SAHLGRENSKA ACADEMY DEPARTMENT OF RADIATION PHYSICS

STUDY OF ULTRAVIOLET C LIGHT

PENETRATION AND DAMAGE IN SKIN

Sofia Saadati

Essay/Thesis: 30 hp

Program and/or course: Medical Physicist Programme

Level: Second Cycle

Semester/year: Spring 2016

Supervisors: David J. Brenner 1 , Eva Forssell-Aronsson 2 , Manuela Buonanno1,

Gerhard Randers-Pehrson1, Nils Rudqvist1

Examiner: Magnus Båth

Report no:

1 Columbia University 2 University of Gothenburg

Abstract

Essay/Thesis: 30 hp

Program and/or course: Medical Physicist Programme

Level: Second Cycle

Semester/year: Spring 2016

Supervisors: David J. Brenner, Eva Forssell-Aronsson, Gerhard Randers-Pehrson,

Manuela Buonanno, Nils Rudqvist

Examiner: Magnus Båth

Report No:

Keyword: UVC, excimer lamp, CPD, epidermis, SKH1 mouse

Purpose: The aim of this work was to give further insight on the surface doses of 222-nm light,

produced by excimer lamp suggested for disinfecting applications, needed to cause

DNA damage in the basal cell layer.

Theory: The use of conventional germicidal lamps for disinfection at the surgical site is

prevented by being both cancerogenic and cataractogenic. Ultraviolet light in the far

UVC range (200-222 nm) produced by excimer lamps are shown to be germicidal,

without being as damaging as conventional lamps to typical human cells in vitro. It

is suggested to be less damaging to the human skin by being highly absorbed by

peptide bonds in protein, giving it limited ability to penetrate biological tissue.

Method: Hairless mice were exposed to two doses of 222 nm light by an excimer lamp. The

other groups consisted of positive (254-nm light) and negative controls. The mice

were killed 48 h post exposure and dorsal skin samples were excised. UV specific

DNA damage cis-syn cyclobutane dimer (CPD) was assessed by

immunohistochemical analysis at different depths in epidermis. The attenuation of

UV light was determined from irradiated phantoms with different protein

concentration.

Result: DNA photodamage was significantly lower in all mouse groups exposed to 222-nm

light in comparison with positive controls. The amount of damaged basal cells was

significantly lower in mice exposed to 222-nm light and negative controls, than those

of positive controls. Attenuation curves indicated contribution of other wavelengths

than the primarily emitted by the lamps. The attenuation coefficient of the 222-nm

light was much higher than for the 254-nm light (6.5 cm2/mg vs. 0.40 cm2/mg). The

correlation of measured attenuation to actual skin is not easily done without further

research and better modeling of the protein content of epidermis.

Table of content

Introduction ............................................................................................................................................. 1

Ultraviolet light ................................................................................................................................... 1

Germicidal ultraviolet lamps ............................................................................................................... 2

The human skin ................................................................................................................................... 2

Attenuation of UV light in skin ........................................................................................................... 4

Aim .......................................................................................................................................................... 6

Materials and methods............................................................................................................................. 7

Assessment of ultraviolet C light attenuation ................................................................................. 7

UV light sources and detectors ............................................................................................................ 7

Transparency measurements ............................................................................................................... 7

Attenuation measurements .................................................................................................................. 7

Depth profile of DNA photodamage in skin .................................................................................... 7

Hairless mice ....................................................................................................................................... 7

Mouse irradiations and immunohistochemical process ....................................................................... 8

Analysis of DNA photodamage with depth in skin ............................................................................. 8

Statistical analysis ................................................................................................................................. 10

Results ................................................................................................................................................... 11

Assessment of ultraviolet C light attenuation ............................................................................... 11

Depth profile of DNA photodamage in skin ................................................................................. 15

Discussion ............................................................................................................................................. 18

Conclusion ............................................................................................................................................. 21

Acknowledgements ............................................................................................................................... 22

Reference list ......................................................................................................................................... 23

1

Introduction

Infections at the surgical site in clean operations can be caused by air borne bacteria and viruses. A

surgical site infection (SSI) may lead to severe complications, spending time in an intensive care unit

(ICU), readmission, increased healthcare costs and death. The number of deaths in the U.S. due to SSI

has been estimated to 8,200 per year (Klevens et al., 2007). The degree of complication of the infections

is exacerbated by the presence of drug-resistant bacteria such as methicillin-resistant Staphylococcus

aureus (MRSA)(Fry and Barie, 2011). One approach would be irradiating the wound with currently

available conventional germicidal UV lamps. These lamps cover the wavelengths 200-400 nm and are

effective against both drug resistant and drug sensitive bacteria. However, being both carcinogenic and

cataractogenic, light with wavelengths produced by these lamps present a health hazard for both patient

and staff (Pfeifer and Besaratinia, 2012; Jose and Pitts, 1985). The need for non-impractical protective

clothing and eye shielding has thus prevented the use of this technique (Buonanno et al., 2013).

