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huCAR) for the constitutive androstane receptor (CAR) and pregnane X receptor (PXR) administered phenobarbitone (PB)
Abstracts / Toxicology Letters 229S (2014) S40–S252
reactive species, and annexin V were also evaluated in HepG2 cells exposed to BDE-153 (0.1–25 mol/L) by 24 and 48 h, and finally, we evaluated the genotoxic potential of the compound through the comet assay using also Hepg2 (0.1–25 mol/L) by 4 and 24 h. It was observed that BDE-153 in concentrations of 25 M and higher, inhibited mitochondrial phosphorylation and induced the dissipation of the , thereby reducing the level of mitochondrial ATP. Additionally, it was observed a decrease of in HepG2, accumulation of reactive species and induces apoptotic cell death since it was observed exposure of phosphatidylserine in the outer cell membrane. Furthermore, BDE-153 is able to induce primary DNA damage observed by comet assay after 4 h and persists after 24 h to exposure. These results demonstrate that BDE-153 lead to a mitochondrial dysfunction causing cell death and induce genotoxic effects. This way, the cell death and mitochondrial impairment may be related to this final hepatotoxicity effect by BDE-153. http://dx.doi.org/10.1016/j.toxlet.2014.06.243 P-1.64 Characterisation of the hepatic effects of pregnenolone 16 alpha-carbonitrile (PCN) in pregnane X receptor (PXR, NR1I2) knockout (KO) rats Audrey Vardy 1 , Mark Chamberlain 1 , John Foster 2 , Clifford Elcombe 1,∗ 1
CXR Biosciences, Dundee, UK, 2 ToxPath Sciences Ltd, Congleton, UK
The administration of PXR activators to rats and mice induces hepatomegaly that is characterised by hepatocellular hypertrophy due to the proliferation of smooth endoplasmic reticulum and concomitant induction of enzymes belonging to the CYP3A and CYP2B families and hyperplasia (increased DNA synthesis and cell proliferation). Studies utilising PXR KO mice have demonstrated that the presence of an active PXR is necessary for the induction of hepatomegaly. This study investigated the effects of the PXR-activator PCN in PAR KO rats (SAGE Labs). Male Wild Type (WT) and PXR KO rats, implanted with osmotic pumps containing BrdU to allow determination of DNA synthesis (Sphase), were administered PCN (100 mg/kg oral gavage) for 7 days. PCN treatment of WT rats resulted in 1.2-fold increases in liver weight and liver/body weight ratios, a 6-fold increase in S-phase labelling index and a 1.9-fold increase in total microsomal P450. The microsomal CYP2B-catalysed reactions pentoxyresorufin-Odepentylation (PROD) and benzyloxyresorufin-O-debenzylation (BROD) were induced 3-fold and benzoquinoline-O-debenzylation (CYP3A catalysed) was induced 4-fold. These changes were accompanied by increases in mRNA expression of CYP2B1 (17-fold), CYP2B2 (18-fold) and CYP3A1 (19-fold). None of these PCN-induced effects were observed in PXR KO rats. In conclusion, the hepatomegaly induced by PCN is dependent on the presence of an active PXR. http://dx.doi.org/10.1016/j.toxlet.2014.06.244
S61
P-1.65 Differential dose–response relationships for hepatic Cyp2b10 induction and cell proliferation in wild type mice and mice humanized (huPXR/huCAR) for the constitutive androstane receptor (CAR) and pregnane X receptor (PXR) administered phenobarbitone (PB) Clifford Elcombe ∗ , Audrey Vardy CXR Biosciences, Dundee, UK The nuclear receptors PXR and CAR regulate hepatomegaly. PB, a nongenotoxic hepatocarcinogen, activates CAR and induces hepatomegaly (hypertrophy/Cyp2b and hyperplasia/cell proliferation). CAR-mediated cell proliferation is the key event in PB-induced hepatocarcinogenesis. To compare the activation of the human and murine receptors PB was administered for 7 days (0, 200, 500, 750 and 1000 ppm diet) to huPXR/huCAR mice and C57BL/6 (WT) mice. Hepatocellular proliferation (S-phase) was determined using implanted BrdU osmotic pumps. This regimen induced hepatic