- Email: [email protected]
Impact of transporter-mediated uptake on statin-induced gene expression in human Huh7 cells
20
Poster Abstracts / Toxicology 262 (2009) 8–26
the characteristics of inhaled substances and exposure assessment methods in the context of EBW.
Reference Kosik, K.S., 2006. Nat Rev Neurosci 7, 911–920. Taylor, E.L., Gant, T.W., 2008. Toxicology 246, 34–39.
Acknowledgements doi:10.1016/j.tox.2009.04.022
Advancing the 3Rs : Exposure-driven risk assessment
Funding for this study was provided by the NC3Rs. Scientific guidance was provided by members of the Working Group of the NC3Rs Regulatory Toxicology Steering Group.
Anna L. Rowbotham ∗ , Rosemary M. Gibson
doi:10.1016/j.tox.2009.04.023
Health Exposures Unit, Health Improvement Group, Health and Safety Laboratory, Harpur Hill, Buxton, Derbyshire SK17 9JN, United Kingdom
P28
P27
E-mail address: [email protected] (A.L. Rowbotham). The National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) provides a focus in the United Kingdom for the promotion, development and implementation of the principles of ‘replacement, reduction and refinement’ (3Rs) in animal research and testing. Through its Regulatory Toxicology Forum, the Centre is exploring how the 3Rs can be advanced in the context of regulatory safety assessment by the chemicals industry. Scope exists across several chemical regulatory schemes to waive or adapt standard hazard data requirements, in order to avoid unnecessary toxicity testing and reduce the number of animals used. For example, the REACH Regulation 1907/2006/EC provides the possibility to waive toxicity testing based on scenarios developed in the exposure assessment. To investigate this further, the NC3Rs established a Working Group of regulatory and industrial toxicologists to explore the feasibility of waiving hazard data requirements under REACH on the basis of exposure data (exposure-based waiving; EBW) for inhaled substances (inhalation presents the primary route of exposure for many chemicals). Under REACH, there are two options for EBW (Fig. 1). In the ‘Column 2 route’ option, a weight of evidence approach justifies waiving tests on the basis that human exposure is low or negligible (e.g. for substances used in a closed system, of low volatility and acute toxicity). In the‘Annex XI route’ option, exposure estimates relevant to the test to be waived are compared with a derived-no-effect level (DNEL) or accepted threshold of toxicological concern (TTC) when a DNEL has not been established. The study explored different approaches for deriving TTC values for the inhalation route and considered typical exposures associated with occupational and consumer uses of chemicals to assess the feasibility of defining non-significant exposures in the context of EBW. The study concluded that exposure-driven risk assessment is at an early stage of development and further exploration of both the hazard and exposure aspects of EBW is required to determine how these can be effectively applied to the regulatory safety assessment of inhaled chemicals. Recommendations for future work to enhance knowledge in this area include exploration of: inhalation toxicity data,
Impact of transporter-mediated uptake on statin-induced gene expression in human Huh7 cells Faizah Sanat a,∗ , Katharine Thumser a , Nick Plant a
Howe b , Tanya
Coleman c , Alfred
a
Centre for Toxicology, FHMS, University of Surrey, Guildford, UK DSRD, Pfizer, Sandwich, Kent, UK c DMPK, AstraZeneca, Alderley Park, Cheshire, UK b
E-mail address: [email protected] (F. Sanat). The statins are a class of HMG-CoA reductase inhibitors that have become frontline treatments for hypercholesteraemia, with the UK becoming the first country to approve non-prescription sales of simvastatin (Zocor) in 2004. This approval was supported by very few observed adverse reactions or drug–drug interactions with the statins. Given their overall good safety profile it is perhaps surprising that many statins are potent transcriptional activators of CYP3A4 expression in vitro (e.g. simvastatin and lovastatin), which would be suggestive of a potential for significant drug–drug interactions. In comparison, pravastatin does not activate CYP3A4 in vitro, although the reason for this differential activation is not clearly understood (El-Sankary et al., 2001). To examine this apparent disparity further, we have characterised the statin-mediated activation of the classical nuclear receptors PXR, CAR and FXR, as well as the PXR target genes CYP3A4, ABCC2 and ABCB1 in the human hepatoma cell line Huh7. For nuclear receptor activation assays, the ligand binding domains (LBD) of PXR, FXR and CAR were cloned into the pBIND reporter plasmid, fusing them to a GAL4 DNA binding domain. These constructs, along with a luciferase reporter gene containing a concatamer of the GAL4 response element were transfected into Huh7 cells and exposed to 0.1, 1 and 10 M agonist for 24 h before nuclear receptor activation was measured. As can be seen from Table 1, at 10 M both simvastatin and lovastatin produced similar fold activations of the PXR LBD, as expected. Interestingly, pravastatin was also able to activate the PXR LBD, and this was confirmed through the use of in silico modelling to demonstrate efficient docking of all three statins into the PXR LBD.
