RESUMO
The nitrofuran antimicrobial agent, furazolidone (FZ), is still used in veterinary medicine in some countries in the Middle and Far Eastern countries. The present study aimed to investigate the effects of successive bolus doses of FZ and its metabolite 3-amino-2-oxazolidinone (AOZ) on cytochrome P450 (CYP)-related activities in the livers of rats and chickens. Female Wistar rats and white Leghorn chickens were orally administered FZ once a day for 4 consecutive days. FZ-treated chickens showed an increase in multiple CYP-related activities, however, rats treated with FZ did not show these changes. In chickens, treatment with FZ also induced production of microsomal CYP2C6-like apoprotein. The present study demonstrated that FZ caused a multiple-type induction of CYP-related activities in chickens, but not in rats.
Assuntos
Galinhas/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Furazolidona/farmacologia , Hidrazinas/farmacologia , Fígado/metabolismo , Oxazolidinonas/farmacologia , Animais , Western Blotting/veterinária , Citocromo P-450 CYP1A1/análise , Citocromo P-450 CYP2B1/análise , Sistema Enzimático do Citocromo P-450/análise , Feminino , Furazolidona/administração & dosagem , Hidrazinas/administração & dosagem , Fígado/enzimologia , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismo , Oxazolidinonas/administração & dosagem , Ratos , Ratos Wistar , Sono/efeitos dos fármacosRESUMO
Pleurotus cornucopiae (PC) mushrooms are found in the field and commonly known in Japan as Tamogidake mushrooms. The present study investigated the protective effects of an aqueous extract of PC on carbon tetrachloride (CCl4)-induced hepatotoxicity and the possible mechanism involved in this protection including cytochrome P450 (CYP) 2E1. Wistar rats were pretreated with aqueous extracts of PC (0, 100, 200, and 400 mg/kg) orally for 8 days prior to the intraperitoneal administration of a single dose of CCl4 (0.5 ml/kg) or corn oil. Pretreatment with PC mushroom extract significantly prevented the increased serum enzyme activities of alanine and aspartate aminotransferases in a dose-dependent manner, and suppressed the expression of CYP2E1. PC mushroom extract also protected hepatocytes from the damage effects of CCl4 as remarked by histological and electromicroscopical findings. It was concluded that repeated daily doses of aqueous extracts of PC mushroom reduced the toxic effects exerted by CCl4 on the liver.
Assuntos
Agaricales , Intoxicação por Tetracloreto de Carbono/prevenção & controle , Tetracloreto de Carbono/toxicidade , Fígado/patologia , Extratos Vegetais/uso terapêutico , Pleurotus , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Citocromo P-450 CYP2E1/genética , Primers do DNA , Amplificação de Genes , Fígado/efeitos dos fármacos , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Peroxynitrite formed by the reaction of superoxide and nitric oxide is a highly reactive species with a role in various pathological processes such as cancer, chronic inflammation, and cardiovascular and neurological diseases. In the present study, the effect of the carotenoids, lycopene and beta-carotene, on peroxynitrite-mediated modifications in plasmid DNA as well as cellular DNA and proteins were investigated. In pUC18 plasmid DNA, these carotenoids strongly inhibited DNA strand breaks caused by peroxynitrite generated from 3-morpholinosydnonimine (SIN-1). SIN-1 was also used to determine effects on DNA damage and protein tyrosine nitration in Chinese hamster lung fibroblasts. SIN-1 dose-dependently increased nitration of proteins in cells above basal levels as determined by Western blotting. This nitration was inhibited in the presence of the uric acid as well as lycopene. Physiological concentrations (0.31-10 microM) of lycopene and beta-carotene also had protective effects on DNA damage, as measured by the comet assay. Lycopene significantly reduced DNA damage particularly, in the median range of concentrations (2.5 microM). The protective effects of lycopene and beta-carotene could be due to their scavenging of reactive oxygen (ROS) and/or nitrogen species (RNS) as they reduce the amount of intracellular ROS/RNS produced following treatment with SIN-1 by as much as 47.5% and 42.4%, respectively. The results obtained in this study suggest that carotenoids may alleviate some of the deleterious effects of peroxynitrite and possibly other reactive nitrogen species as well in vivo.
