Genomics, Epigenetics and Their Application to Elucidate the Mechanism of Efficacious Actives for Personal Care SCC Ontario Education Day Toronto, September 2011 Philip Ludwig Arch Personal Care Outline of the talk . The Human Genome: An Anniversary . Examination of Skin Antioxidants via Human Genomic Microarrays . Examination of Skin Lightening Ingredients via Human GiGenomic Microarrays . Examination of Epigenetic Methylation via Human Epigenomic Arrays . Conclusions Outline of the talk . The Human Genome: An Anniversary . Examination of Skin Antioxidants via Human Genomic Microarrays . Examination of Skin Lightening Ingredients via Human GiGenomic Microarrays . Examination of Epigenetic Methylation via Human Epigenomic Arrays . Conclusions The Human Genome: An Anniversary Science published a four part series celebrating the completion of the human genome sequencing. This occurred 10-years ago this year. The Human Genome 23 Human Chromosomes The Human Genome . Contains approximately 3 billion base pairs . We each vary by only 0.1 %, or approximately 3 million base pairs . The human genome has approximately 25,00025,000--30,00030,000 functioning genes . Approximately 1.5% of the genome codes for protein producing genes ––thethe rest is nonnon--cocoding RNA, in trons, nonnon--cocoding DNA . Our genes are provided to us equally by two parents . They define our physical makeup Outline of the talk . The Human Genome: An Anniversary . Examination of Skin Antioxidants via Human Genomic Microarrays . Examination of Skin Lightening Ingredients via Human GiGenomic Microarrays . Examination of Epigenetic Methylation via Human Epigenomic Arrays . Conclusions Antioxidants and Skin Antioxidants Summary Rosavin is isolated from Rhodiola @ 96% purity Resveratrol was isolated from Japanese Knotweed @ 99% purity Antioxidants Summary EGCG is isolated from Green Tea @ 97% purity Chlorgenic acid was isolated from Coffee @ 99% purity Antioxidants Summary Puerarin is isolated from Kudzu @ 96% purity Genistein is isolated from Soybeans @ >95% purity Antioxidants Summary Pomiferin and Osajin were IlIsolated from Osage Orange @ 95% and 90% purities, respectively Propolis is isolated from Honeycomb @ 80% purity Antioxidants Concentrations The antioxidants were tested at their highest, non‐lethal doses on both cell lines Gene Summary 205 Individual genes felt related to skin function were culled from the over 30,000 genes tested Gene Summary Genes examined included skin functions such as: • Extracellular matrix proteins • Lipid synthesi s • Cellular energy and metabolism • Intrinsic antioxidant synthesis • ROS and DNA repair response proteins • Skin polysaccharide and glycoprotein synthesis • Hormone response • Longevity proteins • Cellular differentiation proteins • RtiRetinoid response protiteins • Circadian rhythm proteins • Skin pigmentation proteins Fibroblasts Responses‐Upregulation To qualify as a significant stimulant of a gene, at least four of the ingredients tested had to show Ratio of Median response greater than 1.3 Summary of Antioxidant Results In fibroblasts and keratinocytes, certain genes were commonly upregulated including: • ATP Citrat e Lyase (ACLY) – fttfatty acid biosynth esi s • Aquaporin 3 (AQP3) – regulate water flow • Cytochrome c Oxidase 1 (COX1) – m.t., making ATP • Nitric Oxide Synthase 3 (NOS3) – signaling molecule • Lysine Hydroxylase 3 (PLOD3) –involved in collagen production In fibroblasts and keratinocytes, only one gene seem to shdhowed common down regultilation: • Progesterone Receptor (PGR) ‐ steroid receptor Summary of Antioxidant Results • The ability of a variety of antioxidants to commonly stimulated the same five genomic targets suggests these targets may be more critical to the effects of these antioxidants than previously anticipated. • The ability of all the treatments to reduce Progesterone Receptor [PR] gene expression in both keratinocytes and fibroblasts suggests an alternative explanation to the standard “estrogen mimicking” effects of these ingredients. • These genomic results will need to be verified by further protein studies including dose responses and time point expansions. Outline of the talk . The Human Genome: An Anniversary . Examination of Skin Antioxidants via Human Genomic Microarrays . Examination of Skin Lightening Ingredients via Human GiGenomic Microarrays . Examination of Epigenetic Methylation via Human Epigenomic Arrays . Conclusions Ingredients Hyyqdroquinone Kojic Acid Niacinamide Ingre dien ts were hig hly pur ifie d an d are we ll established melanin suppressing chemicals Ingredient Toxicities on Melanocytes Hyyqdroquinone ( 0.0001%), ),j Kojic Acid (0.01% ), Niacinamide (0.01% ) Responses for Genes of Interest Summary of ratio of medians for three commercially