RESUMEN
Some phthalate esters alter male rat reproductive development during sexual differentiation by interfering with fetal testis maturation resulting in reduced Leydig Cell synthesis of testosterone and insulin-like 3 (Insl3) hormones. Gene transcripts associated with steroid hormone and cholesterol transport, and cholesterol synthesis and lipid metabolism also are reduced. These alterations cause permanent malformations of hormone-dependent tissues, sperm production and fertility in male offspring; effects known as the "Phthalate Syndrome." We have shown that administration of a high dose of 750 mg diisononyl phthalate (750 mg/kg/d DINP) during sex differentiation reduced fetal testis testosterone production (T Prod), testis gene expression and induced a low incidence of reproductive malformations in male rat offspring. In the current study we administered DINP at even higher dose levels (1.0 and 1.5 g/kg/d) from gestational day (GD) 14 to postnatal (PND) 3 to determine if these effects were dose related and if the magnitude of the effects could be predicted from a statistical model of fetal testosterone production (T Prod) and Insl3 mRNA levels. These models were previously developed using dipentyl phthalate (DPeP) data from fetal T Prod and postnatal studies. We found that the severity of the demasculinizing effects on the androgen-dependent organs and gubernaculum by DINP were accurately predicted from the statistical models of fetal T prod and Insl3 mRNA, respectively. Taken together, our results indicate that reductions fetal T prod and Insl3 predict the severity of demasculinizing effects in utero exposure to the phthalates DINP and DPeP regardless of potency.
Asunto(s)
Dietilhexil Ftalato , Ácidos Ftálicos , Ratas , Masculino , Animales , Testosterona/metabolismo , Citocromo P-450 CYP2B1/metabolismo , Citocromo P-450 CYP2B1/farmacología , Ratas Sprague-Dawley , Semen/metabolismo , Ácidos Ftálicos/toxicidad , Ácidos Ftálicos/metabolismo , Testículo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Colesterol/metabolismoRESUMEN
The rate with which crop yields per hectare increase each year is plateauing at the same time that human population growth and other factors increase food demand. Increasing yield potential ( Y p ) of crops is vital to address these challenges. In this review, we explore a component of Y p that has yet to be optimised - that being improvements in the efficiency with which light energy is converted into biomass ( ε c ) via modifications to CO2 fixed per unit quantum of light (α), efficiency of respiratory ATP production ( ε prod ) and efficiency of ATP use ( ε use ). For α, targets include changes in photoprotective machinery, ribulose bisphosphate carboxylase/oxygenase kinetics and photorespiratory pathways. There is also potential for ε prod to be increased via targeted changes to the expression of the alternative oxidase and mitochondrial uncoupling pathways. Similarly, there are possibilities to improve ε use via changes to the ATP costs of phloem loading, nutrient uptake, futile cycles and/or protein/membrane turnover. Recently developed high-throughput measurements of respiration can serve as a proxy for the cumulative energy cost of these processes. There are thus exciting opportunities to use our growing knowledge of factors influencing the efficiency of photosynthesis and respiration to create a step-change in yield potential of globally important crops.
Asunto(s)
Dióxido de Carbono , Productos Agrícolas , Citocromo P-450 CYP2B1 , Adenosina Trifosfato/metabolismo , Dióxido de Carbono/metabolismo , Productos Agrícolas/fisiología , Citocromo P-450 CYP2B1/metabolismo , Fotosíntesis , Ribulosa-Bifosfato Carboxilasa/metabolismoRESUMEN
Constitutive androstane receptor (CAR) is a nuclear receptor that plays a key role in drug metabolism and disposition and in the development of liver tumors in rodents. CAR is activated by ligands and indirect activators, which do not bind to the receptor but activate it through cellular signaling. In this study, we sought to identify direct and indirect activators of rat CAR (rCAR). Assessment of the influence of mutations on the transcriptional activity of rCAR identified a mutant termed rCAR-3A-G354Q that displays low constitutive activity and high ligand responsiveness. Reporter assays using the mutant were performed with compounds that increased the mRNA levels of Cyp2b1, a CAR target gene, in rat primary hepatocytes. Several compounds activated rCAR-3A-G354Q and were implicated as rCAR ligands. Since indirect CAR activators are considered to display little species differences, we then determined CYP2B6 mRNA levels in human hepatocyte-like HepaRG cells after treatment with compounds that increased Cyp2b1 mRNA levels in rat hepatocytes but did not activate rCAR-3A-G354Q. The results demonstrated six compounds as possible rCAR indirect activators. Taken together, the combined measurement of Cyp2b1 mRNA levels in rat primary hepatocytes and rCAR-3A-G354Q activation in reporter assays can be useful for evaluating rCAR activation by chemicals.