Assuntos
Antioxidantes/farmacologia , Carotenoides/farmacologia , Dano ao DNA/efeitos dos fármacos , Ácido Peroxinitroso/toxicidade , beta Caroteno/farmacologia , Animais , Linhagem Celular , Sobrevivência Celular , Ensaio Cometa , Cricetinae , Cricetulus , Licopeno , Molsidomina/análogos & derivados , Molsidomina/toxicidade , Doadores de Óxido Nítrico/toxicidade , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
Equilenin is one of 10 kinds of estrogens that are found in pregnant mares' urine. It has been used extensively for estrogen replacement therapy in postmenopausal women. Typical inducers of the cytochrome P4501A1 (CYP1A1), such as TCDD, benzo(a)pyrene (B(a)P) and 3-methylcholanthrene, have a planar molecular structure in common and bind to the aryl hydrocarbon receptor (AhR). The structure of equilenin differs from classic estrogens by the presence of two additional double bonds in ring B of the steroid nucleus, and it is planar. This structural similarity of equilenin to the typical AhR agonist prompted us to investigate the capability of equilenin to induce CYP1A1 expression. Administration of equilenin to two mouse strains (C57BL and DBA) that exhibit different degrees of responsiveness to an Ah-receptor agonist and showed that equilenin was capable of dose-dependently increasing both the ethoxyresorufin O-deethylase activity and CYP1a proteins in both strains of mice. Equilenin also induced CYP1A1 mRNA in treated HepG2 cell lines and transcriptional activity in an XRE-directed luciferase reporter gene. Competitive binding studies using C57BL AhR indicated equilenin weakly displaced (3)H-B(a)P from AhR. Together, these data show that equilenin, an equine steroid hormone, served as an AhR ligand in the present study.
Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Citocromo P-450 CYP1A1/biossíntese , Equilenina/farmacologia , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Ligação Competitiva , Carcinoma Hepatocelular/enzimologia , Citocromo P-450 CYP1A1/genética , Indução Enzimática/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica , Cavalos , Humanos , Rim/efeitos dos fármacos , Rim/enzimologia , Ligantes , Luciferases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Gênica , Ativação Transcricional/efeitos dos fármacosRESUMO
D-Mannitol, one of the main phytochemicals of the edible Tamogi-take mushroom (Pleurotus cornucopiae), was found to inhibit an angiotensin I converting enzyme (ACE). The antihypertensive effect of D-mannitol and a hot water extract of Tamogi-take mushroom was demonstrated in spontaneously hypertensive rats (SHR) by oral administration.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Manitol/farmacologia , Pleurotus/química , Animais , Ratos , Ratos Endogâmicos SHRRESUMO
Diazepam was metabolized to three primary metabolites, 3-hydroxy-diazepam, N-desmethyl-diazepam, and p-hydroxy-diazepam. Our previous studies reported metabolic position-specific inter- or intrastrain differences in diazepam metabolism among Sprague-Dawley, Brown Norway, Dark Agouti, and Wistar rats. Especially, there were marked ( approximately 300 fold) inter- or intrastrain differences in diazepam p-hydroxylation activity at low concentration of substrate. In this study, we investigated the enzyme that catalyzes diazepam p-hydroxylation. The activity toward diazepam p-hydroxylation was inhibited by anti-cytochrome P450 2D (CYP2D) antibody, suggesting that this activity was catalyzed by CYP2D isoforms. Comparing the expression levels of the CYP2D subfamily in liver microsomes from various strains of rats using anti-CYP2D2 antibody, we found that there was a band of protein that was consistent with the phenotype of diazepam p-hydroxylation. N-terminal amino acid sequences of the specific protein exactly corresponded to those of CYP2D3, indicating that CYP2D3 might be involved in diazepam p-hydroxylation. Moreover, using rat CYP2D isoforms expressed in yeast, we tested CYP2Ds to catalyze diazepam p-hydroxylation. CYP2D1 and CYP2D2 practically did not participate in diazepam metabolism. On the other hand, diazepam p-hydroxylation was catalyzed by CYP2D3. CYP2D4 had high activity toward diazepam N-desmethylation, but not p-hydroxylation. In conclusion, the polymorphic expression of CYP2D3 caused the inter- or intrastrain differences in diazepam p-hydroxylation among rat strains or individuals.
