- Email: [email protected]
Differential Hepatic microRNA-mediated Translation in Two Independent Models of Erythropoietic Protoporphyria
6
Oral Communications Abstracts / Toxicology 262 (2009) 1–7
adduct formation or topoisomerase inhibition another redox active quinone 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) was tested in a similar manner. DMNQ is redox active but lacks the effects on DNA transcription which are the pharmacological activity of doxorubicin. The data obtained both at the miRNA and mRNA translation levels were very similar to that of doxorubicin, and implicated effects on the ETC as the main route of toxicity with alterations of mRNA translation as a response. miRNA expression changes were correlated bioinformatically (www.targetscan.org) with alteration of individual mRNA translation rates to allow identification of miRNA binding sites within differentially translated mRNAs (Table 1) and allowed derivation of some specific hypotheses for the role of miRNA species in doxorubicin toxicity via the ETC (Table 1).An in vitro model is currently being established utilizing the HL-1 cell line to further test the hypotheses for specific miRNA involvement in mRNA translation of ETC genes in doxorubicin toxicity that can be derived from the data shown in Table 1.
Table 1 Some common differentially expressed miRNAs in Fech and Griseofulvin treated mice. miRNA
Fold change* Fech
Griseofulvin
mmu-miR-21 mmu-miR-27a/b mmu-miR-302a mmu-miR-15a mmu-miR-16
2.4 1.6 1.7 1.6 1.5
2.1 2.1 1.5 1.5 1.5
mmu-miR-9 mmu-miR-215 mmu-miR-101a MM1 mmu-miR-30d mmu-miR-152 MM1
0.2 0.3 0.4 0.5 0.7
0.5 0.4 0.5 0.7 0.7
* Changes are statistically significant (two-sample t test p < 0.05, fold change ≥1.5 or ≤0.7, n ≥ 6) compared to appropriate controls (wild type or vehicle only treated animals).
References Reference Sun, X.H., Zhou, Z.X., Kang, Y.J., 2001. Attenuation of doxorubicin chronic toxicity in metallothionein-overexpressed transgenic mouse heart. Cancer Res. 61, 3382–3387.
Knasmuller, S., Parzefall, W., Helma, C., Kassie, F., Ecker, S., Schulte-Hermann, R., 1997. CRC Crit. Rev. Toxicol. 27, 437–495. Kosik, K.S., 2006. Nat. Rev. Neurosci. 7, 911–920. Tutois, S., Montagutelli, X., Da Silva, V., Jouault, H., Rouyer-Fessard, P., Lerfy-Viard, K., Guénet, J.L., Nordmann, Y., Beuzard, Y., Deybach, J.C., 1991. J. Clin. Invest. 88, 1730–1736.
doi:10.1016/j.tox.2009.04.049 doi:10.1016/j.tox.2009.04.050
P31 Differential Hepatic microRNA-mediated Translation in Two Independent Models of Erythropoietic Protoporphyria Taylor ∗ ,
Emma L. Kate M. Phillips, Joan Riley, Shu-Dong Zhang, Timothy W. Gant MRC Toxicology Unit, University of Leicester, Lancaster Road, Leicester LE1 9HN, United Kingdom E-mail address: [email protected] (E.L. Taylor). microRNAs (miRNAs) are short (18–26 nt), endogenous RNA molecules that regulate mRNA translation (Kosik, 2006). They are transcribed from non-coding regions of the genome under the control of RNA polymerase (Pol II) promoters. Such promoters often contain toxicologically significant enhancer regions, inferring that miRNAs are likely to be of crucial importance in cellular responses to stress and xenobiotics. Here we have investigated and compared the role of miRNAs in the porphyrin-induced hepatotoxicity that is observed in two independent mouse models of Erythropoietic Protoporphyria (EPP). EPP is caused by a block in the final step of haem biosysnthesis. This leads to profound anaemia and the accumulation of porphyrins, particularly in the liver, which ultimately results in hepatocellular carcinoma. The block is caused by decreased ferrochelatase activity and is induced by two different mechanisms in the models studied here. Enzyme activity is reduced by over 90% by either a homozygous SNP mutation in the BALB/cFechm1Pas/m1Pas (Fech) mice (Tutois et al., 1991) or by treatment of wild type BALB/c mice with the ferrochelatase inhibitor Griseofulvin (Knasmuller et al., 1997). Microarray profiling of the livers from Fech mice and mice treated with Griseofulvin revealed some common differentially expressed miRNAs (Table 1). By correlating these altered miRNA profiles with mRNA translational changes in the Fech mice we were able to identify putative functionally relevant miRNA–mRNA interactions. We are currently analysing changes in mRNA translation induced in mice treated with Griseofulvin. This should enable identification of common miRNA-mediated mechanisms of hepatotoxicity between these two very different models.
