Journal of Photochemistry and Photobiology B: Biology 138 (2014) 347–354
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology B: Biology
journal homepage: www.elsevier.com/locate/jphotobiol
Effects of water-filtered infrared-A and of heat on cell death,
inflammation, antioxidative potential and of free radical formation
in viable skin – First results
Helmut Piazena a,⇑, Wolfgang Pittermann b, Werner Müller c, Katinka Jung d, Debra K. Kelleher e,
Thomas Herrling d, Peter Meffert f, Ralf Uebelhack a, Manfred Kietzmann g
a
Charité – University Medicine Berlin, Medical Photobiology Group, Berlin, Germany
Charité – University Medicine Berlin, Medical Photobiology Group, Düsseldorf, Germany
c
Charité – University Medicine Berlin, Medical Photobiology Group, Wetzlar, Germany
d
Gematria-Test Lab, Berlin, Germany
e
University Medical Centre Mainz, Institute of Functional and Clinical Anatomy, Mainz, Germany
f
Ernst Moritz Arndt University of Greifswald, Institute for Community Medicine, Greifswald, Germany
g
University of Veterinary Medicine, Department of Pharmacology, Toxicology and Pharmacy, Hannover, Germany
b
a r t i c l e
i n f o
Article history:
Received 18 February 2014
Received in revised form 27 May 2014
Accepted 3 June 2014
Available online 30 June 2014
Keywords:
Water-filtered infrared-A (wIRA)
Solar irradiance
Heat
In vitro skin
Bovine Udder System (BUS) model
Cell death
Inflammation
Antioxidative potential
Free radicals
a b s t r a c t
The effects of water-filtered infrared-A (wIRA) and of convective heat on viability, inflammation, inducible free radicals and antioxidative power were investigated in natural and viable skin using the ex vivo
Bovine Udder System (BUS) model. Therefore, skin samples from differently treated parts of the udder of
a healthy cow were analyzed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) test, by prostaglandin E2 (PGE2) measurement and by electron spin resonance (ESR) spectroscopy.
Neither cell viability, the inflammation status, the radical status or the antioxidative defence systems of
the skin were significantly affected by wIRA applied within 30 min by using an irradiance of 1900 W m2
which is of relevance for clinical use, but which exceeded the maximum solar IR-A irradiance at the
Earth’s surface more than 5 times and which resulted in a skin surface temperature of about 45 °C without cooling and of about 37 °C with convective cooling by air ventilation. No significant effects on viability and on inflammation were detected when convective heat was applied alone under equivalent
conditions in terms of the resulting skin surface temperatures and exposure time. As compared with
untreated skin, free radical formation was almost doubled, whereas the antioxidative power was reduced
to about 50% after convective heating to about 45 °C.
Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction
Overexposure of skin to short-wavelength infrared radiation
(IR-A, 0.78–1.4 lm) as well as to IR radiation with longer wavelengths (IR-B, 1.4–3.0 lm; IR-C, 3.0 lm–1.0 mm) may cause a
number of unwanted thermal effects such as heat pain, skin burning, thermal urticaria, erythema ab igne and squamous cell carcinoma, so that limitations of irradiance, duration and frequency of
application are warranted [1–7].
In order to achieve an effective reduction in the thermal impact
to the skin, while at the same time enabling the desired thermal
and non-thermal effects, water-filtered infrared-A (wIRA) devices
were developed for therapy and wellness purposes. These devices
⇑ Corresponding author. Tel.: +49 30 97889320, +49 151 17390149.
E-mail address: [email protected] (H. Piazena).
http://dx.doi.org/10.1016/j.jphotobiol.2014.06.007
1011-1344/Ó 2014 Elsevier B.V. All rights reserved.
show a similar spectral distribution to that of solar IR irradiance
at the Earth’s surface at sea level under a cloudless sky at noon during summer in the mid-latitudes or the tropics when the radiation
is filtered by the water vapor and water drops present in the troposphere. Thus, both solar irradiance at the Earth’s surface and the
spectra of wIRA are limited to wavelengths of up to about
2000 nm and show local minima in the emission spectra at wavelengths around the water and water vapor absorption bands.
Today, wIRA devices are being successfully used to apply local
or systemic hyperthermia for therapeutic purposes [8–11]. Skin
exposures of high or moderate irradiance which result in only negligible or limited tissue warming can be used in wound healing and
for the stimulation of regenerative cellular processes due to nonthermal mechanisms [12–23].
