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Home , Genomics

Genomics, 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 : 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 : 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 . This occurred 10-years ago this year. The Human Genome 23 Human 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 . Approximately 1.5% of the genome codes for 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 lines Summary

205 Individual genes felt related to skin were culled from the over 30,000 genes tested Gene Summary Genes examined included skin functions such as: • Extracellular matrix • 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 prottieins • 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 regulltiation: • 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] 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

Ingredi en ts were hi ghl y purifi e 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 ngl y 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 ,,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 callus Shaker Flask Bioreactor

33 Plant Meristematic Cell Cultures Combine Two Current Technologies

Biotech-derived Natural Bioactives compounds (Plant extracts) (fermentation)

GiGrowing organi sms BtBotan ica ls and dthi 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 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 factors and novel plant compounds not produced or produced in minute quantities in whole mature plants . Meristematic cultures enable harvest of proteins and other compounds that would degrade too quickly from traditional harvest plant

Welwitschia mirabilis

Cryptocereus anthonyanus Wollemia nobilis 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 Efficient production of actives needs elicitation . Undifferentiated cells primarily grow, not produce actives . In order to increase secondary metabolite production, elicitation is needed. . Elicitors can include ozone and specific chemicals . Cells containing no actives with have lllittle bbfenefit in topical application Untreated cells Elicited cells Cells primarily grow and Cells produce secondary divide metabolites

40 Elicitation of actives through use of ozone

Ozonized rice suspension culture

Unstressed rice suspension culture 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 The Emerging Evidence of the In fluence of EiEpigent ics on Ag ing

43 What is epigenetics?

. Definition: heritable changes in gene expression that occur by a mechanism other than changes to the DNA sequence . The mechanism by which cells “remember” . How does a cell and its progeny remember that they are skin cells and not nerve cells? Through epigenetics . Epigenetics plays a part in: – Cellular differentiation – Development ––AgingAging ––DiseaseDisease – Differences between identical twins . The “youth switch” What is the ? . Epigenetics is a heritable “switch” that controls how well a gene is able to pass its messages via RNA synthesis.synthesis.

. This controlling switch simply confers a mechanism by which the DNA wraps around its histones and so “pkpacks” into the nucleusnucleus.. 45 Gene expression and differentiation Methylation, Differentiation, and Aging

Cells from different types of tissue have different genes expressed. As aging occurs, methylation of the gene promoters increases, deregulating the cell’s intial gene expression patterns.

47 Aging and epigenetic changes Aging and Epigenetic Changes

As cells age, the regions ofthf the genome k nown as promoters become progressively more methyyglated resulting in diminishment of gene transcription (the ability of the gene to transfers its protein assembling message to the RNA. Aging and Epigenetic Changes Literature References Correlating methylation to protein proddiuction

New active?

AbhldhidlldiAs a gene promoter becomes methylated, the gene is expressed less, leading to a decrease in protein production. A new active was desired to be able to modulate the methylation patterns and decrease methylation, hence increasing protein production.51 Testing Epigenetic Changes

. Fibroblasts were aged both intrinsically (8 population doubling) and extrinsically (UVB). Some cell s were treated with 2% of a meristemtic rice extract (R3). . DNA was extracted and exa mine d for CpG methylation at the promoter regions of the genome and at specific genes e wide, rejuvenating the ns. Shown as ratio to Shown as ratio ns. assay: promoters genome wide romoters p Average CpG methylation at all gene average CpG methylation promoter of GAPDH. Red Rice culture was able to decrease age related CpG promoter methylation genom cells. Reducing methylation ability the increases gene’s protei to express transcribe Young cells, no Young R3 Aged cells, no R3 Aged cells, 2% R3 1 6 0 . 1.2 1.6 1.4 0.8 06 0 0.4 0.2 methylation assay: )

2% + en

Cpg methylation k ture l a ng t i cu o s g

t n enome wide as A i c bl Aged Cells g gp i -Aged Cells ro ays ns i d r Fib blthrough period of 8 tcell culture passages t kwith repeated UBV exposure to produce extrinsic intrinsic and aging t

in vitr x ew – f 1. Non Non- (harvested after a 2. Intrinsic Aging + Extrinsic Aging 3. Intrinsic Aging + f d i f d i l ) EtiE Ai i 2% Red Rice culture. E Treatment Regime: . . .

2% R3 CpG Aged cells,

gene 1A2 related no R3

Aged cells, agen age

promoter ll o Cll 1A2 CllC 1A2

no R3 at Young cells, Average CpG methylation decrease

40 . 1.20 1.00 1.60 140 1 0.80 0.60 0.40 0.20 0.00 to

able

was

2% R3 Aged cells, CpG methylation assay: extract

rice Collagen1A promoters Collagen1A promoters

no R3 Aged cells, promoter Collagen1A1 gene at no R3 Young cells, meristematic Average CpG methylation

1 0 R3 meristematic rice extract was1A1 and 1A2 gene promoters. methylation in the Collagen promoter able to decrease ageof GAPDH gene promoter. as ratio to average CpG methylation Shown related CpG 1.2 0.8 0.6 0.4 0.2 * R3 Aged cells, 2% R3 Aged cells, no R3 Collagen 1A protein levels Young cells, no 1

2 0

1.5

2.5 0.5

ll Protein ll e e C ug per de C-Pepti I e p p Ty ng collagen Protein assay es. kl n i in vitro ess wr l

ith l i kl Expression of is 1A Collagen Type markedly increased in the cells treated with meristematic rice extract. This increase could into skin that translate appears more firm and w Collagen gene methylation and aging; Literature support Summary of epigenetic results

. The emerggging science related to ep pgigenetics is rapidly demonstrating that how we live our lives can actually influence how our skin cells age.

The epigenome is like a switch that can turn a gene on or turn it off. It appears that as we age, the swit ch es withi n our skin cells tend to be more frequently turned "off". However, these changes can be moderated with ingredients that can diminish promoter methylation.

As more knowledge of epigenetic effects on skin aging become known, this will become a target of more intensive research and product development. 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 Overall Conclusions

• Human microarrays can help guide research into the effects of skin ingredients on skin cells • While not always directly matching, gene expression and protein expression usually correlate (Mother doesn’t waste time) • There may be common pathways that certain well known ingredients influence to improve skin health • Sometimes, results can be surprising and somewhat unexppggected such as the skin lighteners influencing tyrosinase and ferritin gene and protein expression • The epigenome is like a switch that can turn a gene on or turn it off. As we age, the switches within our skin cells tend to be more frequentl y turned "off" . However, th ese changes can be moderated with ingredients that can diminish promoter methylation. • New findings can lead to new directions for ingredient developments Acknowledgements . Arch Personal Care Products – Dr. Vince Gruber . Robert Holtz at BioInnovation Laboratories . Society of Cosmetic Chemists THANK YOU