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FEBS 18069 FEBS Letters 401 (1997) 167-170
Ozone depletes tocopherols and tocotrienols topically appliedto
murine skinJens J. Thielea, Maret G. Trabera, Maurizio Podda l a ,
Kenneth Tsanga, Carroll E. Crossb,Lester Packer1'*
^Department of Molecular an d Cell Biology, 251 LSA, University
of California, Berkeley, CA 94720-3200, USAhDepartment of Medicine
and Physiology, University of California School of Medicine, Davis,
CA, USAReceived 6 December 1996
Abstract To evaluate ozone damage to hairless mouse skin,
twoparameters of oxidative damage, vitamin E depletion
andmalondialdehyde (MDA) production, were measured in
vitaminE-enriched and in control skin from mice exposed to ozone
(10ppm). A 5% vitamin E solution (tocotrienol-rich fraction, TRF)in
polyethylene glycol (PEG) was applied to 2 sites on the backof
hairless mice, PEG to 2 sites. After 2 h, the sites were washed,one
of each pair of sites covered and the mice exposed ozone for2 h.
Ozone exposure (compared with covered sites) increasedepidermal MDA
in PEG-treated sites, while vitamin E wasunchanged. In contrast,
ozone exposure significantly depletedvitamin E in TRF-treated
sites, while significant MDAaccumulation was prevented. This is the
first demonstration thatozone exposure causes damage to cutaneous
lipids, an effectwhich can be attenuated by vitamin E
application.Key words: Hairless mice; Vitamin E;
Antioxidant;Malondialdehyde; Epidermis; Cutaneous l ipid
1. IntroductionOzone is the major air pollutant in photochemical
smog.Since half the US population lives in areas exceeding the
USNational Ambient Air Quality Standard (0.12 ppm averagedover a 1
h period ) [1], the presence of oz one in the air po sessignificant
concern [2].Ozone exposure causes oxidation and peroxidation of
bio-molecules both directly and/or via secondary products ofozone
reactions [3-8]. One of the most important mechanismsof ozone
injury is peroxidation of lipids, especially unsatu-rated fatty
acids [5,7,8]; in vitro, vitamin E appears to preventthe prop
agation of this reaction [6]. Altho ugh cc-tocopherolhas the
highest biologic activity of the various vitamin E iso-mers [9],
the properties of some of the other isomers suggest
that they might be more protective against ozone-induceddamage.
For example, a-tocotrienol has a higher antioxida-tive activity
than oc-tocopherol against Fe 2 + /ascorbate andF e 2 +
/NADPH-induced l ipid peroxidation in rat l iver micro-
*Corresponding author. Fax: (1) (510) 642-8313.^Present address:
Zentrum der Dermatologie, Klinikum der J.W.Goethe Universit t,
Theodor-Stern-Kai 7, 60590 Frankfurt am Main,Germany.Abbreviations:
BHT, butylated hydroxytoluene; EDTA, ethylenediamine tetraacetic
acid; HPLC-EC, high pressure liquid chromatog-raphy with
electrochemical detection; PEG, polyethylene glycol-400;TRF,
tocotrienol-rich palm oil fraction
somes [10]. Alternatively, ozon e might attack the c hrom
anolnucleus of - tocotr ienol or - tocopherol in an analogous m
anner to that described for nitrogen dioxide [11].Skin is the organ
the most directly exposed to ozone. Infact, ozone is probably the
most reactive environmental pollutant to which skin is routinely
exposed. Unlike the lung [4],very little attention has been paid to
the potential effects ofenvironmental oxidant pollutants on
cutaneous t issues. This issomewhat surprising since skin contains
peroxidizable lipidsand these constituents are in part responsible
for the cutaneous permeability barrier [12].A variety of enzymatic
and non-enzymatic antioxidantsprotect skin against oxidative stress
[13-17]. Among these isvitamin E. Of the vitamin E isomers, skin
-tocopherol concentrations are much higher than -tocopherol or a- a
n d -tocotrienols [18]. However, skin can be enriched by
topicalapplication of these vitamin E isomers [19].This study
tested the hypotheses that (1) ozone attacks skinlipids and
lipophilic antioxidants and (2) topical applicationof a mixture of
vitamin E forms, including tocopherols andtocotrienols from a
tocotrienol-rich palm oil fraction (TRF),
ameliorates this oxidative damage.2. Materials and methods2.1.