In an in vitro study at Columbia University, researchers tested the hypothesis and provided evidence

that UVC radiation in the range 207-222 nm is cytotoxic to bacteria and viruses but minimally cytotoxic

or premutagenic to typical human cells (Buonanno et al., 2013). The difference in cytotoxicity is due to

the fact that photons with wavelengths around 200 nm is strongly absorbed by proteins, thus have limited

ability to penetrate biological tissue while penetrating bacteria, which are much smaller than typical

human cells. The typical diameter of bacteria is less than 1 µm, in contrast to 10-25 µm in typical human

cells (Metzler and Metzler, 2001). At the time of this thesis work, ongoing in vivo studies show

promising preliminary results regarding confirmation of the in vitro study (Buonanno et al., 2016).

The production of a nearly single-wavelength UV beam is possible using excimer lamps (excilamps)

that contain gas mixtures that determine the spectra emitted. The possibility of using excilamps to

produce UVC light usable for disinfection could lead to a significant reduction in the number of SSI

without humans being subject to hazardous exposure, thus little or no need for protective equipment.

Also, the robustness along with the low costs of this equipment would make it an attractive tool,

especially for low income countries.

Ultraviolet light

Ultraviolet (UV) light is part of the non-ionizing portion of the electromagnetic radiation covering the

wavelengths 100-400 nm. UV light is more specifically part of the optical radiation, and is generally

subdivided into three ranges of wavelengths: 315-400 nm (UVA), 280-315 nm (UVB) and 200-280 nm

(UVC). While most of the UVA light reaches the surface of the earth and penetrate the deeper layers of

our skin, only a few percent of UVB light passes through the ozone layer. Yet, UVB light dominates the

carcinogenic effects of sunlight by being actinic, meaning that it causes photochemical reactions. These

reactions often occur in the nucleic acid, leading to lesions in the DNA which could develop into

mutations. As with UVB light, UVC light possesses even higher energies per photon and is also

considered actinic. UVC light is completely absorbed in the ozone layer and thus not a source of natural

exposure to human beings.

As it passes through medium, UV light is attenuated by exciting particular molecules in the compound.

Depending on the chemical structure of the compound, the gap between the orbitals in the molecule

differs and thus the energy required for an excitation to take place. The energy per photon is inversely

proportional to its wavelength, meaning that light with shorter wavelength is more energetic and capable

2

of exciting molecules with higher excitation gaps. Molecules consisting of UV absorbing groups are

called chromophores and the probability of a photon with a specific wavelength to be absorbed by a

chromophore is typically illustrated with an absorption spectrum.

Ultraviolet light follows the optical laws including reflection and scattering. The irradiance, IR,

(unitW

m2), is the radiant power received by a flat surface per unit area and is often referred to as the

intensity of the light. Irradiance integrated over time of exposure gives the amount of radiant energy

received by a surface which is called the dose (unitJ

m2 ). Dose is often also referred to as fluence.

Germicidal ultraviolet lamps

The term germicidal in germicidal ultraviolet lamp implies that bacteria or viruses are unable to

reproduce themselves following irradiation with ultraviolet light (Kowalski, 2009). This is primarily

achieved with light in the actinic range of wavelength, including UVB and UVC light.

The conventional germicidal lamps used are mercury lamps emitting photons in the range 200-400 nm,

depending on the pressure used in the lamps. While high pressure mercury lamp emits photons in the

mentioned range of wavelength, the low pressure mercury lamp primarily emits light at 254 nm.

Ultraviolet light absorption of DNA peaks at about 200 and 260 nm, close to the primarily emitted

wavelength of the lamp.

Cell death of bacteria and inactivation of viruses can occur due to several factors including changes in

heat and humidity, oxidative stress or unrepaired DNA damage. Extensive loss of vital proteins and/or

enzymes in the cell also has an impact on the viability of the cell.