Fig. 1. Options for exposure-based waiving under REACH.
Poster Abstracts / Toxicology 262 (2009) 8–26
21
Table 1 Fold activation of nuclear receptor by statins. Receptor
Sim (lactone)
Sim (acid)
Lov (lactone)
Lov (acid)
Pravastatin
PXR CAR FXR
1.1 0.6 0.8
2.3 1.0 1.4
2.9 1.1 1.4
2.0 1.8 1.8
2.0 1.3 1.5
*
Results indicate activation of nuclear receptors at 10 M of statin concentration.
Despite the demonstration that all three statins may activate the PXR LBD activation of a CYP3A4 reporter gene showed differential regulation: Whereas simvastatin and lovastatin could increase CYP3A4 transcriptional activity by up to 3000%, with an EC50 of approximately 5 M, pravastatin had no significant effect, even when its obligate uptake transporter (SLCO1B1) was overexpressed in the cell line. However, examination of other known PXR target genes (abbc2 and ABCB1) demonstrated that 10 M pravastatin can increase ABCC2 reporter gene expression by 400%, compared to control, which is in line with activations observed with the other statins; simvastatin and lovastatin. In summary, we have investigated the apparent lack of activation of PXR target genes by pravastatin, compared to other statins currently on the market. Our work demonstrates that pravastatin is capable of activating the PXR LBD to the same extent as both simvastatin and lovastatin, and that whereas this does not result in the activation of some PXR target genes (e.g. CYP3A4), it does increase transcription of ABCC2, also a PXR-target gene. These data demonstrate that pravastatin can activate PXR, although the activated target gene set is more limited than seen with other statins. Funding kindly provided by a BBSRC-CASE award in collaboration with AstraZeneca (KH) And Ministry of Higher Education, Malaysia. Reference El-Sankary, et al., 2001. Drug Metab Dispos 29, 1499–1504.
Fig. 1. Cytotoxicity of EA and QC alone and in combination on cancer cell lines. Values are mean of 3–8 independent experiments ± S.E.M. with 6 replicates per experiment.
decrease cancer cell growth. Our aims were to determine the efficacy of EA and QC, alone and in combination in models of human cancer and establish their effects on polyamine metabolism. Using non-small cell lung cancer (A549), colorectal cancer (HCT116) and breast cancer (MDA-MB-231) cells the cytotoxicity of EA and QC was established, after 96 h of incubation. MTT, Trypan Blue and protein determination assays were used to monitor cell growth. Polyamine content was determined using HPLC. DAPI staining was used to determine whether the cell death induced was due to apoptosis. Exposure to EA and QC alone resulted in dose-dependent cytotoxicity in all cells, measured by MTT (IC50 values; Fig. 1). We combined EA and QC by fixing the concentration of one and varying the other to test this. EA was fixed at 132 M and QC varied from 0 to 370 M and QC was fixed at 118 M and EA varied from 0 to 410 M. The combinations of EA and QC showed increased cytotoxicity compared to EA and QC alone (Fig. 1). Further studies showed that combinations were also more effective in depleting cell number, protein content and intracellular polyamine concentrations by greater than 30% (HCT-116 cells). DAPI staining gave a preliminary indication that the mechanism of cell death was apoptotic. Further investigation is required to determine which part of the polyamine pathway is a target. However, these studies indicate that modulation of polyamine metabolism may, in part, explain some of the cytotoxic effects of EA and QC in cancer cells.
doi:10.1016/j.tox.2009.04.024 P29
Acknowledgement We thank NHS Grampian Endowments Trust for their support.