ChemicalsAll chemicals used were of the highest grade available.
Tocopherolstandards were provided by Henkel Corporation (LaGrange,
IL).TRF was kindly provided by PORIM (Kuala Lumpur,
Malaysia).Tocotrienols for use as standards were purified from TRF
by Dr.Asaf A. Qureshi, University of Wisconsin (Madison, WI). TRF
vitamin E are extracted from palm oil and so are in the 'natural'
configuration - both a- and -tocopherols have 2R, 4'R,
8'R-stereochem-istry; b oth ot- and -tocotrienols have 2R
stereochemistry.2.2. Animals
Animal care, handling, and experimental procedures were
carriedout as described in the animal use protocol approved by the
AnimalCare and Use Committee of the University of California,
Berkeley,CA. Hairless mice (females, between 8 and 10 weeks old,
CharlesRiver Laboratories, Wilmington, MA, USA) were housed
understandard light and temperature conditions. Food (Harlan
TeckladRodent Diet #1846, Madison, WI, USA) and water were
providedad libitum. Mice were anesthetized by an intraperitoneal
injection ofsodium pentobarbital (50 mg/kg body weight, Nembutal,
AbbottLaboratories, North Chicago, IL) and remained anesthetized
duringthe entire experimental period.2.3. Vitamin E applicationA 5%
w/v solution of T RF was prepared in polyethylene glycol-400(PEG;
Sigma, St. Louis, MO). Mice were anesthetized, then 4 polypropylene
plastic rings (1 cm2) were glued onto the animals'
backs,subsequently TR F solution (20 ) was applied to 2 rings and
PEG tothe other 2 rings. After 2 h, the treated areas were washed
as de-
0014-5793/97/S17.00 1997 Federation of Euro pean Biochemical
Societies. All rights reserved.P / / S 0 0 1 4 - 5 7 9 3 ( 9 6 )0 1
4 6 3 - 9
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168 J.J. Thiele et allFEBS Letters 401 (1997) 167-170scribed by
Dupuis et al. [20]. Briefly, the skin was rinsed 3 times with300
ethano l:water (9 5:5), then twice with water alone and driedwith a
cotton tip. After washing, the location of the application sitewas
marked and the plastic rings removed; half of the sites werecovered
with a piece of Kimwipe tissue (Kimberly Clarke, Atlanta,GA) and
sealed with cellophane tape. The Kimwipe prevented injuryto the
skin during the removal of the adhesive tape following exposureto
either air (n = 3, control) or ozone (n = 4) (see below).2.4. Ozone
exposureOzone was produced from oxygen by electric discharge
(Sanderozonizer model IV, Eltze, Germany). The ozone was then
mixedwithfiltered ozone-free) am bient air and allowed to flow into
a stainless steel exposure chamber at a constant rate (200 1/min).
