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Effect of flavonoids on metabolism and excretion of PAHs in Caco-2 cell line
S106
Abstracts / Toxicology Letters 189S (2009) S57–S273
N28 Involvement of caspase-8, -9, and -3 in high glucose-induced apoptosis in PC12 cells Ali M. Sharifi 1,∗ , Habib Eslami 1 , Bagher Larijani 1,2 , Jamsheed Davoodi 1,3,2 1 Iran University of Medical Sciences, Pharmacology, Tehran, Islamic Republic of Iran, 2 Tehran University of Medical Sciences, Endocrine and Metabolism Research Center, Tehran, Islamic Republic of Iran, 3 Tehran University of Medical Sciences, Biochemistry, Tehran, Islamic Republic of Iran
Outline: Hyperglycemia, which occurs under the diabetic condition, is widely recognized as the causal link between diabetes and its serious complications. Diabetic neuropathies, which are among the most frequent complications of diabetes, affect nervous system. The exact mechanisms of high glucose-induced toxicity on neuronal cells, is still unclear. The present study examined the involvement of caspase-3, the executioner of apoptosis, and two initiators of apoptosis, caspase-8 and caspase-9, during high glucose-induced apoptosis in PC12 cells, a neuronal cell line. Cells were exposed to high glucose with or without z-VAD-fmk, a pancaspase inhibitor. Methods: Cell viability was measured by MTT assay. Caspase activity was determined spectrophotometrically using enzyme specific substrates. To correlate the caspase activity with changes in protein expression, procaspase-8, -9, and -3 were evaluated by Western blot analysis. Results: The PC12 cell viability on high glucose exposure was decreased compared to controls, which was reversed by z-VADfmk. The activities of caspase-8, -9, and -3 were significantly increased in treated cells compared to controls. Moreover, high glucose exposure were induced a significant decrease in protein levels of procaspases, indicating conversion of pro-form into the mature caspases. Conclusions: Based on the current data, it could be concluded that high glucose-induced apoptosis in PC12 cells is mediated, in part, by activation of caspase-8, -9, and -3 dependent pathways. doi:10.1016/j.toxlet.2009.06.342
N29 Effect of flavonoids on metabolism and excretion of PAHs in 夽 Caco-2 cell line Hanno Bothe 1,∗ , Jeanette Niestroy 2,∗ 1
Institut für umweltmdizinische Forschung, Molecular toxicology, Düsseldorf, Germany, 2 Leibniz-Institut für Arbeitsforschung, Molecular toxicology, Dortmund, Germany
The arylhydrocarbon receptor (AhR) is a cytosolic protein that mediates the metabolism and excretion of xenobiotic substances like polycyclic aromatic hydrocarbons (PAHs). This process can be modulated by dietary flavonoids. Here we studied the effects of several flavonoids on phase-I and phase-III proteins in Caco-2 cells. Treatment of Caco-2 cells with 1 M B(a)P alone or in combination of 10, 15, 25 and 50 M of luteolin, fisetin, baicalein, epigallocatechingallate, resveratrol and quercetin resulted in a dose-dependent inhibiting effect on the B[a]P induced ERODactivity. These effects were accompanied by dose-dependent inhibition of CYP1A1 mRNA expression with luteolin as the most effective compound. Interestingly, the flavonoids alone showed
agonistic effects on the expression of CYP1A1 mRNA. These flavonoids seem to act as partial agonists. In addition we investigated the effect of the respective flavonoids on the BCRP (breast cancer resistant protein)-mediated efflux of glucuronidated and sulphated B[a]P. Caco-2 cells were treated for 8 h with 5 M hydroxylated B[a]P alone or in combination with 1, 5, 25 and 50 M of the respective flavonoids. Except EGCG, all tested flavonoids inhibited the BCRP driven efflux of conjugated B[a]P dose-dependently. The strongest effect was obtained by luteolin and baicalein. The calculated EC50 value was about 5 M for both substances. The transport was blocked completely by higher doses of flavonoids. The results show that flavonoids can provoke inhibitory effects on the drug metabolizing enzyme CYP1A1 and on the efflux transporter BCRP. Thus, some flavonoids can exert toxifying properties in gut cells. 夽 Selected for Oral Presentation. doi:10.1016/j.toxlet.2009.06.343
