- Email: [email protected]
Covalent modifications of proteins by sensitisers—Far from a random event?
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Abstracts / Toxicology 231 (2007) 104–119
viability over this period. GSH content increased significantly at week 2 (136.58 ± 3.93% of control level) before decreasing to 69.97 ± 0.98% of control levels in week 4. Although, there was a decline in GRd expression to 54.93 ± 1.03% of control levels at week 4, there was sufficient active enzyme to maintain the reaction. In fact, GRd activity was higher in treated cells for all weeks with a sudden increase in activity to 161.23 ± 3.96% of control levels in week 4. The increase in GRd activity is contradictory to previous studies where GRd activity was inhibited, on acute Cr(VI) exposure, in hepatocytes (Gunaratnam and Grant, 2001) and erythrocytes (Koutras et al., 1965). This increase in activity may be part of an adaptive response to Cr(VI), where cells are attempting to protect themselves against Cr(VI) toxicity. In vivo, the serum of patients with orthopaedic implants may contain up to 2 M Cr, and our in vitro findings with macrophages raise serious concern regarding the effect of released Cr(VI) on the immune system. Acknowledgements This research is partially supported by funds from The Wingate foundation and DePuy International Ltd. References Carlberg, I., Mannervik, B., 1985. Methods Enzymol. 113, 484–490. Gunaratnam, M., Grant, M.H., 2001. Chem. Biol. Interact. 134, 191–202. Koutras, G.A., Schneider, A.S., Hattori, M., Valentine, W.N., 1965. Br. J. Haematol. 11, 360–369. Miloˇsev, I., Piˇsot, V., Campbell, P., 2005. J. Orthop. Res. 23, 526–535. Ning, J., Grant, M.H., 2000. Toxicol. In Vitro 14, 329–335.
doi:10.1016/j.tox.2006.11.030 Covalent modifications of proteins by sensitisers— Far from a random event? Maja Aleksic, Camilla K. Pease E-mail address: [email protected] (M. Aleksic). Unilever Research Colworth, Safety & Environmental Assurance Centre, Sharnbrook, Bedford MK44 1LQ, United Kingdom Most known skin sensitisers are electrophiles and show the potential to covalently modify (haptenate) skin proteins via one or more reaction mechanisms generating immunogenic entities. Covalent modification is thought to be a key step in the skin sensitisation process. Chemi-
cals with no intrinsic ability to react with proteins could be converted into protein reactive species by photooxidation, chemical oxidation or cutaneous metabolism. Our understanding of these processes is mainly based on theoretical chemistry. The skin protein targets of sensitising chemicals and/or their metabolites are not known to date. We investigated covalent modifications of a model peptide and a model protein by known sensitisers in vitro to provide experimental evidence for the intrinsic ability of sensitisers to covalently modify protein(s). A model peptide (DS3, sequence VLSPADKTNWGHEYRMFCQIG) and a model protein (human serum albumin) were incubated with known sensitisers (1,4-dinitro-1-chlorobenzene (DNCB) and phenyl salicylate (PS)). Anticipated protein/peptide-sensitiser covalent complexes were digested with trypsin and analysed using matrix assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry, high performance liquid chromatography (HPLC), nanoelectrospray tandem mass spectrometry (ES-MS and -MS/MS) and nano LC-MS and -MS/MS strategies. DNCB modified 10 albumin residues (Lys-190, -195, -212, -225, -351, -414, His-9, Tyr-140, Cys-34 and the N-terminus) whereas PS modified 9 albumin residues (Lys-12, -137, -195, -402, -414, -525, -541, His-3 and Cys-34). DNCB modified six residues of DS3 peptide (Lys, Trp, His, Tyr, Cys and the N-terminus) whereas PS modified Lys, His and Cys. The results suggest that protein modifications are targeted to amino acid residues whose chemical microenvironment within the protein is conducive to reactivity. Same residue types appear to be targeted selectively by DNCB and PS on both models, with an exception of Trp modification by DNCB on DS3 peptide, the equivalent of which was not observed on albumin. Whilst DNCB and PS targeted some common albumin residues (Cys-34, Lys-195 and -414), both chemicals appear to target a distinct pattern of residues, further indicating that protein adduction is selective and dictated by structure and reactivity of the chemical. Previous studies (e.g. Elahi et al., 2004) suggested that chemical modification of skin proteins related to skin sensitisation is extensive and random. However, our results together with other recent evidence from generic peptide–chemical reactivity studies (Gerberick et al., 2004) suggest high selectivity in chemical modification of proteins (not all residues that could theoretically react in a protein do react). In the absence of information on specific protein targets in skin sensitisation and their immunogenic potential, in vitro investigations are limited to the use of model proteins/peptides. Whilst a generic in vitro model of protein haptenation does not have to reflect the in vivo situation, there is a possibility
Abstracts / Toxicology 231 (2007) 104–119
that chosen models may not provide the same spectrum of reactivity to skin sensitisation mechanisms in vivo. It has been suggested that information from in vitro protein reactivity studies could be used as part of a non-animal based approach for the identification of skin sensitisation potential (Jowsey et al., 2006). Such assays should be underpinned with mechanistic understanding of the relevance of covalent modifications and their immunogenic potential. References Elahi, E.N., Wright, Z.M., Hinselwood, D., Hotchkiss, S.A., Basketter, D.A., Pease, C., 2004. Chem. Res. Toxicol. 17, 301–310. Gerberick, G.F., Vassallo, J.D., Bailey, R.E., Chaney, J.G., Morrall, S.W., Lepoittevin, J.P., 2004. Toxicol. Sci. 81 (2), 332–343. Jowsey, I.R., Basketter, D.A., Westmoreland, C., Kimber, I., 2006. J. Appl. Toxicol. 26 (4), 341–350.