Assuntos
Oxirredutases do Álcool/genética , Sistema Enzimático do Citocromo P-450/genética , Diazepam/metabolismo , Oxigenases de Função Mista/genética , Polimorfismo Genético , Oxirredutases do Álcool/química , Oxirredutases do Álcool/metabolismo , Sequência de Aminoácidos , Animais , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Família 2 do Citocromo P450 , Hidroxilação , Cinética , Masculino , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismo , Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , Dados de Sequência Molecular , Ratos , Ratos Wistar , Proteínas Recombinantes/metabolismo , Especificidade da EspécieRESUMO
Catechol estrogens (CEs), such as 4-hydroxyestradiol (4-OHE2), undergo redox cycling during which reactive oxygen species (ROS) such as superoxide (O2*-) and the chemically reactive estrogen semiquinone (CE-SQ) and quinone (CE-Q) intermediates are produced. The quinone's putative mutagenicity may be enhanced by ROS and/or reactive nitrogen species. High concentrations of nitric oxide (NO) present during inflammatory conditions may react with (O2*-) to form peroxynitrite (ONOO-), a potent oxidant implicated in many pathological conditions. In this study, the possible generation of peroxynitrite from the interaction of CEs and NO and its effect on plasmid DNA and intact cells were investigated. A combination of 4-OHE2 and NO increased the level of single strand breaks (SSB) in plasmid DNA by more than 60% compared to vehicle controls in a metal-free buffer system. 4-OHE2 alone or NO alone had no effect. Results obtained from use of different antioxidants and ROS scavengers suggested a role of peroxynitrite in oxidative stress. In cells, 4-OHE2 or NO alone induced dose-dependent DNA damage as assessed by single cell gel electrophoresis. Co-treatment with 4-OHE2 and NO had an additive effect at lower doses. Generation of intracellular ROS was measured by the oxidation of carboxy-2',7'-dichlorofluorescein diacetate to the fluorescent compound carboxy-2',7'-dichlorofluorescein. NO alone, in oxygenated media, generated little ROS whereas 4-OHE2 produced approximately 70% increase in fluorescence. When added together 4-OHE2 and NO, produced a 2-fold increase in ROS. The generation and involvement ofperoxynitrite to this increase was implied since uric acid inhibited it. Generation ofperoxynitrite was also observed by use of dihydrorhodamine 123. Therefore, we conclude that combined treatments with 4-OHE2 and NO generated peroxynitrite seen from increased fluorescence and its inhibition by uric acid or combined SOD and catalase treatments. Results reported here suggest a role of peroxynitrite in causing damage to biomolecules when CEs and NO are present simultaneously. This may have biological relevance as high concentrations of NO formed during inflammatory conditions may exacerbate cancers due to estrogens.
Assuntos
Estrogênios de Catecol/metabolismo , Óxido Nítrico/metabolismo , Estresse Oxidativo/fisiologia , Ácido Peroxinitroso/biossíntese , Neoplasias da Próstata/metabolismo , Antioxidantes/farmacologia , Linhagem Celular Tumoral , Ensaio Cometa , Dano ao DNA/efeitos dos fármacos , Humanos , Masculino , Plasmídeos , Neoplasias da Próstata/fisiopatologia , Espécies Reativas de Oxigênio/análiseRESUMO
The consumption of fruits and vegetables is associated with a reduced risk of various ailments, including cancer and cardiovascular diseases. Carotenoids, such as lycopene and beta-carotene, are natural constituents of edible plants and may protect against disease. In this study, the influence of lycopene and beta-carotene on DNA damage caused by catechol-estrogens in vitro is examined. One possible mechanism by which catechol estrogens such as 4-hydroxyestradiol (4-OHE2) and 2-hydroxyestradiol, which cause DNA damage in naked plasmid DNA as well as in cells, contributing to the process of carcinogenesis, is through the generation of reactive oxygen species. It was found that both carotenoids at concentrations ranging from 0.25 to 10 microM significantly inhibit strand breakage induced by 4-OHE2/copper sulphate by up to approximately 90% in plasmid DNA with beta-carotene being slightly more effective. No prooxidant or cytotoxic effects were observed at the concentrations tested. These carotenoids had a similar, though reduced effect on DNA damage as measured by the comet assay, in Chinese hamster lung fibroblasts. The results obtained show that both lycopene and beta-carotene, most probably and mainly through their potent antioxidant properties, are able to inhibit catechol-estrogen-mediated DNA damage.
