Genomics, Epigenetics and Their
Application to Elucidate the
Mechanism of Efficacious Actives
for Personal Care
SCC Ontario Education Day
Toronto September 2011
Toronto,
Philip Ludwig
Arch Personal Care
Outline of the talk
 The Human Genome: An Anniversary
 Examination of Skin Antioxidants via Human
Genomic Microarrays
y
 Examination of Skin Lightening Ingredients via
H
Human
G
Genomic
i Microarrays
Mi
 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
y
 Examination of Skin Lightening Ingredients via
H
Human
G
Genomic
i Microarrays
Mi
 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
p
pairs
 We each vary by only 0.1 %, or
approximately 3 million base pairs
 The human genome has approximately
25,000--30,000 functioning genes
25,000
 Approximately 1.5% of the genome codes
for protein producing genes – the rest is
non--coding
non
di RNA
RNA, iintrons,
t
non-coding
nondi DNA
 Our genes are provided to us equally by
t
two
p
parents
nts
 They define our physical makeup
Outline of the talk
 The Human Genome: An Anniversary
 Examination of Skin Antioxidants via Human
Genomic Microarrays
y
 Examination of Skin Lightening Ingredients via
H
Human
G
Genomic
i Microarrays
Mi
 Examination of Epigenetic Methylation via Human
Epigenomic Arrays
 Conclusions
Antioxidants and Skin
Antioxidants Summary
Rosavin is isolated from Rhodiola
Rosavin
is isolated from Rhodiola
@ 96% purity Resveratrol was isolated from
Japanese Knotweed @ 99%
purity
Antioxidants Summary
EGCG is isolated from Green Tea
EGCG
is isolated from Green Tea
@ 97% purity Chlorgenic acid was isolated from
Coffee @ 99% purity
Coffee @ 99% purity
Antioxidants Summary
Puerarin is isolated from Kudzu
Puerarin
is isolated from Kudzu
@ 96% purity Genistein is isolated from Soybeans @ >95% purity @ >95% purity
Antioxidants Summary
Pomiferin and Osajin were I l df
Isolated from Osage Orange
O
O
@ 95% and 90% purities, respectively Propolis is isolated from 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 synthesis
Li id
th i
• Cellular energy and metabolism
• Intrinsic antioxidant synthesis
• ROS and DNA repair response proteins
• Skin polysaccharide and glycoprotein synthesis
• Hormone response Hormone response
• Longevity proteins
• Cellular differentiation proteins
• Retinoid response proteins
R ti id
t i
• Circadian rhythm proteins
• Skin pigmentation proteins
Fibroblasts Responses
Fibroblasts Responses‐‐Upregulation
To qualify as a significant stimulant of a gene, at least
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 p
g
Summary of Antioxidant Results
In fibroblasts and keratinocytes, certain genes were commonly upregulated including:
• ATP Citrate Lyase (ACLY) –
ATP Cit t L
(ACLY) fatty acid biosynthesis
f tt
id bi
th i
• 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 showed common down regulation:
h
d
d
l ti
• 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
targets 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 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
point expansions.
Outline of the talk
 The Human Genome: An Anniversary
 Examination of Skin Antioxidants via Human
Genomic Microarrays
y
 Examination of Skin Lightening Ingredients via
H
Human
G
Genomic
i Microarrays
Mi
 Examination of Epigenetic Methylation via Human
Epigenomic Arrays
 Conclusions
Ingredients
Hydroquinone
y q
Kojic Acid
Niacinamide
IIngredients
di t were highly
hi hl purified
ifi d and
d are wellll
established melanin suppressing chemicals
Ingredient Toxicities on Melanocytes
Hydroquinone
y q
((0.0001%),
), Kojic
j 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
well established skin lighteners appear to up-regulate
up regulate
tyrosinase gene and protein expression contrary to anticipated
behavior
•Ferritin
F
np
protein,
n, a p
protein
n that binds
n f
ferric ions
n (F
(Fe+3), is strongly
ng 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
iron induced oxidation step.
step This would reduce the pool
of available hydroxytyrosine available to covert to DOPA, slowing the
tanning response.
