Toronto SCC epigenetics and aginginteresting skin lighteners on melanocytes looking atinteresting...

Genomics, Epigenetics and Their Application to Elucidate theApplication to Elucidate the

Mechanism of Efficacious Actives for Personal Carefor Personal Care

SCC Ontario Education DayToronto September 2011Toronto, September 2011

Philip LudwigPhilip LudwigArch Personal Care

OutlineOutline of the talkof the talk

The Human Genome: An AnniversaryThe Human Genome: An Anniversary

Examination of Skin Antioxidants via Human Examination of Skin Antioxidants via Human Genomic MicroarraysGenomic Microarraysyy

Examination of Skin Lightening Ingredients via Examination of Skin Lightening Ingredients via H G i MiH G i MiHuman Genomic MicroarraysHuman Genomic Microarrays

Examination of Epigenetic Methylation via Human Examination of Epigenetic Methylation via Human Examination of Epigenetic Methylation via Human Examination of Epigenetic Methylation via Human Epigenomic ArraysEpigenomic Arrays

ConclusionsConclusions

The Human The Human Genome: Genome: An AnniversaryAn AnniversaryAn AnniversaryAn Anniversary

Science published a four part series celebratingScience published a four part series celebrating the completion of the human genome sequencing.

This occurred 10-years ago this year.

The Human GenomeThe Human Genome

23 Human Chromosomes23 Human Chromosomes

The Human GenomeThe Human Genome Contains approximately 3 billion base Contains approximately 3 billion base

pairspairspp We each vary by only 0.1 %, or We each vary by only 0.1 %, or

approximately 3 million base pairsapproximately 3 million base pairs The human genome has approximately The human genome has approximately

25,00025,000--30,000 30,000 functioning functioning genesgenes Approximately 1.5% of the genome codes Approximately 1.5% of the genome codes

for protein producing genes for protein producing genes –– the rest is the rest is di RNA i t di RNA i t di DNAdi DNAnonnon--coding RNA, introns, noncoding RNA, introns, non--coding DNAcoding DNA

Our genes are provided to us equally by Our genes are provided to us equally by t p nts t p nts two parents two parents

They define our physical makeupThey define our physical makeup

Antioxidants and SkinAntioxidants and Skin

Antioxidants SummaryAntioxidants Summary

Rosavin is isolated from RhodiolaRosavin is isolated from Rhodiola@ 96% purity

Resveratrol was isolated fromJapanese Knotweed @ 99%purity


EGCG is isolated from Green TeaEGCG is isolated from Green Tea@ 97% purity

Chlorgenic acid was isolated fromCoffee @ 99% purityCoffee @ 99% purity


Puerarin is isolated from KudzuPuerarin is isolated from Kudzu@ 96% purity

Genistein is isolated from Soybeans @ >95% puritySoybeans @ >95% purity


Pomiferin and Osajin were I l d f O OIsolated from Osage Orange@ 95% and 90% purities, respectively

Propolis is isolated fromPropolis is isolated from Honeycomb @ 80% purity

Antioxidants ConcentrationsAntioxidants Concentrations

The antioxidants were tested at their highest,non‐lethal doses on both cell lines

Gene SummaryGene Summary

205 Individual genes felt related to skin function wereculled from the over 30,000 genes tested

Gene SummaryGene SummaryGenes examined included skin functions such as:• Extracellular matrix proteinsLi id th i• Lipid synthesis

• Cellular energy and metabolism• Intrinsic antioxidant synthesis• ROS and DNA repair response proteins• Skin polysaccharide and glycoprotein synthesis• Hormone responseHormone response • Longevity proteins• Cellular differentiation proteinsR ti id t i• Retinoid response proteins

• Circadian rhythm proteins• Skin pigmentation proteins

Fibroblasts ResponsesFibroblasts Responses‐‐UpregulationUpregulation

To qualify as a significant stimulant of a gene at leastTo qualify as a significant stimulant of a gene, at leastfour of the ingredients tested had to show Ratio of Median response greater than 1.3 p g

Summary of Antioxidant ResultsSummary of Antioxidant Results

In fibroblasts and keratinocytes, certain genes were commonly upregulated including:ATP Cit t L (ACLY) f tt id bi th i• ATP Citrate Lyase (ACLY) – fatty acid biosynthesis

• 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 productionproduction

In fibroblasts and keratinocytes, only one gene seem to h d d l tishowed common down regulation:

• Progesterone Receptor (PGR) ‐ steroid receptor

Summary of Antioxidant ResultsSummary of Antioxidant Results• The ability of a variety of antioxidants to commonly stimulated the same five genomic targets suggests thesetargets may be more critical to the effects of thesetargets may be more critical to the effects of these antioxidants than previously anticipated.