P32 Characterization and Quantification of Mercapturate and Glutathione Conjugates of Nevirapine Abhishek Srivastava a,∗ , Dominic P. Williams a , James L. a a a Maggs , Lu-Yun Lian , Mas Chaponda , Iain Gardner b , Munir Pirmohamed a , Kevin Park a a
MRC Centre for Drug Safety Science, Department of Pharmacology and Therapeutics, University of Liverpool, L69 3GE, UK b Pfizer Sandwich, UK
E-mail address: [email protected] (A. Srivastava). Nevirapine (NVP), a non-nucleoside reverse transcriptase inhibitor, is widely used for the treatment of human immunodeficiency virus (HIV) infections. NVP is associated with two serious clinically restrictive side effects: skin reactions and hepatotoxicity (Patel et al., 2004). An immune-mediated mechanism of toxicity has been proposed, together with possible metabolic pathways of hapten formation (Shenton et al., 2007). NVP undergoes P450-dependent metabolism in rat liver microsomes to a reactive intermediate that binds irreversibly to protein (Takakusa et al., 2008). However, no reactive metabolite of NVP trapped as stable thioether conjugates either in vitro or in vivo has hitherto been identified. The aim of this study was to define and quantify the metabolism and bioactivation of NVP with respect to reactive intermediate formation which may play a role in NVP-induced liver and skin toxicity. Human and rat liver microsomes, containing 1–3 mg protein/ml were incubated with NVP (10–25 M) for 1 h at 37 ◦ C in the presence of 1 mM NADPH ± 1 mM glutathione (GSH) or 1 mM N-acetyl cysteine. Freshly isolated rat hepatocytes (2 × 106 ml−1 ) were incubated with NVP (10–50 M) for 4 h. Bile was obtained from male Wistar rats injected with NVP (i.v, 50–150 mg/kg body weight) by cannulating the common bile duct. Urine was collected for 24 h after dosing Wistar rats with NVP (400–600 mg/kg). Human urine samples were obtained from 68 HIV-positive patients from Malawi and Uganda (on 200 mg NVP twice daily). The ethical consent was
Oral Communications Abstracts / Toxicology 262 (2009) 1–7
adduct formation or topoisomerase inhibition another redox active quinone 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) was tested in a similar manner. DMNQ is redox active but lacks the effects on DNA transcription which are the pharmacological activity of doxorubicin. The data obtained both at the miRNA and mRNA translation levels were very similar to that of doxorubicin, and implicated effects on the ETC as the main route of toxicity with alterations of mRNA translation as a response. miRNA expression changes were correlated bioinformatically (www.targetscan.org) with alteration of individual mRNA translation rates to allow identification of miRNA binding sites within differentially translated mRNAs (Table 1) and allowed derivation of some specific hypotheses for the role of miRNA species in doxorubicin toxicity via the ETC (Table 1).An in vitro model is currently being established utilizing the HL-1 cell line to further test the hypotheses for specific miRNA involvement in mRNA translation of ETC genes in doxorubicin toxicity that can be derived from the data shown in Table 1.
Table 1 Some common differentially expressed miRNAs in Fech and Griseofulvin treated mice. miRNA
Fold change* Fech
Griseofulvin
mmu-miR-21 mmu-miR-27a/b mmu-miR-302a mmu-miR-15a mmu-miR-16
2.4 1.6 1.7 1.6 1.5
2.1 2.1 1.5 1.5 1.5
mmu-miR-9 mmu-miR-215 mmu-miR-101a MM1 mmu-miR-30d mmu-miR-152 MM1
0.2 0.3 0.4 0.5 0.7
0.5 0.4 0.5 0.7 0.7
* Changes are statistically significant (two-sample t test p < 0.05, fold change ≥1.5 or ≤0.7, n ≥ 6) compared to appropriate controls (wild type or vehicle only treated animals).
References Reference Sun, X.H., Zhou, Z.X., Kang, Y.J., 2001. Attenuation of doxorubicin chronic toxicity in metallothionein-overexpressed transgenic mouse heart. Cancer Res. 61, 3382–3387.