N30 FOXO transcription factors and their role in overcoming doxorubicin-resistance of cancer cells Yvonni Chovolou ∗ , Agnes Beermann, Regine Lüpertz, Wim Wätjen, Andreas Kampkötter, Regine Kahl Institute of Toxicology, Düsseldorf, Germany Purpose: Forkhead transcription factors (FOXO) play major roles in control of cellular proliferation, oxidative stress and apoptosis and are considered as crucial therapeutic targets in cancer. Our previous work showed FOXO4 sensitizes cancer cells to doxorubicin-mediated cytotoxicity. In this study we further investigated the effects of doxorubicin in various cancer cell lines and the contribution of the FOXO signalling pathway in doxorubicin action and resistance. Methods: FOXO expression on mRNA and protein level was measured by RT-PCR and western blot. Doxorubicin sensitivity was assessed by MTT and neutral red assay. Transient transfection approaches, gel shift assays and immunoblot analysis of subcellular protein fractions will be used to assess the role FOXO transcription factors to doxorubicin resistance. Results: The expression of FOXO1, FOXO3a and FOXO4 was compared in two hepatoma cell lines (HepG2, Huh7), two colon cancer cell lines (Caco2, Hct-116) one breast cancer cell line (MCF7) and two ovarian cancer cell lines (A2780sense, A2780AdR). Interestingly, we found a highly differing expression pattern for the three transcription factors in these cell lines. For example FOXO1 was not expressed in A2780sense cells but was expressed in all other tested cell lines with the highest expression in A2780AdR cells. We next assessed the doxorubicin mediated cytotoxicity in these cell lines found that A2780AdR cells are insensitive to doxorubicinmediated cytotoxicity while most of the other cell lines showed partial resistance. Effects of doxorubicin on the activation status of FOXO transcription factors and their impact in modulating cellular response to doxorubicin are still under investigation. doi:10.1016/j.toxlet.2009.06.344
Abstracts / Toxicology Letters 189S (2009) S57–S273
N28 Involvement of caspase-8, -9, and -3 in high glucose-induced apoptosis in PC12 cells Ali M. Sharifi 1,∗ , Habib Eslami 1 , Bagher Larijani 1,2 , Jamsheed Davoodi 1,3,2 1 Iran University of Medical Sciences, Pharmacology, Tehran, Islamic Republic of Iran, 2 Tehran University of Medical Sciences, Endocrine and Metabolism Research Center, Tehran, Islamic Republic of Iran, 3 Tehran University of Medical Sciences, Biochemistry, Tehran, Islamic Republic of Iran
Outline: Hyperglycemia, which occurs under the diabetic condition, is widely recognized as the causal link between diabetes and its serious complications. Diabetic neuropathies, which are among the most frequent complications of diabetes, affect nervous system. The exact mechanisms of high glucose-induced toxicity on neuronal cells, is still unclear. The present study examined the involvement of caspase-3, the executioner of apoptosis, and two initiators of apoptosis, caspase-8 and caspase-9, during high glucose-induced apoptosis in PC12 cells, a neuronal cell line. Cells were exposed to high glucose with or without z-VAD-fmk, a pancaspase inhibitor. Methods: Cell viability was measured by MTT assay. Caspase activity was determined spectrophotometrically using enzyme specific substrates. To correlate the caspase activity with changes in protein expression, procaspase-8, -9, and -3 were evaluated by Western blot analysis. Results: The PC12 cell viability on high glucose exposure was decreased compared to controls, which was reversed by z-VADfmk. The activities of caspase-8, -9, and -3 were significantly increased in treated cells compared to controls. Moreover, high glucose exposure were induced a significant decrease in protein levels of procaspases, indicating conversion of pro-form into the mature caspases. Conclusions: Based on the current data, it could be concluded that high glucose-induced apoptosis in PC12 cells is mediated, in part, by activation of caspase-8, -9, and -3 dependent pathways. doi:10.1016/j.toxlet.2009.06.342