In contrast, applications of solar radiation are limited by the
ultraviolet (UV) radiation component in the spectrum, and
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therefore protective measures – such as a limitation of exposure
time – are necessary to prevent unwanted UV-related effects. Further limitations arise due to the fact that the solar IR irradiance at
the Earth’s surface is less than about 411 W m2 which was measured extraterrestrially [24]. As a result, the maximum solar IR
irradiance measured at the Earth’s surface in the mid-latitudes at
noontime during summer ranges from values between about
200 W m2 and 250 W m2, whereby maxima of about
350 W m2 can be detected in the tropics [24].
Several articles published over the past years have suggested a
possible intensification and acceleration of skin aging being caused
by IR-A exposure due to direct molecular interaction and formation
of free radicals (see [24]). However, many of these articles presented data which were derived from in vitro and ex vivo experiments with partially erroneous methods, and thus resulted in
unfounded conclusions as a result of the authors overlooking the
occurrence of thermal effects in the exposed sample, the erroneous
extrapolation to the optical and thermal conditions in living tissue
and due to the unfounded use of physical and photobiological basic
laws (for a detailed discussion see [24]). Moreover, some authors
extrapolated their results to the conditions of solar IR skin exposures and recommended active measures such as sunscreens ‘‘with
IR-A protective additions’’ to prevent photo-aging of skin due to
cellular effects and generation of free radicals [25–29]. A discussion of these articles is appropriate since some investigators have
reported possible protective effects of solar IR radiation against
damage caused by the solar UV component, and this could be relevant for an update of the current recommendations and measures
concerning solar UV skin protection [30–38].
Most of the studies as already criticized by Piazena and Kelleher
[24] were performed using in vitro or ex vivo experiments in which
no temperature control of the exposed tissue was undertaken and
in which irradiances of IR-A or wIRA up to 2–7 times higher than
extraterrestrial solar IR irradiance were implemented. The aim of
this study was therefore to determine and to separate the individual acute effects of wIRA and of heat on cell death (cytotoxicity),
inflammation, antioxidative potential and formation of free radicals. Experiments were carried out using the Bovine Udder System
(BUS) which is an accepted ex vivo model of normal aerobic metabolism and is frequently used for testing the impact of physical and
chemical agents on human skin [39–43]. This investigation is a
continuation of a study by Jung et al. [44] who demonstrated that
the generation of reactive oxygen species in dermal fibroblasts was
dependent on heat formation since even an extremely high
wIRA-irradiance of 2450 W m2 failed to result in an increase in
the generation of reactive oxygen species in the absence of heating.
2. Materials and methods
Table 1
Changes in formazan content and in the concentration of prostaglandin-E2 (PGE2) in
untreated tissue of the BUS model (controls) within the periods of 0.5–1 h and 0.5–5 h
according to [47–50].
Number of tested
udders (n)
Perfusion
period (h)
Decrease in
formazan (%)
Increase in
PGE2 (%)
44
44
0.5–1.0
0.5–5.0
1.66 ± 0.44
3.96 ± 1.70
1.27 ± 0.44
0.94 ± 1.99
radiation of different spectra and irradiances showed that all physiological effects such as changes in the steady state surface temperature, appearance of IR-related skin erythema and systemic
regulation were seen within 30 min of treatment [24,46]. It should
however be noted that some of the studies mentioned above
exceeded this time by up to 3 h [24].
In order to assess the effects of wIRA and of heat on cell death
and inflammation, a reference value was first obtained in the form
of a whole skin biopsy (6 mm in diameter and approx. 45.5 mg of
weight, Stiefel Inc., Offenbach, Germany) prior to treatment. Further biopsies were taken 15 min after exposure and immediately
after the end of the 30 min exposure. The values for untreated skin
were found to be in accordance with the results obtained previously in a cohort of 44 untreated BUS skin models [47–50]. As
shown in Table 1, the content of formazan (measured in ng of formazan/lg net weight of tissue) was almost unchanged, as was the
concentration of PGE2 (measured in ng of PGE2/lg net weight of
tissue). The upper limits for these changes (mean values + standard
deviations) were about 2.0% for the decrease in formazan content
within the perfusion period 0.5–1.0 h and about 5.6% within the
period 0.5–5.0 h. Upper limits of the increase in tissue concentration of PGE2 in untreated skin were about 1.7% within the first period and up to about 4% within the period to about 5 h after
commencement of treatment [47–50]. So the historical upper limits of formazan content as well as of the tissue concentration of
PGE2 were 62% each during the relevant time period of 0.5–1.0 h.
On the basis of the values obtained for untreated tissue, treatment-induced changes in the content of formazan or concentration
of PGE2 detected in this study can be assumed to be caused by
wIRA or by heat when the changes found significantly exceed the
upper limits for the untreated tissue.
For analysis of the effects of treatment on free radical formation
and the antioxidative potential, further skin samples of
5 cm 5 cm were taken from the treated area, and for reference,
additionally from an untreated area of the udder, immediately
after each treatment. Untreated skin showed a surface temperature
of 26–27 °C during the total period of all experiments (for details of
skin temperature measurements see below). All skin samples were
immediately frosted in dry ice at 20 °C before laboratory analysis
which was performed within 24 h after the experiments.
