Aging- and Photoaging-Dependent Changes of Enzymic and
Nonenzymic Antioxidants in the Epidermis and Dermis of
Human Skin In Vivo
Gi-eun Rhie, Mi Hee Shin, Jin Young Seo, Won Woo Choi, Kwang Hyun Cho, Kyu Han Kim,
Kyung Chan Park, Hee Chul Eun, and Jin Ho Chung
Department of Dermatology, Seoul National University College of Medicine, and Laboratory of Cutaneous Aging Research, Clinical Research
Institute, Seoul National University Hospital, Seoul, Korea
was signi®cantly lower in the epidermis of photoaged
(56% of young skin level) and aged (61%) skin, but
this was not found to be the case in the dermis.
Ascorbic acid levels were lower in both epidermis
(69% and 61%) and dermis (63% and 70%) of photoaged and naturally aged skin, respectively. Glutathione concentrations were also lower. Uric acid did
not show any signi®cant changes. Our results suggest
that the components of the antioxidant defense
system in human skin are probably regulated in a
complex manner during the intrinsic aging and
photoaging processes. Key words: ascorbic acid/catalase/
glutathione/glutathione peroxidase/glutathione reductase/
superoxide dismutase/a-tocopherol. J Invest Dermatol
117:1212±1217, 2001
This is a comprehensive study of the changes in
major antioxidant enzymes and antioxidant molecules during intrinsic aging and photoaging processes
in the epidermis and dermis of human skin in vivo.
We show that the activities of superoxide dismutase
and glutathione peroxidase are not changed during
these processes in human skin in vivo. Interestingly,
the activity of catalase was signi®cantly increased in
the epidermis of photoaged (163%) and naturally
aged (118%) skin (n = 9), but it was signi®cantly
lower in the dermis of photoaged (67% of the young
skin level) and naturally aged (55%) skin compared
with young (n = 7) skin. The activity of glutathione
reductase was signi®cantly higher (121%) in naturally
aged epidermis. The concentration of a-tocopherol
T
been suggested to play important roles in the intrinsic aging and
photoaging of human skin in vivo (Kawaguchi et al, 1996), and
ROS has been postulated to be responsible for various cutaneous
in¯ammatory disorders and skin cancers (Cross et al, 1987; Record
et al, 1991).
The skin's antioxidant defense system is regulated by an
intimately interlinked network, and it is becoming increasingly
clear that antioxidants interact in a complex fashion, so that changes
in the redox status or concentration of one component may affect
many other components of the system (Packer et al, 1979;
Martensson et al, 1991; Lopez-Torres et al, 1998). Thus, a
comprehensive and integrated antioxidant skin defense mechanism
is considered to be crucial for protecting this organ from ROS, and
consequently for preventing the aging process of skin. Some
researchers have investigated the antioxidant defense mechanisms of
human skin in vivo (Shindo et al, 1994a; Podda et al, 1998) and in
the aged skin of mice (Lopez-Torres et al, 1994). To our
knowledge, however, the levels of the major enzymic and
nonenzymic antioxidants have never been assessed comprehensively during the aging and photoaging processes of human skin
in vivo.
Here, we report the results of the ®rst comprehensive study upon
changes of the major antioxidant enzymes ± superoxide dismutase
(SOD), catalase, glutathione peroxidase (GPx), and glutathione
reductase (GR) ± and the antioxidant molecules-a-tocopherol,
ascorbic acid, uric acid, and glutathione-during the intrinsic aging
and photoaging processes of the epidermis and dermis of human
skin in vivo.
he aging process of skin can be attributed to intrinsic
aging and photoaging. Damage to human skin due to
repeated exposure to ultraviolet (UV) radiation
(photoaging) and damage due to the passage of time
(chronologic aging) are considered to be distinct
entities rather than similar skin aging processes. Clinically, naturally
aged skin is smooth, pale, and ®nely wrinkled. In contrast,
photoaged skin is coarsely wrinkled and associated with dyspigmentation and telangiectasia (Lavker, 1979; Lavker and Kligman,
1988; Gilchrest, 1989).
