RESUMEN
The fungicide fluxapyroxad (BAS 700â¯F) has been shown to significantly increase the incidence of liver tumours in male Wistar rats at dietary levels of 1500 and 3000â¯ppm and in female rats at a dietary level of 3000â¯ppm via a non-genotoxic mechanism. In order to elucidate the mode of action (MOA) for fluxapyroxad-induced rat liver tumour formation a series of in vivo and in vitro investigative studies were undertaken. The treatment of male and female Wistar rats with diets containing 0 (control), 50, 250, 1500 and 3000â¯ppm fluxapyroxad for 1, 3, 7 and 14 days resulted in a dose-dependent increases in relative weight at 1500 and 3000â¯ppm from day 3 onwards in both sexes, with an increase in relative liver weight being also observed in male rats given 250â¯ppm fluxapyroxad for 14 days. Examination of liver sections revealed a centrilobular hepatocyte hypertrophy in some fluxapyroxad treated male and female rats. Hepatocyte replicative DNA synthesis (RDS) was significantly increased in male rats given 1500 and 3000â¯ppm fluxapyroxad for 3 and 7 days and in female rats given 50-3000â¯ppm fluxapyroxad for 7 days and 250-3000â¯ppm fluxapyroxad for 3 and 14 days; the maximal increases in RDS in both sexes being observed after 7 days treatment. The treatment of male and female Wistar rats with 250-3000â¯ppm fluxapyroxad for 14 days resulted in significant increases in hepatic microsomal total cytochrome P450 (CYP) content and CYP2B subfamily-dependent enzyme activities. Male Wistar rat hepatocytes were treated with control medium and medium containing 1-100⯵M fluxapyroxad or 500⯵M sodium phenobarbital (NaPB) for 4 days. Treatment with fluxapyroxad and NaPB increased CYP2B and CYP3A enzyme activities and mRNA levels but had little effect on markers of CYP1A and CYP4A subfamily enzymes and of the peroxisomal fatty acid ß-oxidation cycle. Hepatocyte RDS was significantly increased by treatment with fluxapyroxad, NaPB and 25â¯ng/ml epidermal growth factor (EGF). The treatment of hepatocytes from two male human donors with 1-100⯵M fluxapyroxad or 500⯵M NaPB for 4 days resulted in some increases in CYP2B and CYP3A enzyme activities and CYP mRNA levels but had no effect on hepatocyte RDS, whereas treatment with EGF resulted in significant increase in RDS in both human hepatocyte preparations. Hepatocytes from male Sprague-Dawley wild type (WT) and constitutive androstane receptor (CAR) knockout (CAR KO) rats were treated with control medium and medium containing 1-16⯵M fluxapyroxad or 500⯵M NaPB for 4 days. While both fluxapyroxad and NaPB increased CYP2B enzyme activities and mRNA levels in WT hepatocytes, only minor effects were observed in CAR KO rat hepatocytes. Treatment with both fluxapyroxad and NaPB only increased RDS in WT and not in CAR KO rat hepatocytes, whereas treatment with EGF increased RDS in both WT and CAR KO rat hepatocytes. In conclusion, a series of in vivo and in vitro investigative studies have demonstrated that fluxapyroxad is a CAR activator in rat liver, with similar properties to the prototypical CAR activator phenobarbital. A robust MOA for fluxapyroxad-induced rat liver tumour formation has been established. Based on the lack of effect of fluxapyroxad on RDS in human hepatocytes, it is considered that the MOA for fluxapyroxad-induced liver tumour formation is qualitatively not plausible for humans.