Assuntos
Antioxidantes/farmacologia , Carotenoides/farmacologia , Dano ao DNA/efeitos dos fármacos , Estradiol/análogos & derivados , Antagonistas de Estrogênios/farmacologia , beta Caroteno/farmacologia , Animais , Antimutagênicos , Linhagem Celular , Ensaio Cometa , Cricetinae , Cricetulus , Estradiol/toxicidade , Estrogênios de Catecol , Licopeno , Estresse OxidativoRESUMO
Pleurotus cornucopiae (PC) mushroom with a brilliant yellow pileus is found in the field and known in Japan as Tamogi dake mushroom. The purpose of this paper is to investigate the mechanism of the antimutagenic effect of PC mushroom using both the Ames test and Comet assay. We have found a strong inhibitory effect of both aqueous and organic PC extracts on the mutagenicity elicited by benzo[a]pyrene (B[a]P). This inhibition was dose-dependent in reaction mixtures containing cytosolic and microsomal fractions (S-9) from untreated rat liver as well as in those containing S-9 from aryl hydrocarbon receptor (Ah) ligand of Sudan III-treated rats. Sudan III was a potent inducer of cytochrome P450 1A (CYP1A) activity. We treated rats with Sudan III to enhance the metabolic activation of B[a]P by the S-9 fraction. To explain whether this antimutagenicity was due to the inhibition of CYP1A activity that metabolically activates B[a]P, we tested the effects of the extracts on activities of CYP1A1 and CYP1A2, represented by ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD), respectively. Both aqueous and organic extracts inhibited EROD activity at all dose levels, while the inhibitory effect was only observed at high doses with regard to MROD activity. Furthermore, pre-treatment of Chinese hamster V79cells with PC extracts significantly reduced H2O2-induced-DNA damage, indicating that PC extracts provide a protective effect against oxidative DNA damage. These results indicate that whole-mushroom extracts contain compounds that may inhibit the metabolic activation of B[a]P by CYP1A1 as well as prevent oxidative DNA damage.
Assuntos
Antimutagênicos , Benzo(a)pireno/antagonistas & inibidores , Dano ao DNA , Pleurotus , Salmonella typhimurium/genética , Animais , Benzo(a)pireno/toxicidade , Produtos Biológicos/farmacologia , Linhagem Celular , Cricetinae , Cricetulus , Dano ao DNA/efeitos dos fármacos , Peróxido de Hidrogênio/antagonistas & inibidores , Testes de Mutagenicidade , Salmonella typhimurium/efeitos dos fármacosRESUMO
Green tea contains catechins and caffeine as major constituents. Treatment of rats with green tea (2.5% w/v) significantly increased 7-ethoxycoumarin O-deethylase (7-ECOD), caffeine N-1 demethylase (CN1D) and UDP-glucuronyltransferase (UGT) activities. Treatment with caffeine similarly activated CYP1A2 and related monooxygenases as well as UGT, while treatment with catechins induced UGT activity but not 7-ECOD or CN1D activity. Numbers of benzo[a]pyrene (BP)-induced revertant colonies in an Ames test (mutation assay) with S. typhimurium TA98 as the test strain were markedly larger when BP was preincubated with the liver S-9 (9000 x g supernatant of liver homogenate) from green tea-treated rats than when preincubated with that from control rats. In a modified Ames assay system in which UGT was activated by the addition of UDP-glucuronic acid to the preincubation mixture, the numbers of revertant colonies in the assay using liver S-9 from green tea-treated rats decreased to a similar level to that in the assay using S-9 from controls. The acceleration of two enzymatic reactions may contribute to the rapid elimination of BP; the first step, the formation of a metabolic intermediate (which is mutagenic) by CYP1A2 and the second, the conjugation of active metabolic intermediates by UGT. We speculated that green tea can reduce the amount of time carcinogens reside in the body and the chance that body tissues will be exposed to active metabolites of carcinogens thorough rapid elimination due to the simultaneous induction of CYP1A2 and UGT activities.
Assuntos
Antimutagênicos , Cafeína/farmacologia , Catequina/análogos & derivados , Chá , Animais , Hidrocarboneto de Aril Hidroxilases/biossíntese , Catequina/farmacologia , Indução Enzimática , Glucuronosiltransferase/metabolismo , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Ratos , Ratos WistarRESUMO
Green tea, one of the most popular beverages consumed in Asian countries, has been reported to possess anticarcinogenic and antimutagenic properties. The aim in this study is to test the radical scavenging effect of catechins and caffeine, which were major components of green tea, and if they really prevent oxygen radical-induced mutagenesis. We used TA102 strain of Salmonella typhimurium which is sensitive to hydroxyl radical in the Ames mutation assay. We found that caffeine did not show any effects on mutagenesis in this system, but catechin significantly reduced mutagenesis or genotoxicity caused by hydroxyl radical. This radical-scavenging action of catechins may indeed contribute to the anticarcinogenic activity of green tea as has been proposed.