doi:10.1016/j.tox.2006.11.031 Genomic investigation of the metabolic competency of human cord blood-derived dendritic cells Ruth U. Pendlington, Vicki Summerfield, Andrew White, Camilla K. Pease E-mail address: [email protected] (V. Summerfield). Safety & Environmental Assurance Centre, Unilever Colworth Park, Sharnbrook, Bedford MK44 1LQ, UK With the advent of the 7th Amendment to the European Union’s Cosmetic Directive, risk assessments for cosmetic ingredients will have to be carried out without the generation of new animal data. This represents a major challenge to the Personal Care industry for many endpoints including skin sensitisation. In recent years, many attempts to develop novel in vitro assays for the identification of skin sensitisers have focused on cellbased assays that could model some of the functions of Langerhans cells since these are thought to be a key cellular mediator of contact allergy within the skin. Human monocyte-derived dendritic cells (MD-DCs) have been used by many groups for such work. It is envisaged that these cells could respond to chemicals such that discriminatory biomarkers of sensitisation can be identified. In developing novel cell-based assays to predict skin sensitisation, it is important to characterise the metabolic competency of the system (ultimately at the functional level) to ascertain whether the chemical (and any resulting metabolites) being tested in vitro are comparable to chemicals and metabolites to which the skin is exposed, in order that the data can be interpreted in the context
107
Table 1 Enzyme family
Major xenobiotic metabolising enzyme isoforms detected
Cytochrome P450
CYP1A1, CYP1A2, CYP 2C9, CYP2D6, CYP2E1, CYP3A4 None EPHX1, EPHX2 FMO4, FMO5 GPX1, GPX3, GPX4, GPX7 NAT1, NAT5 GSTA2, GSTA4 and others None SULT1A2, SULT1A3, SULT1C1, SULT4A1
NADPH-P450 oxidoreductase Epoxide hydrolase Flavin-containing monooxygenases Glutathione peroxidase N-Acetyltransferase Glutathione S-transferase UDP-glycosyltransferase Sulfotransferase
of local skin sensitisation effects in man. Ultimately, metabolic activation systems (e.g. S9, keratinocytes, etc.) may need to be added to such test systems, but in the first instance, the basal metabolic competence of the MD-DC cells themselves should be defined. With sparse knowledge about the metabolic competence of MD-DCs (Sieben et al., 1999) a pilot study was initiated to begin to investigate their xenobiotic metabolic competence. These first analyses would then be able to guide more focused functional studies. Preliminary results were obtained from three batches of cord blood-derived dendritic cell mRNA, which were received from MatTek Corporation, MA, USA. mRNA analysis was performed using Codelink whole genome arrays according to the manufacturer’s standard methodology. All samples and arrays passed applied QC thresholds. For each batch of cells, three replicate arrays were generated and analysed. Combining data from the replicates, expression of between 29712 and 32695 genes was detected above background across all three batches. Principal components analysis identified good concordance within replicates of a batch with more significant variation in gene expression levels between batches. Welch ANOVA testing (p = 0.05) with an applied multiple testing correction (Benjamini and Hochberg False Discovery Rate) identified between 300 and 736 genes that showed significant variation between batches. Table 1 below illustrates the observed mRNA expression profile for both Phase I and Phase II xenobiotic metabolising enzymes in these cells. Notably, the key enzyme of the mixed function oxidase system (NADPH-cytochrome P450 oxidoreductase) was absent at the mRNA level. Future studies will assess whether human cord blood-derived dendritic cells express any mixed function oxidase (CYP:NADPH
Abstracts / Toxicology 231 (2007) 104–119
viability over this period. GSH content increased significantly at week 2 (136.58 ± 3.93% of control level) before decreasing to 69.97 ± 0.98% of control levels in week 4. Although, there was a decline in GRd expression to 54.93 ± 1.03% of control levels at week 4, there was sufficient active enzyme to maintain the reaction. In fact, GRd activity was higher in treated cells for all weeks with a sudden increase in activity to 161.23 ± 3.96% of control levels in week 4. The increase in GRd activity is contradictory to previous studies where GRd activity was inhibited, on acute Cr(VI) exposure, in hepatocytes (Gunaratnam and Grant, 2001) and erythrocytes (Koutras et al., 1965). This increase in activity may be part of an adaptive response to Cr(VI), where cells are attempting to protect themselves against Cr(VI) toxicity. In vivo, the serum of patients with orthopaedic implants may contain up to 2 M Cr, and our in vitro findings with macrophages raise serious concern regarding the effect of released Cr(VI) on the immune system. Acknowledgements This research is partially supported by funds from The Wingate foundation and DePuy International Ltd. References Carlberg, I., Mannervik, B., 1985. Methods Enzymol. 113, 484–490. Gunaratnam, M., Grant, M.H., 2001. Chem. Biol. Interact. 134, 191–202. Koutras, G.A., Schneider, A.S., Hattori, M., Valentine, W.N., 1965. Br. J. Haematol. 11, 360–369. Miloˇsev, I., Piˇsot, V., Campbell, P., 2005. J. Orthop. Res. 23, 526–535. Ning, J., Grant, M.H., 2000. Toxicol. In Vitro 14, 329–335.