Asunto(s)
Receptor de Androstano Constitutivo , Citocromo P-450 CYP2B1 , Ratas , Humanos , Animales , Citocromo P-450 CYP2B1/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Hepatocitos/metabolismo , Ligandos , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Chiral polychlorinated biphenyls (PCBs) have atropisomers that have different axial chiralities and exist as racemic mixtures. However, biochemical processes often result in the unequal accumulation of these atropisomers in organisms. This phenomenon leads to enantiospecific toxicity enhancement or reduction because either of the atropisomers mainly affects toxicity expression. Enantioselective accumulation is caused by cytochrome P450 (CYP, P450) monooxygenases, especially the CYP2B subfamilies. Therefore, this study investigates the metabolism of a chiral PCB in vitro. Both atropisomers isolated from racemic 2,2',3,4,4',5',6-heptachlorobiphenyl (CB183) were metabolized by human CYP2B6, but not rat CYP2B1. This may be due to the difference in the size of the substrate-binding cavities of CYP2B6 and CYP2B1. The stable accommodation of (-)-CB183 in the cavity without any steric hindrance explained the preferential metabolism of (-)-CB183 compared to (+)-CB183. Two hydroxylated metabolites, 3'-OH-CB183 and 5-OH-CB183, were identified. The docking study showed that the 3'-position of the trichlorophenyl ring closely approaches the heme of CYP2B6. To our knowledge, this is the first study to elucidate the structural basis of chiral PCB metabolism by P450 isozymes. These results will help promote the precise toxicity evaluation of chiral PCBs and provide an explanation of the structural basis of chiral PCB metabolism.
Asunto(s)
Bifenilos Policlorados , Animales , Citocromo P-450 CYP2B1/metabolismo , Citocromo P-450 CYP2B6/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Hemo , Humanos , Hidroxilación , Isoenzimas/metabolismo , Bifenilos Policlorados/química , Ratas , EstereoisomerismoRESUMEN
Parkinson's disease (PD) is a neurodegenerative disorder that causes uncontrollable movements. Although many breakthroughs in PD therapy have been accomplished, there is currently no cure for PD, and only trials to relieve symptoms have been evaluated. Recently, we reported the total synthesis of cudraisoflavone J and its chiral isomers [Lu et al., J. Nat. Prod. 2021, 84, 1359]. In this study, we designed and synthesized a series of novel cudraisoflavone J derivatives and evaluated their neuroprotective activities in neurotoxin-treated PC12 cells. Among these compounds, difluoro-substituted derivative (13m) and prenylated derivative (24) provided significant protection to PC12 cells against toxicity induced by 6-hydroxydopamine (6-OHDA) or rotenone. Both derivatives inhibited 6-OHDA- or rotenone-induced production of reactive oxygen species and partially attenuated lipid peroxidation in rat brain homogenates, indicating their antioxidant properties. They also increased the expression of the antioxidant enzyme, heme oxygenase (HO)-1, and enhanced the nuclear translocation of Nrf2, the transcription factor that regulates the expression of antioxidant proteins. The neuroprotective effects of 13m and 24 were eliminated by Zn(II)-protoporphyrin IX, an HO-1 inhibitor, demonstrating the critical role of HO-1 in their actions. Moreover, upregulation of HO-1 was abolished by nuclear factor erythroid 2-related factor (Nrf2) knockdown, verifying that Nrf2 is an upstream regulator of HO-1. Compounds 13m and 24 triggered phosphorylation of ERK1/2, JNK, and Akt. Most importantly, 13m- and 24-induced enhancement of Nrf2 translocation and HO-1 expression was reversed by U0126 (an ERK inhibitor), SP600125 (a JNK inhibitor), and LY294002 (an Akt inhibitor). Collectively, our results show that compounds 13m and 24 exert neuroprotective and antioxidant effects through the Nrf2/HO-1 pathway mediated by phosphorylation of ERK1/2, JNK, or Akt in PC12 cells. Based on our findings, both derivatives could serve as potential therapeutic candidates for the neuroprotective treatment of PD.
Asunto(s)
Fármacos Neuroprotectores , Enfermedad de Parkinson , Animales , Ratas , Antioxidantes/farmacología , Citocromo P-450 CYP2B1/metabolismo , Citocromo P-450 CYP2B1/farmacología , Hemo-Oxigenasa 1/metabolismo , Fármacos Neuroprotectores/farmacología , Neurotoxinas/farmacología , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo , Oxidopamina/farmacología , Enfermedad de Parkinson/tratamiento farmacológico , Inhibidores de Agregación Plaquetaria/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Rotenona/farmacologíaRESUMEN
Although polychlorinated biphenyls (PCBs) were commercially banned half a century ago, contamination of the environment and organisms by PCBs is still observed. PCBs show high persistence and bioaccumulation, resulting in toxicity. Among PCBs, chiral PCBs with more than three chlorine atoms at the ortho-position exhibit developmental and neurodevelopmental toxicity. Because toxicity is dependent on the atropisomer, atropisomer-specific metabolism is vital in determining toxicity. However, structural information on enantioselective metabolism remains elusive. Cytochrome P450 (CYP, P450) monooxygenases, particularly human CYP2B6 and rat CYP2B1, metabolize separated atropisomers of 2,2',3,6-tetrachlorobiphenyl (CB45) and 2,2',3,4',6-pentachlorobiphenyl (CB91) to dechlorinated and hydroxylated metabolites. Docking studies using human CYP2B6 predict 4'-hydroxy (OH)-CB45 from (aR)-CB45 as a major metabolite of CB45. Di-OH- and dechlorinated OH-metabolites from human CYP2B6 and rat CYP2B1 are also detected. Several hydroxylated metabolites are derived from CB91 by both P450s; 5-OH-CB91 is predicted as a major metabolite. CB91 dechlorination is also detected by identifying 3-OH-CB51. A stable conformation of PCBs in the substrate-binding cavity and close distance to P450 heme are responsible for high metabolizing activities. As hydroxylation and dechlorination change PCB toxicity, this approach helps understand the possible toxicity of chiral PCBs in mammals.
