ARTICLE
It is now commonly recognized that UVA as well as UVB radiation is involved
in the development of cutaneous cancer. This relation to sun exposure
is particularly clear for squamous cell carcinomas (SCCs) and basal cell
carcinomas (BCCs) in Caucasians [1]. Indeed, the occurrence of cutaneous
cancer is increasing year by year [2] even though the sunscreen market
is increasing. This paradox is probably due to changes in lifestyle [3]
(sun bathing, holidays in sunny countries even in winter...) and to the
delay between cancer induction and its diagnosis. Furthermore, the first
generation of sunscreen products did not provide UVA photoprotection.
The lack of cancer protection by a sunscreen devoid of UVA filtration
was demonstrated, by Wulf et al. [4] in hairless pigmented mice.
Furthermore, the cutaneous SCCs and BCCs carcinogenesis action spectrum
in the albino hairless mouse established by de Gruijl [5] shows two maximum
efficiency wavelengths: one in the UVB and a second in the UVA. The efficiency
at this maximum level is 1,000 times lower than for UVB but this ratio
can be compensated by the higher doses of UVA received. The solar spectrum
comprises approximately 100 to 1,000 times more UVA than UVB radiation.
Moreover, the use of sunbeds strongly increases the cumulative UVA dose
received by some subjects. For these reasons, the cosmetics industry had
to improve sunscreen protection in UVA. Inhibition of immediate pigment
darkening (IPD), the only in vivo macroscopic evidence of UVA exposure,
was chosen to determine UVA protection factors. The other in vivo
methods available have great disadvantages. For example, the method based
on UVA erythema induction requires psoralen [6] and thus is not ethically
acceptable because these compounds are known to interact with DNA. Protection
factors obtained from artificially sensitized skin are probably not relevant
to those determined with normal skin. The pigmentation method is a reproducible
and useful end point for the routine assessment of photoprotection of
normal skin against UVA [7]. However, very little information is available
about the nature and mechanism of this phenomenon. Thus, we felt it of
value to review the literature concerning this question.
Immediate pigment darkening was first observed in 1902 by Meirowski
[8] and was further studied by Guthman in 1927 [9]. As its biological
role remained unrecognized, IPD was neglected by reseachers for nearly
twenty years, between 1960 and 1980.
Description
Clinical description
IPD is a transitory darkening of the skin occurring immediately after
UVA exposure. IPD appears as a grey-brown pigmentation around the swimsuit
after the beach.
Histological description
Jimbow et al. [10] studied melanocyte and keratinocyte ultrastructural
changes occurring during IPD in 19 volunteers (Caucasians, Negroids and
Mongoloids equally represented). The irradiation lamp was a 150 W Xenon
lamp filtered to eliminate radiations bellow 340 nm. The subjects were
exposed for 20 min and received 21.25 J/cm2. A biopsy was performed
on the irradiated area and was studied by electron microscopy. They noticed:
a migration of microtubule and filaments to melanocyte dendrites
[10-12],
an increase in melanosomal dentritic transfer to keratinocytes
[10, 14],
a migration of melanosomes towards nucleous and melanocyte dendrites
[10-12],
no increase in the number of melanocytes [10],
an increase in the number of premature melanosome [10, 13],
DOPA positive reaction in melanocytes [10] showing a tyrosinase
activation.
Other authors did not observe any notable structural changes during
IPD nor tyrosinase activity [13, 15, 16]. Furthermore, Meirowski observed
IPD on dead bodies, suggesting that IPD was (partly) a photochemical reaction
involving substances already present in the epidermis. Photo-oxidation
of melanin should occur, leading to the formation of quinone moiety on
melanin polymer, thus inducing a red-shift in their absorption spectrum
[17-19].
Involvement of melanin precursors has been suggested [17] and could
account for the absence of correlation between subject skin color and
minimal immediate pigment darkening dose (MIPDD) because they are colorless.
However this hypothesis has never been definitely confirmed.
Kinetics
Chardon [20] studied the appearance and disappearance kinetics of IPD
in 15 Caucasian subjects with light or intermediate skin (defined by chromametric
measurement [21]) irradiated with UVASUN-5,000 lamp. The appearance of
IPD induces some notable variations in skin luminance "L*" and to a lesser
extent variations in the red (a*) and the yellow (b*) components of skin
color (chromametry). Figure 1
[20] shows luminosity evolution as a function of the time for different
UVA doses.
deltaL* (L*unexposed skin L*exposed skin)
is maximum immediately after the end of exposure and decreases rapidly
over 2 hrs. The evolution then slows down. Seven days after exposure,
L* is stabilized at a value slightly inferior to the initial one accounting
for the emergence of delayed UVA tanning.