Potentiation of the cyotoxic effects of natural chemopreventative agents, ellagic acid and quercetin Fiona Saunders ∗ , Manju Oommen, Ratchadawadee Lapjitkusol, Edward Amankwatia, Lauren Kaminski, Adele Swanson, Alastair Mitchell, Heather M. Wallace Divison of Applied Medicine, School of Medicine & Dentistry, University of Aberdeen, Polwarth Building, Foresterhill, Aberdeen AB25 2ZD, United Kingdom E-mail address: [email protected] (F. Saunders). Lung and colorectal are among the most prevalent cancers in the world. Current treatments have limited success with 5-year survival (7% and 50%, respectively). So another approach is needed. One strategy is chemoprevention (natural or chemical). The most investigated are notably non-steroidal anti-inflammatory drugs (NSAIDs). However, NSAIDs have side effects and there is growing interest in natural compounds with potentially less side effects. Ellagic acid (EA), a polyphenol, and quercetin (QC), a flavonoid, are naturally occurring compounds, abundant in soft fruits and other foodstuffs (Ren et al., 2003). They are effective in killing cancer cells, but their mechanism of cytotoxicity is unknown. Preliminary studies suggest that inhibition of polyamine biosynthesis may be involved. Polyamine concentrations and metabolism are increased in cancer cells (Kingsnorth et al., 1984). Preventing polyamine accumulation may be one mechanism used by preventative agents to
Reference Ren, W., Qiao, Z., Wnag, H., Zhu, L., Zhang, L., 2003. Med Res Rev 23 (4), 519–534. Kingsnorth, A.N., Lumsden, A.B., Wallace, H.M., 1984. Br J Surg 71, 791–794.
doi:10.1016/j.tox.2009.04.025 P30 How does increasing cytotoxicity affect the accuracy of the GADD45a-GFP genotoxicity screening assay: A comparison of 4 different toxicity testing methods Matthew Tate a,b,∗ , Christopher Jagger b , Nicholas Billinton b , Paul Cahill b , Adam Rabinowitz b , Andrew Knight b , Richard Walmsley a,b a
Faculty of Life Sciences, The University of Manchester, Manchester M13 9PL, UK b Gentronix Ltd, CTF Building, 46 Grafton St, Manchester M13 9NT, UK E-mail address: [email protected] (M. Tate). Recent evaluations of the in vitro mammalian genotoxicity assays have suggested that increased levels of cytotoxicity may lead to an increase in the number of uniquely positive genotoxicity results Kirkland et al. (2005, 2006). Moreover, these assays have
Poster Abstracts / Toxicology 262 (2009) 8–26
the characteristics of inhaled substances and exposure assessment methods in the context of EBW.
Reference Kosik, K.S., 2006. Nat Rev Neurosci 7, 911–920. Taylor, E.L., Gant, T.W., 2008. Toxicology 246, 34–39.
Acknowledgements doi:10.1016/j.tox.2009.04.022
Advancing the 3Rs : Exposure-driven risk assessment
Funding for this study was provided by the NC3Rs. Scientific guidance was provided by members of the Working Group of the NC3Rs Regulatory Toxicology Steering Group.
Anna L. Rowbotham ∗ , Rosemary M. Gibson
doi:10.1016/j.tox.2009.04.023
Health Exposures Unit, Health Improvement Group, Health and Safety Laboratory, Harpur Hill, Buxton, Derbyshire SK17 9JN, United Kingdom
P28
P27
E-mail address: [email protected] (A.L. Rowbotham). The National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) provides a focus in the United Kingdom for the promotion, development and implementation of the principles of ‘replacement, reduction and refinement’ (3Rs) in animal research and testing. Through its Regulatory Toxicology Forum, the Centre is exploring how the 3Rs can be advanced in the context of regulatory safety assessment by the chemicals industry. Scope exists across several chemical regulatory schemes to waive or adapt standard hazard data requirements, in order to avoid unnecessary toxicity testing and reduce the number of animals used. For example, the REACH Regulation 1907/2006/EC provides the possibility to waive toxicity testing based on scenarios developed in the exposure assessment. To investigate this further, the NC3Rs established a Working Group of regulatory and industrial toxicologists to explore the feasibility of waiving hazard data requirements under REACH on the basis of exposure data (exposure-based waiving; EBW) for inhaled substances (inhalation presents the primary route of exposure for many chemicals). Under REACH, there are two options for EBW (Fig. 1). In the ‘Column 2 route’ option, a weight of evidence approach justifies waiving tests on the basis that human exposure is low or negligible (e.g. for substances used in a closed system, of low volatility and acute toxicity). In the‘Annex XI route’ option, exposure estimates relevant to the test to be waived are compared with a derived-no-effect level (DNEL) or accepted threshold of