The human skin

The skin is a multilayered structure consisting of various types of biological components including cells,

fibers, and capillaries, and is the largest organ of the human body. By protecting the body from

chemicals, temperature changes, ultraviolet light, pressure and friction, the skin functions as a barrier

between the body and its external environment. Descending from its surface the three layers of the skin

are epidermis, dermis and hypodermis (also called subcutis) (Figure 1). The thickness of these layers in

most regions of human skin is approximately 80, 1000, and >1000 µm, respectively. Since the

wavelengths investigated in this thesis are suggested to have limited ability to penetrate biological tissue,

focus will lie on the outermost layer of the skin, the epidermis.

The epidermis consists mainly of keratinocytes, hence the structural differences in the tissue within the

epidermis is due to the degree of their stage of differentiation. The innermost sub layer of epidermis,

stratum basale (basal cells layer) consists of keratinocytes and melanocytes. Approximately 10% of the

basal cells are keratinocyte stem cells, constantly providing the epidermis with either new stem cells or

differentiating cells (Sun et al., 2007).

The differentiation proceeds while the cells are moving upward towards the surface of the epidermis,

the stratum corneum (horny cells layer) (Figure 2). During differentiation, the cells change in shape and

structure by losing intracellular water and degrading of organelles. The keratinocytes that reaches the

stratum corneum are called corneocytes, indicating that they have completed their differentiation cycle

in which they have lost their nuclei and cytoplasmic organelles. The thickness of the stratum corneum

is 5-20 µm in human skin (Russell et al., 2008). The corneocytes are randomly and constantly being

3

peeled off, which together with the rapid cell division in the basal cells layer, helps maintain the

thickness of the epidermal skin.

Figure 1: Layers of the skin. From the surface: epidermis, dermis and hypodermis. Hypodermis, not marked in

the picture, is the layer below dermis. (NCI,

https://upload.wikimedia.org/wikipedia/commons/c/c1/Layers_of_the_skin.jpg, 160526)

While the keratinocytes provide the skin with roughness and protection against external stress, the

melanocytes carry the main responsibility for photoprotection by production and distribution of melanin

to the keratinocytes. The melanin is released in the cytoplasm of keratinocytes and accumulated in the

supranuclear region, protecting the underlying DNA in the nucleus from UV light (Kobayashi et al.,

1998). Melanin together with DNA, urocanic acid and tryptophan are the main chromophores in the skin

attenuating UV light (Kowalski, 2009).

4

Figure 2: Cross section of the skin showing epidermis with its different layers. Stratum corneum is the

shallowest and thickest layer with a flattened appearance.

(https://commons.wikimedia.org/wiki/File:Epidermal_layers.png, 160526)

Attenuation of UV light in skin

Attenuation of UV light is due to the optical properties of the skin and thus the different optical

pathways. There is a regular reflectance of the light at the surface of the skin, due to change in refractive

index between air and human stratum corneum. For normal black and white skin, this reflectance is

approximately 5% of the incident light for wavelengths above 250 nm (Anderson and Parrish, 1981).

The decrease in dose of UV light is dependent on the absorption and scattering of light in the epidermis,

which varies between humans.

When absorbed by the DNA in epidermal cells, UV light is capable of inducing premutagenic lesions,

which if not repaired, can be carcinogenic. The two major types of DNA lesions induced by actinic UV

light in the skin are cis-syn cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone

photoproducts (6-4pps) (Pfeifer et al., 2005). Due to quicker repair of 6-4pps, CPDs are believed to be

the major contributor to mutations (Tornaletti and Pfeifer, 1996, Yoon et al., 2000).

The cell has different mechanisms to prevent UV induced DNA damages to become cancerogenous

including DNA repair or apoptosis following cell cycle arrest (Matsumura and Ananthaswamy, 2004).

If these pathways fail to repair or remove the damage, this can result in loss of control of cell

proliferation, allowing a tumor to grow. Aside from the molecular reactions following exposure, the

known acute effects of skin tissue to exposure are hyperplasia and immunosuppression (Matsumura and

Ananthaswamy, 2004). Since premutagenic damages in cells that have withdrawn from the basal layer

do not contribute to the major risk of cancer induction, but rather the dividing stem cells and melanocytes

in the basal cell layer, it is of importance to determine whether any damage can be seen at the basal cell

layer.

Given that not all absorbed photons necessarily cause damage in a cell, the probability of DNA damage

induction increases with the dose of the UV light. Since the optical properties of skin determine the

attenuation of light, it is ideal to find a correlation between dose and biological response. In contrast to

the melanin dependent attenuation of UVA and UVB light, wavelengths in the far UVC region are

mainly attenuated by absorption of peptide bonds and DNA.

