evaluation of skin metabolism of benzo(a)pyrene : ex vivo skin explant model validation c. jacques,...
DESCRIPTION
Xenobiotic Activated molecule Conjugated molecule Phase I enzymes Cytochromes P450, flavines mono-oxygenases, alcohols and aldehydes deshydrogenases, esterases, epoxyde hydrolases Phase II enzymes UDP-glucuronyltransferases, sulfotransferases, glutathion-S-transferases, N-acétyltransferases Functionalization Conjugation I. Background and goalTRANSCRIPT
Evaluation of skin metabolism of Benzo(a)pyrene : ex vivo skin explant model validation
C. Jacques, E.L. Jamin, E. Perdu, S. Bessou-Touya, D. Zalko and H. Duplan
Skin Skin metabolically active organ
(liver reference organ)
Physic and Metabolic Barrier Physic and Metabolic Barrier biotransformation of xenobiotics
Detoxify or bio-activate
I. Background and goal
Xenobiotic
Activated molecule
Conjugated molecule
Phase I enzymesCytochromes P450, flavines mono-oxygenases,alcohols and aldehydes deshydrogenases, esterases, epoxyde hydrolases
Phase II enzymesUDP-glucuronyltransferases, sulfotransferases, glutathion-S-transferases, N-acétyltransferases
Functionalization
Conjugation
I. Background and goal
Pig ear skin model : Pig ear skin model : Valuable alternative ex vivo model to animal experimentValuable alternative ex vivo model to animal experiment
In the objective of complying with the European directive 76/768/CEE and with the 3R (reduce, refine, replace) concept for animal experimentation
Characterization of metabolic activity of ex vivo pig ear Characterization of metabolic activity of ex vivo pig ear skin model coupled with barrier effectskin model coupled with barrier effect
Detailed study of the distribution and metabolism of model xenobiotics
Similarities with human skin (“gold standard”)(Histological and biochemical properties)
Recognized for its use in skin penetration studies
I. Background and goal
Benzo(a)pyrene :
- polycyclic aromatic hydrocarbon
- absorbed following inhalation, oral, and dermal routes
- highly metabolized in the liver into several metabolites
- in particular, the 7,8-diol-9,10-epoxyde could be responsible of the carcinogenic effect of B(a)P
**[B(a)P] Log Kow 6.35
Choice of model xenobiotics
-different physicochemical properties-well-known metabolism-involving Phase I and II enzymes
I. Background and goal
Section 500µm thick
6-well plates Inserts 4.1 cm²
1.5ml culture medium
domestic pig ear skin
(Slaughterhouse)
Schema of skin short-term culture
Incubation 72 hours
37°C, 5% CO2
well
Skin Explant
Insert
Culture medium Changed every 24hrs
radioactive molecules50, 100, 200, 400, 600 and 800 nmol
SamplingCulture medium : 0, 24, 48, 72 hrAll compartments : 72hr
II. Experimental approach
Skin explants
Inserts
WellsSkin surface
Liquid scintillation counting
Pig ear skin model
Culture medium 12 4
5
2
5
1
4Wells
Skin explant
Insert
Culture medium
Skin surface 3
3
II. Experimental approach
Skin explants
Inserts
WellsSkin surface
Liquid scintillation counting
Metabolites detection and quantification : Radio-HPLC
Metabolites identification : Mass spectrometry, NMR
Enzymatic hydrolysis
Pig ear skin model
Culture medium
0 5 1 0 15 2 0 25 30 35 40 45 5 0 5 5 60 650
10 0
20 0
30 0
40 0
50 0
60 0
70 0
80 0
90 0
10 0 0
Te m ps (m in)
DPM
II. Experimental approach
Time : 24, 48 et 72 h
Skin surface Skin Culture Medium Wells Inserts
% o
f rad
ioac
tivity
Skin surface Skin Culture Medium Wells Inserts
% o
f rad
ioac
tivity
- Radioactivity:
Low doses (25 to 100 nmoles): culture media (46 ± 5% to 85 ± 1%) High doses (200 to 800 nmoels): surface + skin (41 ± 8% to 61 ± 2%)
III. Results : quantitative analysis
Compartimental radioactivity levels
B(a)P-OHs
B(a)P-diones
B(a)P-catechols
B(a)P-diols
B(a)P-tetrols
B(a)P
B(a)P-OGlcA
B(a)P-OSO3H
B(a)P-diol-SO3H
B(a)P-diol-GlcA
B(a)P
B(a)P-OGlcA
B(a)P-OSO3H
B(a)P-OHs
B(a)P-diones
B(a)P-catechols
B(a)P-diolsB(a)P-tetrols B(a)P-diol-GlcA
B(a)P-diol-SO3H
Jacques et al. Analytical and Bioanalytical Chemistry (2010). Toxicol Letters (2010)
III. Results : Qualitative analysis
B(a)P-Diones
Benzo(a)pyrene
B(a)P-EpoxydesGSH conjugués
B(a)P-Dihydrodiols
B(a)P-DihydrodiolépoxydesGSH conjugués
Conjugués glucuronides et
sulfates
B(a)P-Phénols
B(a)P-Phénoldiols
B(a)P-Tétrols
GSH conjuguésB(a)P-Catéchols
DNA Adducts
Conjugués glucuronides et
sulfates
Functional enzymes :
CYP1A1, CYP1A2, CYP1B1
Epoxyde hydrolase
Glucuronyltransferases
Sulfotransferases
Jacques et al. Toxicology Letters. (2010).
III. Results : BaP metabolic pathway in our skin model
B(a)P dose applied on skin (nmoles)
B(a
)P m
etab
olite
s (n
mol
es/h
r)
- Major metabolites: glucuronide and sulfates conjugates
- Michaelis Menten curves
- Enzymatic saturation: 200nmoles of B(a)P
III. Results : kinetic of metabolite formation
B(a)PLog Kow 6,5
50 nmoles
400 nmoles 14,5%21.7%
29,1%
60,8%
23,4%
Surface peau
Explant peau
Milieux de culture
Puits
Inserts
Skin surface
Skin
Culture medium
Insert
Wells
IV. Influence of BaP metabolism on its absorption
Epidermis
Dermis
Hydrophobic molecule
Penetration
Stratum Corneum
Reservoir
Biotransformation
Metabolites diffusion
Blood stream transfer
Viable skin
Frozenskin
% o
f rad
ioac
tivity
in c
ultu
re m
ediu
m
IV. Influence of BaP metabolism on its absorption
The percutaneous diffusion rate of a compound depends not only on its physico-chemical properties, but also on the expression of functional enzymes in the skin, especially for hydrophobic compounds like benzo(a)pyrene
Biotransformation at the level of the skin could be an important factor for determining the overall diffusion rate of a xenobiotic
As in the liver, benzo(a)pyrene was metabolized in the skin in several products as diols, tetrols that could induce skin sensitization or skin cancers.
We performed further studies with several known molecules that validated the ex vivo pig ear skin model (testosterone, 7-EC, Caffeine…)Jacques et al; Toxicol In Vitro 2010; SPP 2013 accepted
V. Conclusions
Thanks to :
Carine Jacques Daniel Bacqueville
Sandrine Bessou-TouyaHélène Duplan
www.pierre-fabre.com Daniel ZalkoElisabeth Perdu
Jean-Pierre CravediEmilien Jamin
Laurent DebrauwerINRA UMR 1331