- Email: [email protected]
The evaluation of an in vitro fresh suspension hepatocyte model in the investigation of drug-induced hepatotoxicity
Abstracts / Toxicology 240 (2007) 164–192
would contribute considerably to our understanding of the spatio-temporal kinetics governing the cellular response to genotoxic insult. RAD52 reportedly contributes to the repair of DSB in mammalian cells, through its role in homologous recombination. Although the precise role and importance of RAD52 remains elusive, transcriptional up-regulation has been demonstrated by micro-array analysis and qRTPCR also (Smith et al., 2005). We previously presented a molecular beacon, which demonstrates specific and sensitive binding of human RAD52 mRNA, in a cell free system, resulting in a significant increase in fluorescence (Riches et al., 2006). We have extended these observations to whole cells, and here we present data to support the role of RAD52 in the cellular response to double strand breaks. Our recent work has indicated a dose-dependent increase in molecular beacon fluorescence, following exposure of human TK6 lymphoblastoid cells to gamma irradiation. Irradiated cells were fixed at 1 or 6 h, and samples analysed either for H2AX phosphorylation status, or molecular beacon activation. H2AX phosphorylation is commonly used as a marker of DSB, however the specificity has recently been questioned, since phosphorylation demonstrates a degree of cell cycle dependence. Microscopic analysis has revealed a significant increase in molecular beacon activation, detected as discrete fluorescent foci, at 1 h, which appears diminished
177
by 6 h at low dose (0.5 or 5 Gy), yet continues to increase at high dose (10 Gy) (Fig. 1). This result indicates transcriptional activation of RAD52 and engagement of the cell in homologous recombination repair. We observed a delay in RAD52 mRNA transcription in cells exposed to high dose irradiation (10 Gy), however in contrast with cells receiving a lower dose, this response continued throughout the time course of our experiment, suggestive of an inability of these cells to carry out DSB repair. Supporting data confirmed the presence of DSB by staining for phosphorylated H2AX, and so we propose our RAD52 mRNA specific molecular beacon as an appropriate method for detecting DSB in whole cells. References Riches, L.C., Lynch, A.M., Gooderham, N.J., 2006. Toxicology 226, 59. Smith, C.C., Aylott, M.C., Fisher, K.J., Lynch, A.M., Gooderham, N.J. J. Gene Med., in press.
doi:10.1016/j.tox.2007.06.057 The evaluation of an in vitro fresh suspension hepatocyte model in the investigation of drug-induced hepatotoxicity Sophie Regan 1 , Charlotte Hirst 1 , Daniel Antoine 1 , Gerry Kenna 2 , Kevin Park 1 , Dominic Williams 1 1 Department
of Pharmacology and Therapeutics, University of Liverpool, Liverpool, UK; 2 Safety Assessment, AstraZeneca, Alderley Park, Cheshire, UK E-mail address: [email protected] (S. Regan).
Fig. 1. Analysis of molecular beacon activation in response to DSB formation irradiated TK6 cells were fixed and incubated with molecular beacon for 1 h. Fluorescence was analysed by microscopy and the number of discrete foci scored per cell. The frequency of fluorescent foci is presented here (200 cells per sample). A substantial increase in fluorescence at 1 h, relative to unirradiated samples, is indicative of increased levels of RAD52 mRNA. N = 3 ± S.E.M; * P < 0.01, ** P < 0.001.