microsomal pentoxyresorufin-O-depentylation (Cyp2b10) in WT mice (1.0, 27, 87, 112 and 117 pmol/min/mg at 0, 200, 500, 750 and 1000 ppm respectively) and humanized mice (3.1, 47, 101, 134 and 146 pmol/min/mg at 0, 200, 500, 750 and 1000 ppm respectively). The dose–response curves for Cyp2b10 induction were similar, but not identical, in both strains of mice. However, when dose was expressed as plasma [PB] the curves were superimposable. The S-phase labelling index in WT mice was 0.84, 8.4, 19, 22 and 27% of hepatocytes at 0, 200, 500, 750 and 1000 ppm respectively. In humanized mice the labelling index was 0.23, 0.51, 4.0, 9.2 and 12 0% at 200, 500, 750 and 1000 ppm respectively. These dose–response curves were markedly dissimilar, with the humanised mice being more sensitive. In contrast to Cyp2b10 induction, when dose was expressed as plasma [PB] the curves for S-phase activity were even more divergent. These data demonstrate differences in dose–response relationships for the murine and human receptors, and for enzyme induction and cell proliferation. http://dx.doi.org/10.1016/j.toxlet.2014.06.245 P-1.67 Investigating the roles of lysosomal iron and reactive oxygen species in the onset of mitochondrial toxicity induced through endoperoxide antimalarial drugs James Firman 1,∗ , Kelvin Cain 2 , B. Kevin Park 1 , Marion MacFarlane 2 , Amy Chadwick 1 1
University of Liverpool, Liverpool, UK, 2 University of Leicester, Leicester, UK Derived from the natural product artemisinin, endoperoxide antimalarial drugs form frontline treatment against disease occurring as a consequence of plasmodium Falciparum infection. Although generally well tolerated in man, safety concerns have been raised owing to persistent reports of their ability to induce neurotoxicity and embryotoxicity across animal models. Research performed within our group has identified the onset of reactive oxygen species (ROS) formation, accompanied by mitochondrial dysfunction and the emergence of apoptosis, to be key stages in the pathway through which these drugs induce cytotoxic effects.
reactive species, and annexin V were also evaluated in HepG2 cells exposed to BDE-153 (0.1–25 mol/L) by 24 and 48 h, and finally, we evaluated the genotoxic potential of the compound through the comet assay using also Hepg2 (0.1–25 mol/L) by 4 and 24 h. It was observed that BDE-153 in concentrations of 25 M and higher, inhibited mitochondrial phosphorylation and induced the dissipation of the , thereby reducing the level of mitochondrial ATP. Additionally, it was observed a decrease of in HepG2, accumulation of reactive species and induces apoptotic cell death since it was observed exposure of phosphatidylserine in the outer cell membrane. Furthermore, BDE-153 is able to induce primary DNA damage observed by comet assay after 4 h and persists after 24 h to exposure. These results demonstrate that BDE-153 lead to a mitochondrial dysfunction causing cell death and induce genotoxic effects. This way, the cell death and mitochondrial impairment may be related to this final hepatotoxicity effect by BDE-153. http://dx.doi.org/10.1016/j.toxlet.2014.06.243 P-1.64 Characterisation of the hepatic effects of pregnenolone 16 alpha-carbonitrile (PCN) in pregnane X receptor (PXR, NR1I2) knockout (KO) rats Audrey Vardy 1 , Mark Chamberlain 1 , John Foster 2 , Clifford Elcombe 1,∗ 1
CXR Biosciences, Dundee, UK, 2 ToxPath Sciences Ltd, Congleton, UK