Assuntos
Antimutagênicos/farmacologia , Cafeína/farmacologia , Catequina/farmacologia , Salmonella typhimurium/genética , Animais , Sequestradores de Radicais Livres/farmacologia , Radical Hidroxila , Masculino , Testes de Mutagenicidade , Ratos , Ratos Wistar , Salmonella typhimurium/efeitos dos fármacos , Chá , terc-Butil Hidroperóxido/antagonistas & inibidores , terc-Butil Hidroperóxido/toxicidadeRESUMO
Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates a spectrum of toxic and biological effects of 2,3,7,8-tetrachloro dibenzo-p-dioxin (TCDD) and related compounds. Peroxisome proliferator activated receptor alpha (PPARalpha) is a nuclear receptor involved in the maintenance of lipid and glucose homeostasis. In this study we hypothesized that one of the possible mechanisms for the effect of TCDD and its related chemicals on fat metabolism could be through down regulation of PPARalpha functions. We treated Wistar rats with an AhR ligand, Sudan III (S.III), and/or PPARalpha ligand, Clofibric Acid (CA), for 3 days. We analysed the expression of one of the PPARalpha-target gene products, CYP4A protein and its mRNA. We also tested HepG2 cells with the afore-mentioned treatments and evaluated their effects on PPARalpha and RXRalpha protein. Treatment of Wistar rats with S.III was found to down regulates CYP4A protein expression and reduced its induction with CA. It also decreased mRNA expressions of CYP4A1, CYP4A2, CYP4A3 and PPARalpha. In HepG2 cells, PPARalpha and RXRalpha protein expression was decreased by S.III treatment in a dose dependent manner. Our results suggest that AhR has an inhibitory effect on PPARalpha function and a new pathway by which AhR ligands could disturb lipid metabolism.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , PPAR alfa/metabolismo , Receptores de Hidrocarboneto Arílico/fisiologia , Animais , Compostos Azo/farmacologia , Linhagem Celular Tumoral , Ácido Clofíbrico/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Relação Dose-Resposta a Droga , Regulação para Baixo , Humanos , Fígado/metabolismo , Masculino , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores de Hidrocarboneto Arílico/metabolismo , Receptor X Retinoide alfa/metabolismoRESUMO
In order to examine whether iodine supplements may have caused a global decline in sperm concentrations during the past several decades, the synchronicity of the decline in mean sperm counts and the introduction of iodine supplements was analyzed statistically. A positive correlation between the incidence of thyroid disease and sperm counts has been detected in Europe. In addition, it has been shown that sperm counts began falling around 1965 in the United States, 40 years after iodine supplements were introduced. Mean sperm counts before and after 1965 were 111 x 10(6)/ml and 70 x 10(6)/ml, respectively, in calculations weighted by the number of subjects included in each individual publication. The timing of the declines coincided with the introduction of iodine supplements in the United States, France, and the United Kingdom. The implications are that the global decline in sperm concentrations may be caused by iodine intake. Geographical variation in the types of sperm count decline also appears to be present.
Assuntos
Suplementos Nutricionais/estatística & dados numéricos , Iodo/efeitos adversos , Contagem de Espermatozoides , Espermatozoides/fisiologia , Doenças da Glândula Tireoide/tratamento farmacológico , Adulto , Suplementos Nutricionais/efeitos adversos , Europa (Continente)/epidemiologia , Humanos , Iodo/administração & dosagem , Modelos Lineares , Masculino , Sêmen/fisiologia , Contagem de Espermatozoides/estatística & dados numéricos , Espermatozoides/efeitos dos fármacos , Doenças da Glândula Tireoide/epidemiologia , Doenças da Glândula Tireoide/prevenção & controle , Estados Unidos/epidemiologiaRESUMO
Di-(2-ethylhexyl) phthalate (DEHP), a peroxisome proliferator-activated receptor alpha (PPARalpha) ligand, alters the lipid composition of rat testis, yet the mechanism is unclear. In this study, we investigated the effect of DEHP on the synthesis and metabolism of arachidonic acid (AA), a precursor of eicosanoids, in the testis of prepubertal rats. DEHP (100 and 1,000 mg/kg, 5 days) administration caused a significant reduction in activity of cytosolic phospholipase A2 (cPLA2), the rate-limiting enzyme in the AA and eicosanoid synthesis pathways. DEHP increased the expression of 12-lipoxygenase (12-LOX) in rat testis, whereas cyclooxygenase-2 (COX-2) expression was not altered. Cytochrome P450 4A1 (CYP4A1), a product of a PPARalpha-regulated gene, was markedly increased in the testis by DEHP administration. Taken together, DEHP suppresses cPLA2 activity and induces the AA metabolizing enzymes such as 12-LOX and CYP4A1, resulting in the reduction of AA level. These data suggest that altered AA metabolic cascades may be related to the decrease of testosterone concentration in DEHP-induced testicular atrophy.