5

Earlier studies have demonstrated the depth penetration effects of stained cyclobutane pyrimidine dimer

in human buttock skin for light of wavelengths 260 nm and 300 nm (Chadwick et al., 1995). Being

completely absorbed in the ozone layer, the damage of UVC light in epidermis have not been the main

subject to safety studies. To the best of our knowledge, demonstration of far UVC induced DNA damage

depth profile, is a rather new approach.

6

Aim

The aim of this work was to give further insight on the surface doses of 222-nm light, produced by

excimer lamp suggested for disinfecting applications, needed to cause DNA damage in the basal cells

layer.

This work was performed by 1) obtaining attenuation curves for the wavelengths 222 nm and 254 nm

in a simplified skin model and, 2) quantifying UV induced DNA damage with depth in mouse tissue

using immunohistochemical analysis.

7

Materials and methods

Assessment of ultraviolet C light attenuation

UV light sources and detectors

A krypton-chlorine (Kr-Cl) excimer lamp (High Current Electronics Institute, Tomsk, Russia) was used

for irradiation with 222-nm light. The lamp was either used unfiltered or filtered with a bandpass filter

(Omega Optical, Brattleboro, VT). A conventional mercury germicidal lamp (C-100 Thera-Wand,

Biomation, Almonte, Canada) was used for irradiation with 254-nm light. The dose was measured using

a NIST-traceable light radiometer (ILT1400, International Light Technologies, Peabody, MA). Another

conventional mercury germicidal lamp (G15T8, Sankyo Denki, Kanagawa, Japan) was used for the

attenuation measurement of primarily 254-nm light.

Spectrometry was done to characterize the spectra emitted by the lamps using SpecSoft (Photon Control,

Burnaby, Canada). Calibration of the spectrometer (Photon Control, Burnaby, Canada) was done using

a deuterium lamp standard with NIST-traceable spectral irradiance (Newport Corp, Stratford, CT).

Transparency measurements

The transparency of fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, USA), phosphate buffered

saline (PBS) (Sigma-Aldrich), Dulbecco´s phosphate buffered saline (DPBS) (Sigma-Aldrich) and

Hanks Balanced Salt Solution (HBSS) (Sigma-Aldrich) was assessed by exposing 5 ml of each solution

in a cylindrical (d=50 mm, h=10 mm) quartz dish plate (Technical Glass Products) (approx. 90% UV

transparency). The plate was placed directly on the detector, with the bottom of the dish being 11 cm

from the surface of the lamp. Based on these measurements, the solution with least UV transparency

was used for the attenuation measurements, i.e. FBS. The chosen solution was serially diluted (1:2) with

distilled water, resulting in a total of 8 different concentrations.

Attenuation measurements

The detector was placed 30 cm directly under the excilamp. The time-response stability of the detector

was measured by irradiating 5 ml of distilled water and protein containing solution, respectively. Ten

measurements were done for each solution, each measurement lasting 1 minute. For assessment of

attenuation, the dose measured by the detector through 8 concentrations of protein containing solution

was randomly measured during three sets of measurements. The time of each measurement was 1

minute. The amount of protein per square centimeter was calculated for all concentrations.

Depth profile of DNA photodamage in skin

Hairless mice

Animals used in this study were six to eight weeks old male hairless SKH1 (Elite strain 477) mice from

Charles River Laboratories (Stone Ridge, NY). The mice were housed unrestrained 48 h prior to and

during exposure in an eight compartment mouse irradiation box [W: 60 mm, L: 125 mm, H: 80 mm].

The box was covered with a metal-mesh top with a UV transparency of 74%. Water and Purina

8

Laboratory Chow 5002 diet (St. Louis, MO) were given ad libitum before and during irradiation. The

mice were killed through asphyxiation 48 h post exposure. All animal procedures were performed

according to federal guidelines and protocols approved by the Columbia University Medical Center

Institutional Animal Care and Use Committee (IACUC).

Mouse irradiations and immunohistochemical process

Twelve mice were divided into groups of three; each group was delivered a specific dose and wavelength

of light over a 7-h period. Two groups were exposed to 222-nm light, where one group was exposed to

25.6 mJ/cm2 and the other group to 83.3 mJ/cm2. The positive controls were exposed to 20 mJ/cm2 of

primarily 254-nm light by the germicidal lamp and negative controls were sham irradiated with zero

fluence.

