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The effect of four non-genotoxic carcinogens and four non-carcinogens on NRK-52E cells using a transcriptomics approach
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Abstracts / Toxicology 290 (2011) 1–46
Fig. 1. qRT-PCR characterisation of cell clones differentiated in the presence of vehicle only (VO), 17-estradiol (ES) or di-butyl-phthalate (DBP) for 4 days (D4), 8 days (D8) or 12 days (D12).
Reference Cuzin, F., Rassoulzadegan, M., 2010. Essays Biochem. 48, 101–106. Skinner, M.K., Manikkam, M., Guerrero-Bosagna, C., 2010. Reprod. Toxicol., doi:10.1016/j.reprotox.2010.10.012. West, J.A., Park, I.-H., Daley, G.Q., Geijsen, N., 2006. Nat. Protoc. 4, 2026–2036.
doi:10.1016/j.tox.2011.09.018 P011 The effect of four non-genotoxic carcinogens and four noncarcinogens on NRK-52E cells using a transcriptomics approach Kate M. Bloch 1,∗ , Andrew Evans 1 , Joost van Delft 2 , Edward A. Lock 1 1
School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, Liverpool, UK 2 Department of Toxicogenomics, Maastricht University, Maastricht, The Netherlands E-mail address: [email protected] (K.M. Bloch). This study has used genome-wide transcriptomic analysis, to identify genes or pathways that may predict the carcinogenic potential of non-genotoxic (NGTX) chemicals to the kidney. Normal Rat Kidney cells (NRK-52E) were culture in DMEM supplemented with penicillin/streptomycin solution until >80% confluent in 6-well plates and then exposed individually to 4 NGTX rat renal carcinogens and non-carcinogens (NC) at an IC10 concentration for 72 h (Lock and Hard, 2004). The NGTX chemicals were ochratoxin A (OTA) 0.5 M; monuron (M) 250 M; chlorothalonil (C) 1.1 M or S-1,2-dichlorovinylcysteine (DCVC) 3 M. NC included d-mannitol (10 mM); tolbutamide (0.37 mM); diclofenac sodium (16 M) and nifedipine (35 M). Control cells were exposed to 0.1% DMSO the same as treated cells. The cells were exposed for 6 h, 24 h and 72 h to each chemical in triplicate and RNA was isolated, purified and quality checked. Microarray analysis used Affymetrix rat GeneChips 230, 2.0, while the analysis of gene changes was performed using GeneSpring GX11 software from Agilent. Data was normalised using a GC-RMA algorithm and statistical analysis between control and treated cells conducted on genes with a >2-fold threshold change using a p < 0.001. The DAVID website was used for Kegg pathway analysis (Dennis et al., 2003; Huang et al., 2009). Fig. 1 shows the time course of gene changes following exposure to the four NGTX chemicals. Major pathways affected were: steroid biosynthesis downregulated (↓) by C and OTA at 24 h; terpenoid backbone biosynthesis (↓) by C at 24 h and M at 6 h and cell cycle, by C at 6 h and by DCVC and OTA at 24 h (↓). Other pathways altered were: cell adhesion, by C at 6 h and 24 h; pathways in cancer (renal cell carcinoma and mTOR) by OTA at 24 h and by DCVC and C at
Fig. 1. Number of differentially expressed genes identified after 6, 24, 72 h exposure of NGTX carcinogens.
72 h; aminoacyl t-RNA synthesis by DCVC at 72 h; p53 signalling by OTA at 24 h and DCVC at 72 h and proteolysis by C at 24 h and 72 h.There was no commonly deregulated gene(s) induced by the four NGTX carcinogens at 6 h, 24 h or 72 h. However, there were a number of common pathways affected by some of these NGTX. OTA and C seemed to be the most similar chemicals sharing the highest number of overlapping genes over time (6 genes for 6 h, 20 for 24 h and 26 for 72 h). Studies with 4 NC chemicals showed a very small number of gene changes with only one pathway in common, cell adhesion. These findings indicate that NRK-52E cells are able to detect some of the carcinogenic events associated with NGTX carcinogens in vitro. Acknowledgements: This work was supported by an EU grant, 6th framework on Carcinogenomics. Reference Dennis Jr., G., Sherman, B.T., Hosack, D.A., Yang, J., Gao, W., Lane, H.C., Lempicki, R.A., 2003. Genome Biol. 4, P3. Huang, D.W., Sherman, B.T., Lempicki, R.A., 2009. Nat. Protoc. 4, 44–57. Lock, E.A., Hard, G.C., 2004. Crit. Rev. Toxicol. 34, 211–299.
doi:10.1016/j.tox.2011.09.019
Abstracts / Toxicology 290 (2011) 1–46
Fig. 1. qRT-PCR characterisation of cell clones differentiated in the presence of vehicle only (VO), 17-estradiol (ES) or di-butyl-phthalate (DBP) for 4 days (D4), 8 days (D8) or 12 days (D12).
