- Email: [email protected]
Human constitutive androstane receptor (CAR) supports the hypertrophic but not the hyperplastic response to the murine non-genotoxic carcinogen phenobarbital (pb) in vivo
Poster Abstracts / Toxicology 262 (2009) 8–26
11
Reference Barton, H.A., Pastoor, T.P., Baetcke, K., Chambers, J.E., Diliberto, J., Doerrer, N.G., Driver, J.H., Hastings, C.E., Iyengar, S., Krieger, R., Stahl, B., Timchalk, C., 2006. The acquisition and application of absorption, distribution, metabolism, and excretion (ADME) data in agricultural chemical safety assessments. Crit Rev Toxicol 36, 9–35.
doi:10.1016/j.tox.2009.04.007 P07 Analysis of the effects of cruciferous vegetable consumption on human metabolic phenotype analysed by UPLC-mass spectrometry William M. Edmands ∗ , Florian Geier, Paul Benton, Elaine Holmes, Nigel J. Gooderham Department of Biomolecular Medicine, Imperial College London, SW7 2AZ, United Kingdom E-mail address: [email protected] (W.M. Edmands). Eating cruciferous vegetables (CV) is considered to be a healthy dietary option, the benefit being related to the glucosinolate (-thioglycoside-N-hydroxysulfates) content and their hydrolysis products (Verhoeven et al., 1997). The purpose of this investigation was to use mass spectrometry to investigate whether CV consumption can induce metabolic phenotypic change. Twenty non-smoking Caucasian male subjects were given a high CV dietary intervention. The study was divided into three periods each lasting 14 days; in periods 1 and 3 subjects were maintained on a low CV diet and in period 2 a supervised high CV diet was consumed. Each study was concluded with a red meat meal and a cup of coffee. Urine samples were collected at the end of each phase; pre-dose, 0–10 h.p.d., 10–24 h.p.d. and 24–48h.p.d. (h.p.d. hours post-dose) (Murray et al., 2001). High-resolution 1 H NMR spectrometric analysis coupled with multivariate statistical analysis revealed biomarkers associated with CV consumption and evidence of time-course related metabolic changes related to red-meat consumption. In order to further elucidate metabolic alterations urine samples were also subjected to UPLC-MS analysis (Waters LCT premier). A priori Markerlynx Principal Component Analysis (PCA) of the dataset within positive and negative ionisation modes revealed no clustering based on sample class. Targeted mining of the data for candidate molecules of interest proved limited in characterising the complex metabolic changes observed, however from a list of 86 potential candidate molecules a number were identified for semi-quantitative time-course excretion profiling including Nacetyl-sulforaphane and N-acetyl-allyl isothiocyanate from CV and caffeine and 5-acetylamino-6-formylamino-3-methyluracil from coffee. We also used XCMS a freeware UPLC-MS peak picking programme to identify mass spectral biomarkers of CV consumption (Fig. 1). This programme uses hundreds of endogenous metabolite standards and calculates a non-linear retention time correction profile for each sample. The relative ion intensities of each metabolite are compared to reveal differences and highlight potential biomarkers (Fig. 1). The significance of these biomarkers requires their identification, and MS-MS fragmentation and database mining are being used to establish identity. In summary, we demonstrate the feasibility of this untargeted analytical approach and show that with minimal sample work-up, XCMS can reveal biomarkers of CV consumption within highly complex mass spectral data sets from biofluids.
Fig. 1. Representative extracted ion chromatograms (EIC) of significant feature m/z 276.99–277.19 taken from the XCMS comparison of Phase I pre-dose vs. Phase II predose urine samples. The peak represents a biomarker of CV consumption not present in Phase I (baseline).