Asunto(s)
Bifenilos Policlorados , Animales , Citocromo P-450 CYP2B1/metabolismo , Citocromo P-450 CYP2B6/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Humanos , Hidroxilación , Mamíferos/metabolismo , Bifenilos Policlorados/metabolismo , Ratas , EstereoisomerismoRESUMEN
The present study aimed to investigate the protective effect of quercetin on polychlorinated biphenyls (PCB)-induced liver and embryo damage in pregnant Sprague-Dawley rats. Pregnant rats were divided into five groups, and then were orally gavaged daily with peanut oil (vehicle) or a commercial PCB mixture (Aroclor 1254) - with or without co-treatment with 75, 150, or 300 mg/kg quercetin - on gestation days (GD) 4-7. At GD 9, all rats were euthanized, and their blood, liver, and uterus were collected. Expressions of CYP450 mRNA and protein in liver, cytokines (IFNγ, IL-2, IL-4, and IL-6) and IFNγ/IL-4 ratios in liver and sera, liver morphology, and the status of implanted embryos were analyzed. The results showed Aroclor 1254 treatment alone caused hepatic cord damage (i.e. cell disorganization, swelling, decreased cytoplasm, vacuolization), and that quercetin co-treatment appeared to mitigate this damage. Similarly, levels of CYP1A1 and CYP2B1 mRNA in livers of Aroclor 1254-only-treated rats were significantly higher than those in rats co-treated with quercetin. Hepatic and sera levels of IFNγ, IL-2, IL-6, and IFNγ/IL-4 ratios, and the ratio of delayed-development embryos, all increased in Aroclor 1254-treated rats, but were relatively decreased as a result of quercetin co-treatments. IL-4 levels were decreased by Aroclor 1254 and tended to increase back to normal when quercetin was used. The results indicated that quercetin imparted a protective effect against Aroclor 1254-induced toxicity in pregnant rats, in part, by modulating levels of important pro-inflammatory cytokines and reducing induced CYP1A1 and CYP2B1 expression.
Asunto(s)
Antioxidantes/administración & dosificación , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Lesiones Prenatales/prevención & control , Quercetina/administración & dosificación , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/sangre , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP2B1/metabolismo , Citocinas/sangre , Citocinas/metabolismo , Modelos Animales de Enfermedad , Embrión de Mamíferos/efectos de los fármacos , Embrión de Mamíferos/metabolismo , Femenino , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Embarazo , Lesiones Prenatales/sangre , Lesiones Prenatales/inducido químicamente , Lesiones Prenatales/metabolismo , Ratas , Ratas Sprague-Dawley , Resultado del TratamientoRESUMEN
Chronic kidney disease (CKD) is characterized by progressive reduction in kidney function over time. CKD affects greater than 10% of the population and its incidence is on the rise due to the growing prevalence of its risk factors. Previous studies demonstrated CKD alters nonrenal clearance of drugs in addition to reducing renal clearance. We assessed the function and expression of hepatic CYP2B enzymes using a rat model of CKD. CKD was induced in Wistar rats by supplementing their chow with adenine and confirmed through the detection of elevated uremic toxins in plasma. Liver enzymes AST and ALT were unchanged by the adenine diet. Bupropion was used as a probe substrate for hepatic CYP2B function using rat liver microsomes. The resulting metabolite, hydroxy-bupropion, and bupropion were quantified by ultra-performance liquid chromatography coupled to time-of-flight mass spectrometry. Level of mRNA and protein were determined by RT-PCR and Western blot, respectively. The results of our study demonstrate that CYP2B1 is downregulated in a rat model of CKD. CYP2B1 mRNA level was significantly decreased (88%, P < 0.001) in CKD relative to control. Similarly, maximal enzymatic velocity (Vmax) for CYP2B was decreased by 46% in CKD relative to control (P < 0.0001). Previous studies involving patients with CKD demonstrated altered bupropion pharmacokinetics compared to control. Hence, our results suggest that these alterations may be mediated by attenuated CYP2B hepatic metabolism. This finding may partially explain the alterations in pharmacokinetics and nonrenal drug clearance frequently observed in patients with CKD.
Asunto(s)
Bupropión/farmacocinética , Citocromo P-450 CYP2B1/genética , Citocromo P-450 CYP2B1/metabolismo , Regulación hacia Abajo , Insuficiencia Renal Crónica/inducido químicamente , Adenina/efectos adversos , Animales , Hidrocarburo de Aril Hidroxilasas/genética , Hidrocarburo de Aril Hidroxilasas/metabolismo , Modelos Animales de Enfermedad , Regulación Enzimológica de la Expresión Génica , Masculino , Microsomas Hepáticos/metabolismo , Ratas , Ratas Wistar , Insuficiencia Renal Crónica/genética , Insuficiencia Renal Crónica/metabolismo , Esteroide Hidroxilasas/genética , Esteroide Hidroxilasas/metabolismoRESUMEN
The use of organophosphates phosphate flame retardants, particularly isopropylated triphenyl phosphate (IPTPP), has increased in recent years as replacements for polybrominated diphenyl ethers. This is despite limited understanding of the hazards of IPTPP. To examine the general and endocrine toxicity of IPTPP, adult Wistar rats were fed for 90 days on diets containing IPTPP estimated to deliver daily doses of 5 to 140 mg/kg/d. Exposure to IPTPP caused a dose-related increase in liver and adrenal gland weight in both sexes. Cells in the zona fasciculate (ZF) of the adrenal cortex were observed to be filled with droplets that stained with Nile red, suggesting they contained neutral lipid. Despite marked structural changes, there was no change in basal or stress-induced serum levels of their major secreted ZF product corticosterone (B), suggesting cell function was not altered. There were no effects on responses to glucose or insulin challenge, but serum levels of fructosamine were elevated by IPTPP exposure, suggesting a slight tendency of exposed animals to be hyperglycemic. Serum levels of total cholesterol and high-density lipoprotein cholesterol were significantly elevated in both sexes at the 2 highest doses. This study demonstrates that IPTPP exposure causes hypertrophy and neutral lipid accumulation in adrenal cortex ZF cells but does not result in impaired B production.
