82 ANTI-POLLUTION A


B UPM ■ H-EPS ■ 500 400 300 200 ** 100 ** 0 2h Treatment time (h) 24h * p<0.05 ** p<0.01


Figure 1: Effect of H-EPS and UPM on NRF2 activation. A. Nuclear translocation of NRF2 in keratinocytes treated with either H-EPS or UPM. B. Quantification of NRF2 translocation in keratinocytes treated with H-EPS or UPM for two hours and 24 hours


TABLE 1: EFFECT OF POLLUTION IN MITOCHONDRIAL METABOLISM. THREE PARAMETERS WERE STUDIED: MITOCHONDRIAL MEMBRANE POTENTIAL; O2 CONSUMPTION AND ATP PRODUCTION


Mitochondrial membrane potential 590/520


Ratio (x10-5 per 104


Control UPM


15,52 ± 2,49


4,34 ± 0,90 ) cells Reference -72% p<0,01


1,28 ± 0,14


0,90 ± 0,09 Reference -30% p<0.01 % Change pO2 O2 consumption / minute % Change (nM/106 cell.) % Change ATP


3410 ± 149


2703 ± 168


Reference -21% p<0.01


TABLE 2: EFFECT OF H-EPS ON MITOCHONDRIAL METABOLISM. EFFECT WAS STUDIED IN KERATINOCYTES AND IN THE POLLUTION MODEL


Mitochondrial membrane potential 590/520


Ratio (x10-5 per 104


Control (vehicle)


H-EPS


Control (UPM)


UPM +


H-EPS


12,66 ± 1,34


16,86 ± 2,38


4,34 ± 0,90


6,50 ± 1,26


) cells Reference +33% p<0,01 Reference +50% p<0,01


1,28 ± 0,14


1,59


± 0,20 0,90


± 0,09 1,12


± 0,13 Reference +24% p<0.05 Reference +24% p<0,01 % Change pO2 O2 consumption / minute % Change (nM/106 cell.) % Change ATP


3410 ± 149


6269 ± 778


2703 ± 168


4701 ± 267


Reference +84% p<0,01 Reference +74% p<0,01 ATP production ATP production


NRF2 activation assay HaCaT cells were seeded in 8-compartment glass slides (Labtech) in DMEM medium (Gibco). After 3 days, cells were treated with H-EPS and/ or BaP (benzo[a]pyrene) for up to 24 hours. Cell treatment was stopped at different time points. Cells were then fixed in formaldehyde and immuno-stained for NRF2. NRF2 quantity in the nuclei was determined. Cell density was determined by Hoechst 33258 staining.


Skin treatments Equivalent skin models were produced in-house as described previously. Briefly, for equivalent-dermis, fibroblasts were seeded into collagen-I matrix (Jacques Boy) in six-well plate inserts (Falcon) and kept emerged for 48 hours for dermal contraction. Normal Human keratinocytes (Cellntec) were seeded on top of the equivalent dermis. Models were cultivated for seven days in immersion medium, followed by seven days of air-liquid interphase to obtain a stratified epidermis. H-EPS and/ or UPM were topically applied


for 48 hours. Skins were embedded in freezing media (Leica,) and snap-frozen in liquid nitrogen. 5-7 µm sections were prepared using a cryostat (Leica). Histology was evaluated by H&E (Sigma-


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manufacturer’s instruction. Cells were fixed in histochoice (Interchim) and nuclei number was determined by Hoechst 33258 staining. ATP levels were normalized to cell number.


Oxygen consumption HaCaT cells were seeded in T25 cell flasks (Falcon) in DMEM medium (Gibco). After 48 hours, cells were treated with H-EPS and/ or UPM. The next day cells were detached and added to OxoPlates (PreSens). The medium was covered by a layer of oil


to avoid oxygen exchange with the external environment. Oxygen levels were quantified using fluorescent probe (Ex 540 nm / Em: 650 nm). Oxygen consumption rates were quantified with time.


Quantification of ROS levels HaCaTs cells were seeded in 96 well plates in DMEM medium (Gibco). The next day cells were treated with H-EPS and/or UPM for 4 h. Cells were rinsed with HBSS (Invitrogen) and stained with the CM-H2DCFDA (Invitrogen) probe for 45 minutes. Fluorescence signals were quantified using


ImageXpress (Molecular Devices) (Ex / Em : ∼492–495/517–527 nm). Cells were then fixed in formaldehyde (Sigma-Aldrich) and nuclei number was determined using Hoechst 33258 staining.


NRF2 translocation level (%)


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