2.1. Procedure
2.2. Ex vivo BUS model
To analyze the effects of wIRA and/or of heat on cell death,
inflammation, formation of free radicals and antioxidative power,
different skin areas of the isolated perfused BUS ex vivo model were
successively exposed to wIRA with and without regulation of skin
surface temperature, as well as to heated air flow which solely
transferred convective heat to the skin alone. Experiments were
limited to the use of one udder only which was included into the
BUS model (see Table 1: historical data). All exposures were performed within 1–5 h after the cow was slaughtered. An exposure
time of 15 or 30 min for each treatment was chosen, which is in
accordance with the maximum exposure times of such schedules
with the recommendations for moderate IR therapy, moderate
hyperthermia and for mild whole-body hyperthermia without
thermoregulatory stress as classified by Heckel and von Ardenne
[45]. Additionally, similar exposures of healthy volunteers to IR
Preparation of the udder (total application area: approx.
600 cm2/side) was performed according to Kietzmann et al. [40].
For this, the left and right external pudendal udder arteries of a
healthy cow which had been slaughtered immediately beforehand
were cannulated and infused with heparinized Tyrode’s solution
(7.99 g/l NaCl, 1.09 g/l GlucosexH2O, 1 g/l NaHCO3, 0.26 g/l CaCl2,2H2O, 0.21 g/l MgCl2x6H2O, 0.2 g/l KCl, 0.065 g/l NaH2PO4x2H2O).
The organ was then transported to the laboratory where it was
immediately perfused with oxygenated Tyrode’s solution without
dextran using a peristaltic pump (Masterflex 7553-75; Cole-Palmer
Instruments Co., Vernon Hills, IL, USA). The temperature of the
perfusion fluid was 38.5 °C and the perfused flux was adjusted to
100 ml min1 per side into the A. pudenda ext. of the left/right
sides. Perfusion commenced within 60 min after slaughtering. In
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H. Piazena et al. / Journal of Photochemistry and Photobiology B: Biology 138 (2014) 347–354
this way, both the viability and oxidative metabolism of the isolated udder could be kept stable for about 5 h. This was confirmed
clinically and by whole skin biopsies during the experiment.
Two different methods (cytotoxicity assay and the tissue concentration of eicosanoids) were used for the characterization of
the ‘‘irritation potential’’ and were analyzed in whole skin biopsies
(diameter = 6 mm). The MTT test (methyl tetrazolium salt dye
conversion) indicates the degree of mitochondria impairment in
the cells of the epidermal and dermal layers. The results of the
assessment of prostaglandin E2 induction (inflammation) reveal
the concentration of a mediator substance in the tissue which is
responsible for erythema and edema formation in cases of visible
skin irritation [51,52].
2.3. Treatments, experimental set up and conditions
Four different treatments were applied strictly separated to different areas of the BUS skin model. The treatments were applied consecutively using the following experimental setups (see also Fig. 1):
(1) wIRA exposure to an irradiance of 1900 W m2 with no cooling of the irradiated skin area and resulting in an increase in
skin surface temperature from 27 °C to 45 °C.
(2) wIRA exposure to an irradiance of 1900 W m2 with synchronous cooling of irradiated skin by air ventilation, resulting in an increase in skin surface temperature from 27 °C to
37 °C.
(3) No irradiation of the skin, but convective heating up to a surface temperature of 37 °C using a fan heater.
(4) No irradiation of the skin, but convective heating up to a surface temperature of 45 °C using a fan heater.
Exposure of the skin to wIRA was performed using an irradiator
of type Photodyn 750 (Hydrosun, Müllheim, Germany) which was
equipped with a type RG 780/3 filter (Schott, Mainz, Germany).
In order to ensure relevance for full-body application, the diameter
of each skin area exceeded 10 cm. To prevent cross-talks or superposing effects, skin areas were separated from each other to be
completely outside of the areas which were previously treated
which was controlled by temperature-imaging of the udder surface
using an infrared camera (type VARIOSCAN high resolution, model
3021, Jenoptik, Jena, Germany) (see also Fig. 1).
The spectral distribution of the wIRA irradiance was measured
using a temperature-stabilized double-monochromator spectroradiometer (type SPECTRO 320 D, Instrument Systems, Munich, Germany) with an Ulbricht sphere as the optical entrance window. As
shown in Fig. 2, the spectral slope was in the range of IR-A and
comparable to the solar spectrum depicted, whereby a wIRA irradiance of 1900 W m2 was chosen which was about 6 times higher
than the solar IR-A irradiance shown in the figure, as measured
during summer in the subtropics at noon time, at sea level and
under a cloudless sky. A range of wIRA exposures was extended
to the levels of heat pain induction in humans [24] so that IR exposures causing unfavourable effects are included.
A regular room fan was used for cooling the skin area exposed
in experiment 2, and a fan heater for warming the skin in the
experiments 3 and 4.