Several theoretical mechanisms have been proposed to explain
the aging in skin, and the free radical theory (Harman, 1956, 1981,
1986) is receiving particular attention because skin is constantly
exposed to reactive oxygen species (ROS) from the environment,
such as air, solar radiation, ozone, and other air-borne pollutants, or
from the normal metabolism, primarily from the mitochondrial
respiratory chain wherein excess electrons are donated to molecular
oxygen to generate superoxide anions. Accumulated ROS have
Manuscript received February 20, 2001; revised May 16, 2001; accepted
for publication May 21, 2001.
Reprint requests to: Dr. Jin Ho Chung, Department of Dermatology,
Seoul National University Hospital, 28 Yungon-dong, Chongno-Gu,
Seoul 110-744, Korea. Email: [email protected]
Abbreviations: DTNB, 5,5¢-dithiobis-2-nitrobenzoic acid; GPx, glutathione peroxidase; GR, glutathione reductase; GSH, reduced glutathione;
GSSG, oxidized glutathione; NBT, nitroblue tetrazolium; SOD, superoxide dismutase.
0022-202X/01/$15.00
´ Copyright # 2001 by The Society for Investigative Dermatology, Inc.
1212
VOL. 117, NO. 5 NOVEMBER 2001
MATERIALS AND METHODS
Human skin samples A total of 15 young Koreans (15 men; mean
age 22.9 y; age range 19±28 y) and 15 elderly Koreans (10 men and ®ve
women; mean age 75.8 y; age range 71±86 y) without current or prior
skin diseases provided skin samples. They did not have a recent history
of excessive sun exposure. All elderly Koreans had severely
photodamaged skin, of more than grade 5 by our photographic grading
system (Chung et al, 2001). One percent lidocaine was injected for the
biopsies. Skin samples were obtained from both the forearm and the
upper-inner arm of each subject by punch biopsy (8 mm), and
immediately frozen in liquid nitrogen and stored at ±70°C. All
procedures involving human subjects received prior approval from the
Seoul National University Institutional Review Board, and all subjects
provided written informed consent.
To separate the epidermis from the dermis, the defrosted whole skin
samples were placed dermis-side down on a Petri dish and heated at
55°C for 2 min; they were then separated gently into epidermis and
dermis with a forceps. We con®rmed that these conditions did not
change either four major antioxidant enzyme activities (SOD, catalase,
GPx, and GR) or the oxidation states of antioxidant molecules such as
a-tocopherol, ascorbic acid, uric acid, and glutathione (data not shown).
The separated epidermis and dermis were immediately frozen in liquid
nitrogen, weighed in the frozen state, and used for assays.
Chemicals 5,5¢-Dithiobis-2-nitrobenzoic acid (DTNB), reduced
nicotinamide adenine dinucleotide phosphate, oxidized glutathione
(GSSG), reduced glutathione (GSH), nitroblue tetrazolium (NBT),
triethanolamine, xanthine, butylated hydroxytoluene, a-tocopherol,
ascorbic acid, uric acid, metaphosphoric acid, buttermilk xanthine
oxidase, and yeast glutathione reductase were purchased from Sigma (St.
Louis, MO). Hydrogen peroxide, 2-vinylpyridine, and 2,3-dimercapto1-propanol were purchased from Aldrich Fine Chemicals (Milwaukee,
WI). All other reagents used were of the highest quality generally
available.
Antioxidant enzyme assays Two biopsied skin samples either from
the forearm or the upper-inner arm of each individual were used for the
extraction of proteins required for antioxidant enzyme assays. The
separated epidermis and dermis were homogenized in 300 ml and 600 ml
of buffer A [sodium chloride 130 mM, glucose 5 mM, disodium
ethylenediamine tetraacetic acid (EDTA) 1 mM, and sodium phosphate
10 mM, pH 7.0], respectively. The suspension was rotated at 4°C for
10 min, and centrifuged at 14,000g for 10 min. The supernatant was
kept at ±70°C and used for antioxidant enzyme assays.
