete chrysosporium. Searches of the P. chrysosporium genome revealed five copies of pce1, a ~1,750-nt non-autonomous, class II element. Alleles encoding a putative glucosyltransferase and a cytochrome P450 harbor pce insertions and produce incomplete transcripts. Class I elements included pcret1, an intact 8.14-kb gypsy-like retrotransposon inserted within a member of the multicopper oxidase gene family. Additionally, we describe a complex insertion of nested transposons within another putative cytochrome P450 gene. The disrupted allele lies within a cluster of >14 genes, all of which encode family 64 cytochrome P450s. Components of the insertion include a disjoint copia-like element, pcret3, the pol domain of a second retroelement, pcret2, and a duplication of an extended ORF of unknown function. As in the case of the pce elements, pcret1 and pcret2/3 insertions are confined to single alleles, transcripts of which are truncated. The corresponding wild-type alleles are apparently unaffected. In aggregate, P. chrysosporium harbors a complex array of repetitive elements, at least five of which directly influence expression of genes within families of structurally related sequences.
Assuntos
Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/genética , Genes Fúngicos , Mutagênese Insercional , Phanerochaete/genética , Retroelementos/genética , Alelos , Sequência de Bases , Sistema Enzimático do Citocromo P-450/biossíntese , Sistema Enzimático do Citocromo P-450/genética , Proteínas Fúngicas/biossíntese , Regulação Enzimológica da Expressão Gênica/genética , Glucosiltransferases/biossíntese , Glucosiltransferases/genética , Lignina/metabolismo , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Phanerochaete/enzimologia , Transcrição GênicaRESUMO
CYP3A4, the predominant cytochrome P450 (P450) expressed in human liver and intestine, contributes to the metabolism of approximately half the drugs in clinical use today. CYP3A4 catalyzes the 6beta-hydroxylation of a number of steroid hormones and is involved in the bioactivation of environmental procarcinogens. The expression of CYP3A4 is affected by several stimuli, including environmental factors such as insecticides and pesticides. The o,p'-1,1,1,-trichloro-2,2-bis (p-chlorophenyl)ethane (DDT) isomer of DDT comprises approximately 20% of technical grade DDT, which is an organochloride pesticide. We have recently shown that o,p'-DDT exposure increases CYP3A4 mRNA levels in HepG2 cells. To determine the mechanism by which o,p'-DDT induces CYP3A4 expression, transactivation and electrophoretic mobility shift assays were carried out, revealing that o,p'-DDT activates the CYP3A4 gene promoter through the pregnane X receptor (PXR). CYP3A4 gene promoter activation resulted in both an increase in CYP3A4 mRNA levels and an increase in the total CYP3A4 activity in HepG2 cells. We also observed induction of CYP3A4 and mouse Cyp3a11 mRNA in the intestine of CYP3A4-transgenic mice after exposure to 1 mg/kg o,p'-DDT. At higher doses, a decrease of CYP3A4 inducibility was observed together with an increase in levels of interleukin 6 mRNA, a proinflammatory cytokine that strongly represses CYP3A4 transcription. The present study indicates that regulation of other genes under PXR control may be altered by o,p'-DDT exposure.
Assuntos
Sistema Enzimático do Citocromo P-450/biossíntese , DDT/farmacologia , Praguicidas/farmacologia , Receptores de Esteroides/metabolismo , Animais , Linhagem Celular Tumoral , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Duodeno/metabolismo , Indução Enzimática/efeitos dos fármacos , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Receptor de Pregnano X , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Receptores de Esteroides/genética , Testosterona/metabolismoRESUMO
Hexaclorobenzene (HCB), one of the most persistent environmental pollutants, can cause a wide range of toxic effects including cancer in animals, and hepatotoxicity and porphyria both in humans and animals. In the present study, liver microsomal cytochrome P450 (CYP)-dependent arachidonic acid (AA) metabolism, hepatic PGE production, and cytosolic phospholipase A2 (cPLA2) activity were investigated in an experimental model of porphyria cutanea tarda induced by HCB. Female Wistar rats were treated with a single daily dose of HCB (100 mg kg(-1) body weight) for 5 days and were sacrificed 3, 10, 17, and 52 days after the last dose. HCB treatment induced the accumulation of hepatic porphyrins from day 17 and increased the activities of liver ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD), and aminopyrine N-demethylase (APND) from day 3 after the last dose. Liver microsomes from control and HCB-treated rats generated, in the presence of NADPH, hydroxyeicosatetraenoic acids (HETEs), epoxyeicosatrienoic acids (EETs), 11,12-Di HETE, and omega-OH/omega-1-OH AA. HCB treatment caused an increase in total NADPH CYP-dependent AA metabolism, with a higher response at 3 days after the last HCB dose than at the other time points studied. In addition, HCB treatment markedly enhanced PGE production and release in liver slices. This HCB effect was time dependent and reached its highest level after 10 days. At this time cPLA2 activity was shown to be increased. Unexpectedly, HCB produced a significant decrease in cPLA2 activity on the 17th and 52nd day. Our results demonstrated for the first time that HCB induces both the cyclooxygenase and CYP-dependent AA metabolism. The effects of HCB on AA metabolism were previous to the onset of a marked porphyria and might contribute to different aspects of HCB-induced liver toxicity such as alterations of membrane fluidity and membrane-bound protein function. Observations also suggested that a possible role of cPLA2 in the early increase of AA metabolism cannot be excluded. However, the existence of other pathway(s) for metabolizable AA generation different from cPLA2 activation is also proposed.