interesting skin lighteners on melanocytes looking at Tyrosinase [TYR] and Ferritin [FTH1] gene expression. Treatments were at the highest non-cytotoxic levels for 24 hours Tyrosinase Protein Expression All three skin lighteners appear to increase Tyrosinase protein expression within a 96 hour time frame with the strongest effects being seen in the first 48 hours Ferritin Protein Expression Within 48 hours, all three skin lighteners demonstrated upregulatory influences on ferritin protein expression. These effects diminish at 96 hours, comparable to Tyrosinase protein expression Ferritin in the skin Summary of Skin Lightener Results •Using genomics it is possible to screen skin lightening actives to begin seeking alternative pathways to skin tanning control. •Three well-established skin lighteners appear to up-regulate tyrosinase gene and protein expression contrary to anticipated behavior •FFnpn,pnerritin protein, a protein that bi nfn(Fnds ferric ions (Fe+3), is stro ngly upregulated in melanocytes treated with skin lighteners •All the skin lighteners examined appear to upregulate ferritin protein suggesting their application causes a buildup of a potentially cytotoxic level of iron that must be controlled. •The removal of iron from the melanocytes via ferritin binding may reduce the ability of the cells to create hydroxytyrosine from tyrosine via an iron-induced oxidation step. This would reduce the pool of available hydroxytyrosine available to covert to DOPA, slowing the tanning response. •The role of iron in melanogenesis may be underappreciated Tyrosine Hydroxytyrosine DOPA melanin Outline of the talk . The Human Genome: An Anniversary . Examination of Skin Antioxidants via Human Genomic Microarrays . Examination of Skin Lightening Ingredients via Human GiGenomic Microarrays . Examination of Epigenetic Methylation via Human Epigenomic Arrays . Conclusions Overview of epigenetics section . Review of Plant Meristematic Cell Suspension Culture Technology – a source of unique methylation patterns . Benefits of Meristematic Cell Suspension Cultures . Examination of Epigenetic Methylation via Human Epigenomic Arrays . Summary Uses of plant tissue culture Screening of cells for beneficial characteristics – Plant breeders may look for a high content of an active Meristem tip culture – Produces plant material free from viruses,,pppg often for plants propagated vegetatively Forestry and floriculture – For conservation of rare and endangered plant species Large-scale growth of plant cells as a source of secondary metabolites Review of Meristematic Cell Culture Technology • Meristem – tissue in plants that contain undifferentiated cells, occurs at the shoot and root apex • Callus – Mass of undifferentiated cells Review of Meristematic Cell Culture Technology • Meristem – tissue in plants that contain undifferentiated cells, occurs at the shoot and root apex • Callus – Mass of undifferentiated cells • Totipotent – ability of a cell to produce all of the differentiated cells in an organism • Suspension culture – liquid media in which the plant cells grow Callus separated / Undifferentiated Tissue sample from adult plant is cultured single cells callus forms cultured Plant Meristematic Suspension Culture ScaleScale--UpUp Overview of Product Development Plant callus Shaker Flask Bioreactor 33 Plant Meristematic Cell Cultures Combine Two Current Technologies Biotech-derived Natural Bioactives compounds (Plant extracts) (fermentation) GiGrowing organ isms BtBotan ica ls an dthid their in bioreactors natural bioactives Plant Meristematic Cell Cultures Rice meristem culture: the concept Elicitation: Plant tissue Increases secondary culture: metabolites and Undifferentiated cells actives Rice culture Epigenetic DNA modification: Rejuvenation and renewal of cells Benefits of Meristematic SiSuspension CltCultures Access to rare and hard to obtain plants – Opens frontier to new actives Easier way to procure uniform botanicals – No environmental variation in weather, sunlight, soil and water Very reproducible biomass and Tacca chantrieri concentration of actives – Allantoin ––TeaTea & EGCG Benefits of Meristematic SSsuspensi on CCltultures . More environmentally responsible – Green technology – Prevents depletion of wildwild--growngrown plants that may be scarce . Enables growth of plants under conditions otherwise unttinblnattainable in a fildfield – Defensive stress . Higher concentration of actives . A natural product – Just as yeast fermentation is considered natural, so are plant suspension cultures Trillium Benefits of Meristematic SSiuspension CltCultures . Ability to harvest epigenetic and transcription factors and novel plant compounds not produced or produced in minute quantities in whole mature plants . Meristematic cultures