The activities of SOD (Oberlery and Spitz, 1985), catalase (Claiborne,
1985), GPx (GuÈnzler and Floche, 1985), and GR (Calberg and
Mannervik, 1985), were assayed spectrophotometrically on a Beckman
DU650 spectrophotometer (Beckman Instruments, Fullerton, CA). One
enzyme unit is de®ned as being equivalent to 1 mmol of product
formation or 1 mmol of substrate disappearance per min under the
de®ned conditions, with the exception of SOD, in which case 1 unit
was de®ned as the amount of SOD that inhibited the NBT reduction
rate by 50% under the given assay conditions. Catalase and SOD
activities were measured at room temperature, whereas GPx and GR
activities were measured at 37°C.
Protein concentrations were determined using the Bio-Rad DC
protein assay (Bio-Rad, Hercules, CA) with bovine serum albumin as a
reference protein.
Antioxidant assays One biopsied skin sample was used for each
antioxidant assay. After separating the epidermis from the dermis,
antioxidant molecules were extracted in each extraction buffer.
Glutathione was measured using the DTNB±GR recycling assay
(Anderson, 1985). The separated epidermis and dermis were homogenized in 150 ml and 300 ml of ice-cold 3.3% sulfosalicylic acid, 5 mM
EDTA, and 1.5 mM butylated hydroxytoluene, respectively. This solution had been previously deoxygenated with nitrogen gas. After
homogenization, the samples were immediately centrifuged at 14,000g
for 10 min. The supernatant was used to assay total glutathione (GSH +
GSSG). For the GSSG assay, 1 ml of 2-vinylpyridine and 3 ml of
triethanolamine were added to 50 ml of the above solution. The mixture
was incubated for 1 h to derivatize GSH, thereby rendering it inactive
during the assay.
a-Tocopherol content was determined by high performance liquid
chromatography (HPLC) as described by Lang et al (1986) with slight
modi®cations using UV detection. a-Tocopherol from the separated skin
samples was extracted in reagent alcohol (ethanol:isopropanol 95:5) and
ANTIOXIDANTS IN AGED HUMAN SKIN IN VIVO
1213
hexane (Lang et al, 1986). The extracted a-tocopherol from the
epidermis and dermis was redissolved in 50 ml and 100 ml of methanol:ethanol (70:30), respectively. Then, a 20 ml aliquot was immediately
analyzed by HPLC. Reagent alcohol:methanol (90:10) containing
20 mM LiCl4 was used as the mobile phase.
Ascorbic acid (Dhariwal et al, 1991) and uric acid (Shindo et al, 1994a)
were measured by HPLC with UV detection. Tri¯uoroacetic acid (0.1%)
was used as the mobile phase. The separated epidermis and dermis were
homogenized in 100 ml and 300 ml respectively of ice-cold 5% metaphosphoric acid and 1.5 mM butylated hydroxytoluene solution, previously
deoxygenated with nitrogen gas. After centrifugation at 14,000g for
10 min, a 20 ml aliquot of supernatant was immediately analyzed by
HPLC for ascorbic acid and uric acid. Ascorbic acid (2 mM) and uric
acid (2 mM) standards were freshly prepared.
The concentration of ascorbic acid was determined spectrophotometrically using an extinction coef®cient at 265 nm of 14,500 per m per
cm.
The HPLC system for a-tocopherol, ascorbic acid, and uric acid
consisted of a Gilson Model 302 pump with 20 ml injection valve, a
Gilson 118 UV/VIS detector, and a Gilson Model 802C manometric
module system (Gilson, WI). A Clipeus C18 column, 5 mm,
250 3 4.6 mm (Higgins, CA), was used.