Despite non-clinical and clinical screenings druginduced hepatotoxicity is the most frequent cause for post marketing drug withdrawal. It has been reported that 50% of acute liver failure cases in the United States are as a result of drug-induced liver toxicity, indicating druginduced liver injury is clinically a significant problem. In vitro methods are increasingly used in exploring this area. It has been widely acknowledged that primary cultured hepatocytes represent a suitable in vitro system in toxicological studies. Hepatocytes in culture lose expression of specific liver functions including cytochrome P450, which is a limitation of this system. Drug bioactivation has been reported to play a significant initiating role in the hepatotoxic mechanism of certain xenobiotics, including paracetamol (APAP) and methapyilene (MP). An alternative in vitro model, which enables the investigation of cytotoxicity in parallel with metabolism,
178
Abstracts / Toxicology 240 (2007) 164–192
is freshly isolated hepatocyte suspensions. Hepatocyte suspensions retain specific hepatocyte functions including CYP induction and biosynthesis. We aim to assess the use of fresh hepatocyte suspensions in the investigation of drug-induced liver injury for both model and idiosyncratic toxins. Hepatocytes were isolated from male CD1 mice (20–25 g) and male Wistar rats (125–150 g) by a two-step in situ collagenase perfusion method. Fresh hepatocyte suspensions were incubated for 6 h with direct acting hepatotoxins APAP and MP and the idiosyncratic toxins clozapine (CLZ) and isoniazid (INH) at a concentration range. At this point cytotoxicity was assessed through trypan blue exclusion and MTS reduction. The importance of drug bioactivation was also assessed through the incubation with a non-selective P450 inhibitor 1-aminobenzotriazole (ABT 1 mM). Toxicity was observed with APAP and CLZ in mouse hepatocytes, occurring at 5 mM, and 300 M, respectively, while no toxicity was seen with INH (up to 45 mM) or MP (up to 500 M). In rat hepatocytes both MP and CLZ showed evidence of a dose-related toxicity while APAP and INH caused no toxicity at concentrations up to 5 and 45 mM, respectively. The two-model hepatotoxins are species specific in toxicity with the mouse model more susceptible to APAP induced liver injury and MP causing greater toxicity in rat hepatocytes. CLZ, however, shows no species selectivity in toxicity. With evidence of toxicity in rodent hepatocytes for APAP, MP and CLZ these compounds have been further investigated regarding drug bioactivation. ABT was found to block the toxicity caused by APAP in mouse hepatocytes and MP in rat hepatocytes, yet did not block CLZ-induced cytotoxicity.
Further reading Bissel, D.M., Gores, G.J., Laskin, D.L., Hoofnagle, J.H., 2001. Hepatology 33, 1009–1013. Jaeschke, H., Gores, G.J., Cederbaum, A.I., Hinson, J.A., Pessayre, D., Lemasters, J.J., 2002. Toxicol. Sci. 65, 166–176. Park, B.K., Kitteringham, N.R., Maggs, J.L., Pirmohamed, M., Williams, D.P., 2005. Annu. Rev. Pharmacol. Toxicol. 45, 177–205. Ratra, G.S., Morgan, W.A., Mullervy, J., Powell, C.J., Wright, M.C, 1998. Toxicology 130, 79–93.
doi:10.1016/j.tox.2007.06.058 The effect of in vitro receptor media choice on the percutaneous absorption of methyl paraben Ria Wilkinson 1 , Christopher Jewell 2 , Simon C. Wilkinson 1 , Faith M. Williams 1,2 , Peter G. Blain 1,2 1 Medical
Toxicology Research Centre, Newcastle University NE2 4HH, United Kingdom; 2 Institute for Research on Environment and Sustainability, Newcastle University NE2 4HH, United Kingdom E-mail address: [email protected] (R. Wilkinson). Para-Hydroxybenzoic acid esters (parabens) are widely used as preservatives in both personal care products and topical therapeutics, resulting in significant dermal exposure. Parabens have been reported to be weakly oestrogenic (Pugazhendhi et al., 2005) and may potentiate oxidative stress in keratinocytes (Handa et al., 2006). The aim of this study was to measure percutaneous absorption of methyl paraben (MP) in human skin in vitro using different applied doses and receptor fluids, including ethanol:water (worst case
Viability (% of control) Mouse hepatocytes APAP Trypan blue MTS reduction
28.3 (6.0) 61.6 (3.4)
Rat hepatocytes APAP + ABT (10.3)***
95.3 105.6 (12.2)***
MP
MP + ABT
32.8 (13.06) 52.0 (9.7)
91.9 (10.6)** 97.7 (8.2)**
Values: APAP mean (S.D.) n = 4, *** P < 0.005 compared to APAP alone (Mann–Whitney); MP mean (S.D.) n = 3, ** P < 0.01 compared to MP alone.