Asunto(s)
Corteza Suprarrenal/efectos de los fármacos , Retardadores de Llama/toxicidad , Metabolismo de los Lípidos/efectos de los fármacos , Hígado/efectos de los fármacos , Organofosfatos/toxicidad , Corteza Suprarrenal/metabolismo , Corteza Suprarrenal/patología , Animales , Corticosterona/sangre , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP2B1/metabolismo , Femenino , Hígado/enzimología , Hígado/patología , Masculino , Organofosfatos/química , Ratas WistarRESUMEN
Oxidative stress plays a key role in steatohepatitis induced by both xenobiotic agents and high fat diet (HFD). The present study aimed to evaluate hepatic oxidative stress and anti-oxidant systems response in rats exposed to HFD and/or non-toxic dose of dichlorodiphenyldichloroethylene (DDE), the first metabolite of dichlorodiphenyltrichloroethane. Groups of 8 rats were so treated for 4 weeks: 1- standard diet (N group); 2- standard diet plus DDE (10 mg/kg b.w.) (N+DDE group); 3- HFD (D group); 4- HFD plus DDE (D+DDE group). Oxidative stress was analyzed by determining malondialdehyde as lipid peroxidation product, while the anti-oxidant systems were evaluating by measuring the levels of the principal cytosolic and mitochondrial antioxidant proteins and enzymes, namely superoxide dismutase 1 and 2 (SOD1, SOD2), glutathione peroxidase 1 (GPx1) and uncoupling protein 2 (UCP2) involved in the control of hepatic reactive oxygens species (ROS) accumulation. The results showed malondialdehyde accumulation in livers of all groups, confirming the pro-oxidant effects of both HFD and DDE, but with a greater effect of DDE in absence of HFD. In addition, we found different levels of the analyzed anti-oxidant systems in the different groups. DDE mainly induced UCP2 and SOD2, while HFD mainly induced GPx1. Noteworthy, in the condition of simultaneous exposure to DDE and HFD, the anti-oxidant response was more similar to the one induced by HFD than to the response induced by DDE. Present findings confirmed that both HFD and xenobiotic exposure induced hepatic oxidative stress and showed that the anti-oxidant defense response was not the same in the diverse groups, suggesting that UCP2 induction could be an adaptive response to limit excessive ROS damage, mainly in condition of xenobiotic exposure.
Asunto(s)
Diclorodifenil Dicloroetileno/toxicidad , Dieta Alta en Grasa , Hígado Graso/metabolismo , Hígado Graso/patología , Estrés Oxidativo/efectos de los fármacos , Proteína Desacopladora 2/metabolismo , Xenobióticos/toxicidad , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Antioxidantes/metabolismo , Citocromo P-450 CYP2B1/metabolismo , Hígado Graso/sangre , Regulación de la Expresión Génica/efectos de los fármacos , Disulfuro de Glutatión/metabolismo , Macrófagos del Hígado/efectos de los fármacos , Macrófagos del Hígado/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Lípidos/análisis , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Metaboloma/efectos de los fármacos , Oxidación-Reducción , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Wistar , Proteína Desacopladora 2/genética , Aumento de Peso/efectos de los fármacosRESUMEN
The ubiquitous existence of hexabromocyclododecane (HBCD) in environmental matrices has made it attractive to both field investigators as well as laboratory researchers. However, literature on the biological effects caused by HBCD on aquatic vertebrates seldom exist. This has inevitably increased the difficulty of toxicological assessment in the aquatic environment. Juvenile crucian carp (Carassius carassius) were exposed (flow-through) to different concentrations of technical HBCD (nominal 2, 20, 200 µg L-1) for 7 days to determine the responses of antioxidant and biotransformation enzymes. HBCD was found to be increasingly bioconcentrated in the fish livers as time proceeds. Also, the contribution of α-HBCD exhibited an enhancement from 13% in the exposure solutions to 24% in crucian carp, still much lower than in wild fishes (ca. 80%). HBCD induced activities of antioxidant enzymes in most cases, as well as increased level of lipid peroxidation. In contrast to the weak response of 7-ethoxyresorufin-O-deethylase (EROD), 7-pentoxyresorufin-O-depentylase (PROD) activity was generally induced in a time-dependent manner with peaks at day 2. Phase II enzyme Glutathione-S-transferase (GST) showed a dose-dependent induction with maximums in the 20 µg L-1 treatment at all the four timepoints of 1, 2, 4 and 7 days. Some enzymatic responses showed good associations, indicating coordinated functions. To sum up, tHBCD exposure in the present circumstance had produced an ecological stress to crucian carp. The low levels of biotransformation and slow rates of bioisomerization suggest a possible long-term toxic effect, especially around HBCD point sources.