Skin surface temperatures in both the untreated reference areas
and in the treated areas were measured during experiments as a
function of exposure time using an infrared thermometer (type
FLUKE 574, Fluke Corp., Everett, USA). Measurements were performed considering the recommended distance between skin and
the optical entrance window of the infrared thermometer in order
to ensure correct temperature sampling within the treated area.
The homogeneity of temperature distribution within treated areas
was assessed during exposure and prior to the collection of biopsies using the infrared camera described above. Fig. 3 shows data
for the skin surface temperature which increased during experiment 1 from about 27 °C to about 45 °C (wIRA treatment without
cooling) and during experiment 2 from about 27 °C to about
37 °C (wIRA treatment with convective cooling of skin). Thus, the
target temperatures were achieved only during the last parts of
the experiments 1 and 2, whereas the target temperature values
were achieved within the first few minutes in experiments 3
and 4 (Fig. 3). Thus due to these differences, the comparison of
data between experiments 1 and 4, and experiments 2 and 3 is
limited.
2.4. Laboratory analysis of biopsies
2.4.1. Formazan and PGE2
The contents of formazan and the concentration of prostaglandin E2 in the tissue were determined according to [47,51,52]. Both
variables were independent of one another.
Spectral irradiance [W m -2 nm -1]
6
1
1: Photodyn 750
(with RG 780/3)
5
2: solar irradiance
Tenerife, 4.6.2001
sea level
cloudless sky
noon ( γ = 82 o)
4
3
2
2
1
0
Fig. 1. Experimental setup for the exposure of the BUS model to wIRA or convective
heat at the SIMRED laboratory, Großburgwedel, Germany: The udder was perfused
with heparinized Tyrode’s solution at 38.5 °C, wIRA exposure by using an IR
radiator, type Photodyn 750 (Hydrosun, Müllheim, Germany) equipped with a longpass filter, type RG 780/3 (Schott, Mainz, Germany), and convective heating of udder
skin by using a fan heater. Also shown: infrared camera image with temperature
distribution of the exposed skin area. The red spot seen at the centre of the exposed
area is due to a reference biopsy taken before the start of treatment.
200
500
1000
1500
2000
Wavelength [nm]
Fig. 2. Spectral irradiance of the wIRA radiator (type Photodyn 750) with long-pass
filter (type RG 780/3) and of the sun as a function of wavelength. Solar spectral
irradiance was measured on the island of Tenerife (Spain) on 4 June 2001 during
noon time, at sea level and under a cloudless sky (solar elevation angle above
horizon 82°).
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spectrum. Over a time interval of 5 min, 6 data points were measured and the k-factor (reaction velocity) of the reduction curve
was calculated as k1 for the unprotected skin. The calculation of
the RS (in (%)) was performed by analyzing the data from untreated
and treated skin samples according to the ratio between the number of inducible free radicals in treated skin (NFR,t) and the number
of inducible free radicals in untreated skin (NFR,u):
o
Skin surface temperature [ C]
50
4
45
1
40
3
RS ¼ ðNFR;t =NFR;u Þ 100:
35
2
ð1Þ
Thus, the radical status of untreated skin was defined as
RS = 100%.
30
2.7. Determination of the skin’s antioxidative power (SAP)
25
0
5
10
15
20
25
30
Exposure time [min]
Fig. 3. Skin surface temperature of treated areas of the udder as a function of
exposure time, experiment 1: wIRA exposure without cooling, experiment 2: wIRA
exposure with convective cooling, experiment 3: convective heating to 37 °C and
experiment 4: convective heating to 45 °C (both by using a room heat blower).
Temperatures were measured in the central part of the skin area treated, which
showed a homogeneous temperature distribution as verified by infrared temperature imaging. Whole skin biopsies were taken after 15 and 30 min for the
assessment of cytotoxicity and inflammation.
2.5. Radical status and antioxidative power of the skin
To determine the amount of inducible free radicals (RS) and the
antioxidative power (SAP), samples (5 5 cm) were taken from the
udder and cut into 1 1 cm pieces. The subdermal fat was
removed and the pieces of skin stored at 20 °C prior to analysis
which took place within 24 h. For this, the skin pieces were placed
in cell culture dishes (epidermal side up and in immediate contact
with air) on filter paper soaked in PBS solution containing a
nitroxyl probe as the free radical probe. Skin samples were kept
in the dark for 10 min. A punch biopsy of 4 mm diameter was taken
and inserted into the ESR tissue chamber where the spin probe
concentration was determined before and after various UV doses
by ESR spectroscopy. Detection of radical species was performed
using an X-band ESR spectrometer type MINISCOPE 300 (Magnettech, Berlin, Germany), which was equipped with a special tissue
chamber for skin measurements. Each value resulted from a minimum of 5 independent measurements from 5 different skin biopsy
samples. The amount of inducible free radicals and the antioxidative power was expressed in percentages, relative to the respective
untreated control area values. The medians and 95th percentiles
(P95) were calculated. The methodical experimental error of the
data was approximately ±5%.