Statistics Statistical signi®cance was determined by using the Student t
test. Results are presented as means 6 SEM. All p values quoted are
two-tailed and were accepted as signi®cant for p < 0.05.
RESULTS
Enzymic antioxidants We observed the age-associated changes
in four antioxidant enzymes, SOD, catalase, GPx, and GR. For
intrinsic aging, the enzyme activities of the skin sample from the
sun-protected area (upper-inner arm) of young and old subjects
were compared, and similarly for photoaging the activities of skin
samples from the sun-exposed area (forearm) were compared
(Table I and Fig 1). The activities are reported as units per mg
protein.
The activities of SOD, catalase, and GR were signi®cantly
higher in the epidermis than the dermis by an average of 128% (p
< 0.05), 190% (p < 0.001), and 236% (p < 0.01), respectively, in
young skin, and by 134% (p < 0.01), 436% (p < 0.001), and 238%
(p < 0.001) in old skin. The activity of GPx in the epidermis was
lower than in the dermis, and was 74% of the dermal level in young
skin and 80% of the dermal level in old skin (Table I).
No signi®cant photoaging- or intrinsic aging-related variations
were found for SOD and GPx (Table I, Fig 1). Catalase showed
signi®cant activity increases, however, in the epidermis of
photoaged (forearm) and naturally aged (upper-inner arm) skin
(n = 9) compared with those of young skin (n = 7) by 163%
(p < 0.001) and 118% (p < 0.01), respectively. In the dermis,
catalase showed signi®cantly lower activity in photoaged and
naturally aged skin (n = 9) to 67% (p < 0.01) and 55% (p < 0.001)
of the young skin levels, respectively (Table I, Fig 1). The activity
of GR in the epidermis of naturally aged skin (n = 9) was
signi®cantly increased by 121% (p < 0.05) compared with that of
young skin (n = 7), whereas no signi®cant changes were found in
the photoaged epidermis, or the dermis of aged and photoaged
skin.
To investigate the effect of chronic sun exposure on antioxidant
enzyme activities, the ratios of the antioxidant enzyme activities of
the forearm to the upper-inner arm samples were compared for
young and old volunteers (Table II). These ratios did not show
any signi®cant differences between young and old skin, with the
exception of the catalase activity levels, in which case the forearm
to upper-inner arm ratio was higher for both the epidermis and
dermis of old skin, although only the epidermal difference was
statistically signi®cant (p < 0.05) (Table II).
Nonenzymic antioxidants Changes in the levels of the
antioxidant molecules a-tocopherol, ascorbic acid, uric acid, and
glutathione were also examined. Basically, the same strategy that
was used for the antioxidant enzyme activity analysis was employed.
Antioxidant concentrations are reported as nmol per g skin.
1214
RHIE ET AL
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Table I. Antioxidant enzyme activities in photoaging and aging human skin in vivoa
Youngb (n = 7)
Oldb (n = 9)
Forearm
Superoxide
dismutase
Catalase
Glutathione
peroxidase
Glutathione
reductase
Upper-inner arm
Forearm
Upper-inner arm
Epidermis
Dermis
Epidermis
Dermis
Epidermis
Dermis
Epidermis
Dermis
109.7 6 6.2
88.2 6 6.3h
105.1 6 3.3
80.6 6 6.6i
111.4 6 5.0
88.5 6 6.1i
115.7 6 8.7
81.2 6 5.4i
66.8 6 3.2
0.02 6 0.0
34.6 6 3.6j
0.03 6 0.0
82.6 6 1.6g
0.02 6 0.0
44.1 6 2.7fj
0.0 6 0.0
108.7 6 5.1e
0.02 6 0.0
23.0 6 2.5dj
0.02 6 0.0
97.4 6 3.7df
0.0 6 0.0
24.3 6 3.0ej
0.0 6 0.0
0.03 6 0.0
0.01 6 0.0i
0.0 6 0.0
0.0 6 0.0i
0.0 6 0.0
0.02 6 0.0j
0.04 6 0.0c
0.01 6 0.0j
a
The results are the mean 6 SEM (young mean age, 21.0 y; old mean age, 76.1 y).