Reference Cuzin, F., Rassoulzadegan, M., 2010. Essays Biochem. 48, 101–106. Skinner, M.K., Manikkam, M., Guerrero-Bosagna, C., 2010. Reprod. Toxicol., doi:10.1016/j.reprotox.2010.10.012. West, J.A., Park, I.-H., Daley, G.Q., Geijsen, N., 2006. Nat. Protoc. 4, 2026–2036.
doi:10.1016/j.tox.2011.09.018 P011 The effect of four non-genotoxic carcinogens and four noncarcinogens on NRK-52E cells using a transcriptomics approach Kate M. Bloch 1,∗ , Andrew Evans 1 , Joost van Delft 2 , Edward A. Lock 1 1
School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, Liverpool, UK 2 Department of Toxicogenomics, Maastricht University, Maastricht, The Netherlands E-mail address: [email protected] (K.M. Bloch). This study has used genome-wide transcriptomic analysis, to identify genes or pathways that may predict the carcinogenic potential of non-genotoxic (NGTX) chemicals to the kidney. Normal Rat Kidney cells (NRK-52E) were culture in DMEM supplemented with penicillin/streptomycin solution until >80% confluent in 6-well plates and then exposed individually to 4 NGTX rat renal carcinogens and non-carcinogens (NC) at an IC10 concentration for 72 h (Lock and Hard, 2004). The NGTX chemicals were ochratoxin A (OTA) 0.5 M; monuron (M) 250 M; chlorothalonil (C) 1.1 M or S-1,2-dichlorovinylcysteine (DCVC) 3 M. NC included d-mannitol (10 mM); tolbutamide (0.37 mM); diclofenac sodium (16 M) and nifedipine (35 M). Control cells were exposed to 0.1% DMSO the same as treated cells. The cells were exposed for 6 h, 24 h and 72 h to each chemical in triplicate and RNA was isolated, purified and quality checked. Microarray analysis used Affymetrix rat GeneChips 230, 2.0, while the analysis of gene changes was performed using GeneSpring GX11 software from Agilent. Data was normalised using a GC-RMA algorithm and statistical analysis between control and treated cells conducted on genes with a >2-fold threshold change using a p < 0.001. The DAVID website was used for Kegg pathway analysis (Dennis et al., 2003; Huang et al., 2009). Fig. 1 shows the time course of gene changes following exposure to the four NGTX chemicals. Major pathways affected were: steroid biosynthesis downregulated (↓) by C and OTA at 24 h; terpenoid backbone biosynthesis (↓) by C at 24 h and M at 6 h and cell cycle, by C at 6 h and by DCVC and OTA at 24 h (↓). Other pathways altered were: cell adhesion, by C at 6 h and 24 h; pathways in cancer (renal cell carcinoma and mTOR) by OTA at 24 h and by DCVC and C at
Fig. 1. Number of differentially expressed genes identified after 6, 24, 72 h exposure of NGTX carcinogens.
72 h; aminoacyl t-RNA synthesis by DCVC at 72 h; p53 signalling by OTA at 24 h and DCVC at 72 h and proteolysis by C at 24 h and 72 h.There was no commonly deregulated gene(s) induced by the four NGTX carcinogens at 6 h, 24 h or 72 h. However, there were a number of common pathways affected by some of these NGTX. OTA and C seemed to be the most similar chemicals sharing the highest number of overlapping genes over time (6 genes for 6 h, 20 for 24 h and 26 for 72 h). Studies with 4 NC chemicals showed a very small number of gene changes with only one pathway in common, cell adhesion. These findings indicate that NRK-52E cells are able to detect some of the carcinogenic events associated with NGTX carcinogens in vitro. Acknowledgements: This work was supported by an EU grant, 6th framework on Carcinogenomics. Reference Dennis Jr., G., Sherman, B.T., Hosack, D.A., Yang, J., Gao, W., Lane, H.C., Lempicki, R.A., 2003. Genome Biol. 4, P3. Huang, D.W., Sherman, B.T., Lempicki, R.A., 2009. Nat. Protoc. 4, 44–57. Lock, E.A., Hard, G.C., 2004. Crit. Rev. Toxicol. 34, 211–299.
doi:10.1016/j.tox.2011.09.019