The administration of PXR activators to rats and mice induces hepatomegaly that is characterised by hepatocellular hypertrophy due to the proliferation of smooth endoplasmic reticulum and concomitant induction of enzymes belonging to the CYP3A and CYP2B families and hyperplasia (increased DNA synthesis and cell proliferation). Studies utilising PXR KO mice have demonstrated that the presence of an active PXR is necessary for the induction of hepatomegaly. This study investigated the effects of the PXR-activator PCN in PAR KO rats (SAGE Labs). Male Wild Type (WT) and PXR KO rats, implanted with osmotic pumps containing BrdU to allow determination of DNA synthesis (Sphase), were administered PCN (100 mg/kg oral gavage) for 7 days. PCN treatment of WT rats resulted in 1.2-fold increases in liver weight and liver/body weight ratios, a 6-fold increase in S-phase labelling index and a 1.9-fold increase in total microsomal P450. The microsomal CYP2B-catalysed reactions pentoxyresorufin-Odepentylation (PROD) and benzyloxyresorufin-O-debenzylation (BROD) were induced 3-fold and benzoquinoline-O-debenzylation (CYP3A catalysed) was induced 4-fold. These changes were accompanied by increases in mRNA expression of CYP2B1 (17-fold), CYP2B2 (18-fold) and CYP3A1 (19-fold). None of these PCN-induced effects were observed in PXR KO rats. In conclusion, the hepatomegaly induced by PCN is dependent on the presence of an active PXR. http://dx.doi.org/10.1016/j.toxlet.2014.06.244
S61
P-1.65 Differential dose–response relationships for hepatic Cyp2b10 induction and cell proliferation in wild type mice and mice humanized (huPXR/huCAR) for the constitutive androstane receptor (CAR) and pregnane X receptor (PXR) administered phenobarbitone (PB) Clifford Elcombe ∗ , Audrey Vardy CXR Biosciences, Dundee, UK The nuclear receptors PXR and CAR regulate hepatomegaly. PB, a nongenotoxic hepatocarcinogen, activates CAR and induces hepatomegaly (hypertrophy/Cyp2b and hyperplasia/cell proliferation). CAR-mediated cell proliferation is the key event in PB-induced hepatocarcinogenesis. To compare the activation of the human and murine receptors PB was administered for 7 days (0, 200, 500, 750 and 1000 ppm diet) to huPXR/huCAR mice and C57BL/6 (WT) mice. Hepatocellular proliferation (S-phase) was determined using implanted BrdU osmotic pumps. This regimen induced hepatic microsomal pentoxyresorufin-O-depentylation (Cyp2b10) in WT mice (1.0, 27, 87, 112 and 117 pmol/min/mg at 0, 200, 500, 750 and 1000 ppm respectively) and humanized mice (3.1, 47, 101, 134 and 146 pmol/min/mg at 0, 200, 500, 750 and 1000 ppm respectively). The dose–response curves for Cyp2b10 induction were similar, but not identical, in both strains of mice. However, when dose was expressed as plasma [PB] the curves were superimposable. The S-phase labelling index in WT mice was 0.84, 8.4, 19, 22 and 27% of hepatocytes at 0, 200, 500, 750 and 1000 ppm respectively. In humanized mice the labelling index was 0.23, 0.51, 4.0, 9.2 and 12 0% at 200, 500, 750 and 1000 ppm respectively. These dose–response curves were markedly dissimilar, with the humanised mice being more sensitive. In contrast to Cyp2b10 induction, when dose was expressed as plasma [PB] the curves for S-phase activity were even more divergent. These data demonstrate differences in dose–response relationships for the murine and human receptors, and for enzyme induction and cell proliferation. http://dx.doi.org/10.1016/j.toxlet.2014.06.245 P-1.67 Investigating the roles of lysosomal iron and reactive oxygen species in the onset of mitochondrial toxicity induced through endoperoxide antimalarial drugs James Firman 1,∗ , Kelvin Cain 2 , B. Kevin Park 1 , Marion MacFarlane 2 , Amy Chadwick 1 1
University of Liverpool, Liverpool, UK, 2 University of Leicester, Leicester, UK Derived from the natural product artemisinin, endoperoxide antimalarial drugs form frontline treatment against disease occurring as a consequence of plasmodium Falciparum infection. Although generally well tolerated in man, safety concerns have been raised owing to persistent reports of their ability to induce neurotoxicity and embryotoxicity across animal models. Research performed within our group has identified the onset of reactive oxygen species (ROS) formation, accompanied by mitochondrial dysfunction and the emergence of apoptosis, to be key stages in the pathway through which these drugs induce cytotoxic effects.