Asunto(s)
Hidrocarburos Bromados/farmacología , Hígado/efectos de los fármacos , Contaminantes Químicos del Agua/farmacología , Animales , Biotransformación , Carpas , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP2B1/metabolismo , Femenino , Proteínas de Peces/metabolismo , Glutatión Transferasa/metabolismo , Hígado/metabolismo , Masculino , Estrés Oxidativo/efectos de los fármacosRESUMEN
Cyclophosphamide (CPA) is a pro-drug commonly used in the chemotherapeutic schemes for glioma treatment but has high toxicity and the side effects include brain damage and even death. Since CPA is activated mainly by CY2B6, over-expression of the enzyme in the tumor cells has been proposed to enhance CPA activation. In this study, we explored the induction of the Cyp2b1 (homologous to CYP2B6) by nicotine in an animal rat model with glioma. Gene expression and protein levels were analyzed by RT-PCR and Western blot. Nicotine treatment increased CYP2B1 protein levels in the healthy animals' brain tissue. In the brain tissue of animals with glioma, the CYP2B1 showed a high expression, even before nicotine treatment. Nicotine did not increase significantly the CYP2B1 protein expression in the tumor, but increased its expression in the tumor vicinity, especially around blood vessels in the cortex. We also explored CY2B6 expression in glioma samples derived from pediatric patients. Tumor tissue showed a variable expression of the enzyme, which could depend on the tumor malignancy grade. Induction of the CYP2B6 in pediatric gliomas with lower expression of the enzyme, could be an alternative to improve the antitumoral effect of CPA treatment.
Asunto(s)
Neoplasias Encefálicas/genética , Citocromo P-450 CYP2B1/genética , Citocromo P-450 CYP2B6/genética , Glioma/genética , Nicotina/farmacología , Agonistas Nicotínicos/farmacología , Adolescente , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Niño , Citocromo P-450 CYP2B1/metabolismo , Citocromo P-450 CYP2B6/metabolismo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Glioma/metabolismo , Glioma/patología , Humanos , Masculino , Ratas , Ratas Endogámicas F344RESUMEN
BACKGROUND AND OBJECTIVES: Gambogenic acid (GNA), which possesses diverse antitumor activities both in vitro and in vivo, is regarded as a potential anticancer compound. Cytochrome P450 (CYP) enzymes play an important role in the metabolism of most xenobiotics; constitutive androstane receptor (CAR), a nuclear receptor that might be activated by xenobiotics and associated with the expression of some CYPs. In this study, we determined the effect of GNA on multiple rat liver CYP isoforms (CYP1A2, 2B1, and 2E1) and CAR as well as the potential of GNA to interact with co-administered drugs. METHODS: Male SD rats were randomly divided into the control, and the low (5 mg/kg)-, medium (25 mg/kg)-, and high- (100 mg/kg) dose GNA groups. After the intragastric administration of GNA for 14 consecutive days, a cocktail method was adopted to evaluate the activities of CYP1A2, 2B1, and 2E1. The liver expression of CYP1A2, 2B1, and 2E1 and CAR was analyzed by Western blotting (WB) and quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR). RESULTS: The 14-day administration of GNA significantly increased both the mRNA and protein expressions and the activity of CYP2E1. Additionally, the mRNA and protein expressions of CYP1A2 were clearly induced, while only the high GNA dose increased the activity of liver CYP1A2. Moreover, the mRNA expression levels of CYP2B1 and CAR were increased, but their protein levels and the activity parameters of CYP2B1 did not show significant changes. CONCLUSIONS: The obtained results suggest that the CYP1A2 and CYP2E1 enzymes could be induced in rats after treatment with GNA. Therefore, when GNA is administrated with other drugs, potential drug-drug interactions (DDI) mediated by CYP1A2 and CYP2E1 induction should be taken into consideration.
Asunto(s)
Citocromo P-450 CYP1A2/biosíntesis , Citocromo P-450 CYP2B1/biosíntesis , Citocromo P-450 CYP2E1/biosíntesis , Fenacetina/farmacocinética , Receptores Citoplasmáticos y Nucleares/biosíntesis , Xantenos/farmacología , Animales , Bupropión/sangre , Bupropión/farmacocinética , Clorzoxazona/sangre , Clorzoxazona/farmacocinética , Receptor de Androstano Constitutivo , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2B1/metabolismo , Citocromo P-450 CYP2E1/metabolismo , Inductores de las Enzimas del Citocromo P-450/sangre , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Hígado/metabolismo , Masculino , Fenacetina/sangre , RatasRESUMEN
Short-chain chlorinated paraffins (SCCPs) are known to disturb thyroid hormone (TH) homeostasis in rodents. However, the mechanism remains to be fully characterized. In this study, male Sprague Dawley rats received SCCPs (0, 1, 10, or 100mg/kg/day) via gavage once a day for consecutive 28days. Plasma and hepatic TH concentrations, thyrocyte structure, as well as thyroid and hepatic mRNA and protein levels of genes associated with TH homeostasis were examined. Moreover, we performed molecular docking to predict interactions between constitutive androstane receptor (CAR), a key regulator in xenobiotic-induced TH metabolism, with different SCCP molecules. Exposure to SCCPs significantly decreased the circulating free thyroxine (T4) and triiodothyronine (T3) levels, but increased thyroid-stimulating hormone (TSH) levels by a feedback mechanism. Decreased hepatic T4 and increased hepatic T3 levels were also seen after 100mg/kg/day SCCPs exposure. SCCPs didn't show any significant effects on the expression of thyroid TH synthesis genes or thyrocyte structure. However, stimulation effects were observed for mRNA and protein levels of hepatic uridine diphosphoglucuronosyl transferase (UGT) 1A1 and organic anion transporter 2, suggesting an accelerated TH metabolism in rat liver. The increased cytochrome P450 2B1 but not 1A1 mRNA and protein levels indicated that the CAR signaling was activated by SCCPs exposure. According to docking analysis, SCCPs form hydrophobic interactions with CAR and the binding affinity shows dependency on chlorine content. Overall, our data showed that CAR implicated enhancement of hepatic TH influx and degradation could be the main cause for SCCPs induced TH deficiency in male rats.