The concentration in the exposure chamber was adjusted to 10 ppm
andcontinuously monitored with an ozone analyzer (Dasibi model
1003-AH, Glendale, CA). The ozone chamber provided a maximum
spacefor 4 animals during the 2 h ozone exposure. Control mice
weretreated identically in terms of anesthesia and animal handling,
butwere kept under air (0 ppm ozone).After air or ozone exposure,
the mice were given another dose ofpentobarbital, allowed to rest
for 30 min, then were killed by cervicaldislocation. The skin was
excised, the subcutaneous fat removed witha scalpel, then punch
biopsies were taken and the samples immediately frozen in liquid
nitrogen. The skin samples were stored for nolonger than a week at
80C.2.5. Vitamin E analysisTocopherols and tocotrienols were
extracted from full thicknessskin, as described [21,22]. Briefly,
the skin sample was weighed (approximately 20 mg), ground under
liquid nitrogen, then homogenizedin a Potter-Elvehjem homogenizing
tube with 2 ml buffer (10 mMphosphate, 130 mM NaCl, 1 mM EDTA, pH
7.0) and 50 ml BHT(1 mg/ml) and extracted after addition of 1 ml of
0.1 M SDS, 2 mlethanol and 2 ml hexane. An appropriate aliquot of
hexane was usedfor HPLC analysis, as described [23]. The
electrochemical detectorwas operated with a 0.5 V potential, full
recorder scale at 50 nAfor quantitation of a- and -tocopherols and
a- and -tocotrienols.Authentic compounds were used to generate
standard curves.2.6. LipidperoxidationFluorimetric detection of the
malondialdehyde-thiobarbituric acidadduct (MDA-TBA) was performed
after HPLC separation of theTBA-reactive substances, based on
methods for MDA determinationin plasma and other body tissues
[19-21]. After weighing, the scrapedmouse epidermis was extracted
with 2 ml methanol, 2 ml 15% SDSsolution, 50 10% BHT in ethanol and
4 ml chloroform. An aliquotof the chloroform was dried and the
residue resuspended in 400 15% SDS and incubated w ith 250 0.375%
TBA and 200 1.22 Mphosphoric acid for 30 min at 100C, followed by
addition of 380 methanol and 20 1 N N aOH . After centrifugation,
100 supernatant were injected into the HPLC system, which consisted
of a 114M Solvent Delivery Module pump (Beckman, Fullerton, CA),
anAlltima C18 column and a Hitachi (Hitachi Ltd. Tokyo, Japan)
F-105fluorescencespectrophotometer (excitation 532 nm and
emission553 nm). The mobile phase consisted of 60% methanol and 40%
50mM NaH 2P0 4 , pH adjusted to 5.5, at a rate of 0.9 ml/min.
MDAstandards (ranging from 0.5 to 10 pmol) were prepared using
dilutionsof 1,1,3,3-tetramethoxypropane. Samples and stand ards
were analyzed in duplicate.2.7. Statistical analysisAll statistical
analyses were carried out using SuperAnova (AbacusConcepts, Inc.,
Berkeley, CA) for the Macintosh (Apple Computers,Cupertino, CA).
Analyses included: one-factor ANOVA (air vs.ozone); two-factor
ANOVA with 2 within groups repeated measures
(PEG vs. TRF, and covered vs. exposed) with least square
meanscomparisons. Data were log-transformed to equalize variances
between TR F- and PEG-treated sites. A P-value < 0.05 was
consideredstatistically significant. Values are given as means
S.D.
3 . Results
3.1. Antioxidants in murin e skinTo investigate the
susceptibility of skin to ozone damage, astudy design was planned
using covered and uncovered skinsites, such that each animal would
serve as its own control.Therefore, we first evaluated whether
covering the skinchanged the antioxidant composition. PEG was
applied to 2sites and TRF to 2 si tes, the compounds were allowed
topenetrate for 2 h, then the skin was washed. Subsequently,half of
the sites were covered with tissue paper and sealed withcellophane
tape for 2 h during which time the mice were exposed to air.
Tocopherols and tocotrienols in PEG-treated air-exposed skin were
within the range of previous reports fromour laboratory [19,24],
while covering the skin significantlyincreased vitamin E contents
(Table 1). TRF treatment resulted in significant increases in the
concentrations of all vitamin E forms, as reporte d previously
[19]. How ever, coveringthe TRF-treated sites resulted in
significantly lower concentrations of vitamin E than in the
uncovered sites.
To evaluate whether the various vitamin E forms penetrated
murine skin differently, the percentage distribution ofeach of the
vitamin E homologues in the TRF suspension wascompared to i ts
percentage distribution in skin treated withTRF or vehicle (PEG)
alone (Fig. 1). The percentage distribution of vitamin E forms that
penetrated the skin (above background concentrations) was
significantly different from theirdistribution in the TRF
suspension - a higher percentage of - tocopherol was found in TRF-t
rea ted skin than was presentin TRF suspens ion (P
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170 J.J. Thiele et allFEBS Letters 401 (1997) 167-17020
1 6-= 1 2 -< z
E 8 -
H T EG - PEG- TRF- TRF-covered exposed covered exposedFig. 3 .