toxicological concern (TTC) when a DNEL has not been established. The study explored different approaches for deriving TTC values for the inhalation route and considered typical exposures associated with occupational and consumer uses of chemicals to assess the feasibility of defining non-significant exposures in the context of EBW. The study concluded that exposure-driven risk assessment is at an early stage of development and further exploration of both the hazard and exposure aspects of EBW is required to determine how these can be effectively applied to the regulatory safety assessment of inhaled chemicals. Recommendations for future work to enhance knowledge in this area include exploration of: inhalation toxicity data,
Impact of transporter-mediated uptake on statin-induced gene expression in human Huh7 cells Faizah Sanat a,∗ , Katharine Thumser a , Nick Plant a
Howe b , Tanya
Coleman c , Alfred
a
Centre for Toxicology, FHMS, University of Surrey, Guildford, UK DSRD, Pfizer, Sandwich, Kent, UK c DMPK, AstraZeneca, Alderley Park, Cheshire, UK b
E-mail address: [email protected] (F. Sanat). The statins are a class of HMG-CoA reductase inhibitors that have become frontline treatments for hypercholesteraemia, with the UK becoming the first country to approve non-prescription sales of simvastatin (Zocor) in 2004. This approval was supported by very few observed adverse reactions or drug–drug interactions with the statins. Given their overall good safety profile it is perhaps surprising that many statins are potent transcriptional activators of CYP3A4 expression in vitro (e.g. simvastatin and lovastatin), which would be suggestive of a potential for significant drug–drug interactions. In comparison, pravastatin does not activate CYP3A4 in vitro, although the reason for this differential activation is not clearly understood (El-Sankary et al., 2001). To examine this apparent disparity further, we have characterised the statin-mediated activation of the classical nuclear receptors PXR, CAR and FXR, as well as the PXR target genes CYP3A4, ABCC2 and ABCB1 in the human hepatoma cell line Huh7. For nuclear receptor activation assays, the ligand binding domains (LBD) of PXR, FXR and CAR were cloned into the pBIND reporter plasmid, fusing them to a GAL4 DNA binding domain. These constructs, along with a luciferase reporter gene containing a concatamer of the GAL4 response element were transfected into Huh7 cells and exposed to 0.1, 1 and 10 M agonist for 24 h before nuclear receptor activation was measured. As can be seen from Table 1, at 10 M both simvastatin and lovastatin produced similar fold activations of the PXR LBD, as expected. Interestingly, pravastatin was also able to activate the PXR LBD, and this was confirmed through the use of in silico modelling to demonstrate efficient docking of all three statins into the PXR LBD.
Fig. 1. Options for exposure-based waiving under REACH.
Poster Abstracts / Toxicology 262 (2009) 8–26
21
Table 1 Fold activation of nuclear receptor by statins. Receptor
Sim (lactone)
Sim (acid)
Lov (lactone)
Lov (acid)
Pravastatin
PXR CAR FXR
1.1 0.6 0.8
2.3 1.0 1.4
2.9 1.1 1.4
2.0 1.8 1.8
2.0 1.3 1.5
*
Results indicate activation of nuclear receptors at 10 M of statin concentration.
Despite the demonstration that all three statins may activate the PXR LBD activation of a CYP3A4 reporter gene showed differential regulation: Whereas simvastatin and lovastatin could increase CYP3A4 transcriptional activity by up to 3000%, with an EC50 of approximately 5 M, pravastatin had no significant effect, even when its obligate uptake transporter (SLCO1B1) was overexpressed in the cell line. However, examination of other known PXR target genes (abbc2 and ABCB1) demonstrated that 10 M pravastatin can increase ABCC2 reporter gene expression by 400%, compared to control, which is in line with activations observed with the other statins; simvastatin and lovastatin. In summary, we have investigated the apparent lack of activation of PXR target genes by pravastatin, compared to other statins currently on the market. Our work demonstrates that pravastatin is capable of activating the PXR LBD to the same extent as both simvastatin and lovastatin, and that whereas this does not result in the activation of some PXR target genes (e.g. CYP3A4), it does increase transcription of ABCC2, also a PXR-target gene. These data demonstrate that pravastatin can activate PXR, although the activated target gene set is more limited than seen with other statins. Funding kindly provided by a BBSRC-CASE award in collaboration with AstraZeneca (KH) And Ministry of Higher Education, Malaysia. Reference El-Sankary, et al., 2001. Drug Metab Dispos 29, 1499–1504.