Asunto(s)
Hígado/metabolismo , Parafina/toxicidad , Glándula Tiroides/efectos de los fármacos , Hormonas Tiroideas/sangre , Animales , Receptor de Androstano Constitutivo , Citocromo P-450 CYP2B1/metabolismo , Glucuronosiltransferasa/metabolismo , Homeostasis/efectos de los fármacos , Masculino , Simulación del Acoplamiento Molecular , Ratas , Ratas Sprague-Dawley , Receptores Citoplasmáticos y Nucleares/metabolismo , Glándula Tiroides/fisiopatologíaRESUMEN
Cytochrome P450 (CYP) 1A and 2B subfamily enzymes are important drug metabolizing enzymes, and are highly conserved across species in terms of sequence homology. However, there are major to minor structural and macromolecular differences which provide for species-selectivity and substrate-selectivity. Therefore, species-selectivity of CYP1A and CYP2B subfamily proteins across human, mouse and rat was analyzed using molecular modeling, docking and dynamics simulations when the chiral molecules quinine and quinidine were used as ligands. The three-dimensional structures of 17 proteins belonging to CYP1A and CYP2B subfamilies of mouse and rat were predicted by adopting homology modeling using the available structures of human CYP1A and CYP2B proteins as templates. Molecular docking and dynamics simulations of quinine and quinidine with CYP1A subfamily proteins revealed the existence of species-selectivity across the three species. On the other hand, in the case of CYP2B subfamily proteins, no role for chirality of quinine and quinidine in forming complexes with CYP2B subfamily proteins of the three species was indicated. Our findings reveal the roles of active site amino acid residues of CYP1A and CYP2B subfamily proteins and provide insights into species-selectivity of these enzymes across human, mouse, and rat.
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Citocromo P-450 CYP1A1/metabolismo , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Animales , Sitios de Unión , Dominio Catalítico , Citocromo P-450 CYP1A1/química , Citocromo P-450 CYP2B1/química , Citocromo P-450 CYP2B1/metabolismo , Humanos , Enlace de Hidrógeno , Ligandos , Ratones , Conformación Molecular , Quinidina/química , Quinidina/metabolismo , Quinina/química , Quinina/metabolismo , Ratas , Programas Informáticos , Especificidad de la EspecieRESUMEN
Liver fibrosis is a consequence of chronic liver disease that can progress to liver cirrhosis or even hepatocarcinoma. Fuzheng Huayu (FZHY), a Chinese herbal formula, has been shown to exert anti-fibrotic effects. To better understand the molecular mechanisms underlying the anti-fibrotic effects of FZHY, we analyzed transcriptomic and proteomic combination profiles in CCl4-induced liver fibrosis in rats, which were treated with extracted FZHY powder (0.35 g·kg-1·d-1, ig) for 3 weeks. We showed that FZHY administration significantly improved liver function, alleviated hepatic inflammatory and fibrotic changes, and decreased the hydroxyproline content in the livers of CCl4-treated rats. When their liver tissues were examined using microarray and iTRAQ, we found 255 differentially expressed genes (fold change ≥1.5, P<0.05) and 499 differentially expressed proteins (fold change ≥1.2, P<0.05) in the FZHY and model groups. Functional annotation with DAVID (The Database for Annotation, Visualization and Integrated Discovery) showed that 15 enriched gene ontology terms, including drug metabolic process, response to extracellular stimulus, response to vitamins, arachidonic acid metabolic process, response to wounding, and oxidation reduction might be involved in the anti-fibrotic effects of FZHY; whereas KEGG pathway analysis revealed that eight enriched pathways, including arachidonic acid metabolism, retinol metabolism, metabolism of xenobiotics by cytochrome P450, and drug metabolism might also be involved. Moreover, the protein-protein interaction network demonstrated that 10 core genes/proteins overlapped, with Ugt2a3, Cyp2b1 and Cyp3a18 in retinol metabolism pathway overlapped to a higher degree. Compared to the model rats, the livers of FZHY-treated rats had significantly higher mRNA and protein expression levels of Ugt2a3, Cyp2b1 and Cyp3a18. Furthermore, the concentration of retinoic acid was significantly higher in the FZHY-treated rats compared with the model rats. The results suggest that the anti-fibrotic effects of FZHY emerge through multiple targets, multiple functions, and multiple pathways, including FZHY-regulated retinol metabolism, xenobiotic metabolism by cytochrome P450, and drug metabolism through up-regulated Ugt2a3, Cyp2b1, and Cyp3a18. These genes may play important anti-fibrotic roles in FZHY-treated rats.