N29 Effect of flavonoids on metabolism and excretion of PAHs in 夽 Caco-2 cell line Hanno Bothe 1,∗ , Jeanette Niestroy 2,∗ 1
Institut für umweltmdizinische Forschung, Molecular toxicology, Düsseldorf, Germany, 2 Leibniz-Institut für Arbeitsforschung, Molecular toxicology, Dortmund, Germany
The arylhydrocarbon receptor (AhR) is a cytosolic protein that mediates the metabolism and excretion of xenobiotic substances like polycyclic aromatic hydrocarbons (PAHs). This process can be modulated by dietary flavonoids. Here we studied the effects of several flavonoids on phase-I and phase-III proteins in Caco-2 cells. Treatment of Caco-2 cells with 1 M B(a)P alone or in combination of 10, 15, 25 and 50 M of luteolin, fisetin, baicalein, epigallocatechingallate, resveratrol and quercetin resulted in a dose-dependent inhibiting effect on the B[a]P induced ERODactivity. These effects were accompanied by dose-dependent inhibition of CYP1A1 mRNA expression with luteolin as the most effective compound. Interestingly, the flavonoids alone showed
agonistic effects on the expression of CYP1A1 mRNA. These flavonoids seem to act as partial agonists. In addition we investigated the effect of the respective flavonoids on the BCRP (breast cancer resistant protein)-mediated efflux of glucuronidated and sulphated B[a]P. Caco-2 cells were treated for 8 h with 5 M hydroxylated B[a]P alone or in combination with 1, 5, 25 and 50 M of the respective flavonoids. Except EGCG, all tested flavonoids inhibited the BCRP driven efflux of conjugated B[a]P dose-dependently. The strongest effect was obtained by luteolin and baicalein. The calculated EC50 value was about 5 M for both substances. The transport was blocked completely by higher doses of flavonoids. The results show that flavonoids can provoke inhibitory effects on the drug metabolizing enzyme CYP1A1 and on the efflux transporter BCRP. Thus, some flavonoids can exert toxifying properties in gut cells. 夽 Selected for Oral Presentation. doi:10.1016/j.toxlet.2009.06.343
N30 FOXO transcription factors and their role in overcoming doxorubicin-resistance of cancer cells Yvonni Chovolou ∗ , Agnes Beermann, Regine Lüpertz, Wim Wätjen, Andreas Kampkötter, Regine Kahl Institute of Toxicology, Düsseldorf, Germany Purpose: Forkhead transcription factors (FOXO) play major roles in control of cellular proliferation, oxidative stress and apoptosis and are considered as crucial therapeutic targets in cancer. Our previous work showed FOXO4 sensitizes cancer cells to doxorubicin-mediated cytotoxicity. In this study we further investigated the effects of doxorubicin in various cancer cell lines and the contribution of the FOXO signalling pathway in doxorubicin action and resistance. Methods: FOXO expression on mRNA and protein level was measured by RT-PCR and western blot. Doxorubicin sensitivity was assessed by MTT and neutral red assay. Transient transfection approaches, gel shift assays and immunoblot analysis of subcellular protein fractions will be used to assess the role FOXO transcription factors to doxorubicin resistance. Results: The expression of FOXO1, FOXO3a and FOXO4 was compared in two hepatoma cell lines (HepG2, Huh7), two colon cancer cell lines (Caco2, Hct-116) one breast cancer cell line (MCF7) and two ovarian cancer cell lines (A2780sense, A2780AdR). Interestingly, we found a highly differing expression pattern for the three transcription factors in these cell lines. For example FOXO1 was not expressed in A2780sense cells but was expressed in all other tested cell lines with the highest expression in A2780AdR cells. We next assessed the doxorubicin mediated cytotoxicity in these cell lines found that A2780AdR cells are insensitive to doxorubicinmediated cytotoxicity while most of the other cell lines showed partial resistance. Effects of doxorubicin on the activation status of FOXO transcription factors and their impact in modulating cellular response to doxorubicin are still under investigation. doi:10.1016/j.toxlet.2009.06.344