doi:10.1016/j.tox.2006.11.030 Covalent modifications of proteins by sensitisers— Far from a random event? Maja Aleksic, Camilla K. Pease E-mail address: [email protected] (M. Aleksic). Unilever Research Colworth, Safety & Environmental Assurance Centre, Sharnbrook, Bedford MK44 1LQ, United Kingdom Most known skin sensitisers are electrophiles and show the potential to covalently modify (haptenate) skin proteins via one or more reaction mechanisms generating immunogenic entities. Covalent modification is thought to be a key step in the skin sensitisation process. Chemi-
cals with no intrinsic ability to react with proteins could be converted into protein reactive species by photooxidation, chemical oxidation or cutaneous metabolism. Our understanding of these processes is mainly based on theoretical chemistry. The skin protein targets of sensitising chemicals and/or their metabolites are not known to date. We investigated covalent modifications of a model peptide and a model protein by known sensitisers in vitro to provide experimental evidence for the intrinsic ability of sensitisers to covalently modify protein(s). A model peptide (DS3, sequence VLSPADKTNWGHEYRMFCQIG) and a model protein (human serum albumin) were incubated with known sensitisers (1,4-dinitro-1-chlorobenzene (DNCB) and phenyl salicylate (PS)). Anticipated protein/peptide-sensitiser covalent complexes were digested with trypsin and analysed using matrix assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry, high performance liquid chromatography (HPLC), nanoelectrospray tandem mass spectrometry (ES-MS and -MS/MS) and nano LC-MS and -MS/MS strategies. DNCB modified 10 albumin residues (Lys-190, -195, -212, -225, -351, -414, His-9, Tyr-140, Cys-34 and the N-terminus) whereas PS modified 9 albumin residues (Lys-12, -137, -195, -402, -414, -525, -541, His-3 and Cys-34). DNCB modified six residues of DS3 peptide (Lys, Trp, His, Tyr, Cys and the N-terminus) whereas PS modified Lys, His and Cys. The results suggest that protein modifications are targeted to amino acid residues whose chemical microenvironment within the protein is conducive to reactivity. Same residue types appear to be targeted selectively by DNCB and PS on both models, with an exception of Trp modification by DNCB on DS3 peptide, the equivalent of which was not observed on albumin. Whilst DNCB and PS targeted some common albumin residues (Cys-34, Lys-195 and -414), both chemicals appear to target a distinct pattern of residues, further indicating that protein adduction is selective and dictated by structure and reactivity of the chemical. Previous studies (e.g. Elahi et al., 2004) suggested that chemical modification of skin proteins related to skin sensitisation is extensive and random. However, our results together with other recent evidence from generic peptide–chemical reactivity studies (Gerberick et al., 2004) suggest high selectivity in chemical modification of proteins (not all residues that could theoretically react in a protein do react). In the absence of information on specific protein targets in skin sensitisation and their immunogenic potential, in vitro investigations are limited to the use of model proteins/peptides. Whilst a generic in vitro model of protein haptenation does not have to reflect the in vivo situation, there is a possibility
Abstracts / Toxicology 231 (2007) 104–119
that chosen models may not provide the same spectrum of reactivity to skin sensitisation mechanisms in vivo. It has been suggested that information from in vitro protein reactivity studies could be used as part of a non-animal based approach for the identification of skin sensitisation potential (Jowsey et al., 2006). Such assays should be underpinned with mechanistic understanding of the relevance of covalent modifications and their immunogenic potential. References Elahi, E.N., Wright, Z.M., Hinselwood, D., Hotchkiss, S.A., Basketter, D.A., Pease, C., 2004. Chem. Res. Toxicol. 17, 301–310. Gerberick, G.F., Vassallo, J.D., Bailey, R.E., Chaney, J.G., Morrall, S.W., Lepoittevin, J.P., 2004. Toxicol. Sci. 81 (2), 332–343. Jowsey, I.R., Basketter, D.A., Westmoreland, C., Kimber, I., 2006. J. Appl. Toxicol. 26 (4), 341–350.