Assuntos
Ácido Araquidônico/biossíntese , Dietilexilftalato/toxicidade , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Fosfolipases A/metabolismo , Testículo/efeitos dos fármacos , Testículo/patologia , Animais , Araquidonato 12-Lipoxigenase/metabolismo , Atrofia/induzido quimicamente , Cromatografia Líquida de Alta Pressão , Sistema Enzimático do Citocromo P-450/metabolismo , Família 4 do Citocromo P450 , Citosol/metabolismo , Immunoblotting , Masculino , Fosfolipases A2 , Ratos , Ratos WistarRESUMO
Knowledge of strain differences in drug metabolism is important for the selection of animals for pharmacokinetic, pharmacodynamic, and toxicological studies. Hepatic microsomes from Sprague-Dawley (SD) and Brown Norway (BN) rats had 300-fold higher diazepam p-hydroxylation activity than Dark Agouti (DA) and Wistar (W) rats at a low diazepam concentration (3 microM). Kinetic studies indicated that diazepam p-hydroxylation in SD and BN rats proceeded with lower K(m) and higher V(max) values than it did in DA and W rats. However, the expression levels of cytochrome P450 CYP2D1, the reported enzyme for diazepam p-hydroxylation, did not cosegregate with the activity. These results suggest the presence of a new high-affinity diazepam p-hydroxylation enzyme other than CYP2D1 in SD and BN rats. DA rats showed 3- and 2-fold higher diazepam 3-hydroxylation and N-desmethylation activities, respectively, than the other rat strains. In agreement with this, DA rat liver microsomes had a higher expression of CYP3A2, which is responsible for diazepam 3-hydroxylation and partly responsible for N-desmethylation. Values of CL(int) (V(max)/K(m)) indicated that p-hydroxy-diazepam is the major metabolite in SD and BN rats, whereas 3-hydroxy-diazepam is the major metabolite in DA and W rats. The sum of the CL(int) in each strain was in the order of DA > SD = BN >> W. Strain differences in the pharmacodynamics of diazepam between SD and DA rats may be due to these differences in diazepam metabolism. We found that both the rate of elimination of diazepam and the major metabolic pathways in diazepam metabolism differed among the different rat strains due to polymorphic expression of the two enzymes involved in diazepam metabolism.
Assuntos
Diazepam/metabolismo , Polimorfismo Genético/genética , Especificidade da Espécie , Temazepam/análogos & derivados , Oxirredutases do Álcool , Animais , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/imunologia , Hidrocarboneto de Aril Hidroxilases/metabolismo , Western Blotting/métodos , Cromatografia Líquida de Alta Pressão/métodos , Citocromo P-450 CYP3A , Família 2 do Citocromo P450 , Diazepam/antagonistas & inibidores , Diazepam/farmacologia , Hidroxilação/efeitos dos fármacos , Soros Imunes/imunologia , Soros Imunes/metabolismo , Cinética , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , NADP/metabolismo , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos , Ratos Mutantes , Ratos Sprague-Dawley , Ratos Wistar , Esteroide 16-alfa-Hidroxilase/genética , Esteroide 16-alfa-Hidroxilase/imunologia , Esteroide 16-alfa-Hidroxilase/metabolismo , Temazepam/metabolismoRESUMO
Fibrates, hypolipidemic drugs, have been reported to suppress the metabolic activities of cytochrome P450 1A1 and 1A2 in rats but the mechanism has not been elucidated. In the present study we tested the hypothesis that the inhibitory effect of fibrates on arylhydrocarbon receptor (AhR) function may be due to their stimulatory effects on PPARalpha. Sudan III (S.III) treatment induced CYP 1A1 and CYP 1A2 protein expression, mRNA and their metabolic activities, methoxyresorufin-O-demethylase (MROD) and ethoxyresorufin-O-deethylase (EROD), in Wistar rats higher than those in the control. Co-treatment of rats with S.III and clofibric acid (CA) caused a 40-50% decrease in the induced levels of CYP1A1 and CYP1A2 protein, mRNA expression and their metabolic activities and reduced AhR protein expression. When we treated HepG2 cells with S.III and/or CA, no suppressive effect on S.III-induced CYP1A1 protein expression due to CA was found. HepG2 cells were transiently transfected with increasing concentrations of PPARalpha mammalian expression vector and exposed to the same treatment. CA co-treatment with S.III decreased AhR protein and S.III-induced CYP1A1 protein expression with increasing dose of PPARalpha transfected into HepG2 cells. Our results demonstrate that the suppressive effect of fibrates on CYP1A is PPARalpha-dependent and suggest that PPARalpha has an inhibitory effect on AhR function.