Funded by the Food Standards Agency. Reference Murray, S., Lake, B.G., Gray, S., Edwards, A.J., Springhall, C., Bowey, E.A., Williamson, G., Boobis, A.R., Gooderham, N.J., 2001. Carcinogenesis 22, 1413–1420. Verhoeven, T.H., Verhagen, H., Goldbohm, R.H., Van der Brandt, P.A., Van Poppel, G., 1997. Chem Biol Interact 103, 79–129.
doi:10.1016/j.tox.2009.04.008 P08 Human constitutive androstane receptor (CAR) supports the hypertrophic but not the hyperplastic response to the murine non-genotoxic carcinogen phenobarbital (pb) in vivo Clifford R. Elcombe a,∗ , Jillian Ross a , Nico Scheer b , Anja Rode b , C. Roland Wolf a a b
CXR Biosciences, Dundee, United Kingdom TaconicArtemis GmbH, Cologne, Germany
E-mail address: [email protected] (C.R. Elcombe). PB is a non-genotoxic carcinogen that in mice induces hepatomegaly (characterised by hypertrophy and hyperplasia) and, following long-term treatment, hepatocellular tumours, possibly due to its ability to increase cell proliferation. The relevance of these tumours to human health is controversial due to the lack of a clear molecular mechanism and suitable human-like models. PB has been shown to activate the murine and human constitutive androstane receptors (CAR) and pregnane X receptors (PXR). The nuclear receptor CAR mediates specific xenobiotic induction of drug metabolism. CAR knockout mice cannot activate the Cyp2b10 gene in response to PB, nor do the hypertrophic and hyperplastic responses elicited by PB occur (Wei et al., 2000). PB (80 mg/kg i.p., 4 days) was administered to double “humanised” CAR and PXR (huPXR/huCAR) mice and wild type C57BL/6J mice to investigate whether hyperplastic responses to chemicals observed in rodents are relevant to humans. Mice which were devoid of both receptors (PXRKO/CARKO) were used as controls. Mice (n = 10 per group) were implanted with osmotic pumps containing BrdU to allow determination of replicative DNA synthesis. Relative liver weights were increased (to about 120% of controls) by PB in the wild type (WT) mice and “human-
12
Poster Abstracts / Toxicology 262 (2009) 8–26
ised” mice but not in the PXRKO/CARKO animals. Cytochrome P450 induction was observed in WT and huPXR/huCAR mice but not in the PXRKO/CARKO mice, as determined by catalytic activity measurements (pentoxyresorufin-O-depentylation and 7benzyloxyquinoline-O-debenzylation)) and immunoblotting (for Cyp2b10 and Cyp3a11). The nuclear incorporation of BrdU was determined as a measure of cell proliferation (S-phase). PB increased the hepatocellular labelling index (S-phase, % of cells labelled) by approximately 5-fold in the WT mice (WT control, 1.75 ± 1.10; WT PB-treated, 9.30 ± 3.64, P < 0.001). However, no change in S-phase was detected in either the huPXR/huCAR (control, 1.93 ± 0.67; PB-treated, 2.00 ± 1.18) or PXRKO/CARKO (control, 1.96 ± 1.04; PB-treated, 2.83 ± 1.07) mice following PB treatment. In conclusion, these data demonstrate that the human receptors are able to support the chemically induced hypertrophic response but not the hyperplastic response. These data, obtained using novel human response models, suggest that it is unlikely that exposure to PB poses a hepatocarcinogenic hazard to humans. Acknowledgement This work was supported by ITI Life Sciences. Reference Wei, et al., 2000. Nature 407, 920–923.