The mice were killed 48 h after delivery of the dose. Samples of dorsal skin were then excised and

assayed for DNA photodamage. The tissues were fixed overnight in 10% neutral buffered formalin. The

tissue was then embedded in paraffin and cut in 6 µm thick sections. After being deparaffinized and

rehydrated, the samples were put in target retrieval solution at 96 ⁰C for 20 min using DAKO Ancillary

system K1499 (DAKO, Carpinteria, CA). Thereafter, the samples were cooled off for 20 minutes. The

samples were then washed three times in 1X phosphate buffered saline (PBS) and blocked in protein

blocking solution (1% bovine serum albumine (BSA) in PBS) for 1 h. They were incubated for 1 h in a

humid chamber using 1:1000 mouse anti-CPD antibody (Cosmo, Bioscience USA, Carlsbad, CA) in

protein blocking solution. The samples were washed, and then incubated with biotinylated secondary

antibody at room temperature for 45 min. A mouse-specific HRP/DAB (ABC) detection kit (Abcam,

Cambridge, MA) was used for signal detection. A coverslip mounting medium containing 4',6-

diamidino-2-phenylindole (DAPI) (Vectashield, Burlingam, CA), a flourescent stain that binds to nuclei

of cells, was used on each sample slide.

Analysis of DNA photodamage with depth in skin

The samples were examined using a microscope (Olympus IX70 microscope, USA) with high efficiency

digital camera. The analysis was done using Image-Pro Plus 6.0 (Media Cybernetics, Rockville, USA).

Twelve randomly selected 60x field-of-view (FOV) of the epidermis for each group were examined.

The stained cells with a potential CPD were shown as grayish or black in the microscope. The amount

of CPD in the epidermis was counted relative to the total number of cells, i.e. DAPI-stained cells. In

addition, DAPI-stained cells were examined to optimize the localization of the cells and verification of

the photodamage. Healthy cells were shown as very bright in this field and damages could be seen as

black dots in the nuclei.

After being counted as either healthy or damaged cell, the depth of each cell relative to innermost layer

of stratum corneum was measured. This was done using a tool measuring the perpendicular distance in

Image Pro Plus 6.0. The stratum corneum could be distinguished from the other parts of the epidermis

by its non-nucleated flattened appearance (cf. Figure 3). Because of the wavy appearance of stratum

corneum, the skin was divided into separate columns, in which the perpendicular distance from the inner

part of the stratum corneum to the center of each cell in that column was measured. The number of

damaged cells relative to the number of total cells (i.e. cells with DAPI-stained nuclei) was counted for

5-µm intervals.

9

'

Figure 3: Cross-section of the skin of hairless mice in different views of microscope (60x). The left hand

picture represents the bright field view. The epidermis is the light gray part of the tissue and the dermis is the lower

left hand part of the picture. The flat layers on the top of the skin represent the non-nucleated stratum corneum,

followed by the nucleated epidermis. The basal cells layer is the last layer of cells above the dermis. The right

hand panel is a picture taken in a fluorescent mode. After being scored as healthy or damaged in the bright field

(left) the confirmation of localization and damage was done by looking at the fluorescent view (right). Each cell

was marked as healthy or damaged in the bright field and the depth of each cell was determined by measuring the

perpendicular distance from the inner part of the stratum corneum to the center of the cell.

10

Statistical analysis

The uncertainties in the data from stability measurements of the detector were determined using CV

(coefficient of variation):

𝐶𝑉 =𝑆𝐷

𝑋,

where SD is the standard deviation of the original distribution and X is the mean value of a series of

measurements.

The uncertainties in the data from attenuation measurements were calculated using SEM (Standard Error

of the Mean),

𝑆𝐸𝑀𝑥 =𝑆𝐷

√𝑛,

where x is the mean value of the samples, SD is the standard deviation of the of the original distribution

and n is the number of samples.

The uncertainties in the data from tissue measurements were determined by error propagation of

Poisson-distributed values using a 95% confidence interval.

11

Results

Assessment of ultraviolet C light attenuation

Spectrometry of the unfiltered excimer lamp showed emitted photons of primarily 222 nm and lower

fluences from photons of higher wavelengths around 260 nm (Figure 4a). Using the filter, the higher

wavelengths were not visible. Spectrometry of the germicidal lamp showed photon peaks at primarily

254 nm together with photons with wavelengths above 300 nm (Figure 4b).

Fetal bovine serum (FBS) showed lowest transparency in comparison with the rest of the solutions and

was used as the protein containing solution in the phantom.

The stability of the detector was tested using liquids with or without FBS and the samples were irradiated

repeatedly. The results are shown in Figure 5. The CV of the response of the detector was 8% at low

fluences and 1.8% at high fluences.