Asunto(s)
Receptor de Androstano Constitutivo , Fungicidas Industriales , Hepatocitos , Ratas Wistar , Receptores Citoplasmáticos y Nucleares , Animales , Masculino , Femenino , Ratas , Fungicidas Industriales/toxicidad , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Humanos , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hepatocitos/patología , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Relación Dosis-Respuesta a Droga , Tamaño de los Órganos/efectos de los fármacos , Neoplasias Hepáticas Experimentales/inducido químicamente , Neoplasias Hepáticas Experimentales/patología , Neoplasias Hepáticas Experimentales/metabolismo , Replicación del ADN/efectos de los fármacos , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Neoplasias Hepáticas/inducido químicamente , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologíaRESUMEN
Basimglurant (RG7090), a small molecule under development to treat certain forms of depression, demonstrated foci of altered hepatocytes in a long-term rodent-toxicity study. Additional evidence pointed toward the activation of the constitutive androstane receptor (CAR), an established promoter of nongenotoxic and rodent-specific hepatic tumors. This mode of action and the potential human relevance was explored in vivo using rodent and cynomolgus monkey models and in vitro using murine and human liver spheroids. Wild type (WT) and CAR/pregnane X receptor (PXR) knockout mice (CAR/PXR KO) were exposed to RG7090 for 8 consecutive days. Analysis of liver lysates revealed induction of Cyp2b mRNA and enzyme activity, a known activation marker of CAR, in WT but not in CAR/PXR KO animals. A series of proliferative genes were upregulated in WT mice only, and immunohistochemistry data showed increased cell proliferation exclusively in WT mice. In addition, primary mouse liver spheroids were challenged with RG7090 in the presence or absence of modified antisense oligonucleotides inhibiting CAR and/or PXR mRNA, showing a concentration-dependent Cyp2b mRNA induction only if CAR was not repressed. On the contrary, neither human liver spheroids nor cynomolgus monkeys exposed to RG7090 triggered CYP2B mRNA upregulation. Our data suggested RG7090 to be a rodent-specific CAR activator, and that CAR activation and its downstream processes were involved in the foci of altered hepatocytes formation detected in vivo. Furthermore, we demonstrated the potential of a new in vitro approach using liver spheroids and antisense oligonucleotides for CAR knockdown experiments, which could eventually replace in vivo investigations using CAR/PXR KO mice.
Asunto(s)
Imidazoles/farmacología , Piridinas/farmacología , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores de Esteroides , Animales , Receptor de Androstano Constitutivo , Hepatocitos , Humanos , Hígado , Macaca fascicularis , Ratones , Ratones Endogámicos C57BL , OrganoidesRESUMEN
In a 79 week bioassay the pesticide synergist piperonyl butoxide (PBO) was shown to significantly increase the incidence of hepatocellular adenoma (but not hepatocellular carcinoma) in male CD-1 mice at dietary levels of 100 and 300 mg/kg/day PBO and in female mice at a dietary level of 300 mg/kg/day. As PBO is not a genotoxic agent, a series of investigative studies were undertaken to elucidate the mode of action (MOA) for PBO-induced mouse liver tumour formation. Male CD-1 mice were fed diets to provide intakes of 0 (control), 30, 100 and 300 mg/kg/day PBO and for purposes of comparison 500 ppm sodium phenobarbital (NaPB), a known constitutive androstane receptor (CAR) activator, for 7 and 14 days. Treatment with 100 and 300 mg/kg/day PBO and 500 ppm NaPB increased relative liver weight which was associated with hepatocyte hypertrophy, with hepatocyte replicative DNA synthesis (RDS) being increased after 7 days treatment. The treatment of CD-1 mice with 30-300 mg/kg/day PBO for 14 days resulted in significant dose-dependent increases in hepatic microsomal cytochrome P450 (CYP) content and 7-pentoxyresorufin O-depentylase (PROD) activity and in hepatic Cyp2b10 mRNA levels. In contrast, PBO produced a biphasic effect on markers of activation of the peroxisome proliferator-activated receptor alpha (PPARα), with small increases in microsomal lauric acid 12-hydroxylase activity and hepatic Cyp4a10 mRNA levels being observed in mice given 100 mg/kg/day with PBO, with either no increase or a significant inhibition being observed in mice given 300 mg/kg/day PBO. The hepatic effects of PBO in male CD-1 mice were generally similar to those produced by NaPB and were reversible after the cessation of treatment for 28 days. Studies were also performed in male C57BL/6J (wild type) mice and in hepatic CAR and pregnane X receptor (PXR) knockout mice (CAR KO/PXR KO mice), where in the CAR KO/PXR KO mice PBO had little effect on markers of CAR activation, but produced some increases in markers of PPARα activation. The treatment of male CD-1 mouse hepatocytes for 4 days with 5-50 µM PBO, 10-1000 µM NaPB and 25 ng/mL epidermal growth factor (EGF) resulted in significant increases in hepatocyte RDS. While treatment of hepatocytes from one male and one female human donor with 5-500 µM PBO and 10-1000 µM NaPB for 4 days had no effect on hepatocyte RDS, treatment with EGF resulted in significant increases in RDS in both human hepatocyte preparations. In summary, PBO is predominantly a hepatic CAR activator at carcinogenic dose levels in CD-1 mice, with activation of hepatic CAR resulting in a suppression of the effect of PBO on hepatic PPARα. A robust MOA for PBO-induced mouse liver tumour formation has been established, this MOA being similar to that previously identified for NaPB and some other rodent liver CAR activators. Based on the lack of effect of PBO on RDS in human hepatocytes, it is considered that the MOA for PBO-induced mouse liver tumour formation is qualitatively not plausible for humans.