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Tetracloruro de Carbono , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Medicamentos Herbarios Chinos/farmacología , Perfilación de la Expresión Génica/métodos , Cirrosis Hepática Experimental/prevención & control , Hígado/efectos de los fármacos , Proteómica/métodos , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Citocromo P-450 CYP2B1/genética , Citocromo P-450 CYP2B1/metabolismo , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Redes Reguladoras de Genes/efectos de los fármacos , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/metabolismo , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática Experimental/genética , Cirrosis Hepática Experimental/metabolismo , Cirrosis Hepática Experimental/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteoma , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , TranscriptomaRESUMEN
Human hepatic cytochromes P450 (CYP) are integral to xenobiotic metabolism. CYP2B6 is a major catalyst of biotransformation of environmental toxicants, including polybrominated diphenyl ethers (PBDEs). CYP2B substrates tend to contain halogen atoms, but the biochemical basis for this selectivity and for species specific determinants of metabolism has not been identified. Spectral binding titrations and inhibition studies were performed to investigate interactions of rat CYP2B1, rabbit CYP2B4, and CYP2B6 with a series of phenoxyaniline (POA) congeners that are analogues of PBDEs. For most congeners, there was a <3-fold difference between the spectral binding constants (KS) and IC50 values. In contrast, large discrepancies between these values were observed for POA and 3-chloro-4-phenoxyaniline. CYP2B1 was the enzyme most sensitive to POA congeners, so the Val-363 residue from that enzyme was introduced into CYP2B4 or CYP2B6. This substitution partially altered the protein-ligand interaction profiles to make them more similar to that of CYP2B1. Addition of cytochrome P450 oxidoreductase (POR) to titrations of CYP2B6 with POA or 2'4'5'TCPOA decreased the affinity of both ligands for the enzyme. Addition of cytochrome b5 to a recombinant enzyme system containing POR and CYP2B6 increased the POA IC50 value and decreased the 2'4'5'TCPOA IC50 value. Overall, the inconsistency between KS and IC50 values for POA versus 2'4'5'TCPOA is largely due to the effects of redox partner binding. These results provide insight into the biochemical basis of binding of diphenyl ethers to human CYP2B6 and changes in CYP2B6-mediated metabolism that are dependent on POA congener and redox partner identity.
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Hidrocarburo de Aril Hidroxilasas/antagonistas & inhibidores , Citocromo P-450 CYP2B1/antagonistas & inhibidores , Citocromo P-450 CYP2B6/efectos de los fármacos , Inhibidores Enzimáticos del Citocromo P-450/farmacología , Éteres Difenilos Halogenados/farmacología , Alquilación/efectos de los fármacos , Sustitución de Aminoácidos , Compuestos de Anilina , Animales , Hidrocarburo de Aril Hidroxilasas/química , Hidrocarburo de Aril Hidroxilasas/genética , Hidrocarburo de Aril Hidroxilasas/metabolismo , Derivados del Benceno/farmacología , Citocromo P-450 CYP2B1/química , Citocromo P-450 CYP2B1/genética , Citocromo P-450 CYP2B1/metabolismo , Citocromo P-450 CYP2B6/química , Citocromo P-450 CYP2B6/genética , Citocromo P-450 CYP2B6/metabolismo , Inhibidores del Citocromo P-450 CYP2B6/metabolismo , Inhibidores del Citocromo P-450 CYP2B6/farmacología , Inhibidores Enzimáticos del Citocromo P-450/metabolismo , Familia 2 del Citocromo P450/antagonistas & inhibidores , Familia 2 del Citocromo P450/química , Familia 2 del Citocromo P450/genética , Familia 2 del Citocromo P450/metabolismo , Citocromos b5/metabolismo , Contaminantes Ambientales/metabolismo , Éteres Difenilos Halogenados/metabolismo , Humanos , Hidrocarburos Halogenados/metabolismo , Concentración 50 Inhibidora , Estructura Molecular , Mutagénesis Sitio-Dirigida , NADPH Oxidasas/metabolismo , Oxidación-Reducción , Conejos , Ratas , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
High dietary levels of the non-genotoxic synthetic pyrethroid momfluorothrin increased the incidence of hepatocellular tumors in male and female Wistar rats. Mechanistic studies have demonstrated that the mode of action (MOA) for momfluorothrin-induced hepatocellular tumors is constitutive androstane receptor (CAR)-mediated. In the present study, to evaluate the potential human carcinogenic risk of momfluorothrin, the effects of momfluorothrin (1-1,000 µM) and a major metabolite Z-CMCA (5-1,000 µM) on hepatocyte replicative DNA synthesis and CYP2B mRNA expression were examined in cultured rat and human hepatocyte preparations. The effect of sodium phenobarbital (NaPB), a prototypic rodent hepatocarcinogen with a CAR-mediated MOA, was also investigated. Human hepatocyte growth factor (hHGF) produced a concentration-dependent increase in replicative DNA synthesis in rat and human hepatocytes. However, while NaPB and momfluorothrin increased replicative DNA synthesis in rat hepatocytes, NaPB, momfluorothrin and Z-CMCA did not increase replicative DNA synthesis in human hepatocytes. NaPB, momfluorothrin and Z-CMCA increased CYP2B1/2 mRNA levels in rat hepatocytes. NaPB and momfluorothrin also increased CYP2B6 mRNA levels in human hepatocytes. Overall, while momfluorothrin and NaPB activated CAR in cultured human hepatocytes, neither chemical increased replicative DNA synthesis. Furthermore, to confirm whether the findings observed in vitro were also observed in vivo, a humanized chimeric mouse study was conducted. Replicative DNA synthesis was not increased in human hepatocytes of chimeric mice treated with momfluorothrin or its close structural analogue metofluthrin. As human hepatocytes are refractory to the mitogenic effects of momfluorothrin, in contrast to rat hepatocytes, the data support the hypothesis that the MOA for momfluorothrin-induced rat liver tumor formation is not relevant for humans.