MDA in murine skin. The MDA concentrat ions(mean S.D.) in mou se
epidermis following application w ith P EGor TRF, covered or not,
and exposed to ozone (n = 4) . Only PEGtreatment followed by ozone
exposure significantly increased MDA( P < 0 . 0 1 ) .
c o n t a i n e d lower v i t a mi n E c o n c e n t r a t i o n
s t h a n a i r - e x p o s e dsk in (Tab le 1 ) . Th e f ind ing
th a t cove r in g the sk in ch ang edi t s a n t i o x i d a n t c
o m p o s i t i o n wa s u n e x p e c t e d . M o s t l i k e ly ,
c o v e r ing the sk in occ lude d i t, i nc reas in g it s m o is
tu re con ten t , a ndc h a n g i n g i t s p e n e t r a t i o n c
h a r a c t e r i s t i c s [ 27 ] . No n e t h e l e s s , t h es
k i n v i t a mi n E c h a n g e s o b s e r v e d d i d n o t a l
t e r t h e c o n c l u s i o n so f t h e s t u d y .
T h e o z o n e c o n c e n t r a t i o n ( 1 0 p p m ) u s e d
in t h i s s t u d y , g i v e nfo r a longe r exposure t ime ,
causes l e tha l damage to the r e s p i r a t o r y s y s t e m. I
n u r b a n a i r p o l l u t i o n , l o we r o z o n e c o n c e
n t r a t i o n s ( 0 . 1 - 0 . 8 p p m ) a r e e n c o u n t e r e
d [ 4 ]. S i n c e n o o t h e rd a t a c o n c e r n i n g c u t a
n e o u s e f f e c t s o f o z o n e e x p o s u r e a r e a v a i
l a b l e , the p resen t s tudy was des igned to a s sess r e
sponses toh i g h l e v e l s o f o z o n e e x p o s u r e . F u r
t h e r i n v e s t i g a t i o n s a r en o w n e e d e d t o e v
a l u a t e wh e t h e r s i mi l a r b u t l es s ma r k e d m o l
e c u l a r s k i n d a ma g e t a k e s p l a c e a t l o we r ,
mo r e r e l e v a n t o z o n ec o n c e n t r a t i o n s a n d
wh e r e t h i s d a m a g e o c c u r s . T h i s m o s tl i k e l
y wo u l d r e q u i r e c h r o n i c o r i n t e r mi t t e n t e
x p o s u r e s a n d /o r mor e sens i t ive t ech n iqu es to d i
f f e ren t i a t e and ana l yze theu p p e r m o s t e p i d e r
m a l s k i n l a y e r s , i n p a r t i c u l a r t h e s t r a t
u mc o r n e u m .
In sum m ary , the ma jo r f ind ing o f th i s s tudy was tha t
to p i c a l l y a p p l i e d v i t a mi n E f o r ms a r e d r a
ma t i c a l l y d e p l e t e d b ya c u t e , s h o r t - t e r m
e x p o s u r e t o o z o n e . T h i s is t h e f ir s t r e p o r
ts h o w i n g t h a t o z o n e i s c a p a b l e o f in i t i a t
i n g o x i d a t i v e p r o c e s s e si n c u t a n e o u s t i
s s u e s . W h e t h e r o z o n e - r e l a t e d o x i d a t i v
e s t r e s scon t r ibu te s to sk in d i so rde r s a s a r e su
l t o f l i f e t ime exposurer e ma i n s t o b e d e t e r mi n e
d .Acknowledgements: Nathalie Espuno provided excellent technical
assistance. We gratefully acknowledge the efforts of Dr. Asaf A.