• The ability of all the treatments to reduce Progesterone• The ability of all the treatments to reduce ProgesteroneReceptor [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 byfurther protein studies including dose responses and time point expansionspoint expansions.

IngredientsIngredients

Hydroquinone y q

Kojic Acid

Niacinamide

I di t hi hl ifi d d ll

Niacinamide

Ingredients were highly purified and are wellestablished melanin suppressing chemicals

Ingredient Toxicities on MelanocytesIngredient Toxicities on Melanocytes

Hydroquinone (0.0001%), Kojic Acid (0.01%), Niacinamide (0.01%)y q ( ), j ( ), ( )

Responses for Genes of InterestResponses for Genes of Interest

Summary of ratio of medians for three commerciallyinteresting skin lighteners on melanocytes looking atinteresting 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 ExpressionTyrosinase Protein Expression

All three skin lighteners appear to increase Tyrosinase protein expression within a 96Tyrosinase protein expression within a 96

hour time frame with the strongest effects beingseen in the first 48 hours

Ferritin Protein ExpressionFerritin Protein Expression

Within 48 hours, all three skin lighteners demonstrated upregulatory influences on ferritindemonstrated upregulatory influences on ferritinprotein expression. These effects diminish at 96

hours, comparable to Tyrosinase protein expression

Ferritin in the skinFerritin in the skin

Summary of Skin Lightener ResultsSummary 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 •Three well-established skin lighteners appear to up-regulate tyrosinase gene and protein expression contrary to anticipated behavior•Ferritin protein, a protein that binds ferric ions (Fe+3), is strongly F n p n, p n n f n (F ), ng yupregulated 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 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 The role of iron in melanogenesis may be underappreciated

Tyrosine Hydroxytyrosine DOPA melanin

Overview of epigenetics sectionOverview of epigenetics sectionOverview of epigenetics sectionOverview of epigenetics section

Review of Plant Meristematic Cell Suspension Culture Review of Plant Meristematic Cell Suspension Culture pp

Technology Technology –– a source of unique methylation patterns a source of unique methylation patterns

Benefits of Meristematic Cell Suspension CulturesBenefits of Meristematic Cell Suspension Cultures Benefits of Meristematic Cell Suspension CulturesBenefits of Meristematic Cell Suspension Cultures

Examination of Epigenetic Methylation via Human Examination of Epigenetic Methylation via Human

EpigenomicEpigenomic ArraysArrays

SummarySummarymm ymm y

Uses of plant tissue culture Uses of plant tissue culture Screening of cells for beneficial Screening of cells for beneficial

characteristicscharacteristics–– Plant breeders may look for a high Plant breeders may look for a high

content of an activecontent of an activeMeristem tip cultureMeristem tip cultureMeristem tip cultureMeristem tip culture

–– Produces plant material free from Produces plant material free from viruses, often for plants propagated viruses, often for plants propagated , p p p g, p p p gvegetativelyvegetatively

Forestry and floricultureForestry and floriculture–– For conservation of rare and For conservation of rare and

endangered plant speciesendangered plant speciesLargeLarge scale growth of plant cells as a scale growth of plant cells as a LargeLarge--scale growth of plant cells as a scale growth of plant cells as a source of secondary metabolitessource of secondary metabolites

Review of Meristematic Cell Review of Meristematic Cell Culture TechnologyCulture Technology

• Meristem – tissue in plants that contain undifferentiated cells, occurs at the shoot and root apex

Culture TechnologyCulture Technology

p• Callus – Mass of undifferentiated cells

Review of Meristematic Cell Review of Meristematic Cell Culture TechnologyCulture Technology

• Meristem – tissue in plants that contain undifferentiated cells, occurs at the shoot and root apex