The attenuation curves of the excimer and germicidal lamp were obtained by irradiating water containing

different concentrations of FBS (Figure 6). As seen in Figure 6, the log-linear plot shows the attenuation

of light of the two lamps as bi-exponential curves. The attenuation coefficient for the UV light in the

first exponential part was assessed using an exponential trend line for the measured values. The

attenuation coefficient for the first part of the plot of the filtered excilamp was 6.51 cm2/mg and 0.060

cm2/mg for the second part of the curve. The corresponding values for the germicidal lamp (Figure 5b)

were 0.40 cm2/mg and 0.15 cm2/mg.

12

a)

b)

Figure 4: Spectrometry of the lamps used in the experiments. a) Unfiltered and filtered Krypton-Chlorine (Kr-

Cl) lamp. In addition to the expected high intensity of light at primarily 222 nm, photons with wavelengths of

around 240-260 nm were also found. These wavelengths were completely absorbed by the filter, which is shown

as dark gray. The plot to the right shows the spectrum with different scale on the y-axis. b) Conventional low-

pressure germicidal lamp. A peak at 254 nm was seen. Photons with wavelengths in the UVA (320-400 nm) and

UVB (280-320 nm) range were detected.

0

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Figure 5: Investigation of time-response stability of the detector for different doses over time. The values

that are presented show the dose measured by the detector for each measurement. Two concentrations (0% and

100%) of fetal bovine serum were irradiated repeatedly 10 times each. The irradiation time was 1 minute for each

measurement. The detector showed good stability over time at low and high doses

1

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Do

se (m

J/cm

2)

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Low dose

14

a)

b)

Figure 6: Log-linear plot of the attenuation of light from a) an excimer lamp emitting 222-nm light and b)

a conventional germicidal lamp emitting primarily 254-nm light. The dose is given as function of the areal

density of protein in each sample containing specific concentration of FBS. For 222-nm light from the filtered

excimer lamp, the attenuation factor of the first exponential part of the plot was much higher (6.5 cm2/mg) than

that of the second exponential part (0.060 cm2/mg). For the conventional germicidal lamp, the biexponential curve

was much less steep, with corresponding attenuation factors of 0.40 cm2/mg and 0.15 cm2/mg. The data are

represented as mean values. Error bars indicate SEM.

y = 29922e-0,40x

R² = 0,998

y = 3102,e-0,15x

R² = 1

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se (m

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Areal density of protein (mg/cm2)

y = 11597e-6,51x

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y = 9,109e-0,06x

R² = 0,912

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Areal density of protein (mg/cm2)

15

Depth profile of DNA photodamage in skin

Representative sections of epidermis of all groups are shown in the left panel of Figure 7. The right hand

column shows corresponding fluorescent view of the microscope. The sham irradiated sections show

little staining in the bright view, and bright DAPI-stained nuclei in the fluorescent view, indicating

healthy cells. Little damage can be seen in the two middle rows, showing the sections of epidermis

exposed to 25.6 mJ/cm2 and 83.3 mJ/cm2. The positive control in the bottom row shows many damaged

cells with dark cell nuclei in both views.

As shown in Figure 8, cells with photodamage in mice exposed to 222-nm light were only observed in

the first part (0-5 µm) of the epidermis. The amount of cells showing damage in this interval was 2.7%,

in comparison with the positive controls with 80% damage throughout the epidermis. No statistically

significant difference in damage was seen between the groups exposed to 222-nm light and negative

controls. However, a higher mean value for mice exposed to the higher dose indicates that a greater

sample size could be needed to evaluate a potential difference between the groups.

The total amount of damaged basal cells is presented in Figure 9. The amount of damaged basal cells

was significantly higher for the positive controls (approximately 68%) in comparison with other groups

(<1%). There was no statistically significant difference in amount of damaged basal cells between mice

exposed to 222 nm and negative controls.

16

Figure 7: Sections of epidermis from mice. A)-D) shows representative tissue in bright view and E)-H) shows

in fluorescent view. A&E) sham, B&F) 25.6 mJ/cm2 222 nm, C&G) 83.3 mJ/cm2 222 nm, and D&H) 254-nm

light. The yellow bars seen in A)-D) indicate 20.2 µm.