Qure-shi, University of Wisconsin (Madison, WI), who isolated
tocotrienols
for use as standards for this study. This study was supported in
partby NIH Grant HL47628, gifts from the Colgate Palmolive and
thePalm Oil Research Institute of Malaysia. J.T. was supported by
afellowship of the Fritz Thyssen Stiftung, Germany (AZ
21295008).References
[1] Office of Technology and Assessment (1989) U.S.
GovernmentPrinting Office, Washington, DC.[2] Lippmann, M. (1989)
J. Abnorm. Psychol. 39, 672-695.[3] Menzel, D.B. (1984) J. Toxicol.
Environ. Health 13, 183-204.[4] Mustafa, M.G. (1990) Free Radical
Biol. Med. 9, 245-265.[5] Pryor, W.A. and Church, D.F. (1991) Free
Radical Biol. Med.11 ,41^16 .[6] Pryor, W.A. (1991) Am. J. Clin.
Nutr. 53, 702-722.[7] Cross, C.E., Motchnik, P.A., Bruener, B.A.,
Jones, D.A., Kaur,Ft., Ames, B.N. and Halliwell, B. (1992) FEBS
Lett. 298, 269-272.[8] Pryor, W.A ., Squadrito , G.L. and Fried ma
n, M . (1995) FreeRadical Biol. Med. 19, 935-941 .[9] Traber, M.G.
and Sies, H. (1996) Annu. Rev. Nutr. 16, 321-347.[10] Serbinova,
E., Kagan, V., Han, D. and Packer, L. (1991) FreeRadical Bio.l Med.
10, 263-27 5.[11] Cooney, R.W., France, A.A., Harwood, P.J . ,
Hatch-Pigot t , V.,Custer, L.J. and Mordan, L.J. (1993) Proc. Natl.
Acad. Sei. USA90 , 1771-1775.[12] Elias, P.M. and Feingold, K.R.
(1992) Semin. Dermatol. 11, 176-82 .[13] Shindo, Y., Witt, E., Han,
D., Epstein, W. and Packer, L. (1994)J. Invest. Dermatol. 102,
122-124.[14] Shindo, Y., Witt, E., Han, D. and Packer, L. (1994) J
Invest.Derm atol . 102, 470-475.[15] Shindo, Y., Witt, E., Han, D.,
Tzeng, B., Aziz, T., Nguyen, L.and Packer , L. (1994) Photoderm.
Photoim munol . Photomed . 10,183-191.[16] Fuchs, J., Mehlhorn,
R.J. and Packer, L. (1989) J. Invest. Dermatol . 93, 633-640.[17]
Fuchs, J., Huflejt, M.E., Rothfuss, L.M., Wilson, D.S., Carcamo,G.
and P acker, L. (1989) J. Invest. Derm atol. 93, 769-77 3.[18] Podd
a, M., Weber, C , Tra ber, M . and Packer, L. (1996) J. LipidRes.
37, 893-901.
[19] Webe r, C , Pod da, M., Rallis, M., Traber, M .G . and
Packer, L.(1996) Free Radical Biol. Med. (in press).[20] Dupuis,
D., Rougier, A., Roguet, R., Lotte, C. and Kalopissis,G. (1984) J.
Invest. Dermatol. 82, 353-356.[21] Burton, G.W., Webb, A. and
Ingold, K.U. (1985) Lipids 20, 29-39 .[22] Lang, J.K., Gohil, K.
and Packer, L. (1986) Anal. Biochem. 157,106-116.[23] Podd a, M.,
Web er, C , T raber, M .G . and Packer, L. (1996)J. Lipid Res. 37,
893-901 .[24] Shindo, Y., Witt, E. and Packer, L. (1993) J. Invest.
Dermatol.100, 260-265.[25] Pryor, W.A. (1992) Free Radical Biol.
Med. 12, 83-88.[26] Frei, B., Stocker, R. and Ames, B.N. (1988)
Proc. Natl. Acad.Sei USA 85, 9748-9752.[27] Shaw, J.E., Prevo, M.,
R, G. and Yum, S.I. (1991) in: Physiology, Biochemistry, and
Molecular Biology of the Skin (L.A.Goldsmith, Ed.) Oxford
University Press, New York.