Asunto(s)
Neoplasias Hepáticas Experimentales/inducido químicamente , Sinergistas de Plaguicidas/toxicidad , Butóxido de Piperonilo/toxicidad , Animales , Tamaño de la Célula , Replicación del ADN/efectos de los fármacos , Dieta , Regulación de la Expresión Génica/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Hepatocitos/patología , Humanos , Hígado/efectos de los fármacos , Hígado/enzimología , Pruebas de Función Hepática , Neoplasias Hepáticas Experimentales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fenobarbital/toxicidad , Receptores Sensibles al Calcio/genéticaRESUMEN
Nongenotoxic chemicals can produce liver tumours in rats and mice by a mitogenic mode of action involving activation of the constitutive androstane receptor (CAR). The aim of this study was to evaluate the usefulness of cultured hepatocytes from normal (wild type; WT) and CAR knockout (KO) rats to screen compounds as potential activators of rat CAR and to validate this test system. Cultured hepatocytes from male Sprague-Dawley WT and CAR KO rats were treated with either 100 and 1000 µM sodium phenobarbital (NaPB), 3-100 µM fluquinconazole (FQZ), or 3-300 µM 3-(difluoromethyl)-1-methyl-N-(3´,4´,6-trifluoro[1,1´-biphenyl]-2-yl)-1H-pyrazole-4-carboxamide (TI1) for 96 h. Induction of cytochrome P450 (CYP) enzymes was monitored by measurement of 7-pentoxyresorufin O-depentylase (PROD), 7-benzyloxyresorufin O-debenzylase (BROD) and 7-benzyloxyquinoline O-debenzylase (BQ) activities. Hepatocytes undergoing replicative DNA synthesis (RDS) were labelled by adding 10 µM 5-bromo-2´-deoxyuridine to the culture medium for determination of the hepatocyte labelling index. The treatment of WT, but not of CAR KO, rat hepatocytes with NaPB, FQZ and TI1 increased hepatocyte RDS and induced CYP2B-dependent PROD activity. In contrast, all three compounds increased CYP2B/3A-dependent BROD and CYP3A-dependent BQ activities in both WT and CAR KO rat hepatocytes. Hepatocyte RDS was increased in both WT and CAR KO rat hepatocytes by treatment with 25 ng/ml epidermal growth factor as a positive control. Overall, these results demonstrate that the effects of three CAR activators on RDS and CYP2B enzyme induction are abolished in cultured CAR KO rat hepatocytes. As demonstrated by this validation study, the CAR KO hepatocyte model is a useful in vitro mechanistic tool for the rapid screening of chemicals as potential activators of rat CAR.