2.6. Determination of the radical status (RS)
The skin’s free radical system exhibited a sensitive interaction
with external and internal impacts. This response was used to
characterize the radical status (RS), which was defined as a normalized ratio [53]. The determination of RS started with the generation of free radicals by a defined number of photons characterized
by irradiance and exposure time of UV radiation, followed by the
measurement of the radical response of the skin. The skin was
marked with the radical indicator nitroxide 2,2,5,5 tetramethylpyrrolidine-N-oxyl (PCA) which was obtained from Sigma–Aldrich,
Munich, Germany, at the highest grade of purity available. This
particular spin probe was chosen because of its stability in biological tissues and its photostability. The various free radicals (e.g.,
O
2 , OH , L , LO , LOO ) were generated by UV irradiation, and these
in turn reacted with PCA and decreased the amplitude of its ESR
Due to the enzymatic and non-enzymatic antioxidant systems
present in the epidermis which make up the skin’s antioxidative
power (SAP), the skin has an intrinsic antioxidative protection. This
variable – which quantitatively defines the antioxidant activity
inside the epidermal and dermal layers of the skin – was measured
using ESR spectroscopy [54]. For this, the skin was labeled with the
spin marker TEMPO (2,2,6,6 tetramethylpiperidine-N-oxyl),
obtained from Sigma–Aldrich, Munich, Germany, at the highest
purity grade available. This particular spin probe is reduced by
the non-enzymatic antioxidants of the skin (i.e. tocopherols, carotinoids, glutathione, etc.) and it is therefore suitable to detect the
activity of the antioxidative systems of the skin. Each of the punch
biopsies were individually inserted into an ESR tissue cell and the
decay of signal intensity SR (t) of a semi-stable organic free radical
was monitored for tm = 10 min. The resulting data were fitted
mono-exponentially according to:
SR ðtÞ ¼ SR0 ðt0 Þ þ A expðkR tÞ
ð2Þ
where SR = signal intensity of the test radical (arbitrary units),
SR0 = signal intensity of the test radical at t = 0, t = time during monitoring (0 6 t 6 tm), A 1 and kR = the characteristic constant of
decay. SAP was defined by both the kinetic parameter of the treated
skin sample kRt and of the untreated sample kRu by:
SAP ¼ ðkRt =kRu Þ 100:
ð3Þ
Thus, the SAP was equivalent to 100% for untreated skin samples. In the cases of SAP > 100%, the topically applied agents were
able to penetrate and were effective against free radical injury in
the skin. A SAP value of below 100% indicated that the topical
treatment (chemical or physical stress such as sun exposure
or detergents) had diminished the skin’s antioxidant defence
system.
2.8. Statistic analysis
The one-way ANOVA method with Scheffé correction for multiple tests was used to assess significance of the differences observed
for data of the radical status (RS) and of the antioxidative power
(SAP) between the experiments 1–4.
3. Results
3.1. Cell death (cytotoxicity)
When compared with the values obtained for the untreated reference samples and the historical data, the results presented in
Fig. 4 indicate a decrease in the relative number of living cells in
the tissue with increasing exposure time and a dependency of
the extent of the decrease on the treatment applied. With each
treatment, the decreases were almost doubled by the extension
of the exposure time from 15 min to 30 min. At a first glance, the
use of wIRA with or without cooling seems to be more cytotoxic
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H. Piazena et al. / Journal of Photochemistry and Photobiology B: Biology 138 (2014) 347–354
20
threshold of biological relevance
15
wIRA & temperature up to
Change of PGE2 [%]
than the use of heat alone. The maximum decrease of about 7.7%
was seen at the end of experiment 2 in which the skin surface temperature was regulated to a maximum of 37 °C, whereas wIRA
exposure without temperature control and a skin surface temperature of up to 45 °C resulted in a decrease of about 5.5% at the end
of experiment 1. In contrast, decreases in living cells of about 2.2%
were found at the end of convective heating of the skin surface to
about 37 °C (experiment 3) and of about 4.4% in the case of convectively heated skin surface to about 45 °C (experiment 4). However,
as noted in Table 1, the spontaneous decrease in living cells as
detected in untreated tissue was found to be up to about 2% during
the perfusion period of 30 min at the beginning of the experiments.
The observed rates of deviation may indicate a cytotoxic potential of the wIRA exposure compared with the results of the convectively heated skin. It must however be noted that these changes are
not biologically or clinically relevant, since after such a short exposure period the threshold of relevance is generally about 15% or
more [52]. With this in mind, the changes in the number of living
cells as detected in all experiments in this study were below the
threshold of minimal cytotoxicity and therefore of no biological
relevance.