Assuntos
Ácido Araquidônico/antagonistas & inibidores , Ácido Araquidônico/metabolismo , Hexaclorobenzeno/efeitos adversos , Fígado/química , Fígado/metabolismo , Animais , Ácido Araquidônico/química , Citocromo P-450 CYP1A1/biossíntese , Sistema Enzimático do Citocromo P-450/biossíntese , Modelos Animais de Doenças , Feminino , Hexaclorobenzeno/administração & dosagem , Ácidos Hidroxieicosatetraenoicos/biossíntese , Intubação Gastrointestinal , Métodos , Microssomos Hepáticos/química , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , NADP/metabolismo , Oxirredutases/biossíntese , Fosfolipases A/metabolismo , Fosfolipases A2 , Porfirias Hepáticas/induzido quimicamente , Prostaglandinas E/biossíntese , Ratos , Ratos Wistar , Fatores de TempoRESUMO
Technical grade DDT, a commonly used pesticide, contains 20% of the o,p'-DDT isomer. It alters the activity of hepatic mixed function oxidase and induces expression of cytochrome P450 such as 3A subfamilies. In the present study, we analyzed the effects of o,p'-DDT on CYP3A4 gene expression in HepG2 cells. Treatment with o,p'-DDT dose-dependently increased levels of CYP3A4 mRNA and the immunoreactive protein, by 13- and 3.8-fold, respectively, relative to untreated cultures. Treatment with actinomycin D blocked the o,p'-DDT-induced increase in CYP3A4 mRNA levels. These findings demonstrate that this insecticide is able to induce CYP3A4 transcription and may play an important role in the modulation of endogenous hormones and xenobiotic metabolism.
Assuntos
Sistema Enzimático do Citocromo P-450/biossíntese , DDT/farmacologia , Estrogênios não Esteroides/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Citocromo P-450 CYP3A , Relação Dose-Resposta a Droga , Humanos , Neoplasias Hepáticas/patologia , Transcrição Gênica , Células Tumorais CultivadasRESUMO
The effect on liver cytochrome P450 (CYP) by i.p. injections of methoxychlor (MXC) in corn oil at 0, 100, 150, 200 or 250 mg/kg twice daily for 3 days was investigated in adult male and female Wistar rats. The MXC injection (100 mg/kg b.w.) caused a similar increase of total CYP content in males and females as compared with controls who received the vehicle only. In males, this increase continued up to 250 mg/kg. As to the induction of specific CYP activities, the effect of MXC was found to be sex dependent with three different patterns. Males showed the greatest increases of ethoxy- and methoxyresorufin-O-dealkylase (EROD and MROD, respectively), two CYP1A1/1A2-related activities. On the contrary, females were more responsive than males for pentoxyresorufin-O-dealkylase (PROD) and benzyloxyresorufin-O-dearylase (BROD), two CYP2B-related activities. Finally, p-nitrophenol hydroxylase (PNPH), a CYP2E1-related activity, showed a similar small, although statistically significant, increase for both sexes. As to CYP apoprotein levels, CYP1A1 and CYP2B1/2B2 showed greater increases in females than in males; whereas, interestingly, CYP2E1 induction was higher in males than in females. These results indicate overall that gender modulates CYP expression after MXC injection both qualitatively and quantitatively, and, therefore, this pesticide is not a pure PB inducer. Moreover, the statistically significant increase of CYP3A2 apoprotein expression observed in females and also, to a lower extent, in males, and the decrease of CYP2C11 apoprotein found in males, two sex-related enzymes, may explain the reported endocrine disrupting effect of MXC. The relevance of the different patterns of rat liver CYP induction observed after MXC treatment, in relationship to the speculated endocrine disrupting potential of MXC in humans potentially exposed to this pesticide, needs further investigation.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Inseticidas/farmacologia , Fígado/enzimologia , Metoxicloro/farmacologia , Animais , Western Blotting , Citocromo P-450 CYP1A1/biossíntese , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP2B1/biossíntese , Citocromo P-450 CYP2B1/metabolismo , Citocromo P-450 CYP2E1/biossíntese , Citocromo P-450 CYP2E1/metabolismo , Sistema Enzimático do Citocromo P-450/biossíntese , Indução Enzimática/efeitos dos fármacos , Feminino , Indicadores e Reagentes , Inseticidas/toxicidade , Isoenzimas/metabolismo , Fígado/efeitos dos fármacos , Masculino , Metoxicloro/toxicidade , Oxirredutases/biossíntese , Oxirredutases/metabolismo , Ratos , Ratos Wistar , Caracteres SexuaisRESUMO
CYP3A4, the predominant cytochrome P450 (CYP) expressed in human liver, contributes to the metabolism of approximately half the drugs in use today. In general, human-derived cell lines fail to express CYPs. It was previously shown that CYP3A4 mRNA and CYP3A immunoreactive protein are induced by 1alpha,25-dyhydroxyvitamin D(3) (1alpha,25-(OH)(2)D(3)) in the human colon carcinoma cell line Caco-2. The aim of the present study was to examine whether 1alpha,25-(OH)(2)D(3) regulates CYP3A4 gene expression in HepG2 cells, a human hepatocarcinoma cell line. Treatment with 1alpha,25-(OH)(2)D(3) resulted in an induction of CYP3A4 mRNA and CYP3A4 immunoreactive protein, 1.5-fold and 4.0-fold respectively, when compared to control cultures, in a time-dependent fashion. These observations are in agreement with previous reports suggesting a role of 1alpha,25-(OH)(2)D(3) on CYP3A4 transcription regulation, and demonstrate that this hormone, as in Caco-2 cells, increase CYP3A4 levels in HepG2 cells. In conclusion, HepG2 cell cultures treated with 1alpha,25-(OH)(2)D(3), provides a useful model to study the function of CYP3A4 and its role in drug liver metabolism.