Units per mg protein.
Young different from old, p < 0.05.
d
Young different from old, p < 0.01.
e
Young different from old, p < 0.001.
f
Forearm different from upper-inner arm, p < 0.05.
g
Forearm different from upper-inner arm, p < 0.01.
h
Epidermis different from dermis, p < 0.05.
i
Epidermis different from dermis, p < 0.01.
j
Epidermis different from dermis, p < 0.001.
b
c
Figure 1. The comparison of epidermal and
dermal antioxidant enzyme activities in the
forearm and upper-inner arm of young and
old volunteers. Values are expressed as a
percentage of the activity in young volunteers
(Table I). (a, b) Epidermis; (c, d) dermis; (a, c)
forearm; (b, d) upper-inner arm. Results are
quoted as mean 6 SEM. ***p < 0.001, **p
< 0.01, *p < 0.05.
Table II. The ratios of the antioxidant enzyme activities of the forearm to the upper-inner arm in aging humana
Epidermisb
Superoxide dismutase
Catalase
Glutathione peroxidase
Glutathione reductase
Dermisc
Young (n = 7)
Old (n = 9)
Young (n = 7)
Old (n = 9)
1.1
0.8
0.1
1.0
0.1
1.1
1.1
0.9
1.1
0.8
1.0
1.0
1.1
1.0
1.1
1.0
6
6
6
6
0.1
0.0
0.1
0.1
6
6
6
6
0.1
0.1d
0.2
0.0
6
6
6
6
0.1
0.1
0.2
0.1
6
6
6
6
0.1
0.1
0.1
0.1
a
The results are the mean 6 SEM (young mean age, 21.0 y; old mean age, 76.1 y).
The ratio of activity of the epidermis of forearm to that of upper-inner arm.
The ratio of activity of the dermis of forearm to that of upper-inner arm.
d
The ratio of young different from old, p < 0.05.
b
c
Nonenzymic antioxidant levels were signi®cantly higher in the
epidermis than the dermis. The concentration of the lipophilic
antioxidant a-tocopherol was signi®cantly higher in the epidermis
than the dermis, by an average of 456% (p < 0.001) in young skin
and 303% (p < 0.01) in old skin. The concentrations of the
hydrophilic antioxidants ascorbic acid, uric acid, and glutathione
were signi®cantly higher in the epidermis, by 227% (p < 0.001),
181% (p < 0.01), and 151% (p < 0.01) for young skin, and by 225%
(p < 0.01), 173% (p < 0.01), and 129% (p < 0.05) for old skin
(Table III), respectively.
ANTIOXIDANTS IN AGED HUMAN SKIN IN VIVO
6
6
6
6
6
6
6
4.6
107.1
219.1
521.8
13.1
250.5
5.5
1.3d
20.1e
32.5
46.2
6.2
49.1
1.4
6
6
6
6
6
6
6
Epidermis
Epidermis
Epidermis
13.1
218.5
380.6
334.8
23.9
358.7
6.7
Epidermis
Forearm
Upper-inner arm
Forearm
6
6
6
6
6
6
6
4.9
102.1
202.9
267.7
15.8
283.5
6.2
1.9d
19.4d
28.8
24.6e
5.7
24.2e
2.1
14.6
252.2
351.1
307.6
18.8
326.4
6.7
6
6
6
6
6
6
6
DISCUSSION
The results are the mean 6 SEM (young mean age, 21.0 y; old mean age, 76.1 y).
nmol per g skin (Wet weight).