Dose-response curves
Chardon et al. [20] exposed 19 Caucasian healthy volunteers to
a series of 9 UVA exposures (from 0.3 to 16 J/cm2). The color
of the skin was monitored by chromametry. The dose-response curves (skin
luminance variations deltaL* as a function of the UVA dose received) are
linear for doses higher than 4 J/cm2 and non linear for smaller
ones [20]. The critical value of 4 J/cm2 is close to MIPDD
for subjects with very dark skin.
Action spectrum
The first IPD induction action spectrum reported in the literature showed
an increase in efficiency from 300 to 450 nm and a decrease at higher
wavelengths [22]. More recently, Irwin et al. [23] reexamined this
action spectrum in the UVA region. Thirty volunteers with phototype III,
IV, V were exposed to 10 nm bandwidth UVA radiation. MIPDD was determined
for each subject and each band between 310 and 400 nm. The spectrum obtained
(Fig. 2 [23]) shows a
maximum induction efficiency at 340 nm.
In a similar study involving Japanese volunteers irradiated with a monochomator,
Kawada [24] estimated the maximum efficiency wavelength at 320 nm. This
value is 20 nm above that observed by Irwin. This shift could be due to
some specificity of mongoloid skin. Furthermore, Kawada investigated 4
wavelengths with an interval of 20 nm (320, 340, 360 nm) thus the precision
on the obtained value is of 20 nm range. Rosen et al. [25] examined
longwave UVA induction of IPD and concluded that it was maximum below
365 nm. This confirms Irwin's conclusions.
Variations
Influence of oxygen concentration in tissue on IPD
The dependence of IPD on oxygen concentration was first described by
Henschke in 1939 [26]. Later, Tegner et al. [27] reported a hypopigmentation
on compressed (thus hypoxic) zones of subjects irradiated on sunbeds [28,
29]. This effect was inhibited by local application of hydrogen peroxide.
A quantitative study of the influence of oxygen on IPD was made by Auletta
et al. [30] on 20 volunteers. The forearm was exposed to UVA and
oxygen pressure was monitored using a sphygmomanometer cuff and a Clark
detector. Minimum induction doses of IPD and delayed tanning increased
with the imposed pressure (Fig.
3) [30]. No similar influence was noted for UVB pigmentation and
erythema [30]. IPD inhibition in the absence of oxygen suggests the oxidative
nature of the reactions involved.
Flux of the irradiation source
Kaidbey et Barnes showed [31] that the flux of the light does not influence
the value of MIPDD source if it is lower than 50 mW/cm2. Césarini
[32] made similar observations for flux between 7.5 and 66 mW/cm2
(Table I) [32]. On the
other hand, Kagetsu et al. [33] observed an important increase
in the value of MIPDD for very low flux (< 10 mW/cm2) (Table
I). That means that the reciprocity law (the same physiological
response is obtained with the same UV dose, regardless of flux and time)
for the IPD is not respected. Given that the UVA solar flux on June 21st
1987 in Toulon (6.8 mW/cm2, 43°07'N, South of France)
and La Baule (5.5 mW/cm2, 47°17'N, North of France) [34]
are far from the Xenon light source flux (> 20 mW/cm2),
it is doubtful whether sunscreen UVA protection could be forecast by an
IPD protection factor method.
Individual variation of
MIPDD
Phototype and MIPDD. Some authors have found no IPD response
in phototype I and II [35], while others have reported IPD visible for
all skin types [27]. In an extensive study (1,300 Caucasian volunteers),
Poh Agin et al. [36] found that 30% of volunteers with phototype
I do not develop IPD and 64% required equivalent or greater energy than
their minimal erythemal dose (MED) to produce IPD. As far as ethics are
concerned, it is not acceptable to determine UVA protecting factor on
phototype I subjects using the IPD in vivo method since the UVA
dose required would be extremely high. This is a paradox because light
skins are probably those which need an adapted UVA photoprotection the
most. Indeed, these subjects, erythema sensitive, tend to use high sun
protective factor products which allow a longer sun exposure without burning.
Among phototype II subjects [36], 3.5% did not develop IPD and the proportion
was 0.8% for phototype III and 0% for phototype IV. The absence of IPD
in phototype II and III occurred mostly among subjects with green or blue
eyes (87%).