10
convective heating to
o
o
45 C
5
37 C
untreated
samples
o
o
37 C
45 C
0
a
-5
b
exposure time
-10
a: 15 min
b: 30 min
-15
threshold of biological relevance
-20
0
1
2
3
4
Experiment
Fig. 5. Changes in prostaglandin-E2 (PGE2) concentration after treatment of 15 min
(a) and of 30 min (b) duration in the experiments 1–4 as compared to reference
samples taken before the start of experiments (experiment 0). The biologically
relevant threshold was marked at a change increasing the total of |±15%| according
to [52].
3.2. Inflammation
Tissue inflammation correlates with an increase in the concentration of prostaglandin-E2 (PGE2) [51,52]. The threshold of inflammation was defined as an increase in the concentration of PGE2 of
more than 20% after short periods of exposure of the order of about
30 min [52]. As seen in Fig. 5, the changes in PGE2 concentration
found during or after wIRA exposure with or without temperature
control were all below 1.7%, so that the PGE2 concentrations did
not differ from the reference values by more than 2.0% (see
Fig. 5, experiments 1 and 2).
When convective heating of the skin was applied, an increase of
1.8% and a decrease of 5.4% in the PGE2 concentration were found
in experiments 3 and 4 respectively. These changes exceeded the
range of variability of PGE2 levels in untreated tissue as noted in
Table 1 by 1.27 ± 0.44% during the perfusion period of 0.5–1.0 h
to an irrelevant extent. It should be noted that convective heating
up to 45 °C induced irregular cellular conditions and reactions
which differed from the results obtained by wIRA-induced heating
up to 45 °C. However regarding inflammation, neither wIRA exposure up to 1900 W m2, nor convective heating of the tissue to
about 45 °C resulted in significant differences within the chosen
exposure time.
3.3. Radical status (RS)
According to the data in Fig. 6, the amount of inducible free radicals quantified by RS increased from a median of 100% and from
P95 = 105% in untreated samples at a surface temperature of 26–
27 °C to a median of 198% (P95 = 212%) following convective heating of the skin surface to 45 °C for 30 min (experiment 4). In contrast, the formation of inducible free radicals increased to a
median of RS of 117% (P95 = 126%) after wIRA treatment without
temperature regulation, which resulted in a rise in the skin surface
temperature to about 45 °C (experiment 1), whereas no increase in
free radical formation was found when wIRA was applied with
temperatures up to a maximum of 37 °C using synchronous convective cooling of the skin (median of RS = 98% and P95 = 102%,
Decrease of MTT (living cells) [%]
20
threshold of biological relevance
15
10
5
exposure time
a: 15 min
b: 30 min
wIRA
&
temperature
o
up to 37 C
wIRA
&
temperature
o
up to 45 C
convective
heating
o
to 45 C
b
convective
heating
o
to 37 C
untreated
samples
a
Formation of Free Radicals [%]
260
240
220
200
wIRA
&
temperature
o
up to 37 C
convective
heating
to 37 O C
180
160
140
120
convective
heating
o
to 45 C
wIRA
&
temperature
o
up to 45 C
untreated
samples
100
80
60
median
P 95
40
20
0
0
1
2
3
4
Experiment
0
0
1
2
3
4
Experiment
Fig. 4. Changes in the number of living cells after treatment of 15 min (a) and of
30 min (b) duration in the experiments 1–4 in comparison to the number of living
cells in the reference sample before the start of experiments (experiment 0). The
biologically relevant threshold was marked at a decrease of P15% according to [52].
Fig. 6. The radical status (RS) as determined after treatment of 30 min duration in
the experiments 1–4, as compared to values in untreated skin at a surface
temperature of 26–27 °C. Both treated samples and the respective reference
samples were taken by biopsy immediately after the end of each experiment.
Determination of medians (forward slash bars) and of 95th percentiles (P95,
backslash bars) were based on five independent measurements of inducible free
radicals in five different areas of each skin sample.
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H. Piazena et al. / Journal of Photochemistry and Photobiology B: Biology 138 (2014) 347–354
Fig-7.fpw
Antioxidative potential (SAP) [%]
240
wIRA
&
temperature
o
up to 37 C
220
200
180
wIRA
&
temperature
o
up to 45 C
160
140
120
convective
heating
o
to 37 C
untreated
samples
100
convective
heating
o
to 45 C
80
60
40
median
P 95
20
0
0
1
2
3
4
Experiment
Fig. 7. Antioxidative power (SAP) measured after treatment of 30 min duration in
the experiments 1–4 in comparison to untreated skin with a surface temperature of
26–27 °C. Both the treated samples and the corresponding untreated reference
samples were taken by biopsy immediately after the end of each experiment.