Knasmuller, S., Parzefall, W., Helma, C., Kassie, F., Ecker, S., Schulte-Hermann, R., 1997. CRC Crit. Rev. Toxicol. 27, 437–495. Kosik, K.S., 2006. Nat. Rev. Neurosci. 7, 911–920. Tutois, S., Montagutelli, X., Da Silva, V., Jouault, H., Rouyer-Fessard, P., Lerfy-Viard, K., Guénet, J.L., Nordmann, Y., Beuzard, Y., Deybach, J.C., 1991. J. Clin. Invest. 88, 1730–1736.
doi:10.1016/j.tox.2009.04.049 doi:10.1016/j.tox.2009.04.050
P31 Differential Hepatic microRNA-mediated Translation in Two Independent Models of Erythropoietic Protoporphyria Taylor ∗ ,
Emma L. Kate M. Phillips, Joan Riley, Shu-Dong Zhang, Timothy W. Gant MRC Toxicology Unit, University of Leicester, Lancaster Road, Leicester LE1 9HN, United Kingdom E-mail address: [email protected] (E.L. Taylor). microRNAs (miRNAs) are short (18–26 nt), endogenous RNA molecules that regulate mRNA translation (Kosik, 2006). They are transcribed from non-coding regions of the genome under the control of RNA polymerase (Pol II) promoters. Such promoters often contain toxicologically significant enhancer regions, inferring that miRNAs are likely to be of crucial importance in cellular responses to stress and xenobiotics. Here we have investigated and compared the role of miRNAs in the porphyrin-induced hepatotoxicity that is observed in two independent mouse models of Erythropoietic Protoporphyria (EPP). EPP is caused by a block in the final step of haem biosysnthesis. This leads to profound anaemia and the accumulation of porphyrins, particularly in the liver, which ultimately results in hepatocellular carcinoma. The block is caused by decreased ferrochelatase activity and is induced by two different mechanisms in the models studied here. Enzyme activity is reduced by over 90% by either a homozygous SNP mutation in the BALB/cFechm1Pas/m1Pas (Fech) mice (Tutois et al., 1991) or by treatment of wild type BALB/c mice with the ferrochelatase inhibitor Griseofulvin (Knasmuller et al., 1997). Microarray profiling of the livers from Fech mice and mice treated with Griseofulvin revealed some common differentially expressed miRNAs (Table 1). By correlating these altered miRNA profiles with mRNA translational changes in the Fech mice we were able to identify putative functionally relevant miRNA–mRNA interactions. We are currently analysing changes in mRNA translation induced in mice treated with Griseofulvin. This should enable identification of common miRNA-mediated mechanisms of hepatotoxicity between these two very different models.
P32 Characterization and Quantification of Mercapturate and Glutathione Conjugates of Nevirapine Abhishek Srivastava a,∗ , Dominic P. Williams a , James L. a a a Maggs , Lu-Yun Lian , Mas Chaponda , Iain Gardner b , Munir Pirmohamed a , Kevin Park a a
MRC Centre for Drug Safety Science, Department of Pharmacology and Therapeutics, University of Liverpool, L69 3GE, UK b Pfizer Sandwich, UK
E-mail address: [email protected] (A. Srivastava). Nevirapine (NVP), a non-nucleoside reverse transcriptase inhibitor, is widely used for the treatment of human immunodeficiency virus (HIV) infections. NVP is associated with two serious clinically restrictive side effects: skin reactions and hepatotoxicity (Patel et al., 2004). An immune-mediated mechanism of toxicity has been proposed, together with possible metabolic pathways of hapten formation (Shenton et al., 2007). NVP undergoes P450-dependent metabolism in rat liver microsomes to a reactive intermediate that binds irreversibly to protein (Takakusa et al., 2008). However, no reactive metabolite of NVP trapped as stable thioether conjugates either in vitro or in vivo has hitherto been identified. The aim of this study was to define and quantify the metabolism and bioactivation of NVP with respect to reactive intermediate formation which may play a role in NVP-induced liver and skin toxicity. Human and rat liver microsomes, containing 1–3 mg protein/ml were incubated with NVP (10–25 M) for 1 h at 37 ◦ C in the presence of 1 mM NADPH ± 1 mM glutathione (GSH) or 1 mM N-acetyl cysteine. Freshly isolated rat hepatocytes (2 × 106 ml−1 ) were incubated with NVP (10–50 M) for 4 h. Bile was obtained from male Wistar rats injected with NVP (i.v, 50–150 mg/kg body weight) by cannulating the common bile duct. Urine was collected for 24 h after dosing Wistar rats with NVP (400–600 mg/kg). Human urine samples were obtained from 68 HIV-positive patients from Malawi and Uganda (on 200 mg NVP twice daily). The ethical consent was