•The
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
y
 Examination of Skin Lightening Ingredients via
H
Human
G
Genomic
i Microarrays
Mi
 Examination of Epigenetic Methylation via Human
Epigenomic Arrays
 Conclusions
Overview of epigenetics section
Overview of epigenetics section
 Review of Plant Meristematic Cell Suspension
p
Culture
Technology – a source of unique methylation patterns
 Benefits of Meristematic Cell Suspension Cultures
 Examination of Epigenetic Methylation via Human
Epigenomic Arrays
 Summary
mm y
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,, often for plants
p
propagated
p p g
vegetatively
Forestry and floriculture
– For conservation of rare and
endangered plant species
Large-scale growth of plant cells as a
Largesource of secondary metabolites
Review of Meristematic Cell
Culture Technology
•
•
Meristem – tissue in plants that contain undifferentiated cells,
occurs at the shoot and root apex
p
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
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
cultured
Plant Meristematic Suspension Culture
Scale--Up
Scale
Overview of Product Development
Plant callus
Shaker Flask
Bioreactor
33
Plant Meristematic Cell Cultures
Combine Two Current Technologies
Biotech-derived
compounds
(fermentation)
Growing
G
i organisms
i
in bioreactors
Natural Bioactives
(Plant extracts)
Botanicals
B
t i l and
d their
th i
natural bioactives
Plant Meristematic Cell Cultures
Rice meristem culture: the concept
Elicitation:
Increases secondary
metabolites and
actives
Plant tissue
culture:
Undifferentiated cells
Rice culture
Epigenetic DNA
modification:
Rejuvenation and
renewal of cells
Benefits of Meristematic
S
Suspension
i C
Cultures
lt
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
concentration of actives
– Allantoin
– Tea & EGCG
Tacca chantrieri
Benefits of Meristematic
S s
si Cultures
C lt
s
Suspension
 More environmentally responsible – Green technology
– Prevents depletion of wild
wild--grown plants that may
be scarce
 Enables growth of plants under conditions otherwise
unattainable
n tt in bl in a fi
field
ld
– Defensive stress
 Higher concentration of actives
 A naturall product
d
– Just as yeast fermentation is considered natural,
so are plant suspension cultures
Trillium
Benefits of Meristematic
S
i C
lt
Suspension
Cultures
 Ability to harvest epigenetic and transcription 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:
Increases secondary
metabolites and
actives
Plant tissue
culture:
Undifferentiated cells
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
ll containing no actives with
h have
h
little
l l benefit
b
f in
topical application
Untreated cells
Cells primarily grow and
divide
Elicited cells
Cells produce secondary
metabolites
40
Elicitation of actives through use of
ozone
Ozonized rice suspension culture
Unstressed rice suspension culture
Overview of epigenetics section
Overview of epigenetics section
 Review of Plant Meristematic Cell Suspension
p
Culture
Technology – a source of unique methylation patterns
 Benefits of Meristematic Cell Suspension Cultures
 Examination of Epigenetic Methylation via Human
Epigenomic Arrays
 Summary
mm y
The Emerging Evidence of the
I fl
fE
i
i on Aging
i
Influence
of
Epigentics
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
y 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
– Aging
– Disease
– Differences between identical twins
 The “youth switch”
What is the epigenome
epigenome?
?
 Epigenetics is a
heritable “switch”
that controls how
well a gene is able
to p
pass its messages
g
via RNA synthesis.
synthesis.
 This controlling
switch simply
confers a mechanism
by which the DNA
wraps around its
histones and so
“p
packs
k ” into
int th
the
nucleus..
nucleus
45
Gene expression and differentiation
Methylation, Differentiation, and Aging
Methylation,
Cells from different
types
yp 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
off the
th genome known
k
as
promoters become
progressively more
methylated
y
resulting
g 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
d
i
production
New active?
As a gene promoter b
A
becomes methylated,
h l d the
h gene iis expressed
d lless, lleading
di 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).
(UVB) Some
S
cells
ll
were treated with 2%
of a meristemtic rice
extract (R3).
 DNA was extracted
and
nd examined
x min d f
for CpG
methylation at the
promoter regions of
the genome and at
specific genes
in vitro Cpg methylation assay:
genome wide promoters
g
p
Treatment Regime:
 1. NonNon-Aged Cells
(harvested after a
f
few
d
days iin culture)
l
)
 2. Intrinsic Aging +
Extrinsic Aging
–
Fib bl t ttaken
Fibroblasts
k
through period of 8
cell culture passages
with repeated UBV
exposure to produce
intrinsic and extrinsic
aging
 3. Intrinsic Aging +
Extrinsic
E t i i A
Aging
i + 2%
Red Rice culture.
Average CpG methylation at all gene
promoters genome wide
1.6
1.4
1.2
1
0.8
06
0.6
0.4
0.2
0
Young cells, no R3
Aged cells, no R3
Aged cells, 2% R3
Red Rice culture was able to decrease age related CpG
promoter methylation genome wide, rejuvenating the
cells. Reducing methylation increases the gene’s ability
to express transcribe proteins. Shown as ratio to
average CpG promoter methylation of GAPDH.
in vitro CpG methylation assay:
Collagen1A promoters
Average CpG methylation
at Collagen1A1 gene
promoter
1.2
Average CpG methylation
at Collagen1A2
C ll
1A2 gene
promoter
1.60
1 40
1.40
1
1.20
0.8
1.00
0.6
0.80
0.60
0.4
0.40
0.2
0.20
0
Young cells, Aged cells,
no R3
no R3
Aged cells,
2% R3
0.00
Young cells, Aged cells, Aged cells,
no R3
no R3
2% R3
R3 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 vitro collagen Protein assay
Collagen 1A protein levels
Expression of
Collagen Type 1A is
markedly increased in
the cells treated with
meristematic rice
extract.
This increase could
translate into skin that
appears more firm and
with
ith lless wrinkles.
i kl
ng Typ
pe I C-Peptide per ug Ce
ell Protein
2.5
2
*
1.5
1
0.5
0
Young cells, no Aged cells, no Aged cells, 2%
R3
R3
R3
Collagen gene methylation and aging;
Literature support
Summary of epigenetic results
g g science related to epigenetics
pg
 The emerging
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,
th switches
the
s it h s within
ithi our skin
ski cells
lls tend
t d to
t b
be more
frequently turned "off". However, these changes
can be moderated with ingredients
g
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
y
 Examination of Skin Lightening Ingredients via
H
Human
G
Genomic
i Microarrays
Mi
 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,
matching gene expression and
protein expression usually correlate (Mother Nature 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
unexpected
p
such as the skin lighteners
g
influencing
g
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
d to be
b more frequently
f
l turned
d "off".
" ff" However,
H
these
h
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.
Dr Vince Gruber
 Robert Holtz at BioInnovation
Laboratories
 Society of Cosmetic Chemists
THANK YOU