Determination of medians (forward slash bars) and of 95th percentiles (P95,
backslash bars) were based on five independent measurements of antioxidative
power at five different areas of each skin sample.
experiment 2). Moreover, application of convective heating alone
to the skin up to a temperature of about 37 °C resulted in a slight
decrease in the amount of inducible free radicals to about a median
of RS = 86% (P95 = 90%) as compared to untreated reference samples which showed a variability which appeared to be due to both
natural variability and the methodical error of determination
(experiment 3).
3.4. Antioxidative power (SAP)
After convective heating of the skin to about 45 °C, the antioxidative potential decreased to a median of SAP of 54% and to
P95 = 55% as compared to the untreated reference sample with a
median of 102% (P95 = 108%) at a surface temperature of 26–
27 °C, for which the samples were obtained immediately at the
end of each experiment (see Fig. 7). In contrast, increases in SAP
were found for the experiments 1–3 where the median of SAP
increased up to 143% (P95 = 152%) after wIRA treatment with a
temperature control to a maximum of 37 °C (experiment 2). An
increase in SAP of 122% (P95 = 132%) was measured after wIRA
exposure without temperature control and a maximum temperature of the exposed skin of about 45 °C (experiment 1). This
increase was similar to the effect of convective heating of the tissue
up to about 37 °C, resulting in a median of SAP of 123% and in
P95 = 127% as compared with the reference value (experiment 3).
4. Discussion and conclusions
The experiments presented here were performed after a relatively short exposure period and showed neither clinically relevant
decreases in the formazan concentration nor relevant increases in
the PGE2 concentration and therefore did not match or exceed the
thresholds of minimal biological relevance in terms of cytotoxicity
or minimal inflammation. Instead, the data showed a natural variability and the effects seen were comparable to the effects
observed in the analysis of commonly used cosmetic consumer
products for skin care [55]. The formation of free radicals almost
doubled when the convective heating was applied to the skin up
to a surface temperature of about 45 °C as compared to untreated
skin areas of about 26–27 °C at the surface (p < 0.001). In contrast,
convective heating of the skin to a surface temperature of about
37 °C resulted in a (statistically insignificant) decrease in the number of inducible free radicals of about 14% (p = 0.337) in comparison with the reference sample which should however be
considered to be due to natural variability and the methodical
error of determination ranging within ±5% as described in [56].
Moreover, the amounts of inducible free radicals were insignificantly less (63%) in comparison to those found in the untreated
skin area as detected following treatment with wIRA and convective cooling, such that the skin surface temperature was maintained at 37 °C or below (p = 0.975). In the case of wIRA
treatment without convective cooling of the skin surface such that
a surface temperature of 45 °C was reached at the end of exposure,
the increase in free radicals was about 17%, thus showing a trend
toward significance (p = 0.061). These data suggest that the formation of free radicals in skin, as detected after wIRA exposure, was
due to thermal factors and not to the non-thermal action of IR-A
radiation as has been assumed by a number of authors [25–29].
Two further observations could indicate a possible protective
action of wIRA radiation in terms of thermal skin damage and a
resulting increase in free radical production in the skin:
(1) the reduced increase in the number of inducible free radicals
of only 17% (p = 0.061) after wIRA exposure as compared to
the increase of about 98% (p < 0.001) due to convective heating to the same temperature of 45 °C and
(2) the increase of the antioxidative potential (SAP) after wIRA
exposure of about 41% (p < 0.001) when the temperature
was regulated to about 37 °C and of about 20% (p < 0.001)
when the skin temperature increased to 45 °C, whereas
SAP decreased by about 48% (p < 0.001) after convective
heating of the skin to 45 °C and increased by only about
21% (p < 0.001) after convective heating to 37 °C.
It has to be noted however, that this interpretation has to be
considered cautiously since the respective temperatures were not
identical as can be seen in Fig. 3. This consequently results in a limitation in the comparability of the experiments 1 vs. 4 and 2 vs. 3.
Nevertheless, data from the experiments 1–4 clearly show that the
increased induction of free radicals during infrared skin exposures
was predominantly caused by overheating of the skin which
resulted in cellular damage, but was not – or only to an insignificant amount – directly due to short-wavelength IR radiation. This
conclusion is supported by a consideration of the quantum energy
of infrared-A radiation according to Planck’s Law, which ranges
from values of 1.59 eV at 780 nm to 0.89 eV at 1400 nm. It is
well-known that these levels of quantum energy may cause molecular movements such as vibrations and rotations, whereas photochemical effects in photobiology due to one-photon absorption
require photon energies which exceed the order of about 1 eV,
even in the case of interaction with a photosensitizer [57–60], with
generation of singlet oxygen only being achieved by direct radiation transfer of about 1 eV using a laser [61].