Young different from old, p < 0.05.
d
Young different from old, p < 0.01.
e
Young different from old, p < 0.001.
f
Epidermis different from dermis, p < 0.05.
g
Epidermis different from dermis, p < 0.01.
h
Epidermis different from dermis, p < 0.001
c
b
a
a-Tocopherol
Ascorbic acid
Uric acid
Reduced glutathione
Oxidized glutathione
Total glutathione
GSSG/GSH(%)
26.3
363.7
419.0
237.4
28.7
550.5
5.4
6
6
6
6
6
6
6
3.4
24.8
21.7
11.5cf
6.9
43.4
1.2
4.2
162.4
193.2
286.8
19.2
306.0
6.4
6
6
6
6
6
6
6
0.5h
18.6h
24.1h
17.5h
3.2
20.1h
0.8
23.1
354.7
420.5
427.9
19.5
492.3
4.1
6
6
6
6
6
6
6
2.7
23.5
28.7
64.9
4.2
63.7
1.4
3.9
152.0
260.7
286.8
21.7
308.5
7.5
6
6
6
6
6
6
6
00.6h
19.0h
31.4g
17.7g
3.6
19.2g
1.1f
Dermis
Dermis
Dermis
1215
The concentration of a-tocopherol was signi®cantly lower in
photoaged and naturally aged epidermis to 56% (p < 0.01) and 61%
(p < 0.01) of the young skin levels. No signi®cant difference in the
concentration of a-tocopherol, however, was found in the dermis
of young and aged skin. The level of ascorbic acid was lower in
photoaged and aged epidermis to 69% (p < 0.01) and 61% (p
< 0.001) of the corresponding young skin levels, respectively.
Photoaged and naturally aged dermis contained 63% (p < 0.05) and
70% (p < 0.05) of the ascorbic acid levels of young dermis. Uric
acid did not show any signi®cant changes due to the aging process.
The levels of total (GSH + GSSG) glutathione were lower in
photoaged and aged epidermis at 59% (p < 0.001) and 73% of the
young skin levels, respectively. In photoaged and aged dermis, the
total glutathione level was lower at 93% and 81% (p < 0.05) of the
young skin levels, respectively. The ratio of GSSG to GSH did not
show any difference for young and old epidermis, or dermis
(Table III and Fig 2).
Antioxidant concentration changes due to chronic sun exposure
were investigated by comparing the ratio of the concentration of
antioxidant molecules in the forearm skin to that of the inner arm
skin for young and old skin. This ratio did not show any signi®cant
differences between the young and old volunteers (data not
shown).
0.8g
22.8ch
36.1g
21.7
2.5
22.2
1.0
Upper-inner arm
Oldb (n = 9)
Youngb (n = 9)
Table III. Antioxident molecules concentrations in photoaging and aging human skin in vivoa
Dermis
0.7h
10.0c
29.1g
40.7
2.1
11.8cf
0.7
VOL. 117, NO. 5 NOVEMBER 2001
In this study, we demonstrated that the activities of SOD and GPx
were not changed during the aging and photoaging processes in
human skin in vivo. Interestingly, however, the activity of catalase
was signi®cantly higher in the epidermis, whereas it was signi®cantly lower in the dermis, of photoaged and naturally aged human
skin in vivo compared with young skin. GR activity was higher in
naturally aged epidermis. It was also found that the concentration
of the nonenzymic antioxidant a-tocopherol was lower in the
epidermis, but not in the dermis, of photoaged and naturally aged
skin. Ascorbic acid levels were lower in the epidermis and dermis
of photoaged and naturally aged skin. Uric acid levels were similar
in the epidermis and dermis, however, during the photoaging and
the natural aging processes. The concentration of glutathione was
lower in photoaged and naturally aged epidermis and dermis,
although statistical signi®cance was only found in the photoaged
epidermis and aged dermis. To our knowledge, this is the ®rst
comprehensive study of changes in the levels of four major
antioxidant enzymes and nonenzymic antioxidants during the
photoaging and aging processes of human skin in vivo.