Culture TechnologyCulture Technology

p• 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

Tissue sample from adult plant is cultured Undifferentiated callus forms

Callus separated / single cells culturedcultured

Plant Meristematic Suspension Culture Plant Meristematic Suspension Culture Plant Meristematic Suspension Culture Plant Meristematic Suspension Culture ScaleScale--UpUp

Overview of Product Development

Plant callus Shaker Flask Bioreactor

3333

Plant callus Shaker Flask Bioreactor

Plant Meristematic Cell Cultures Plant Meristematic Cell Cultures Combine Two Current TechnologiesCombine Two Current Technologies

Biotech-derived compounds

(fermentation)

Natural Bioactives (Plant extracts)

G i i B t i l d th i

(fermentation)

Growing organisms in bioreactors

Botanicals and their natural bioactives

Plant Meristematic Cell Cultures

Rice meristem culture: the conceptRice meristem culture: the concept

Elicitation:Plant tissue culture:Undifferentiated cells

Elicitation:Increases secondary metabolites and actives

Rice culture

Epigenetic DNA modification:Rejuvenation and renewal of cells

Benefits of Meristematic Benefits of Meristematic S i C ltS i C ltSuspension CulturesSuspension Cultures

Access to rare and hard to obtain Access to rare and hard to obtain plantsplants

–– Opens frontier to new activesOpens frontier to new actives

Easier way to procure uniform Easier way to procure uniform botanicalsbotanicals

–– No environmental variation in No environmental variation in –– No environmental variation in No environmental variation in weather, sunlight, soil and waterweather, sunlight, soil and water

Very reproducible biomass and Very reproducible biomass and Tacca chantriericoncentration of activesconcentration of actives

–– AllantoinAllantoin

Tacca chantrieri

–– Tea & EGCGTea & EGCG

Benefits of Meristematic Benefits of Meristematic S s si C lt sS s si C lt sSuspension CulturesSuspension Cultures

More environmentally responsible More environmentally responsible –– Green technologyGreen technology–– Prevents depletion of wildPrevents depletion of wild--grown plants that may grown plants that may

be scarcebe scarce Enables growth of plants under conditions otherwise Enables growth of plants under conditions otherwise

n tt in bl in fi ldn tt in bl in fi ldunattainable in a fieldunattainable in a field–– Defensive stressDefensive stress Higher concentration of activesHigher concentration of actives

A l dA l d A natural productA natural product–– Just as yeast fermentation is considered natural, Just as yeast fermentation is considered natural,

so are plant suspension culturesso are plant suspension cultures

TrilliumTrillium

Benefits of Meristematic Benefits of Meristematic S i C ltS i C ltSuspension CulturesSuspension Cultures

Ability to harvest epigenetic and transcription factors Ability to harvest epigenetic and transcription factors and novel plant compounds not produced or produced in and novel plant compounds not produced or produced in minute quantities in whole mature plants minute quantities in whole mature plants

Meristematic cultures enable harvest of proteins and Meristematic cultures enable harvest of proteins and Meristematic cultures enable harvest of proteins and Meristematic cultures enable harvest of proteins and other compounds that would degrade too quickly from other compounds that would degrade too quickly from traditional harvest planttraditional harvest plant

Welwitschia mirabilis Cryptocereus anthonyanus Wollemia nobilis

Rice meristem culture: the conceptRice meristem culture: the concept

Elicitation:Plant tissue culture:Undifferentiated cells

Elicitation:Increases secondary metabolites and actives

Rice culture

Epigenetic DNA modification:Rejuvenation and renewal of cells

Efficient production of actives Efficient production of actives needs elicitationneeds elicitationneeds elicitationneeds elicitation

Undifferentiated cells primarily grow, not produce Undifferentiated cells primarily grow, not produce activesactives

In order to increase secondary metabolite production, In order to increase secondary metabolite production, elicitation is needed.elicitation is needed.