Asunto(s)
Hepatocitos/efectos de los fármacos , Fenobarbital/farmacología , Quinazolinonas/farmacología , Receptores Citoplasmáticos y Nucleares/genética , Triazoles/farmacología , Animales , Receptor de Androstano Constitutivo , Inductores de las Enzimas del Citocromo P-450/administración & dosificación , Inductores de las Enzimas del Citocromo P-450/farmacología , Sistema Enzimático del Citocromo P-450/efectos de los fármacos , Sistema Enzimático del Citocromo P-450/metabolismo , ADN/metabolismo , Relación Dosis-Respuesta a Droga , Técnicas de Inactivación de Genes , Masculino , Ratones Noqueados , Fenobarbital/administración & dosificación , Quinazolinonas/administración & dosificación , Ratas , Ratas Sprague-Dawley , Triazoles/administración & dosificaciónRESUMEN
In a 2-year study the herbicide metazachlor (BAS 479H) was shown to significantly increase the incidence of liver tumours in female Wistar rats at a dietary level of 8000â¯ppm. As metazachlor is not a genotoxic agent, a series of in vivo and in vitro investigative studies were undertaken to elucidate the mode of action (MOA) for metazachlor-induced female rat liver tumour formation. Male and female Wistar rats were given diets containing 0 (control), 200 and 8000â¯ppm metazachlor for 3, 7, 14 and 28 days. The treatment of male rats with 200 and 8000â¯ppm metazachlor and female rats with 8000â¯ppm metazachlor resulted in significant increases in relative liver weight, which was associated with a centrilobular hepatocyte hypertrophy. Hepatocyte replicative DNA synthesis (RDS) was significantly increased in male rats given 8000â¯ppm metazachlor for 3 and 7 days and in female rats given 200â¯ppm metazachlor for 7-28 days and 8000â¯ppm metazachlor for 3-28 days. Significant increases in relative liver weight, centrilobular hepatocyte hypertrophy and hepatocyte RDS were also observed in male and female Wistar rats given and 500â¯ppm sodium phenobarbital (NaPB) for 3-28 days. The treatment of female Wistar rats with either 8000â¯ppm metazachlor for 7 days or with 500â¯ppm NaPB for 3 and 7 days resulted in the nuclear translocation of the hepatic constitutive androstane receptor (CAR). Treatment of male and female Wistar rats with 8000â¯ppm metazachlor for 14 days resulted in significant increases in hepatic microsomal total cytochrome P450 (CYP) content, CYP2B subfamily-dependent enzyme activities and mRNA levels, together with some increases in CYP3A enzyme activity and mRNA levels. The treatment of male Wistar rat hepatocytes with metazachlor (concentration range 0.5-50⯵M) and NaPB (500 µM) for 4 days resulted in increased CYP2B enzyme activities and mRNA levels; with metazachlor and NaPB also producing significant increases in hepatocyte RDS levels. Studies were also performed with hepatocytes from male Sprague-Dawley wild type (WT) rats and CAR knockout (CAR KO) rats. While both treatment with metazachlor and NaPB for 4 days increased CYP2B enzyme activities and mRNA levels in WT rat hepatocytes, only minor effects were observed in CAR KO rat hepatocytes. Treatment with both metazachlor and NaPB only increased RDS in WT but not in CAR KO rat hepatocytes. The treatment of hepatocytes from two male human donors with 0.5-25⯵M metazachlor or 500⯵M NaPB for 4 days resulted in increases in CYP2B6 and CYP3A4 mRNA levels but had no effect on hepatocyte RDS. EGF as concurrently used positive control demonstrated the expected RDS response in all rat and human hepatocyte cultures. In conclusion, a series of in vivo and in vitro investigative studies have demonstrated that metazachlor is a CAR activator in rat liver, with similar properties to the prototypical CAR activator phenobarbital. A robust MOA for metazachlor-induced female rat liver tumour formation has been established. Based on the lack of effect of metazachlor on RDS in human hepatocytes, it is considered that the MOA for metazachlor-induced rat liver tumour formation is qualitatively not plausible for humans.
Asunto(s)
Acetamidas/toxicidad , Herbicidas/toxicidad , Neoplasias Hepáticas Experimentales/inducido químicamente , Animales , Células Cultivadas , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus/efectos de los fármacos , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus/metabolismo , Replicación del ADN/efectos de los fármacos , Femenino , Técnicas de Inactivación de Genes , Hepatocitos/efectos de los fármacos , Hepatocitos/patología , Humanos , Hígado/patología , Neoplasias Hepáticas Experimentales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Tamaño de los Órganos/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Translocación Genética/efectos de los fármacosRESUMEN
The metabolism of the pyrethroids deltamethrin (DLM), cis-permethrin (CPM) and trans-permethrin (TPM) was studied in human expressed cytochrome P450 (CYP) and carboxylesterase (CES) enzymes. DLM, CPM and TPM were metabolised by human CYP2B6 and CYP2C19, with the highest apparent intrinsic clearance (CLint) values for pyrethroid metabolism being observed with CYP2C19. Other CYP enzymes contributing to the metabolism of one or more of the three pyrethroids were CYP1A2, CYP2C8, CYP2C9*1, CYP2D6*1, CYP3A4 and CYP3A5. None of the pyrethroids were metabolised by CYP2A6, CYP2E1, CYP3A7 or CYP4A11. DLM, CPM and TPM were metabolised by both human CES1 and CES2 enzymes. Apparent CLint values for pyrethroid metabolism by CYP and CES enzymes were scaled to per gram of adult human liver using abundance values for microsomal CYP enzymes and for CES enzymes in liver microsomes and cytosol. TPM had the highest and CPM the lowest apparent CLint values for total metabolism (CYP and CES enzymes) per gram of adult human liver. Due to their higher abundance, all three pyrethroids were extensively metabolised by CES enzymes in adult human liver, with CYP enzymes only accounting for 2%, 10% and 1% of total metabolism for DLM, CPM and TPM, respectively.