Usually, enzymic activity is expressed as units per milligram of
protein to determine the degree of purity during separation and
puri®cation (Hussain et al, 1995; Mo et al, 1995; Pansarasa et al,
1999). Some investigators have suggested that units per gram of
skin would be more reasonable (Shindo et al, 1993; 1994a). LopezTorres et al (1994), however, demonstrated that there was no
signi®cant difference between using these two units of measurement, except for GPx when measuring the antioxidant enzyme
activities in young and old mice skin. It is also possible that many
factors such as biopsy procedures and water accumulation might
introduce artifacts into the determined skin weight values, and
these factors might cause changes in quoted values (Lopez-Torres
et al, 1994). Thus, we expressed enzyme activity in units per
milligram of protein.
As epidermis is directly exposed to various oxidative stresses
from the environment, it is presumed to possess a higher
antioxidant defense capacity than dermis, to maintain the redox
balance of the skin (Lopez-Torres et al, 1994; Shindo et al, 1994a,
b). As expected, we observed that the activities of SOD, catalase,
and GR were higher in the epidermis than the dermis in both
young and aged skin in vivo. The concentrations of the
nonenzymic antioxidants a-tocopherol, ascorbic acid, uric acid,
and glutathione were also higher in the epidermis than in the
dermis of both young and old skin in vivo. Our results are similar to
those of Shindo et al (1994a) in terms of the activities of catalase
1216
RHIE ET AL
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Figure 2. Comparison of the concentrations
of epidermal and dermal antioxidant
molecules in the forearm and upper-inner
arm of young and old volunteers. Values are
expressed as percentages of the concentrations
found in the young volunteers (Table III). (a, b)
Epidermis; (c, d) dermis; (a, c) forearm; (b, d)
upper-inner arm. Data are mean 6 SEM. ***p
< 0.001, **p < 0.01, *p < 0.05.
and GR (expressed as units per mg protein), and of the levels of the
nonenzymic antioxidants a-tocopherol, ascorbic acid, and glutathione, which were all higher in the epidermis than the dermis.
We demonstrated that the antioxidant enzyme catalase is
regulated differently in the epidermis and dermis during the
photoaging and aging processes of skin. The activity of catalase
increased signi®cantly in photoaged and aged epidermis, and
increased more in photoaged epidermis than aged epidermis. This
increased catalase level in the epidermis during the photoaging
process is of interest, as it is known that catalase is destroyed by
visible light (Cheng and Packer, 1979). It has also been reported
that murine epidermal and dermal catalase showed a marked drop
in activity after acute UVA and UVB irradiation (Shindo et al,
1994b). The reason for this increased activity of epidermal catalase
in photoaged and aged human skin is not clear at this point. It is
possible that the induction of catalase in the epidermis may be a
defense response to environmental oxidative stress. On the other
hand, chronic oxidative stress over a lifetime might stimulate the
skin cells to produce certain cytokines that upregulate catalase
expression. Further investigations on this topic are being undertaken.
In contrast to the epidermis, the activity of catalase decreased
signi®cantly in the dermis of photoaged and aged skin, and it
tended to decrease less in the photoaged dermis than in the aged
dermis. As UVR can reach into the upper dermis, cells in the upper
dermis may respond to chronic UV exposure by inducing catalase.
Our quanti®cations of catalase in the dermis are consistent with
those of Shindo et al (1991), who compared skin ®broblasts derived
from old people with those derived from young people. They also
described a remarkable decrease of catalase activity on aging,
whereas SOD and GR remained unchanged. The reason for
decreased catalase activity in the aged dermis is not clear, though it
might be due to the aging process. Our results indicate that catalase
activity may be differently regulated in the epidermis and dermis
during photoaging and intrinsic aging, and that catalase might be a
key enzyme that ful®lls an important role in the protection of skin
from oxidative damage.