In summary, it appears that fresh hepatocyte suspensions represent a suitable model for investigating drug-induced hepatotoxicity while retaining species differences seen in vivo.
scenario). This would provide information on contribution of skin absorption to systemic exposure the extent of metabolism into p-hydroxybenzoic acid (PHBA) by cutaneous esterases, which have been detected in previously frozen skin. MP was chosen as it is usually present in the highest concentration in topical products.
would contribute considerably to our understanding of the spatio-temporal kinetics governing the cellular response to genotoxic insult. RAD52 reportedly contributes to the repair of DSB in mammalian cells, through its role in homologous recombination. Although the precise role and importance of RAD52 remains elusive, transcriptional up-regulation has been demonstrated by micro-array analysis and qRTPCR also (Smith et al., 2005). We previously presented a molecular beacon, which demonstrates specific and sensitive binding of human RAD52 mRNA, in a cell free system, resulting in a significant increase in fluorescence (Riches et al., 2006). We have extended these observations to whole cells, and here we present data to support the role of RAD52 in the cellular response to double strand breaks. Our recent work has indicated a dose-dependent increase in molecular beacon fluorescence, following exposure of human TK6 lymphoblastoid cells to gamma irradiation. Irradiated cells were fixed at 1 or 6 h, and samples analysed either for H2AX phosphorylation status, or molecular beacon activation. H2AX phosphorylation is commonly used as a marker of DSB, however the specificity has recently been questioned, since phosphorylation demonstrates a degree of cell cycle dependence. Microscopic analysis has revealed a significant increase in molecular beacon activation, detected as discrete fluorescent foci, at 1 h, which appears diminished
177
by 6 h at low dose (0.5 or 5 Gy), yet continues to increase at high dose (10 Gy) (Fig. 1). This result indicates transcriptional activation of RAD52 and engagement of the cell in homologous recombination repair. We observed a delay in RAD52 mRNA transcription in cells exposed to high dose irradiation (10 Gy), however in contrast with cells receiving a lower dose, this response continued throughout the time course of our experiment, suggestive of an inability of these cells to carry out DSB repair. Supporting data confirmed the presence of DSB by staining for phosphorylated H2AX, and so we propose our RAD52 mRNA specific molecular beacon as an appropriate method for detecting DSB in whole cells. References Riches, L.C., Lynch, A.M., Gooderham, N.J., 2006. Toxicology 226, 59. Smith, C.C., Aylott, M.C., Fisher, K.J., Lynch, A.M., Gooderham, N.J. J. Gene Med., in press.
doi:10.1016/j.tox.2007.06.057 The evaluation of an in vitro fresh suspension hepatocyte model in the investigation of drug-induced hepatotoxicity Sophie Regan 1 , Charlotte Hirst 1 , Daniel Antoine 1 , Gerry Kenna 2 , Kevin Park 1 , Dominic Williams 1 1 Department
of Pharmacology and Therapeutics, University of Liverpool, Liverpool, UK; 2 Safety Assessment, AstraZeneca, Alderley Park, Cheshire, UK E-mail address: [email protected] (S. Regan).
Fig. 1. Analysis of molecular beacon activation in response to DSB formation irradiated TK6 cells were fixed and incubated with molecular beacon for 1 h. Fluorescence was analysed by microscopy and the number of discrete foci scored per cell. The frequency of fluorescent foci is presented here (200 cells per sample). A substantial increase in fluorescence at 1 h, relative to unirradiated samples, is indicative of increased levels of RAD52 mRNA. N = 3 ± S.E.M; * P < 0.01, ** P < 0.001.
