16 ANTI-POLLUTION n Control n Urban dust n Urban dust + 0.03% artichoke extract 200 150 * p<0.05 versus particulate matter control
150 100 100 50 50 * * *
0 H3K27me2 H3K4me2
Figure 2: Artichoke extract protects against urban dust-induced epigenetic changes on histone 3.
the DNA and thus whether a gene is active or not. Meanwhile, there are many publications about the effects of air pollution on the epigenetic pattern, indicating an epigenetic mechanism involved in the health problems induced by air pollution.1
pollution-induced modifications of the histone H3 protein.2
Recent studies showed air So far, the epigenetic
effect of pollution on skin cells has not yet been elucidated.
In this work, human epidermal keratinocytes were used to study the effects of urban dust and benzo[a]pyrene on the epigenetic pattern of the histone H3 protein. The assay was then used to screen a series of plant extracts in order to find cosmetic actives that protect the skin against air pollution.
Materials and methods
Pollution-induced epigenetic changes in keratinocytes l Cultures and treatments Normal human epidermal keratinocytes (2nd passage) were seeded in 175 cm2
flasks
(125 000 cells) and cultured in Keratinocyte- SFM supplemented with EGF (0.25 ng/ml), pituitary extract (25 µg/ml) and Gentamycin (25 µg/ml) for 24 hours. The medium was then replaced with culture medium containing or not (control) the pollutants
Concentration of cress sprout extract
Detox enzymes NADPH: quinone reductase 1 Heme oxygenase 1
Thioredoxin reductase 1 PERSONAL CARE EUROPE
Pollution-induced protein carbonylation in fibroblasts Normal human dermal fibroblasts were seeded in 6 well plates and cultured for 24 hours in culture medium. The medium was removed and replaced by the assay medium
Enzyme expression relative to control (%) 0.05%
0.2% 75
212 184
Figure 3: Cress sprout extracts activates detoxification enzymes in keratinocytes. 214
4182 2316
0
Particulate matter (PM) control
PM + N-Acetyl- cysteine
Particulate matter (PM) control
PM + 1% Cress sprout extract
Figure 4: Cress sprout extract protects fibroblasts against pollution-induced protein carbonylation.
and the test compounds. The urban dust (Ref. NIST, Standard Reference Material (SRM) 1649b) was used as stock solution (100 mg/ml in ethanol/water, 2:1) at 0.01 mg/ml in the culture medium. Cells were then subcultured from 2nd
to 6th passage
every week with treatment after each subculture. At the 7th
passage, the cells
were seeded in 12-well plates (350 000 cells/well) and the treatment was renewed. Cells were then incubated for 24 hours. At the end of the incubation, the medium was discarded and the cells were washed in phosphate buffered saline (PBS) solution.
l Quantification of histone modifications The quantification of total histone H3 and 21 histone H3 modifications was performed using the EpiQuick Histone H3 Modification Multiplex Assay Kit (Colorimetric) from Epigentek (Ref. P-3100-96) following the supplier’s instructions. For each experimental condition, 100 ng of total histone extract was deposited per well.
containing or not (control) the reference N- Acetylcysteine (NAC), or cress sprout extract. After 7 hours, the medium was removed and the particulate matter (PM10
-like) added to
the assay medium. 16 hours after treatment, cells were washed with PBS and proteins were extracted from HDF for further analysis. All experimental conditions were performed by triplicate (n=3). Extracted proteins were quantified by the Bradford method and split into equal amounts for analysis. Oxidatively damaged (carbonylated) proteins were labelled with specific functionalised fluorescent probes and samples were resolved by high-resolution electrophoresis separation via 4-20% gradient SDS-PAGE. Total proteins were post-stained with SyproRubyTM protein gel stain. Image acquisition for carbonylated and total proteins was performed using the Ettan® DIGE imager. Image processing and analysis was performed using ImageJ.
Results Preventing epigenetic changes caused by pollution To test whether air pollution influences the epigenome of skin cells, a novel assay was designed that combined the long-term treatment of keratinocytes with pollution with the subsequent analysis of histone modifications. The EpiQuick Histone H3 modification assay is designed for measuring 21 histone H3 modifications simultaneously. Each histone H3 modified at specific sites is captured by an antibody that is coated on the strip wells and specifically targets the respective histone modification pattern. The captured histone sites are detected with a detection antibody, followed by a colour development reagent. Exposure of the keratinocytes to urban dust or benzo[a]pyrene was found to strongly modulate methylation, acetylation
November 2018
Change in histone modification level compared to control (=100) in %
Increase in protein carbonylation compared to control without particulate matter in %
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