Asunto(s)
Carboxilesterasa/química , Sistema Enzimático del Citocromo P-450/química , Nitrilos/química , Permetrina/química , Piretrinas/química , Carboxilesterasa/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Humanos , Nitrilos/farmacocinética , Permetrina/farmacocinética , Piretrinas/farmacocinética , EstereoisomerismoRESUMEN
The metabolism of deltamethrin (DLM), cis-permethrin (CPM) and trans-permethrin (TPM) was studied in liver microsomes, liver cytosol and plasma from male Sprague-Dawley rats aged 15, 21 and 90 days and from adult humans. DLM and CPM were metabolised by rat hepatic microsomal cytochrome P450 (CYP) enzymes and to a lesser extent by microsomal and cytosolic carboxylesterase (CES) enzymes, whereas TPM was metabolised to a greater extent by CES enzymes. In human liver, DLM and TPM were mainly metabolised by CES enzymes, whereas CPM was metabolised by CYP and CES enzymes. The metabolism of pyrethroids by cytosolic CES enzymes contributes to the overall hepatic clearance of these compounds. DLM, CPM and TPM were metabolised by rat, but not human, plasma CES enzymes. This study demonstrates that the ability of male rats to metabolise DLM, CPM and TPM by hepatic CYP and CES enzymes and plasma CES enzymes increases with age. In all instances, apparent intrinsic clearance values were lower in 15 than in 90 day old rats. As pyrethroid-induced neurotoxicity is due to the parent compound, these results suggest that DLM, CPM and TPM may be more neurotoxic to juvenile than to adult rats.
Asunto(s)
Citosol/metabolismo , Hígado/metabolismo , Microsomas Hepáticos/metabolismo , Nitrilos/metabolismo , Permetrina/metabolismo , Plasma/metabolismo , Piretrinas/metabolismo , Animales , Humanos , Cinética , Masculino , Ratas Sprague-DawleyRESUMEN
The hepatic and thyroid gland effects of the constitutive androstane receptor (CAR) activator sodium phenobarbital (NaPB) and the pregnane X receptor (PXR) activator pregnenolone-16α-carbonitrile (PCN) were examined in male Sprague-Dawley wild-type (WT) and knockout (KO) rats lacking both hepatic CAR and PXR receptors (CAR KO/PXR KO rats). The treatment of WT rats for 7 d with 500 ppm NaPB in the diet and 100 mg/kg/d PCN by gavage resulted in increased relative liver weight, hepatocyte hypertrophy, increased hepatocyte replicative DNA synthesis (RDS) and induction of cytochrome P450 CYP2B and CYP3A subfamily enzymes. NaPB and PCN also induced thyroid gland follicular cell RDS and hepatic microsomal UDP-glucuronosyltransferase activity towards thyroxine as substrate. These effects were not observed in the liver and thyroid gland of CAR KO/PXR KO rats. Male C57BL/6 J (WT) and CAR KO/PXR KO mice were given 1000 ppm NaPB in the diet for 7 d. In WT, but not in CAR KO/PXR KO, mice NaPB treatment resulted in liver hypertrophy and induction of hepatocyte RDS and Cyp2b enzymes. These results suggest that the CAR KO/PXR KO rat and mouse models are useful experimental models for mode of action studies with rodent CAR activators.