Despite non-clinical and clinical screenings druginduced hepatotoxicity is the most frequent cause for post marketing drug withdrawal. It has been reported that 50% of acute liver failure cases in the United States are as a result of drug-induced liver toxicity, indicating druginduced liver injury is clinically a significant problem. In vitro methods are increasingly used in exploring this area. It has been widely acknowledged that primary cultured hepatocytes represent a suitable in vitro system in toxicological studies. Hepatocytes in culture lose expression of specific liver functions including cytochrome P450, which is a limitation of this system. Drug bioactivation has been reported to play a significant initiating role in the hepatotoxic mechanism of certain xenobiotics, including paracetamol (APAP) and methapyilene (MP). An alternative in vitro model, which enables the investigation of cytotoxicity in parallel with metabolism,
178
Abstracts / Toxicology 240 (2007) 164–192
is freshly isolated hepatocyte suspensions. Hepatocyte suspensions retain specific hepatocyte functions including CYP induction and biosynthesis. We aim to assess the use of fresh hepatocyte suspensions in the investigation of drug-induced liver injury for both model and idiosyncratic toxins. Hepatocytes were isolated from male CD1 mice (20–25 g) and male Wistar rats (125–150 g) by a two-step in situ collagenase perfusion method. Fresh hepatocyte suspensions were incubated for 6 h with direct acting hepatotoxins APAP and MP and the idiosyncratic toxins clozapine (CLZ) and isoniazid (INH) at a concentration range. At this point cytotoxicity was assessed through trypan blue exclusion and MTS reduction. The importance of drug bioactivation was also assessed through the incubation with a non-selective P450 inhibitor 1-aminobenzotriazole (ABT 1 mM). Toxicity was observed with APAP and CLZ in mouse hepatocytes, occurring at 5 mM, and 300 M, respectively, while no toxicity was seen with INH (up to 45 mM) or MP (up to 500 M). In rat hepatocytes both MP and CLZ showed evidence of a dose-related toxicity while APAP and INH caused no toxicity at concentrations up to 5 and 45 mM, respectively. The two-model hepatotoxins are species specific in toxicity with the mouse model more susceptible to APAP induced liver injury and MP causing greater toxicity in rat hepatocytes. CLZ, however, shows no species selectivity in toxicity. With evidence of toxicity in rodent hepatocytes for APAP, MP and CLZ these compounds have been further investigated regarding drug bioactivation. ABT was found to block the toxicity caused by APAP in mouse hepatocytes and MP in rat hepatocytes, yet did not block CLZ-induced cytotoxicity.
Further reading Bissel, D.M., Gores, G.J., Laskin, D.L., Hoofnagle, J.H., 2001. Hepatology 33, 1009–1013. Jaeschke, H., Gores, G.J., Cederbaum, A.I., Hinson, J.A., Pessayre, D., Lemasters, J.J., 2002. Toxicol. Sci. 65, 166–176. Park, B.K., Kitteringham, N.R., Maggs, J.L., Pirmohamed, M., Williams, D.P., 2005. Annu. Rev. Pharmacol. Toxicol. 45, 177–205. Ratra, G.S., Morgan, W.A., Mullervy, J., Powell, C.J., Wright, M.C, 1998. Toxicology 130, 79–93.
doi:10.1016/j.tox.2007.06.058 The effect of in vitro receptor media choice on the percutaneous absorption of methyl paraben Ria Wilkinson 1 , Christopher Jewell 2 , Simon C. Wilkinson 1 , Faith M. Williams 1,2 , Peter G. Blain 1,2 1 Medical
Toxicology Research Centre, Newcastle University NE2 4HH, United Kingdom; 2 Institute for Research on Environment and Sustainability, Newcastle University NE2 4HH, United Kingdom E-mail address: [email protected] (R. Wilkinson). Para-Hydroxybenzoic acid esters (parabens) are widely used as preservatives in both personal care products and topical therapeutics, resulting in significant dermal exposure. Parabens have been reported to be weakly oestrogenic (Pugazhendhi et al., 2005) and may potentiate oxidative stress in keratinocytes (Handa et al., 2006). The aim of this study was to measure percutaneous absorption of methyl paraben (MP) in human skin in vitro using different applied doses and receptor fluids, including ethanol:water (worst case
Viability (% of control) Mouse hepatocytes APAP Trypan blue MTS reduction
28.3 (6.0) 61.6 (3.4)
Rat hepatocytes APAP + ABT (10.3)***
95.3 105.6 (12.2)***
MP
MP + ABT
32.8 (13.06) 52.0 (9.7)
91.9 (10.6)** 97.7 (8.2)**
Values: APAP mean (S.D.) n = 4, *** P < 0.005 compared to APAP alone (Mann–Whitney); MP mean (S.D.) n = 3, ** P < 0.01 compared to MP alone.
In summary, it appears that fresh hepatocyte suspensions represent a suitable model for investigating drug-induced hepatotoxicity while retaining species differences seen in vivo.
scenario). This would provide information on contribution of skin absorption to systemic exposure the extent of metabolism into p-hydroxybenzoic acid (PHBA) by cutaneous esterases, which have been detected in previously frozen skin. MP was chosen as it is usually present in the highest concentration in topical products.