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Androstanos , Carcinoma Hepatocelular/inducido químicamente , Carcinoma Hepatocelular/patología , Transformación Celular Neoplásica/genética , Hepatocitos/patología , Neoplasias Hepáticas/inducido químicamente , Neoplasias Hepáticas/patología , Piretrinas/toxicidad , Receptores Androgénicos/fisiología , Animales , Células Cultivadas , Citocromo P-450 CYP2B1/genética , Citocromo P-450 CYP2B1/metabolismo , Replicación del ADN/efectos de los fármacos , Femenino , Factor de Crecimiento de Hepatocito/farmacología , Hepatocitos/metabolismo , Humanos , Masculino , Ratones , Fenobarbital/toxicidad , ARN Mensajero/metabolismo , Ratas WistarRESUMEN
Efavirenz is an anti-HIV drug that presents relevant short- and long-term central nervous system adverse reactions. Its main metabolite (8-hydroxy-efavirenz) was demonstrated to be a more potent neurotoxin than efavirenz itself. This work was aimed to understand how efavirenz biotransformation to 8-hydroxy-efavirenz is related to its short- and long-term neuro-adverse reactions. To access those mechanisms, the expression and activity of Cyp2b enzymes as well as the thiolomic signature (low molecular weight thiols plus S-thiolated proteins) were longitudinally evaluated in the hepatic and brain tissues of rats exposed to efavirenz during 10 and 36days. Efavirenz and 8-hydroxy-efavirenz plasma concentrations were monitored at the same time points. Cyp2b induction had a delayed onset in liver (p<0.001), translating into increases in Cyp2b activity in liver and 8-hydroxy-efavirenz plasma concentration (p<0.001). Moreover, an increase in S-cysteinyl-glycinylated proteins (p<0.001) and in free low molecular weight thiols was also observed in liver. A distinct scenario was observed in hippocampus, which showed an underexpression of Cyp2b as well as a decrease in S-cysteinylated and S-glutathionylated proteins. Additionally, the observed changes in tissues were associated with a marked increase of S-glutathionylation in plasma. Our data suggest that the time course of efavirenz biotransformation results from different mechanisms for its short- and long-term neurotoxicity. The difference in the redox profile between liver and hippocampus might explain why, despite being mostly metabolized by the liver, this drug is neurotoxic. If translated to clinical practice, this evidence will have important implications in efavirenz short- and long-term neurotoxicity prevention and management.
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Fármacos Anti-VIH/farmacocinética , Benzoxazinas/farmacocinética , Síndromes de Neurotoxicidad/metabolismo , Alquinos , Animales , Fármacos Anti-VIH/efectos adversos , Fármacos Anti-VIH/sangre , Hidrocarburo de Aril Hidroxilasas/genética , Hidrocarburo de Aril Hidroxilasas/metabolismo , Benzoxazinas/efectos adversos , Benzoxazinas/sangre , Benzoxazinas/metabolismo , Biotransformación , Ciclopropanos , Citocromo P-450 CYP2B1/genética , Citocromo P-450 CYP2B1/metabolismo , Hipocampo/metabolismo , Hígado/metabolismo , Masculino , Síndromes de Neurotoxicidad/sangre , Corteza Prefrontal/metabolismo , Ratas Wistar , Esteroide Hidroxilasas/genética , Esteroide Hidroxilasas/metabolismo , Compuestos de Sulfhidrilo/metabolismoRESUMEN
Despite widespread use of bromuconazole as a pesticide for food crops and fruits, limited studies have been done to evaluate its toxic effects. Here, we evaluated the hepatotoxic effect of bromuconazole using classical toxicological (biochemical analysis and histopathological examination) and gene-based molecular methods. Male rats were treated either orally or topically with bromuconazole at doses equal to no observed adverse effect level (NOAEL) and 1/10 LD50 for 90 d. Bromuconazole increased activities of liver enzymes (ALT, AST, ALP, and ACP), and levels of bilirubin. It also induced hepatic oxidative stress as evidenced by significant decrease in the activities of superoxide dismutase (SOD), and significant increase in levels of malondialdehyde (MDA) in liver. In addition, bromuconazole caused an increase in liver weights and necrobiotic changes (vacuolation and hepatocellular hypertrophy). It also strongly induced the expression of PXR and its downstream target CYP3A1 gene as well as the activity of CYP3A1. However, it inhibited the expression of CAR and its downstream target CYP2B1 gene without significant changing in CYP2B1 activity. Overall, the oral route showed higher hepatotoxic effect and molecular changes than the dermal route and all changes were dose dependent. This is the first investigation to report that bromuconazole-induced liver oxidative damage is accompanied by upregulation of PXR/CYP3A1 and downregulation of CAR/CYP2B1.