Asunto(s)
Hígado/efectos de los fármacos , Fenobarbital/farmacología , Receptor X de Pregnano/genética , Carbonitrilo de Pregnenolona/farmacología , Receptores Citoplasmáticos y Nucleares/genética , Glándula Tiroides/efectos de los fármacos , Animales , Receptor de Androstano Constitutivo , Replicación del ADN/efectos de los fármacos , Técnicas de Inactivación de Genes , Masculino , Ratones Endogámicos C57BL , Ratas , Ratas Sprague-DawleyRESUMEN
A number of chemicals produce liver and thyroid gland tumours in rodents by nongenotoxic modes of action (MOAs). In this study the hepatic and thyroid gland effects of the constitutive androstane receptor (CAR) activator sodium phenobarbital (NaPB) were examined in male Sprague-Dawley wild type (WT) rats and in CAR knockout (CAR KO) rats and the effects of the pregnane X receptor (PXR) activator pregnenolone-16α-carbonitrile (PCN) were examined in WT and PXR knockout (PXR KO) rats. Rats were either fed diets containing 0 (control) or 500â¯ppm NaPB or were dosed with 0 (control) or 100â¯mg/kg/day PCN orally for 7â¯days. The treatment of WT rats with NaPB and PCN for 7â¯days resulted in increased relative liver weight, increased hepatocyte replicative DNA synthesis (RDS) and the induction of cytochrome P450 CYP2B and CYP3A subfamily enzyme, mRNA and protein levels. In marked contrast, the treatment of CAR KO rats with NaPB and PXR KO rats with PCN did not result in any increases in liver weight and induction of CYP2B and CYP3A enzymes. The treatment of CAR KO rats with NaPB had no effect on hepatocyte RDS, while PCN produced only a small increase in hepatocyte RDS in PXR KO rats. Treatment with NaPB had no effect on thyroid gland weight in WT and CAR KO rats, whereas treatment with PCN resulted in an increase in relative thyroid gland weight in WT, but not in PXR KO, rats. Thyroid gland follicular cell RDS was increased by the treatment of WT rats with NaPB and PCN, with NaPB also producing a small increase in thyroid gland follicular cell RDS in CAR KO rats. Overall, the present study with CAR KO rats demonstrates that a functional CAR is required for NaPB-mediated increases in liver weight, stimulation of hepatocyte RDS and induction of hepatic CYP enzymes. The studies with PXR KO rats demonstrate that a functional PXR is required for PCN-mediated increases in liver weight and induction of hepatic CYP enzymes; with induction of hepatocyte RDS also being largely mediated through PXR. The hepatic effects of NaPB in CAR KO rats and of PCN in PXR KO rats are in agreement with those observed in other recent literature studies. These results suggest that CAR KO and PXR KO rats are useful experimental models for liver MOA studies with rodent CAR and PXR activators and may also be useful for thyroid gland MOA studies.
Asunto(s)
Hepatocitos/metabolismo , Fenobarbital/farmacología , Receptor X de Pregnano/deficiencia , Carbonitrilo de Pregnenolona/farmacología , Receptores Citoplasmáticos y Nucleares/deficiencia , Glándula Tiroides/metabolismo , Animales , Receptor de Androstano Constitutivo , Hepatocitos/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Receptor X de Pregnano/genética , Ratas , Ratas Sprague-Dawley , Ratas Transgénicas , Receptores Citoplasmáticos y Nucleares/genética , Glándula Tiroides/efectos de los fármacosRESUMEN
Phenobarbital (PB), a constitutive androstane receptor (CAR) activator, produces liver tumours in rodents by a mitogenic mode of action involving CAR activation. In this study, the hepatic effects of sodium phenobarbital (NaPB) were compared in male C57BL/6J wild type (WT) mice and in humanized mice, where both the mouse CAR and pregnane X receptor (PXR) have been replaced by their human counterparts (hCAR/hPXR mice). Investigations were also performed in cultured male C57BL/6J and CD-1 mouse, male Sprague-Dawley rat and male and female human hepatocytes. The treatment of WT and hCAR/hPXR mice with 186-984â¯ppm NaPB in the diet for 7â¯days resulted in increased relative liver weight, hypertrophy and induction of cytochrome P450 (CYP) enzyme activities. Treatment with NaPB also produced dose-dependent increases in hepatocyte replicative DNA synthesis (RDS), with the effect being more marked in WT than in hCAR/hPXR mice. While the treatment of cultured C57BL/6J and CD-1 mouse, Sprague-Dawley rat and human hepatocytes with 100 and/or 1000⯵M NaPB for 4â¯days induced CYP enzyme activities, increased RDS was only observed in mouse and rat hepatocytes. However, as a positive control, epidermal growth factor increased RDS in hepatocytes from all three species. In summary, although human hepatocytes are refractory to the mitogenic effects of NaPB, treatment with NaPB induced RDS in vivo in hCAR/hPXR mice, which is presumably due to the human CAR and PXR receptors operating in a mouse hepatocyte regulatory environment. As the response of the hCAR/hPXR mouse to the CAR activator NaPB differs markedly from that of human hepatocytes, the hCAR/hPXR mouse is thus not a suitable animal model for studies on the hepatic effects of nongenotoxic rodent CAR activators.