Fig. 1. Cytotoxicity of EA and QC alone and in combination on cancer cell lines. Values are mean of 3–8 independent experiments ± S.E.M. with 6 replicates per experiment.
decrease cancer cell growth. Our aims were to determine the efficacy of EA and QC, alone and in combination in models of human cancer and establish their effects on polyamine metabolism. Using non-small cell lung cancer (A549), colorectal cancer (HCT116) and breast cancer (MDA-MB-231) cells the cytotoxicity of EA and QC was established, after 96 h of incubation. MTT, Trypan Blue and protein determination assays were used to monitor cell growth. Polyamine content was determined using HPLC. DAPI staining was used to determine whether the cell death induced was due to apoptosis. Exposure to EA and QC alone resulted in dose-dependent cytotoxicity in all cells, measured by MTT (IC50 values; Fig. 1). We combined EA and QC by fixing the concentration of one and varying the other to test this. EA was fixed at 132 M and QC varied from 0 to 370 M and QC was fixed at 118 M and EA varied from 0 to 410 M. The combinations of EA and QC showed increased cytotoxicity compared to EA and QC alone (Fig. 1). Further studies showed that combinations were also more effective in depleting cell number, protein content and intracellular polyamine concentrations by greater than 30% (HCT-116 cells). DAPI staining gave a preliminary indication that the mechanism of cell death was apoptotic. Further investigation is required to determine which part of the polyamine pathway is a target. However, these studies indicate that modulation of polyamine metabolism may, in part, explain some of the cytotoxic effects of EA and QC in cancer cells.
doi:10.1016/j.tox.2009.04.024 P29
Acknowledgement We thank NHS Grampian Endowments Trust for their support.
Potentiation of the cyotoxic effects of natural chemopreventative agents, ellagic acid and quercetin Fiona Saunders ∗ , Manju Oommen, Ratchadawadee Lapjitkusol, Edward Amankwatia, Lauren Kaminski, Adele Swanson, Alastair Mitchell, Heather M. Wallace Divison of Applied Medicine, School of Medicine & Dentistry, University of Aberdeen, Polwarth Building, Foresterhill, Aberdeen AB25 2ZD, United Kingdom E-mail address: [email protected] (F. Saunders). Lung and colorectal are among the most prevalent cancers in the world. Current treatments have limited success with 5-year survival (7% and 50%, respectively). So another approach is needed. One strategy is chemoprevention (natural or chemical). The most investigated are notably non-steroidal anti-inflammatory drugs (NSAIDs). However, NSAIDs have side effects and there is growing interest in natural compounds with potentially less side effects. Ellagic acid (EA), a polyphenol, and quercetin (QC), a flavonoid, are naturally occurring compounds, abundant in soft fruits and other foodstuffs (Ren et al., 2003). They are effective in killing cancer cells, but their mechanism of cytotoxicity is unknown. Preliminary studies suggest that inhibition of polyamine biosynthesis may be involved. Polyamine concentrations and metabolism are increased in cancer cells (Kingsnorth et al., 1984). Preventing polyamine accumulation may be one mechanism used by preventative agents to
Reference Ren, W., Qiao, Z., Wnag, H., Zhu, L., Zhang, L., 2003. Med Res Rev 23 (4), 519–534. Kingsnorth, A.N., Lumsden, A.B., Wallace, H.M., 1984. Br J Surg 71, 791–794.
doi:10.1016/j.tox.2009.04.025 P30 How does increasing cytotoxicity affect the accuracy of the GADD45a-GFP genotoxicity screening assay: A comparison of 4 different toxicity testing methods Matthew Tate a,b,∗ , Christopher Jagger b , Nicholas Billinton b , Paul Cahill b , Adam Rabinowitz b , Andrew Knight b , Richard Walmsley a,b a
Faculty of Life Sciences, The University of Manchester, Manchester M13 9PL, UK b Gentronix Ltd, CTF Building, 46 Grafton St, Manchester M13 9NT, UK E-mail address: [email protected] (M. Tate). Recent evaluations of the in vitro mammalian genotoxicity assays have suggested that increased levels of cytotoxicity may lead to an increase in the number of uniquely positive genotoxicity results Kirkland et al. (2005, 2006). Moreover, these assays have