Assuntos
Ácido Clofíbrico/toxicidade , Citocromo P-450 CYP1A1/biossíntese , Citocromo P-450 CYP1A2/biossíntese , Fígado/efeitos dos fármacos , PPAR alfa/metabolismo , Administração Oral , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto , Compostos Azo/farmacologia , Western Blotting , Linhagem Celular Tumoral , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Proteínas de Ligação a DNA/biossíntese , Indução Enzimática/efeitos dos fármacos , Humanos , Fígado/enzimologia , Fígado/metabolismo , PPAR alfa/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores de Hidrocarboneto Arílico/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/biossíntese , TransfecçãoRESUMO
We observed variations in the metabolism of diazepam in Wistar rats. We studied these variations carefully, and found that the variations are dimorphic and about 17% of male rats of Wistar strain we examined showed two times higher diazepam metabolic activities in their liver microsomes than the rest of animals at the substrate concentrations less than 5 microM. We classified them as extensive metabolizer (EM) and poor metabolizer (PM) of diazepam. No sex difference was observed in the frequency of appearance of EM. Activities of the primary metabolic pathways of diazepam were examined to elucidate the cause of this polymorphism in male Wistar rats. No significant differences were observed in activities of neither diazepam 3-hydroxylation or N-desmethylation between EM and PM rats, while activity of diazepam p-hydroxylation was markedly (more than 200 times) higher in EM rats, indicating that this reaction is responsible for the polymorphism of diazepam metabolism in Wistar rats. We examined the expression levels of CYP2D1, which was reported to catalyze diazepam p-hydroxylation in Wistar rats to find no differences in the expression levels of CYP2D1 between EM and PM rats. The kinetic study on diazepam metabolism in male Wistar rats revealed that EM rats had markedly higher V(max) and smaller K(m) in diazepam p-hydroxylation than those of PM rats, indicating the presence of high affinity high capacity p-hydroxylase enzyme in EM rats. As a consequence, at low concentrations of diazepam, major pathways of diazepam metabolism were p-hydroxylation and 3-hydroxylation in male EM rats, while in male PM rats, 3-hydroxylation followed by N-desmethylation. Due to this kinetic nature of p-hydroxylase activity, EM rats had markedly higher total CL(int) of diazepam than that of PM rats. Polymorphism in diazepam metabolism in humans is well documented, but this is the first report revealing the presence of the polymorphism in diazepam metabolism in rats. The current results infer polymorphic expression of new diazepam p-hydroxylating enzyme with lower K(m) than CYP2D1 in EM Wistar rats.
Assuntos
Diazepam/metabolismo , Polimorfismo Genético , Animais , Diazepam/farmacologia , Relação Dose-Resposta a Droga , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Polimorfismo Genético/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
Exposure of pubertal rats to di-(2-ethylhexyl) phthalate (DEHP) for 14 days was reported to result in reduced testosterone (T) biosynthesis by altering androstenedione 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activity. However, our study indicated that shorter period exposure of DEHP (100 or 1000 mg/kg for 5 days) to 4-week-old male rats did not affect the activity of 17beta-HSD, the rate-limiting enzyme of T biosynthesis in the testis. Testosterone 5alpha-reductase (T5alpha-R) activity in the testis was significantly enhanced, while aromatase mRNA was significantly reduced by increasing doses of DEHP. The expressions of cytochrome P450 (CYP) isoforms, CYP2C11 and CYP3A, in the testis increased along with their enzymatic activities, T16alpha- and T6beta-hydroxylation, respectively. Thus, the current study clearly indicates that the short period exposure to DEHP alters T metabolism through altering activities of T5alpha-R, aromatase and CYP2C11/3A2 in the testis of prepubertal rats, and that they are more sensitive marker enzymes to the DEHP exposure than those of biosynthetic enzymes of T from androstenedione.