doi:10.1016/j.tox.2009.04.009 P09 Involvement of drug-specific CD4+ trimethoprim-mediated hepatitis El-Ghaiesh a,∗ ,
Lavergne a ,
and CD8+
T-cells in
Farell a ,
Syn b ,
and T-cell receptor Vs), function (proliferation, cytokine secretion and cytolytic activity), mechanism of TMP presentation (role of metabolism, covalent binding and processing) and cross reactivity with structurally related compounds. Approval for the study was obtained from Liverpool local research ethics committee. Lymphocytes from the patient, but not TMP-exposed volunteers, were stimulated to proliferate with TMP (3801 ± 1174.4 cpm, [no drug]; (25368 ± 3900.5 cpm, [TMP 25 g/ml]; P < 0.05). 35 and 14 CD4+ and CD8+ T-cell clones that proliferated and secreted high levels of INF-(, TNF-( & IP-10 following TMP stimulation were generated from the allergic patient. High levels of CXCR3, CCR6, CLA and CD11a were found on the surface of clones expressing a variety of V receptors. Interestingly, CD4+, but not CD8+, clones killed autologous target cells, assessed using a 4 h [51 Cr] release assay and by measuring a transient increase in CD107a expression on the surface of TMP-stimulated clones. TMP activated specific T-cell receptors on T-cell clones via two pathways: the first involved direct drug binding to MHC and the T-cell receptor; the second involved protein complex formation and antigen processing. Processing-dependent TMP presentation to T-cells was blocked with methimazole (MTZ) and 1-aminobenzotriazole (ABT), inhibitors of peroxidise and cytochrome P450 enzymes. TMPspecific T-cells were highly specific in terms of drug structure; clones were stimulated to proliferate with TMP, pyrimethamine, and diaveridine, but not the other structurally related compounds. These data indicate that T cells responsive towards the parent drug and drug metabolites are present in the circulation of a patient with TMP-mediated hepatitis. Secretion of various cytokines and expression of tissue homing chemokine receptors on the surface of T-cells are likely responsible for the organ-selective tissue damage. Acknowledgements
Sabah Sidonie John Wing Munir Pirmohamed a , B. Kevin Park a , Dean J. Naisbitt a a
MRC Centre for Drug Safety Science, Department of Pharmacology, Ashton Street, University of Liverpool, Liverpool L69 3GE, United Kingdom b Liver Research Group, University of Birmingham, B15 2TT United Kingdom
E-mail address: [email protected] (S. El-Ghaiesh). Drug-induced liver injury is a cause of significant patient mortality and morbidity; hepatic adverse events are a major cause of drug withdrawal. Development of clinical manifestations in many instances is associated with reactive metabolite formation and subsequent protein complex formation. Increasing evidence indicates that the release of endogenous mediators from dying cells stimulates an innate immune response that promotes tissue damage; however, to date, the involvement of antigen-specific T-cells in drug-induced liver injury has not been defined (Antoine et al., 2008). In this study, the nature of the T-cell response in a patient who experienced severe hepatitis following trimethoprim (TMP) exposure was delineated by means of in vitro stimulation of lymphocytes and T-cell clones. The patient, a 20-year-old female, developed a maculopapular rash two weeks after initiating trimethoprim treatment. She was admitted to hospital with worsening rash, fever, eosinophilia and AST levels above 6000 IU/l. The patient was discharged eight days after admission and liver function tests were normal at two-month follow-up. Lymphocytes were isolated from peripheral blood (at the time of the reaction and monthly thereafter) and incubated with TMP (0.1–500 g/ml) for 6 days (37 ◦ C; 5% CO2 ). Proliferation was measured by addition of [3 H]thymidine for the last 16 h of the experiment. T-cells from responding cultures were cloned by serial dilution to evaluate their phenotype (CD, chemokine receptor
This work was supported by the Medical Research Council [grant number G0700654]. SEG is a PhD student receiving funding from Tanta University, Egyptian Government. Reference Antoine, D.J., Williams, D.P., Park, B.K., 2008. Expert Opin Drug Metab Toxicol 4, 1415–1427.
doi:10.1016/j.tox.2009.04.010 P011 Phylogenetic analysis of the ATP-binding cassette super-family: Implications for the development of multiple drug resistance phenotype Ciaran Fisher a,∗ , Tanya Coleman b , Nick Plant a a
Centre for Toxicology, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey GU2 7XH, United Kingdom b DMPK, AstraZeneca, Alderley Park, Macclesfield, Cheshire SK10 4TF, United Kingdom E-mail address: c.p.fi[email protected] (C. Fisher). The increasing availability of genomic data, coupled with accessible tools to analyse these data, offers the opportunity to study the evolution of important gene families. The ATP-binding cassette (ABC) super-family of genes encodes 48 proteins in humans, divided into seven sub-families. These sub-families are represented in all eukaryotic genomes thus far studied, and are also expressed in a number of prokaryotic genomes, suggesting they confer an important evolutionary advantage. Of these seven sub-families, five encode for membrane exporter proteins with an extensive and diverse range of substrate specificities including both endobiotics
11
Reference Barton, H.A., Pastoor, T.P., Baetcke, K., Chambers, J.E., Diliberto, J., Doerrer, N.G., Driver, J.H., Hastings, C.E., Iyengar, S., Krieger, R., Stahl, B., Timchalk, C., 2006. The acquisition and application of absorption, distribution, metabolism, and excretion (ADME) data in agricultural chemical safety assessments. Crit Rev Toxicol 36, 9–35.