Various wavelengths within the range of short-wavelength IR-A
radiation have been identified as being effective in improving cellular metabolic processes, as well as in stimulating cellular repair
processes [12–18,20–23]. Moreover, protective effects of shortwavelength IR-A radiation against cytotoxic effects of ultraviolet
radiation have been found by a number of different authors [33–
37]. Thus, these beneficial effects could possibly be due to a protective action of short-wavelength IR-A against free radical production as suggested by the results of the study presented here.
Whereas the experiments in the present study were performed
using wIRA with a spectrum between about 780 nm and 1400 nm
(cf. Fig. 2), Zastrow et al. [62] exposed ex vivo skin biopsies to
broad-band radiation with a spectrum which started at about
H. Piazena et al. / Journal of Photochemistry and Photobiology B: Biology 138 (2014) 347–354
600 nm and increased with a continuous slope to a maximum at
about 1100 nm, but also – as specified by the authors - was
extended to 1600 nm and into the spectral range of infrared-B
(1400–3000 nm). The authors reported the production of free radicals in the samples which increased with irradiance (which in turn
varied from 400–800 W m2), exposure time/dose (which varied
from 100 to 1600 s, e.g. between 4 and 128 J cm2) and with steady
state temperatures which were measured inside the samples and
which resulted from heating with a lamp (from about 35 °C at
400 W m2 to about 45 °C at 800 W m2) [62]. From their data,
the authors concluded firstly that the production of free radicals
was caused by short-wavelength IR radiation and was substantially
affected by the heating-up of the tissue due to absorption of radiation. Secondly, intensification of radical production was described
whenever the initiated tissue temperature exceeded about 37 °C.
Whereas the latter result was in agreement with the result of thermally-induced free radical production derived from the BUS experiments, the first conclusion was not supported by these data. Thus,
it seems likely that non-thermal production of free radicals by
near-infrared radiation as claimed by Zastrow et al. [62] could be
caused by visible radiation with wavelengths between 600 and
780 nm which was emitted by the lamp used.
The irradiance of wIRA chosen in the present study exceeded
the extraterrestrial solar irradiance in the range of IR-A by a factor
of about 4.6 and the maximum IR-A irradiance of the sun at the
Earth’s surface by a factor of more than 5. In addition, convective
heating of the skin up to a skin surface temperature of 37 °C was
used, which characterized moderate warming, as well as to the
threshold skin temperature of the transition from heat pain to
thermal burning of about 45 °C according to DIN 33403/2 [63].
Since the experimental conditions in the present study exceeded
both the possible irradiances due to solar IR-A radiation on the
Earth, as well as tolerable skin temperatures, the results are also
in contradiction to the findings of other authors who inappropriately applied the Bunsen-Roscoe law of proportionality and who
extrapolated data from IR skin exposures performed using high
irradiances without temperature control of the skin and who also
claimed skin damage caused by free radical induction due to solar
IR exposure athermally [25–29]. Due to similarities between the
spectra of solar IR irradiance at noon and wIRA, experimental data
showing the dependence of steady state skin surface temperature
on incident wIRA irradiance under in vivo conditions are of interest
in this context [24]. According to these measurements, steady state
temperatures on the backs of resting, healthy volunteers were limited to values between about 37 °C and 41 °C and depended on the
air temperature of the surrounding environment and of thermal
regulatory responses stimulated in volunteers whereas chosen
wIRA irradiance of 400 W m2 was already above the possible
maximum of solar IR-A irradiance on the Earth’s surface [24,64].
The results found in the present study are of relevance for the
risk assessment of skin-damaging effects resulting from wIRA
application for moderate therapeutic and wellness purposes due
to the selected exposure times and irradiances. The chosen wIRA
irradiance of 1900 W m2 exceeded both the upper limit recommended by DIN 5031/10 [65] for therapeutic application of (even
unfiltered) IR-A as well as the recommended and usually applied
levels of wIRA irradiance for local therapy as well as for moderate
hyperthermia by a factor of about 1.58.
The exposure times of 15 min or 30 min chosen in this study
were in accordance with current recommendations and the usual
schedules for moderate IR therapy and for wellness applications.
They were, however, lower in comparison to the usual durations
of persons spending prolonged times outdoors for occupational
or leisure purposes. Future investigations should be extended to
durations of up to several hours to include possible effects of solar
IR radiation and heat due to prolonged exposure, as well as to the
353
effects of solar IR upon the impact of solar UV and visible radiation
to human skin based on the analysis of a representative number of
udders in order to include effects of individual variability of skin
properties into the analysis.
5. Abbreviations
wIRA
Water filtered infrared-A
Acknowledgements
The authors are grateful to the Dr. med. h.c. Erwin Braun Foundation, Basle (Switzerland) for generous support of this study. We
would like to thank Dr. vet. med. B. Blume, SIMRED, Großburgwedel (Germany) for technical support during the BUS experiments
and M. Seifert, GEMATRIA-Test Lab, Berlin (Germany) for skilful
assistance.
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