Asunto(s)
Hepatocitos/efectos de los fármacos , Hipnóticos y Sedantes/farmacología , Fenobarbital/farmacología , Receptores Citoplasmáticos y Nucleares/efectos de los fármacos , Receptores de Esteroides/efectos de los fármacos , Animales , Células Cultivadas , Receptor de Androstano Constitutivo , Citocromos/metabolismo , Replicación del ADN/efectos de los fármacos , Factor de Crecimiento Epidérmico/metabolismo , Femenino , Humanos , Hipnóticos y Sedantes/sangre , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Tamaño de los Órganos , Fenobarbital/sangre , Receptor X de Pregnano , Ratas , Ratas Sprague-DawleyRESUMEN
In a National Toxicology Program (NTP) bioassay, inhalation of tetrahydrofuran (THF) induced liver tumors in female B6C3F1 mice but not in male mice or rats of either sex. Since THF is not genotoxic, the NTP concluded this carcinogenic activity was likely mediated via non-genotoxic modes of action (MOA). Based on evidence that THF and phenobarbital share a similar MOA, female Car/Pxr knock-out mice were orally exposed to THF to evaluate the potential role of CAR activation in the MOA for THF-induced liver tumors. Because data from this oral study with Car/Pxr knock-out mice (C57Bl/6) and the inhalation studies with wild type mice (B6C3F1) reported by NTP and others were derived from different strains, oral studies with wild type B6C3F1 and C57Bl/6 mice were conducted to ensure THF responses in both strains were comparable. As seen in inhalation studies with THF, oral exposure of wild type female mice to a maximum tolerated dose of THF increased total P450 content, CAR-related P450 activities, and hepatocyte proliferation; these effects were not observed in Car/Pxr knock-out female mice. This finding supports the hypothesis THF-induced carcinogenicity is likely mediated via CAR activation that has limited, if any, relevance to humans.
Asunto(s)
Carcinógenos/toxicidad , Furanos/toxicidad , Neoplasias Hepáticas Experimentales/inducido químicamente , Hígado/efectos de los fármacos , Administración Oral , Animales , Carcinógenos/administración & dosificación , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica/inducido químicamente , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Receptor de Androstano Constitutivo , Sistema Enzimático del Citocromo P-450/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Furanos/administración & dosificación , Genotipo , Humanos , Hígado/metabolismo , Hígado/patología , Neoplasias Hepáticas Experimentales/genética , Neoplasias Hepáticas Experimentales/metabolismo , Neoplasias Hepáticas Experimentales/patología , Dosis Máxima Tolerada , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Receptor X de Pregnano , Receptores Citoplasmáticos y Nucleares/deficiencia , Receptores Citoplasmáticos y Nucleares/genética , Receptores de Esteroides/deficiencia , Receptores de Esteroides/genética , Medición de Riesgo , Especificidad de la EspecieRESUMEN
Registration of new plant protection products (e.g., herbicide, insecticide, or fungicide) requires comprehensive mammalian toxicity evaluation including carcinogenicity studies in two species. The outcome of the carcinogenicity testing has a significant bearing on the overall human health risk assessment of the substance and, consequently, approved uses for different crops across geographies. In order to understand the relevance of a specific tumor finding to human health, a systematic, transparent, and hypothesis-driven mode of action (MoA) investigation is, appropriately, an expectation by the regulatory agencies. Here, we describe a novel approach of prospectively generating the MoA data by implementing additional end points to the standard guideline toxicity studies with sulfoxaflor, a molecule in development. This proactive MoA approach results in a more robust integration of molecular with apical end points while minimizing animal use. Sulfoxaflor, a molecule targeting sap-feeding insects, induced liver effects (increased liver weight due to hepatocellular hypertrophy) in an initial palatability probe study for selecting doses for subsequent repeat-dose dietary studies. This finding triggered the inclusion of dose-response investigations of the potential key events for rodent liver carcinogenesis, concurrent with the hazard assessment studies. As predicted, sulfoxaflor induced liver tumors in rats and mice in the bioassays. The MoA data available by the time of the carcinogenicity finding supported the conclusion that the carcinogenic potential of sulfoxaflor was due to CAR/PXR nuclear receptor activation with subsequent hepatocellular proliferation. This MoA was not considered to be relevant to humans as sulfoxaflor is unlikely to induce hepatocellular proliferation in humans and therefore would not be a human liver carcinogen.