doi:10.1016/j.tox.2009.04.007 P07 Analysis of the effects of cruciferous vegetable consumption on human metabolic phenotype analysed by UPLC-mass spectrometry William M. Edmands ∗ , Florian Geier, Paul Benton, Elaine Holmes, Nigel J. Gooderham Department of Biomolecular Medicine, Imperial College London, SW7 2AZ, United Kingdom E-mail address: [email protected] (W.M. Edmands). Eating cruciferous vegetables (CV) is considered to be a healthy dietary option, the benefit being related to the glucosinolate (-thioglycoside-N-hydroxysulfates) content and their hydrolysis products (Verhoeven et al., 1997). The purpose of this investigation was to use mass spectrometry to investigate whether CV consumption can induce metabolic phenotypic change. Twenty non-smoking Caucasian male subjects were given a high CV dietary intervention. The study was divided into three periods each lasting 14 days; in periods 1 and 3 subjects were maintained on a low CV diet and in period 2 a supervised high CV diet was consumed. Each study was concluded with a red meat meal and a cup of coffee. Urine samples were collected at the end of each phase; pre-dose, 0–10 h.p.d., 10–24 h.p.d. and 24–48h.p.d. (h.p.d. hours post-dose) (Murray et al., 2001). High-resolution 1 H NMR spectrometric analysis coupled with multivariate statistical analysis revealed biomarkers associated with CV consumption and evidence of time-course related metabolic changes related to red-meat consumption. In order to further elucidate metabolic alterations urine samples were also subjected to UPLC-MS analysis (Waters LCT premier). A priori Markerlynx Principal Component Analysis (PCA) of the dataset within positive and negative ionisation modes revealed no clustering based on sample class. Targeted mining of the data for candidate molecules of interest proved limited in characterising the complex metabolic changes observed, however from a list of 86 potential candidate molecules a number were identified for semi-quantitative time-course excretion profiling including Nacetyl-sulforaphane and N-acetyl-allyl isothiocyanate from CV and caffeine and 5-acetylamino-6-formylamino-3-methyluracil from coffee. We also used XCMS a freeware UPLC-MS peak picking programme to identify mass spectral biomarkers of CV consumption (Fig. 1). This programme uses hundreds of endogenous metabolite standards and calculates a non-linear retention time correction profile for each sample. The relative ion intensities of each metabolite are compared to reveal differences and highlight potential biomarkers (Fig. 1). The significance of these biomarkers requires their identification, and MS-MS fragmentation and database mining are being used to establish identity. In summary, we demonstrate the feasibility of this untargeted analytical approach and show that with minimal sample work-up, XCMS can reveal biomarkers of CV consumption within highly complex mass spectral data sets from biofluids.
Fig. 1. Representative extracted ion chromatograms (EIC) of significant feature m/z 276.99–277.19 taken from the XCMS comparison of Phase I pre-dose vs. Phase II predose urine samples. The peak represents a biomarker of CV consumption not present in Phase I (baseline).
