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Construction and characterization of stable transfected hepatocyte-like cells
S140
Abstracts / Toxicology Letters 211S (2012) S43–S216
A major interest in current risk assessment studies is to reconstruct exposure doses of individuals from measurements of appropriate biomarkers in accessible biological matrices such as urine. However, to establish a one-to-one relation between the xenobiotic exposure doses and biomarkers, a thorough knowledge of the kinetics of the chemical substance and its route of exposure in the subjects under study is needed. In the present study, we have built a physiologically-based pharmacokinetic (PBPK) model of benzo(a)pyrene and its 3hydroxybenzo(a)pyrene from in vivo experimental data in rats and, thereafter, we have extrapolated the model to simulate human kinetics following real exposure scenarios. All the typical physiological parameters (i.e. organ weights, blood flows, etc.) corresponded to commonly known values in rats and humans. The partition coefficients, metabolism rates, absorption fractions, and other route-of-entry and excretion parameters were obtained directly from in vivo rat time courses in various organs available from published works. The parameters required were best-fitted by least square procedures and Monte Carlo simulations. Subsequently, sensitivity analyses were carried out to ensure the stability of the model and the crucial degrees of freedom driving the model. Finally, we have found that the rat-to-human extrapolation is a proper way to model the kinetics of the polycyclic aromatic hydrocarbons studied. Furthermore, our in vivo PBPK model has allowed us to clearly identify the storage tissues and its leading importance in the extrapolation to human exposure scenarios. In conclusion, our simulations have appropriately simulated independent sets of rat and human experimental data. doi:10.1016/j.toxlet.2012.03.510
P21-06 Construction and characterization of stable transfected hepatocyte-like cells Zdenek Dvorak 1 , Petr Pavek 2 , Aneta Novotna 1 Palacky University Olomouc, Czech Republic, 2 Charles University Prague, Czech Republic
1
Lack of hepatocyte-specific function and altered phenotype of cancer cell lines derived from hepatocytes is at least partly caused by under-expression of transcription factors such as peroxisome proliferator-activated receptor gamma co-activator 1␣ (PGC-1␣), steroid receptors co-activator 1 (SRC1), hepatocyte nuclear factor 4␣ (HNF4␣) and several others. Transient transfection of HNF4␣ in hepatoma cells revealed improved hepatospecific functions, including the expression of drug-metabolizing enzymes Transient transfection of PGC-1␣ in HepG2 partly restored the expression of drug-metabolizing enzymes through co-activation of hepatocyte nuclear factor HNF4␣. We established human cell lines derived from HepG2 cells stably transfected with human HNF4␣ (HepG2–HNF4␣) and PGC1␣ (HepG2–PGC-1␣). We tested resulted cell lines for hepatospecific markers. We found increased secretion of albumin, fibrinogen and plasminogen but not ␣1-antitrypsin in HepG2–HNF4␣ cell line and increased secretion of fibrinogen in HepG2–PGC-1␣. Expression of xenoreceptors pregnane X receptor (PXR) and aryl hydrocarbon receptor (AhR) proteins was increased, but not mRNAs in both HepG2–HNF4␣ and HepG2–PGC-1␣ cell lines. Basal expression of CYP3A4 protein was increased, but rifampicin-induced expression of CYP3A4 protein was lower than in parent HepG2 in both HepG2–HNF4␣ and HepG2–PGC-1␣ cell lines. Expression of TCDDinducible CYP1A1 protein was augmented in HepG2–HNF4␣ cells
compared to parent HepG2 cells but we did not found increased TCDD-inducible expression CYP1A1 protein in HepG2–PGC-1␣ cells compared to parent HepG2 cells. We concluded that stable over-expression of HNF4␣ and PGC-1␣ in HepG2 cells restores some of the hepatospecific functions, but it has only minor effect on the expression of xenobiotic-metabolizing enzymes and their regulators. doi:10.1016/j.toxlet.2012.03.511 P22: Metals