A)

))

B)

C)

E)

D)

))

G)

F)

)

H)

17

Figure 8: Amount of epidermal cells with cis-syn cyclobutane pyrimidine dimers (CPDs) in skin biopsies

from mice exposed to different doses of 222 nm, negative controls (sham-exposed) and positive controls (254

nm). Exposure was performed with 25.6 and 83.3 mJ/cm2 of 222-nm light by the filtered excilamp and 20 mJ/cm2

of primarily 254 nm by a broad range germicidal lamp. The histogram shows the relationship between the amount

of cells with CPD and depth in epidermis. No significant difference was shown between different doses of 222-

nm light and negative controls. Enlarged plot shown to the right where all amount of damage is shown. The

uncertainties are given as 95% confidence intervals.

Figure 9: Basal cells with cis-syn cyclobutane pyrimidine dimer (CPD) in mice exposed to different doses of

222-nm light in comparison with negative and positive control groups. Enlarged plot is shown to the right. The

amount of damage was significantly higher in the positive controls (254 nm) in comparison with the values for

doses of 222-nm light. No damage was observed for the low dose of 222-nm light. The uncertainties are given as

95% confidence interval.

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6

7

0-5 5.1-10 10.1-15 15.1-20 20.1-25 25.1-30

Am

ou

nt o

f e

pid

erm

al c

ell

s w

ith

CP

D (

%)

Epidermal depth (µm)

254 nm low dose222 nm high dose222 nm low doseSham

D) H)

18

Discussion

Spectrometry of the excilamp and germicidal lamp showed peaks of photons with other wavelengths

than the primarily emitted. For the excilamp, the main peak was at 222 nm but with a broader range

around 225-270 nm and with contribution also from photons with higher wavelengths. When the

bandpass filter was used for the excilamp almost complete absorption of light other than around 222 nm

was obtained, which is ideal when investigating the effect of the energy deposit of a single wavelength

in tissue. The filter did, however, also reduce high amounts of the 222-nm light, which will increase the

exposure time to receive a specific dose. Furthermore, the germicidal lamp showed approximately a 2-

fold number of counts for the primarily emitted wavelength in comparison with the unfiltered excilamp.

In order to have approximately the same surface dose as with the excilamp, the lamp was collimated

and the lamp-detector distance was increased.

The contribution of higher wavelengths in the spectrum of the germicidal lamp is typical for

conventional low pressure mercury lamps, which emit light at a broader range of wavelengths in the UV

spectrum. Since germicidal lamps are usually used unfiltered, all experiments were performed without

any filter. The small peak of the germicidal lamp in the UVB region could have a minor effect on the

formation of CPD in observed skin. Not being actinic, light in the UVA region shown in the spectrum

of the germicidal lamp should not have contributed much to the CPD observed.

No investigation was done in the stability of the lamps over time and potential rising time. To minimize

any risk of unnecessary exposure during experiments, the lamps were turned off between each

measurement. These effects should, however, be limited, since the measurements of the dose were done

a few seconds after turning on the lamp. Since all measurements were done following the same

procedure at the same pace, effects should be similar for all measurements and most probably only

increase the uncertainty of each measured parameter.

The time response of the detector-lamp setup in attenuation measurements showed good stability with

a CV of maximum 8% in the lowest range of doses used in the experiments. The measurements of the

different concentrations of protein were done randomly in order to minimize any systematic

uncertainties.

The fitting to the depth curves for both lamps were good with R2-values > 0.9. The attenuation of UV

light by absorption of proteins was much higher for the germicidal lamp than the excimer lamp. The

first part of the curve was steeper for both lamps, with the logical explanation that these are from the

primarily (shorter) emitted wavelength (254 nm and 222 nm) of the lamps. The attenuation of 222-nm

light was much higher for the same areal density of protein than that of 254-nm light. In accordance

with the spectrum of the germicidal lamp, the other exponential part of the curve is due to photons with

higher wavelengths. The attenuation coefficient of the second part of the excimer lamp was much lower

than the corresponding coefficient of the germicidal lamp, indicating presence of photons with even

higher wavelengths. Showing no other peaks in the ultraviolet range in the spectra, there is reason to

believe that this part of the attenuation curve is due to light above the ultraviolet spectrum, i.e. visible

or infrared light.

The idea behind the phantom used in the attenuation measurements was a very simplified model of

actual skin, based on the theory that the protein content throughout the epidermis determines the dose

19

of UV light at different depths and therefore also the amount of damage. Fetal bovine serum was chosen

due to its high protein content, mainly the globular protein bovine serum albumin. The focus of this

work was to study effects of various protein amounts on the attenuation of light. The actual composition

of proteins throughout the epidermis cannot easily be estimated, why there is no easy correlation

between different amounts of protein used in this model and that in human skin. More research is needed

in order to translate the energy deposition from the data presented in this work to the situation in actual

skin types of the human body. Then a more complex model with various materials, more similar to

actual cell layers in epidermis, should be used. Another approach would be a more advanced physical

and biological modeling of the skin and UV light penetration. However, this approach also needs better

knowledge of interaction of UV light with different molecules in tissue, as well as variation in molecular

density within epidermis.