Elicitors can include ozone and specific chemicalsElicitors can include ozone and specific chemicalsll h h l l b f ll h h l l b f Cells containing no actives with have little benefit in Cells containing no actives with have little benefit in

topical applicationtopical application

Untreated cells Elicited cells

Cells primarily grow and divide

Cells produce secondary metabolites

4040

Elicitation of actives through use of Elicitation of actives through use of ozone ozone ozone ozone

Ozonized rice suspension culture

Unstressed rice suspension culture

Overview of epigenetics sectionOverview of epigenetics sectionOverview of epigenetics sectionOverview of epigenetics section

Review of Plant Meristematic Cell Suspension Culture Review of Plant Meristematic Cell Suspension Culture pp

Technology Technology –– a source of unique methylation patterns a source of unique methylation patterns

Benefits of Meristematic Cell Suspension CulturesBenefits of Meristematic Cell Suspension Cultures Benefits of Meristematic Cell Suspension CulturesBenefits of Meristematic Cell Suspension Cultures

Examination of Epigenetic Methylation via Human Examination of Epigenetic Methylation via Human

EpigenomicEpigenomic ArraysArrays

SummarySummarymm ymm y

The Emerging Evidence of theThe Emerging Evidence of theI fl f E i i iI fl f E i i iInfluence of Epigentics on AgingInfluence of Epigentics on Aging

4343

What is epigenetics?What is epigenetics?What is epigenetics?What is epigenetics?

Definition: heritable changes in gene expression that Definition: heritable changes in gene expression that occur by a mechanism other than changes to the DNA occur by a mechanism other than changes to the DNA sequencesequence

The mechanism by which cells “remember”The mechanism by which cells “remember”yy How does a cell and its progeny remember that they are How does a cell and its progeny remember that they are

skin cells and not nerve cells? Through epigeneticsskin cells and not nerve cells? Through epigenetics Epigenetics plays a part in:Epigenetics plays a part in:Epigenetics plays a part in:Epigenetics plays a part in:

–– Cellular differentiationCellular differentiation–– DevelopmentDevelopment–– AgingAging–– DiseaseDisease–– Differences between identical twinsDifferences between identical twinsDifferences between identical twinsDifferences between identical twins

The “youth switch”The “youth switch”

What is the What is the epigenomeepigenome??What is the What is the epigenomeepigenome?? Epigenetics is Epigenetics is a a

heritable heritable “switch” “switch” that controls that controls how how well a well a gene gene is able is able to pass its to pass its messages messages pp ggvia via RNA synthesisRNA synthesis..

This controlling This controlling This controlling This controlling switch simply switch simply confers confers a mechanism a mechanism by by which the which the DNA DNA by by which the which the DNA DNA wraps wraps around its around its histones and histones and so so “p k“p k ” int th ” int th packspacks into the into the nucleusnucleus..

4545

Gene expression and differentiationGene expression and differentiationGene expression and differentiationGene expression and differentiation

MethylationMethylation, Differentiation, and Aging, Differentiation, and AgingMethylationMethylation, Differentiation, and Aging, Differentiation, and Aging

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

4747

Aging and epigenetic changesAging and epigenetic changesAging and epigenetic changesAging and epigenetic changes

Aging and Epigenetic ChangesAging and Epigenetic Changes

As cells age, the regionsf th kof the genome known as

promoters become progressively more methylated resulting iny gdiminishment of genetranscription (the ability of the gene to transfers its proteintransfers its proteinassembling messageto the RNA.

Aging and Epigenetic ChangesAging and Epigenetic ChangesLiterature ReferencesLiterature ReferencesLiterature ReferencesLiterature References

Correlating methylation to protein Correlating methylation to protein d id iproductionproduction

New active?

A b h l d h i d l l di

5151

As 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.

Testing Epigenetic ChangesTesting Epigenetic Changes

Fibroblasts were aged Fibroblasts were aged both intrinsically (8 both intrinsically (8 both intrinsically (8 both intrinsically (8 population doubling) population doubling) and extrinsically and extrinsically (UVB) S ll (UVB) S ll (UVB). Some cells (UVB). Some cells were treated with 2% were treated with 2% of a meristemtic rice of a meristemtic rice extract (R3).extract (R3).