doi:10.1016/j.tox.2006.11.031 Genomic investigation of the metabolic competency of human cord blood-derived dendritic cells Ruth U. Pendlington, Vicki Summerfield, Andrew White, Camilla K. Pease E-mail address: [email protected] (V. Summerfield). Safety & Environmental Assurance Centre, Unilever Colworth Park, Sharnbrook, Bedford MK44 1LQ, UK With the advent of the 7th Amendment to the European Union’s Cosmetic Directive, risk assessments for cosmetic ingredients will have to be carried out without the generation of new animal data. This represents a major challenge to the Personal Care industry for many endpoints including skin sensitisation. In recent years, many attempts to develop novel in vitro assays for the identification of skin sensitisers have focused on cellbased assays that could model some of the functions of Langerhans cells since these are thought to be a key cellular mediator of contact allergy within the skin. Human monocyte-derived dendritic cells (MD-DCs) have been used by many groups for such work. It is envisaged that these cells could respond to chemicals such that discriminatory biomarkers of sensitisation can be identified. In developing novel cell-based assays to predict skin sensitisation, it is important to characterise the metabolic competency of the system (ultimately at the functional level) to ascertain whether the chemical (and any resulting metabolites) being tested in vitro are comparable to chemicals and metabolites to which the skin is exposed, in order that the data can be interpreted in the context
107
Table 1 Enzyme family
Major xenobiotic metabolising enzyme isoforms detected
Cytochrome P450
CYP1A1, CYP1A2, CYP 2C9, CYP2D6, CYP2E1, CYP3A4 None EPHX1, EPHX2 FMO4, FMO5 GPX1, GPX3, GPX4, GPX7 NAT1, NAT5 GSTA2, GSTA4 and others None SULT1A2, SULT1A3, SULT1C1, SULT4A1
NADPH-P450 oxidoreductase Epoxide hydrolase Flavin-containing monooxygenases Glutathione peroxidase N-Acetyltransferase Glutathione S-transferase UDP-glycosyltransferase Sulfotransferase
of local skin sensitisation effects in man. Ultimately, metabolic activation systems (e.g. S9, keratinocytes, etc.) may need to be added to such test systems, but in the first instance, the basal metabolic competence of the MD-DC cells themselves should be defined. With sparse knowledge about the metabolic competence of MD-DCs (Sieben et al., 1999) a pilot study was initiated to begin to investigate their xenobiotic metabolic competence. These first analyses would then be able to guide more focused functional studies. Preliminary results were obtained from three batches of cord blood-derived dendritic cell mRNA, which were received from MatTek Corporation, MA, USA. mRNA analysis was performed using Codelink whole genome arrays according to the manufacturer’s standard methodology. All samples and arrays passed applied QC thresholds. For each batch of cells, three replicate arrays were generated and analysed. Combining data from the replicates, expression of between 29712 and 32695 genes was detected above background across all three batches. Principal components analysis identified good concordance within replicates of a batch with more significant variation in gene expression levels between batches. Welch ANOVA testing (p = 0.05) with an applied multiple testing correction (Benjamini and Hochberg False Discovery Rate) identified between 300 and 736 genes that showed significant variation between batches. Table 1 below illustrates the observed mRNA expression profile for both Phase I and Phase II xenobiotic metabolising enzymes in these cells. Notably, the key enzyme of the mixed function oxidase system (NADPH-cytochrome P450 oxidoreductase) was absent at the mRNA level. Future studies will assess whether human cord blood-derived dendritic cells express any mixed function oxidase (CYP:NADPH