Funded by the Food Standards Agency. Reference Murray, S., Lake, B.G., Gray, S., Edwards, A.J., Springhall, C., Bowey, E.A., Williamson, G., Boobis, A.R., Gooderham, N.J., 2001. Carcinogenesis 22, 1413–1420. Verhoeven, T.H., Verhagen, H., Goldbohm, R.H., Van der Brandt, P.A., Van Poppel, G., 1997. Chem Biol Interact 103, 79–129.
doi:10.1016/j.tox.2009.04.008 P08 Human constitutive androstane receptor (CAR) supports the hypertrophic but not the hyperplastic response to the murine non-genotoxic carcinogen phenobarbital (pb) in vivo Clifford R. Elcombe a,∗ , Jillian Ross a , Nico Scheer b , Anja Rode b , C. Roland Wolf a a b
CXR Biosciences, Dundee, United Kingdom TaconicArtemis GmbH, Cologne, Germany
E-mail address: [email protected] (C.R. Elcombe). PB is a non-genotoxic carcinogen that in mice induces hepatomegaly (characterised by hypertrophy and hyperplasia) and, following long-term treatment, hepatocellular tumours, possibly due to its ability to increase cell proliferation. The relevance of these tumours to human health is controversial due to the lack of a clear molecular mechanism and suitable human-like models. PB has been shown to activate the murine and human constitutive androstane receptors (CAR) and pregnane X receptors (PXR). The nuclear receptor CAR mediates specific xenobiotic induction of drug metabolism. CAR knockout mice cannot activate the Cyp2b10 gene in response to PB, nor do the hypertrophic and hyperplastic responses elicited by PB occur (Wei et al., 2000). PB (80 mg/kg i.p., 4 days) was administered to double “humanised” CAR and PXR (huPXR/huCAR) mice and wild type C57BL/6J mice to investigate whether hyperplastic responses to chemicals observed in rodents are relevant to humans. Mice which were devoid of both receptors (PXRKO/CARKO) were used as controls. Mice (n = 10 per group) were implanted with osmotic pumps containing BrdU to allow determination of replicative DNA synthesis. Relative liver weights were increased (to about 120% of controls) by PB in the wild type (WT) mice and “human-
12
Poster Abstracts / Toxicology 262 (2009) 8–26
ised” mice but not in the PXRKO/CARKO animals. Cytochrome P450 induction was observed in WT and huPXR/huCAR mice but not in the PXRKO/CARKO mice, as determined by catalytic activity measurements (pentoxyresorufin-O-depentylation and 7benzyloxyquinoline-O-debenzylation)) and immunoblotting (for Cyp2b10 and Cyp3a11). The nuclear incorporation of BrdU was determined as a measure of cell proliferation (S-phase). PB increased the hepatocellular labelling index (S-phase, % of cells labelled) by approximately 5-fold in the WT mice (WT control, 1.75 ± 1.10; WT PB-treated, 9.30 ± 3.64, P < 0.001). However, no change in S-phase was detected in either the huPXR/huCAR (control, 1.93 ± 0.67; PB-treated, 2.00 ± 1.18) or PXRKO/CARKO (control, 1.96 ± 1.04; PB-treated, 2.83 ± 1.07) mice following PB treatment. In conclusion, these data demonstrate that the human receptors are able to support the chemically induced hypertrophic response but not the hyperplastic response. These data, obtained using novel human response models, suggest that it is unlikely that exposure to PB poses a hepatocarcinogenic hazard to humans. Acknowledgement This work was supported by ITI Life Sciences. Reference Wei, et al., 2000. Nature 407, 920–923.
