The Center for Alternatives to Animal Testing is an academic center affiliated with the Division of Toxicological Sciences in the Department of Environmental Health Sciences of the Johns Hopkins University Bloomberg School of Public Health.
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CAAT 20th Anniversary Symposium
September 10-11, 2001
PIER 5 HOTEL
711 Eastern Avenue
Baltimore, Maryland
Sponsors: 3M, Avon, Charles River Laboratories, Inc., The Cosmetic, Toiletries, and Fragrance Association, Covance, ExxonMobil Biomedical Sciences, Inc., In Vitro Technologies, Johnson & Johnson, Mary Ann Liebert, Inc., Procter & Gamble Company, Revlon
Proceedings for CAAT 20th Anniversary Symposium
Expression Systems
Ian Kimber
Syngenta
A more detailed understanding of the genome, combined with the advent of microarray and related technologies, has the potential to revolutionise our approach to molecular toxicology and safety assessment. In principle, microarray methods allow the simultaneous analysis of induced changes in expression of many hundreds or many thousands of genes. Of particular importance is the fact that this in turn encourages consideration of genes (and the biological responses they orchestrate) that had not been associated previously with toxic processes. Genomics (and proteomics) is now being applied widely in investigative toxicology, including in the characterisation of chemical- and protein-induced allergy. Evaluation of allergen-induced changes in gene expression will be described and will be used to exemplify the ways in which judicious application of microarray technology can provide important new research leads and create the basis for alternative approaches to toxicity testing and safety assessment.
Overview
- Skin sensitization
- Local Lymph Node Assay
- Transcript profiling of allergen-activated mouse lymph node cells
- In vitro identification of contact allergens
- Transcript profiling of allergen-activated cultured human dendritic cells
Skin Sensitization
Immune Activation
Local Lymph Node Assay
Requirements for Induction of Skin Sensitization
- Chemical must gain access to the viable epidermis via the stratum corneum.
- Chemical must cause sufficient local trauma to induce/augment cutaneous cytokine production.
- Chemical must be protein-reactive or metabolized to a protein-reactive species.
- Chemical must be inherently antigenic and recognized by responsive T lymphocytes.
Local Lymph Node Assay
Advantages of the LLNA
- Animal welfare (reduction and refinement)
- Relevant, objective and quantitative endpoint
- Exposure via relevant route
- Analysis of coloured chemicals
- Analysis of irritant chemicals
- Speed/cost/accuracy
LLNA and relative contact allergenic potency
| chemical | human classification | LLNA EC3 value | |
| CMIT | strong | 0.05% | |
| DNCB | strong | 0.08% | |
| DPC | strong | 0.05% | |
| PPD | strong | 0.06% | |
| cinnamic aldehyde | moderate | 2.0% | |
| glutaraldehyde | moderate | 0.2% | |
| isoeugenol | moderate | 1.3% | |
| tetramethylthiuram disulfide | moderate | 6.0% | |
| citral | weak | 13% | |
| eugenol | weak | 13% | |
| HCA | weak | 8.0% | |
| hydroxycitronellal | weak | 20% | |
| ethyleneglycol dimethylacrylate | extremely weak | 35% | |
| isopropyl myristate | extremely weak | 44% | |
| propyl paraben | extremely weak | >50% | |
| propylene glycol | extremely weak | >100% | |
| glycerol | non-sensitizing | >100% | |
| hexane | non-sensitizing | >100% | |
| diethyl phthlate | non-sensitizing | >100% | |
| tween 80 | non-sensitizing | >100% |
Epidermal cytokine environment
GlyCAM-1 is a robust and relatively sensitive marker of early changes in the lymph node induced by contact allergen
- GlyCAM-1 expression was down-regulated by DNFB exposure at all times (18 to 120h) examined
- GlyCAM-1 expression was down-regulated by DNFB exposure at all concentrations (0.05 to 0.5 %) examined
- threshold for positivity for DNFB (EC3 value) in the standard lymph node assay was calculated to be 0.03%
What is Transcript Profiling?
GlyCAM-1 is a robust and relatively sensitive marker of early changes in the lymph node induced by contact allergen
- GlyCAM-1 expression was down-regulated by DNFB exposure at all times (18 to 120h) examined
- GlyCAM-1 expression was down-regulated by DNFB exposure at all concentrations (0.05 to 0.5 %) examined
- threshold for positivity for DNFB (EC3 value) in the standard lymph node assay was calculated to be 0.03%
Ongoing studies:
- Validation of marker genes for their ability to provide a specific, sensitive and robust correlate of contact allergenicity
(correlation with EC3 values derived from standard LLNA)
In vitro identification of chemical allergens
Metabolism
- DNA metabolism
- Protein metabolism
- Lipid metabolism
- Monosaccharide Metabolismpolysaccharide and Glycoconjugate metabolism
- Amino acid biosynthesis
- Amino acid degradation
- Urea metabolism
- Energy metabolism
- Cofactor metabolism
- Sulphate metabolism
- Antioxidant metabolism
- Drug and Xenobiotic metabolism
- Secondary metabolism
Signal transduction and regulation
- Extracellular messengers receptors
- Intracellular signalling
- Transcription factors
Electron transfer
- Cytochromes
- Flavoproteins
Oxidoreductases
- Acting on a peroxide as acceptor (peroxidases)
- Carbon-sulfur lyases
Protein modification and maintenance
- Proteases
- Protease inhibitors
- Chaperones
Localised and structural proteins
- Secreted and extracellular
- Cytoskeleton
- Organelle
Growth and Development
- Cell division
- Reproduction
Differentiation and proliferation
- Apoptosis
- Aging and senescence
Kinesis
- Motility
Membrane transport
- Channels and porins
- Transporters and pumps
Immunity and defence
- Antigen processing and presentation
- Inflammatory response
- Defence and resistance
- Cell adhesion molecules
- Cell surface receptors
- Chemokines
- Chemokine receptors
- Extracellular matrix
- Heat shock proteins
- Interleukins
- Metalloproteinases
Adhesion and molecular recognition
- Adhesion
- Antigen recognition
Epithelial cell biology
- Epithelial cell transporters
Environmental responses
- Metal tolerances
Others
- Phosphotransferase system
- a-ketoglutarate dH complex
- Glutathione cycle enzymes
- Proteoglycans
- Orphan receptors
- Opioid receptors
- Development
Ongoing studies:
- screening of additional human donors for responder / non-responder status with respect to DNFB-induced changes in IL-1b m RNA expression
- transcript profiling of additional responders / non-responders exposed to DNFB and other contact allergens
- Validation of marker genes for their ability to provide a specific, sensitive and robust correlate of contact allergenicity
Summary and perspectives
Acknowledgements
Syngenta Central Toxicology Laboratory
Immunology
- Catherine Betts
- Marie Cumberbatch
- Rebecca Dearman
Toxicogenomics
- Jonathan Moggs
- George Orphanides
- Bill Pennie