DNA was extracted DNA was extracted nd x min d f CpG nd x min d f CpG and examined for CpG and examined for CpG

methylation at the methylation at the promoter regions of promoter regions of the genome and at the genome and at specific genesspecific genes

in vitroin vitro Cpg Cpg methylationmethylation assay: assay: genome wide promotersgenome wide promotersg pg p

Average CpG methylation at all gene

1.4

1.6

Average CpG methylation at all gene promoters genome wideTreatment Regime:Treatment Regime:

1. Non1. Non--Aged Cells Aged Cells (harvested after a (harvested after a f d i l )f d i l )

0 6

0.8

1

1.2few days in culture)few days in culture) 2. Intrinsic Aging + 2. Intrinsic Aging +

Extrinsic AgingExtrinsic AgingFib bl t t k Fib bl t t k

0

0.2

0.4

0.6–– Fibroblasts taken Fibroblasts taken through period of 8 through period of 8 cell culture passages cell culture passages with repeated UBV with repeated UBV exposure to produce exposure to produce 0

Young cells, no R3 Aged cells, no R3 Aged cells, 2% R3exposure to produce exposure to produce intrinsic and extrinsic intrinsic and extrinsic agingaging

3. Intrinsic Aging + 3. Intrinsic Aging + E t i i A i 2% E t i i A i 2%

Red Rice culture was able to decrease age related CpG promoter methylation genome wide, rejuvenating the cells. Reducing methylation increases the gene’s ability

Extrinsic Aging + 2% Extrinsic Aging + 2% Red Rice culture.Red Rice culture.

to express transcribe proteins. Shown as ratio to average CpG promoter methylation of GAPDH.

in vitroin vitro CpG methylation assay: CpG methylation assay: Collagen1A promotersCollagen1A promotersCollagen1A promotersCollagen1A promoters

Average CpG methylation at Collagen1A1 gene

Average CpG methylation C ll 1A2

1

1.2

at Collagen1A1 gene promoter

1 40

1.60

at Collagen1A2 gene promoter

0.6

0.8

1

0.80

1.00

1.20

1.40

0

0.2

0.4

0.20

0.40

0.60

0

Young cells, no R3

Aged cells, no R3

Aged cells, 2% R3

0.00

Young cells, no R3

Aged cells, no R3

Aged cells, 2% R3

R3 meristematic rice extract was able to decrease age related CpGR3 meristematic rice extract was able to decrease age related CpG promoter methylation in the Collagen 1A1 and 1A2 gene promoters. Shown as ratio to average CpG methylation of GAPDH gene promoter.

in vitroin vitro collagen Protein assay collagen Protein assay

2.5

Collagen 1A protein levels

2

ell P

rote

in *Expression of Collagen Type 1A is

1.5

de p

er u

g Cemarkedly increased in

the cells treated with meristematic rice extract.

1pe

I C

-Pep

tiextract.This increase could translate into skin that appears more firm and

ith l i kl

0

0.5

ng T

ypwith less wrinkles.

Young cells, no R3

Aged cells, no R3

Aged cells, 2% R3

Collagen gene methylation and aging;Collagen gene methylation and aging;Literature supportLiterature supportLiterature supportLiterature support

Summary of epigenetic resultsSummary of epigenetic resultsSummary of epigenetic resultsSummary of epigenetic results

The emerging science related to epigenetics is g g p grapidly 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, th s it h s ithi ski lls t d t b the switches within our skin cells tend to be more frequently turned "off". However, these changes can be moderated with ingredients that can gdiminish promoter methylation.

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

Overall ConclusionsOverall Conclusions

• Human microarrays can help guide research into the effects of skin ingredients on skin cells

• While not always directly matching gene expression and • While not always directly matching, gene expression and protein expression usually correlate (Mother Nature doesn’t waste time)

• There may be common pathways that certain well known There may be common pathways that certain well known ingredients influence to improve skin health

• Sometimes, results can be surprising and somewhat unexpected such as the skin lighteners influencing p g gtyrosinase 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

d b f l d " ff" H h tend to be more frequently turned "off". However, these changes can be moderated with ingredients that can diminish promoter methylation.New findings can lead to new directions for ingredient • New findings can lead to new directions for ingredient developments

AcknowledgementsAcknowledgementsAcknowledgementsAcknowledgements Arch Personal Care ProductsArch Personal Care Products

Dr Vince GruberDr Vince Gruber–– Dr. Vince GruberDr. Vince Gruber Robert Holtz at Robert Holtz at BioInnovationBioInnovation

LaboratoriesLaboratoriesLaboratoriesLaboratories Society of Cosmetic ChemistsSociety of Cosmetic Chemists

THANK YOUTHANK YOUTHANK YOUTHANK YOU

Toronto SCC epigenetics and aginginteresting skin lighteners on melanocytes looking atinteresting...

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