The observed effects of aging on antioxidants in human skin
in vivo were quite different from those found in murine skin
(Lopez-Torres et al, 1994). In the murine epidermis and dermis, no
signi®cant differences were found in the activities of SOD, catalase,
and GR (expressed as units per mg protein) of young and old
animals. Only epidermal GPx showed decreased activity with age.
In addition, the hydrophilic (ascorbic acid, glutathione, and uric
acid) and lipophilic (a-tocopherol) antioxidants did not change as a
function of age in mouse skin.
Figure 3. Aging- and photoaging-dependent changes in the
antioxidants of human skin in vivo and their possible roles in skin
aging. In aged and photoaged human skin, the components of the
antioxidant defense system are regulated differently. In the epidermis,
SOD and GPx remain completely unchanged, whereas catalase and GR
tend to increase. In the dermis, of the four antioxidant enzymes
examined, only catalase activity was signi®cantly decreased. Nonenzymic
antioxidants such as a-tocopherol, ascorbic acid, and glutathione were
decreased in the epidermis and/or dermis. The decreased antioxidant
capacity of aged skin may cause an increased accumulation of ROS,
which will affect cell signaling pathways and lead to skin aging.
Ascorbic acid levels were lower in both the epidermis and
dermis, whereas a-tocopherol was lower in the epidermis but not
in the dermis, of both photoaged and aged skin. The reasons for this
difference are not known. a-Tocopherol provides protection
against oxidative membrane damage caused by various environmental factors, such as UV, presumably by scavenging ROS and
free radicals (Burton and Traber, 1990). When skin is exposed to
oxidative stress, such as UVR, a large number of tocophenoxy
radicals are formed by the oxidation of a-tocopherol, and the
antioxidative properties of a-tocopherol are closely linked with its
continual regeneration by other antioxidants such as glutathione
and ascorbic acid (Vessey, 1993). As ascorbate is present in the skin
in much higher amounts than a-tocopherol, it acts as a large
VOL. 117, NO. 5 NOVEMBER 2001
reservoir of antioxidative potential, which may be delivered more
speci®cally by a-tocopherol (Steenvoorden and Beijersbergen van
Henegouwen, 1997). It is also possible that a-tocopherol in the
dermis can be regenerated by other antioxidants supplied by the
blood.
It has been demonstrated that, when exposed to UV radiation,
levels of a-tocopherol, ascorbate, and glutathione in the skin are
depleted (Fuchs et al, 1989; Shindo et al, 1993). As a-tocopherol
and ascorbic acid were decreased in both sun-exposed (forearm)
and sun-protected (upper-inner arm) skin to a similar extent, other
variables, perhaps nutritional factors, rather than chronic UV
irradiation seem to play more important roles in the decrease of
nonenzymic antioxidants in the skin of the elderly.
According to the free radical theory of aging, ROS increases
with aging due to the reduced activity of the antioxidant defense
enzymes (Harman, 1956, 1981, 1986). Our results are compatible
with this free radical theory of aging, as the decreased activity of
catalase in the dermis, and the decreased levels of nonenzymic
antioxidants in epidermis and dermis of photoaged and aged human
skin in vivo, are believed to cause an age-associated increase in
oxidative stress (ROS) in aged skin. Consequently, ROS, such as
hydrogen peroxide, in aged skin may increase and be accumulated;
these ROS will affect signaling pathways and ®nally lead to aged
and photoaged skin in vivo (Fisher et al, 1997; Kang et al, 1997)
(Fig 3). In our laboratory, higher levels of hydrogen peroxide were
found in skin ®broblasts derived from old people than those of
younger subjects (personal observation). Thus, the induction and
regulation of endogenous antioxidant defense mechanisms may
offer a good strategy for the treatment and prevention of aging and
photoaging in human skin.
This study was supported in part by a grant ``Life Phenomena and Function
Research Group Program (2000)'' from the Ministry of Science and Technology,
and a research agreement with the Paci®c Corporation. We thank Mi Ran Kwon for
her excellent technical assistance.
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