P22-01 Effect of cadmium chloride on interleukin-2, lymphocyte and DNA in adult male albino rats Kawther Elhady, Ayman Abdelhamid, Rania Elgawish Faculty of Vet Medicine, Suez Canal Univ, Egypt Purpose: The objective of this study is to evaluate the effect of different doses of cadmium chloride in drinking water for 90 days on interleukin-2, lymphocyte transformation, DNA fragmentation and thymus histopathology in adult male albino rats. Material and method: Forty adult male albino rats were divided randomly into 4 equal groups (one control and three treated groups). The control group received only dis. water, while the treated groups received 5, 50 and 100 ppm of cadmium chloride (CdCl2 ) for consecutive 90 days in drinking water. Blood samples were collected just before scarification of the rats and serum was separated. The level of interleukin-2 in the serum of control and treated male albino rats was measured by ELISA. Lymphocyte transformation and DNA fragmentation assay in blood lymphocyte of rats were evaluated. At Day 90 of the experiment, rats were sacrificed by cervical dislocation and thymus was collected, weighted and stored in 10% neutral buffered formalin for histopathology. Data were analyzed by one-way analysis of variance using GraphPad Prism software. Result and conclusion: The interleukin-2 level was declined in a dose-dependent manner in treated rats with different doses (5, 50 and 100 ppm) of CdCl2 . Obviously the rats treated with 100 ppm of CdCl2 showed a significant decrease (P < 0.001) in the level of interleukin-2 compared to control. Moreover, rats administrated 100 ppm of CdCl2 showed a significantly (P < 0.05) high rate of lymphocyte transformation compared to control. Rats treated with 5, 50 and 100 ppm of CdCl2 showed certain levels of DNA strand breaks in blood lymphocyte. Male rats exposed to100 ppm (P < 0.001) and 50 ppm (P < 0.05) of CdCl2 showed a significant decrease in the relative thymus weight than that of control ones. The thymus of rats treated with 50 and 100 ppm of CdCl2 showed a reduction in the medulla’s diameter and interlobular fat globules compared to control. doi:10.1016/j.toxlet.2012.03.513
P22-02 Early-life exposure to lithium and boron from drinking water Florencia Harari Freire 1 , Ana María Ronco 2 , Gabriela Concha 3 , Miguel Llanos 2 , Francisca Castro 2 , Barbro Nermell 1 , Margaretha Grandér 1 , Brita Palm 1 , Marie Vahter 1 Karolinska Institutet, Sweden, 2 Inst. of Nutrition and Food Technology, Chile, 3 National Food Administration, Sweden
1
Abstracts / Toxicology Letters 211S (2012) S43–S216
A major interest in current risk assessment studies is to reconstruct exposure doses of individuals from measurements of appropriate biomarkers in accessible biological matrices such as urine. However, to establish a one-to-one relation between the xenobiotic exposure doses and biomarkers, a thorough knowledge of the kinetics of the chemical substance and its route of exposure in the subjects under study is needed. In the present study, we have built a physiologically-based pharmacokinetic (PBPK) model of benzo(a)pyrene and its 3hydroxybenzo(a)pyrene from in vivo experimental data in rats and, thereafter, we have extrapolated the model to simulate human kinetics following real exposure scenarios. All the typical physiological parameters (i.e. organ weights, blood flows, etc.) corresponded to commonly known values in rats and humans. The partition coefficients, metabolism rates, absorption fractions, and other route-of-entry and excretion parameters were obtained directly from in vivo rat time courses in various organs available from published works. The parameters required were best-fitted by least square procedures and Monte Carlo simulations. Subsequently, sensitivity analyses were carried out to ensure the stability of the model and the crucial degrees of freedom driving the model. Finally, we have found that the rat-to-human extrapolation is a proper way to model the kinetics of the polycyclic aromatic hydrocarbons studied. Furthermore, our in vivo PBPK model has allowed us to clearly identify the storage tissues and its leading importance in the extrapolation to human exposure scenarios. In conclusion, our simulations have appropriately simulated independent sets of rat and human experimental data. doi:10.1016/j.toxlet.2012.03.510
P21-06 Construction and characterization of stable transfected hepatocyte-like cells Zdenek Dvorak 1 , Petr Pavek 2 , Aneta Novotna 1 Palacky University Olomouc, Czech Republic, 2 Charles University Prague, Czech Republic
1