The animal experiments showed statistically significant higher amount of damage in the epidermis of

mice exposed to the germicidal lamp i.e. 254 nm, in comparison with 222-nm light by the excilamp. The

amount of damage was statistically higher throughout the epidermis. In mice exposed to 222 nm, DNA

damage was only observed in the first 5 µm of the nucleated epidermis and not statistically different

from the sham irradiated mice. No statistically significant difference was shown between the damage

depth profile of mice exposed to 25.6 mJ/cm2 and 83.3 mJ/cm2for 222-nm light.

Statistically significant higher amount of damage was observed in the basal cells of the epidermis of

mice exposed to 254 nm in comparison with 222-nm light. Mice exposed to the investigated doses of

222-nm light in this work showed no difference in amount of damage than sham irradiated mice. These

results indicate that peptide bonds in the proteins of the stratum corneum attenuate 222-nm light to such

a high extent that no significant damage can be seen in the nucleated epidermis in comparison with

unexposed mice.

Comparison between mice exposed to 222 nm and 254-nm light was done only for the lower dose, 25.6

mJ/cm2. This is due to the uncertainties in the number of total cells following UV induced

hyperproliferation of the keratinocytes. This effect, together with cell cycle arrest of damaged cells,

leads to epidermal thickening. The thickness of the epidermis was shown to be three times higher

(Buonannoet al, 2016.) in mice exposed 254 nm in comparison with the sham irradiated mice 48 h post

exposure. Cells that were present at the time of irradiation could have been delocalized due to

hyperproliferation and cell cycle arrest of damaged cells. It is difficult to distinguish the cells that were

not present at the time of irradiation with the rest. For the lower dose, the epidermis was shown to be

approximately 50% thicker than that of sham irradiated (Buonanno et al, 2016.). This might have an

impact on the certainty in the positioning of the irradiated cells and overall scoring of the total cells.

Since the total number of cells will be more than the actual number during irradiation, there is a risk of

underestimation of the damage in each interval. This is an issue if one wants to correlate the fluence and

damage at a specific depth in epidermis.

Stratum corneum, due to higher areal protein density, greatly attenuates the incoming light. Therefore,

it is of great importance to take into account that the thickness of the stratum corneum is dependent on

location of the actual skin. In human beings, the inner mouth has no stratum corneum whereas the sole

of the foot has the thickest one. This might clearly affect the depth profile of the photodamage in

epidermis.

20

The correlation of the attenuation of light at various depths in real epidermis to observed photodamage

would be ideal to find. This would give further insight in the surface fluences needed to cause damage

in the basal cells layer. Based on the results of this work, doses of up to 83.3 mJ/cm2 of 222-nm light,

are suggested to not be premutagenic. This dose is approximately 25 times higher than the dose for 90%

inactivation (D90,) of Staphylococcus aeureus exposed to 254 nm germicidal lamp (Kowalski, 2009).

21

Conclusion

The difference in attenuation coefficient for 222 nm and 254-nm light verifies that there is great

difference in attenuation of light for the same areal density of proteins. No statistically significant

difference in amount of photodamage was observed between investigated doses of 222-nm light and

sham irradiated mice. The translation of data on measured attenuation, due to protein absorption, to dose

at different depth in actual skin is not easily done and requires further research and better knowledge

and modeling of the protein content of different parts of epidermis.

22

Acknowledgements

I would like to express my gratitude to Dr. Brenner for giving me the honour of being a part of this

interesting work.

Thanks to my supervisor Dr. Forssell-Aronsson for your support and constructive comments on this

report along the way.

Special thanks to Dr. Buonanno for providing me with the mouse samples of the in vivo experiments.

Also thank you for all help and guidance regarding the practical parts of the analysis of mouse tissue.

Many thanks to Dr. Randers-Pehrsson for your fruitful ideas and help throughout this project.

Thanks to Dr. Rudqvist for our many discussions, comments on the report and your big support

throughout this project.

I wish to thank Dr. Yanping Xu for your help with the dosimetry and technical help with the attenuation

measurements.

Many thanks to my friends and family for their endless love and encouraging mood. Last but not least,

thanks to Arman for always believing in me.

23

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