Assuntos
Ácidos Ftálicos/toxicidade , Testículo/efeitos dos fármacos , Testosterona/metabolismo , 17-Hidroxiesteroide Desidrogenases/metabolismo , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/metabolismo , Androstenodiona/metabolismo , Animais , Hidrocarboneto de Aril Hidroxilases/metabolismo , Biomarcadores/análise , Western Blotting , Citocromo P-450 CYP3A , Família 2 do Citocromo P450 , Isoenzimas/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Microssomos/metabolismo , Ácidos Ftálicos/administração & dosagem , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esteroide 16-alfa-Hidroxilase/metabolismo , Testículo/metabolismo , Testosterona/biossíntese , Testosterona/sangueRESUMO
Accumulations of polychlorinated dibenzo-p-dioxins, dibenzofurans and coplanar polychlorinated biphenyls were analyzed in common carp (Cyprinus carpio) collected in the Hikiji River, Kanagawa Prefecture, Japan in which dioxin contaminated effluent was released during the period starting from November 1992 to March 2000. Higher levels of 2,3,7,8-tetrachlorinated dibenzo-p-dioxin toxic equivalents were observed in carps collected downstream to the dioxin release site (contaminated site) than the reference site. Modulations of cytochrome p450 (CYP) enzyme in liver, serum estrogen concentration and gonadal somatic index (GSI) were also measured as biomarkers for the contaminants. Total CYP content in livers was markedly higher in male and female carps from the contaminated site relative to the reference site fish. The expression level of the cytochrome p450 1A and Ethoxyresorufin O-deethylase activity were significantly higher in female carps from the contaminated site than from the reference site. A lower level of plasma estrogen was observed in carps from the contaminated site. The GSI in female carps from the contaminated site was smaller than that recorded at the reference site. The present study indicates that dioxins released to the Hikiji River might induce the CYP enzyme and inhibit the reproductive functions in common carps dwelling downstream from the release site.
Assuntos
Carpas/fisiologia , Dioxinas/efeitos adversos , Ovário/fisiologia , Testículo/fisiologia , Poluentes Químicos da Água/efeitos adversos , Animais , Citocromo P-450 CYP1A1/farmacologia , Dioxinas/farmacocinética , Indução Enzimática , Estrogênios/sangue , Feminino , Japão , Masculino , Ovário/efeitos dos fármacos , Fatores Sexuais , Testículo/efeitos dos fármacos , Poluentes Químicos da Água/farmacocinéticaRESUMO
From 16 largha seals (Phoca largha) and 15 ribbon seals (Phoca fasciata) in the coastal waters of Hokkaido, Japan, blubber chlorinated hydrocarbon (CHC) levels and hepatic cytochrome P450 (CYP) catalytic activities and their immunochemically detected protein content levels were measured. Concentrations of DDTs (2,2-bis(4-chlorophenyl)-1,1-dichloroethylene,p,p'-DDE; 2,2-bis(4-chlorophenyl)-1,1-dichloroethane, p,p'-DDD; dichlorodiphenyltrichloroethane, p,p'-DDT), polychlorinated biphenyl congeners (PCBs), and chlordane compounds (oxychlordane, chlordanes, and nonachlors) in both species were in the range of 290 to 5,300, 420 to 4,000, and 130 to 1,500 ng/g lipid weight, respectively. Aryl hydrocarbon receptor (AhR) agonists, non-ortho (IUPAC 77 and 126) and mono-ortho (IUPAC 105, 118, and 156) coplanar PCB congeners, were also detected, and the 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD) toxic equivalents (TEQs) were 4.9 to 120 pg TEQ/g lipid weight. Cross-reactive proteins with polyclonal antibodies against rat CYP1A1 and CYP3A2 were notably detected in seal liver microsomes. Interestingly, a polyclonal antibody against rat CYP2B1 recognized proteins only at trace levels. In largha seals, both levels of alkoxyresorufin- (methoxy-, ethoxy-, pentoxy-, and benzyloxyresorufin) O-dealkylase (AROD) activities and proteins detected by polyclonal antibodies against rat CYP1A1 were significantly correlated with the concentrations of individual coplanar PCB congeners, total TEQs, and total PCBs. Threshold concentrations for TEQs in blubber of the largha seal to induce hepatic CYP1A protein and EROD activity were estimated to be 8.5 and 19 pg TEQ/g fat weight, respectively. In ribbon seals, similar correlations were not detected, although the TEQ levels were not significantly lower than those in largha seals. These results suggest that AROD activity and CYP1A1 protein in the liver of the largha seal could be a biomarker for the exposure to AhR agonists such as coplanar PCB congeners. This study also indicates differences in AhR-mediated responses to the CHC exposures between largha and ribbon seals.