The higher the phototype is, the lower the MIPDD and the higher the
intensity of pigment darkening will be [36, 37] (Table
II). MIPDD values are clearly higher for phototype II than for
phototype IV [36, 37, 41]. Average values of MIPDD for a given phototype
differ, according to the authors, probably owing to variations in visual
assessments, source spectra and dosimetry. This is particularly flagrant
for authors in reference [36] who used a radiometer with a large spectrum
band path.
Melanotype and MIPDD. Classification of Caucasian subjects into
6 melanotypes in order to predict their solar reactions has been used
by Césarini [32, 38]. The melanotype of the subject depends on
his constitutive skin color, tanned skin color, eye and hair color, presence
of freckles, ability to tan and is determined by interviewing the subject.
Césarini failed to demonstrate a better correlation MIPDD-group
than with the usual phototypes described by Fitzpatrick's classification
[39].
MED, skin color and MIPDD. The study previously cited [32] showed
the absence of correlation between MED and MIPDD. The same conclusion
was made for Thaï subjects of phototype III, IV and V [40]. More
surprisingly, no correlation between skin color and MIPDD was found in
this same study (skin color was defined as the melanin index determined
by spectroreflectometry).
On the contrary, classification of Caucasian subjects using chromametrically
defined skin color allows a good color-MIPDD correlation (Table
III, according to [21]). MIPDD is higher for light skin.
Biological role of IPD
IPD and delayed UVA tanning
Descamps et al. [41] studied delayed UVA tanning in 10 Caucasian
subjects irradiated with UVA on two areas, one of them being subjected
to hypoxia by skin compression, which did not develop IPD. The mimimal
dose for induction of delayed UVA tanning was higher (average + 124%)
for 8 subjects out of 10 on the hypoxic zone. According to these results
it is possible that IPD may be implicated in the appearance of delayed
UVA-tanning.
IPD and erythemal protection
Black et al. [42] exposed 11 volunteers with phototype II and
III to UVA followed by UVB three hours later. In 8 subjects out of 11
pre-exposure to UVA increases MED (average 25%). Conversely, Kaidbey et
Kligman [43] did not observe any IPD protection against UVB-erythema.
CONCLUSION
The cellular mechanism of immediate pigment darkening is still poorly
understood. It seems to be related to a spatial rearrangement of melanosomes
in keratinocytes and melanocytes and a photo-oxidation of pre-existing
melanin. IPD is inhibited in the absence of oxygen. The maximum induction
efficiency wavelength is about 340 nm. Dose-response curves are linear
for doses superior to 4 J/cm2 and a MIPDD-flux dependence has
been observed.
This implies some caution when using IPD for the determination of UVA
protection factor: irradiation of subjects must be carried out without
imposing any pressure on the skin during exposure to the solar simulator.
Doses provided must be situated in the dose-response linearity zone. IPD
dependence on the source fluence can be at the origin of some inconsistency
between indoor and outdoor protection factor measurements. However, it
has been studied by very few researchers, using different lamps and dosimetry
so that the given flux values could not be compared directly. Furthermore,
few flux have been studied above the supposed critical value of 10 mW/cm2.
More information is needed before drawing any conclusions. No clear correlation
between MIPDD and phototype, melanotype and UVB-MED was observed. Indeed,
phototype or melanotype discrimination are essentially based on erythemal
reactions of the subject and are not the most suitable for IPD experiments.
Skin color (defined by chromametry) seems to be the best predictive criterion
for MIPDD values. A possible role for IPD development could be the activation
of melanogenesis in so far as IPD seems to promote UVA-delayed tanning.
The biological role of IPD has not yet been clearly defined but one
obviously exists. An accessory side effect, photo-oxidation of melanin,
could be a realistic hypothesis but ultrastructural changes must be linked
to a well defined-process.
Concerning UVA protection factor evaluated in humans, IPD method is
a simple reproducible method for routine UVA photoprotection assessment.
It gives good indications of the UVA absorption properties of a sunscreen.
Nevertheless, the aim of UVA photoprotection is not to inhibit IPD as
a biological phenomenon. So, this method does not give actual information
about a "tumor protection factor" which would be one of the main parameters
to evaluate sunscreen efficacy. Of course it is unthinkable to perform
a test on this protection factor in vivo in humans.
Different parameters deduced from methods such as inhibition of photo-immunosuppression
in vivo, DNA photoprotection (comet assay) and spectral analysis
of sunscreens (in vitro), are probably the first steps in a long
process to forecast UVA photoprotection with an actual "tumor protection
factor".
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