doi:10.1016/j.tox.2009.04.009 P09 Involvement of drug-specific CD4+ trimethoprim-mediated hepatitis El-Ghaiesh a,∗ ,
Lavergne a ,
and CD8+
T-cells in
Farell a ,
Syn b ,
and T-cell receptor Vs), function (proliferation, cytokine secretion and cytolytic activity), mechanism of TMP presentation (role of metabolism, covalent binding and processing) and cross reactivity with structurally related compounds. Approval for the study was obtained from Liverpool local research ethics committee. Lymphocytes from the patient, but not TMP-exposed volunteers, were stimulated to proliferate with TMP (3801 ± 1174.4 cpm, [no drug]; (25368 ± 3900.5 cpm, [TMP 25 g/ml]; P < 0.05). 35 and 14 CD4+ and CD8+ T-cell clones that proliferated and secreted high levels of INF-(, TNF-( & IP-10 following TMP stimulation were generated from the allergic patient. High levels of CXCR3, CCR6, CLA and CD11a were found on the surface of clones expressing a variety of V receptors. Interestingly, CD4+, but not CD8+, clones killed autologous target cells, assessed using a 4 h [51 Cr] release assay and by measuring a transient increase in CD107a expression on the surface of TMP-stimulated clones. TMP activated specific T-cell receptors on T-cell clones via two pathways: the first involved direct drug binding to MHC and the T-cell receptor; the second involved protein complex formation and antigen processing. Processing-dependent TMP presentation to T-cells was blocked with methimazole (MTZ) and 1-aminobenzotriazole (ABT), inhibitors of peroxidise and cytochrome P450 enzymes. TMPspecific T-cells were highly specific in terms of drug structure; clones were stimulated to proliferate with TMP, pyrimethamine, and diaveridine, but not the other structurally related compounds. These data indicate that T cells responsive towards the parent drug and drug metabolites are present in the circulation of a patient with TMP-mediated hepatitis. Secretion of various cytokines and expression of tissue homing chemokine receptors on the surface of T-cells are likely responsible for the organ-selective tissue damage. Acknowledgements
Sabah Sidonie John Wing Munir Pirmohamed a , B. Kevin Park a , Dean J. Naisbitt a a
MRC Centre for Drug Safety Science, Department of Pharmacology, Ashton Street, University of Liverpool, Liverpool L69 3GE, United Kingdom b Liver Research Group, University of Birmingham, B15 2TT United Kingdom
E-mail address: [email protected] (S. El-Ghaiesh). Drug-induced liver injury is a cause of significant patient mortality and morbidity; hepatic adverse events are a major cause of drug withdrawal. Development of clinical manifestations in many instances is associated with reactive metabolite formation and subsequent protein complex formation. Increasing evidence indicates that the release of endogenous mediators from dying cells stimulates an innate immune response that promotes tissue damage; however, to date, the involvement of antigen-specific T-cells in drug-induced liver injury has not been defined (Antoine et al., 2008). In this study, the nature of the T-cell response in a patient who experienced severe hepatitis following trimethoprim (TMP) exposure was delineated by means of in vitro stimulation of lymphocytes and T-cell clones. The patient, a 20-year-old female, developed a maculopapular rash two weeks after initiating trimethoprim treatment. She was admitted to hospital with worsening rash, fever, eosinophilia and AST levels above 6000 IU/l. The patient was discharged eight days after admission and liver function tests were normal at two-month follow-up. Lymphocytes were isolated from peripheral blood (at the time of the reaction and monthly thereafter) and incubated with TMP (0.1–500 g/ml) for 6 days (37 ◦ C; 5% CO2 ). Proliferation was measured by addition of [3 H]thymidine for the last 16 h of the experiment. T-cells from responding cultures were cloned by serial dilution to evaluate their phenotype (CD, chemokine receptor
This work was supported by the Medical Research Council [grant number G0700654]. SEG is a PhD student receiving funding from Tanta University, Egyptian Government. Reference Antoine, D.J., Williams, D.P., Park, B.K., 2008. Expert Opin Drug Metab Toxicol 4, 1415–1427.
doi:10.1016/j.tox.2009.04.010 P011 Phylogenetic analysis of the ATP-binding cassette super-family: Implications for the development of multiple drug resistance phenotype Ciaran Fisher a,∗ , Tanya Coleman b , Nick Plant a a
Centre for Toxicology, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey GU2 7XH, United Kingdom b DMPK, AstraZeneca, Alderley Park, Macclesfield, Cheshire SK10 4TF, United Kingdom E-mail address: c.p.fi[email protected] (C. Fisher). The increasing availability of genomic data, coupled with accessible tools to analyse these data, offers the opportunity to study the evolution of important gene families. The ATP-binding cassette (ABC) super-family of genes encodes 48 proteins in humans, divided into seven sub-families. These sub-families are represented in all eukaryotic genomes thus far studied, and are also expressed in a number of prokaryotic genomes, suggesting they confer an important evolutionary advantage. Of these seven sub-families, five encode for membrane exporter proteins with an extensive and diverse range of substrate specificities including both endobiotics