Asunto(s)
Insecticidas/toxicidad , Hígado/efectos de los fármacos , Piridinas/toxicidad , Compuestos de Azufre/toxicidad , Pruebas de Toxicidad/métodos , Animales , Proliferación Celular/efectos de los fármacos , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP2B1/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Insecticidas/administración & dosificación , Hígado/enzimología , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Noqueados , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Microsomas Hepáticos/metabolismo , Estudios Prospectivos , Piridinas/administración & dosificación , ARN/química , ARN/genética , Ratas , Ratas Endogámicas F344 , Reacción en Cadena en Tiempo Real de la Polimerasa , Compuestos de Azufre/administración & dosificaciónRESUMEN
Certain metabolites of progesterone and deoxycorticosterone are established as potent and selective positive allosteric modulators of the gamma-aminobutyric acid type A (GABA(A)) receptor. Upon administration these steroids exhibit clear behavioural effects that include anxiolysis, sedation and analgesia, they are anticonvulsant and at high doses induce a state of general anaesthesia, a profile consistent with an action to enhance neuronal inhibition. Physiologically, peripherally synthesised pregnane steroids derived from endocrine glands such as the adrenals and ovaries function as hormones by crossing the blood brain barrier to influence neuronal signalling. However, the demonstration that certain neurons and glial cells within the central nervous system (CNS) can synthesize these steroids either de novo, or from peripherally derived progesterone, has led to the proposal that these steroids (neurosteroids) can additionally function in a paracrine manner, to locally influence GABAergic transmission. Steroid levels are known to change dynamically, for example in stress and during pregnancy. Given that GABA(A) receptors are ubiquitously expressed throughout the central nervous system, such changes in steroid levels would be predicted to cause a global enhancement of inhibitory neurotransmission throughout the brain, a scenario that would seem incompatible with a physiological role as a selective neuromodulator. Here, we will review emerging evidence that the GABA-modulatory actions of the pregnane steroids are highly selective, with their actions being brain region and indeed neuron dependent. Furthermore, the sensitivity of GABA(A) receptors is not static but can dynamically change. The molecular mechanisms underpinning this neuronal specificity will be discussed with particular emphasis being given to the role of GABA(A) receptor isoforms, protein phosphorylation and local steroid metabolism and synthesis.
Asunto(s)
Sistema Nervioso Central/fisiología , Receptores de GABA-A/efectos de los fármacos , Receptores de GABA-A/metabolismo , Esteroides/metabolismo , Esteroides/farmacología , Animales , Humanos , Plasticidad Neuronal/fisiología , Fosforilación , Isoformas de Proteínas , Receptores de GABA-A/química , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiologíaRESUMEN
The ability of differing subunit combinations of 5-HT3 receptors to form functional cell surface receptors was analyzed by a variety of approaches. The results revealed that 5-HT3 receptor assembly occurred within the endoplasmic reticulum (ER) and involved the interaction with chaperone proteins. The 5-HT3A subunit could assemble into functional homomeric receptors that were expressed on the cell surface. In contrast, the 5-HT3B subunit did not exhibit 5-hydroxytryptamine binding or function, could not assemble, and was efficiently retained and degraded within the ER. However, upon the coexpression of the 5-HT3A subunit, 5-HT3B could be "rescued" from the ER and transported to the cell surface to form functional heteromeric receptors with distinct functional characteristics. In support of the existence of homomeric 5-HT3 receptors in vivo, recombinantly expressed 5-HT3A receptors were capable of clustered cell surface expression in cortical neurons.