Lack of hepatocyte-specific function and altered phenotype of cancer cell lines derived from hepatocytes is at least partly caused by under-expression of transcription factors such as peroxisome proliferator-activated receptor gamma co-activator 1␣ (PGC-1␣), steroid receptors co-activator 1 (SRC1), hepatocyte nuclear factor 4␣ (HNF4␣) and several others. Transient transfection of HNF4␣ in hepatoma cells revealed improved hepatospecific functions, including the expression of drug-metabolizing enzymes Transient transfection of PGC-1␣ in HepG2 partly restored the expression of drug-metabolizing enzymes through co-activation of hepatocyte nuclear factor HNF4␣. We established human cell lines derived from HepG2 cells stably transfected with human HNF4␣ (HepG2–HNF4␣) and PGC1␣ (HepG2–PGC-1␣). We tested resulted cell lines for hepatospecific markers. We found increased secretion of albumin, fibrinogen and plasminogen but not ␣1-antitrypsin in HepG2–HNF4␣ cell line and increased secretion of fibrinogen in HepG2–PGC-1␣. Expression of xenoreceptors pregnane X receptor (PXR) and aryl hydrocarbon receptor (AhR) proteins was increased, but not mRNAs in both HepG2–HNF4␣ and HepG2–PGC-1␣ cell lines. Basal expression of CYP3A4 protein was increased, but rifampicin-induced expression of CYP3A4 protein was lower than in parent HepG2 in both HepG2–HNF4␣ and HepG2–PGC-1␣ cell lines. Expression of TCDDinducible CYP1A1 protein was augmented in HepG2–HNF4␣ cells
compared to parent HepG2 cells but we did not found increased TCDD-inducible expression CYP1A1 protein in HepG2–PGC-1␣ cells compared to parent HepG2 cells. We concluded that stable over-expression of HNF4␣ and PGC-1␣ in HepG2 cells restores some of the hepatospecific functions, but it has only minor effect on the expression of xenobiotic-metabolizing enzymes and their regulators. doi:10.1016/j.toxlet.2012.03.511 P22: Metals
P22-01 Effect of cadmium chloride on interleukin-2, lymphocyte and DNA in adult male albino rats Kawther Elhady, Ayman Abdelhamid, Rania Elgawish Faculty of Vet Medicine, Suez Canal Univ, Egypt Purpose: The objective of this study is to evaluate the effect of different doses of cadmium chloride in drinking water for 90 days on interleukin-2, lymphocyte transformation, DNA fragmentation and thymus histopathology in adult male albino rats. Material and method: Forty adult male albino rats were divided randomly into 4 equal groups (one control and three treated groups). The control group received only dis. water, while the treated groups received 5, 50 and 100 ppm of cadmium chloride (CdCl2 ) for consecutive 90 days in drinking water. Blood samples were collected just before scarification of the rats and serum was separated. The level of interleukin-2 in the serum of control and treated male albino rats was measured by ELISA. Lymphocyte transformation and DNA fragmentation assay in blood lymphocyte of rats were evaluated. At Day 90 of the experiment, rats were sacrificed by cervical dislocation and thymus was collected, weighted and stored in 10% neutral buffered formalin for histopathology. Data were analyzed by one-way analysis of variance using GraphPad Prism software. Result and conclusion: The interleukin-2 level was declined in a dose-dependent manner in treated rats with different doses (5, 50 and 100 ppm) of CdCl2 . Obviously the rats treated with 100 ppm of CdCl2 showed a significant decrease (P < 0.001) in the level of interleukin-2 compared to control. Moreover, rats administrated 100 ppm of CdCl2 showed a significantly (P < 0.05) high rate of lymphocyte transformation compared to control. Rats treated with 5, 50 and 100 ppm of CdCl2 showed certain levels of DNA strand breaks in blood lymphocyte. Male rats exposed to100 ppm (P < 0.001) and 50 ppm (P < 0.05) of CdCl2 showed a significant decrease in the relative thymus weight than that of control ones. The thymus of rats treated with 50 and 100 ppm of CdCl2 showed a reduction in the medulla’s diameter and interlobular fat globules compared to control. doi:10.1016/j.toxlet.2012.03.513
P22-02 Early-life exposure to lithium and boron from drinking water Florencia Harari Freire 1 , Ana María Ronco 2 , Gabriela Concha 3 , Miguel Llanos 2 , Francisca Castro 2 , Barbro Nermell 1 , Margaretha Grandér 1 , Brita Palm 1 , Marie Vahter 1 Karolinska Institutet, Sweden, 2 Inst. of Nutrition and Food Technology, Chile, 3 National Food Administration, Sweden
1