Research ArticleOxidative Damage to Nucleic Acids andBenzo(a)pyrene-78-diol-910-epoxide-DNA Adducts andChromosomal Aberration in Children with Psoriasis RepeatedlyExposed to Crude Coal Tar Ointment and UV Radiation

Lenka Borska1 Ctirad Andrys2 Jan Krejsek2 Vladimir Palicka3 Marcela Chmelarova3

Kvetoslava Hamakova4 Jan Kremlacek1 and Zdenek Fiala5

1 Institute of Pathological Physiology Faculty of Medicine in Hradec Kralove Charles University in Prague50038 Hradec Kralove Czech Republic

2 Institute of Clinical Immunology and Allergology Faculty of Medicine in Hradec Kralove Charles University in PragueSimkova 870 50038 Hradec Kralove Czech Republic

3 Institute of Clinical Biochemistry and Diagnosis Faculty of Medicine in Hradec Kralove Charles University in Prague50038 Hradec Kralove Czech Republic

4 Clinic of Dermal and Venereal Diseases University Hospital Hradec Kralove Novy Hradec Kralove500 05 Hradec Kralove Czech Republic

5 Institute of Hygiene and Preventive Medicine Charles University in Prague Faculty of Medicine in Hradec Kralove50038 Hradec Kralove Czech Republic

Correspondence should be addressed to Lenka Borska borkalfhkcunicz

Received 13 June 2014 Revised 22 July 2014 Accepted 22 July 2014 Published 12 August 2014

Academic Editor Narasimham L Parinandi

Copyright copy 2014 Lenka Borska et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The paper presents a prospective cohort study Observed group was formed of children with plaque psoriasis (n = 19) treated byGoeckerman therapy (GT) The study describes adverse (side) effects associated with application of GT (combined exposure of3 crude coal tar ointment and UV radiation) After GT we found significantly increased markers of oxidative stress (8-hydroxy-21015840-deoxyguanosine 8-hydroxyguanosine and 8-hydroxyguanine) significantly increased levels of benzo[a]pyrene-78-diol-910-epoxide (BPDE) DNA adducts (BPDE-DNA) and significantly increased levels of total number of chromosomal aberrationsin peripheral lymphocytes We found significant relationship between (1) time of UV exposure and total number of aberratedcells and (2) daily topical application of 3 crude coal tar ointment ( of body surface) and level of BPDE-DNA adducts Thefindings indicated increased hazard of oxidative stress and genotoxic effects related to the treatment However it must be notedthat the oxidized guanine species and BPDE-DNA adducts also reflect individual variations in metabolic enzyme activity (differentextent of bioactivation of benzo[a]pyrene to BPDE) and overall efficiency of DNARNA repair system The study confirmed goodeffectiveness of the GT (significantly decreased PASI score)

1 Introduction

Oxidative stress characterized by a high degree of reactiveoxygen species (ROS) is known to induce damage to cellularcomponents including strand breaks and base modificationsin nucleic acids [1] Oxidative damage to nucleic acids hasbeen found to be associated with a variety of diseases

including cancer and aging but the precise mechanisms arestill remaining to be elucidated [2 3]

Psoriasis is a multifactorial relapsing and remittinginflammatory skin diseaseTheprevalence of psoriasis rangedfrom 14 to 33 in North America and Europe [4]The disease represents about 4 of all dermatoses whichoriginally manifested at 16 years of age or younger often as

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2014 Article ID 302528 10 pageshttpdxdoiorg1011552014302528

2 Oxidative Medicine and Cellular Longevity

plaque psoriasis [5 6] Psoriasis can adversely affect qualityof life of children [7]

Goeckerman therapy (GT) represents effective treatmentof plaque psoriasis in children [8 9] GT is based on dailydermal application of pharmaceutical grade crude coal tar(CCT) ointment with subsequent whole body exposure toUV radiation (UVR) Duration of the treatment is usually2-3 weeks [10] Retrospective study demonstrated that GT iseffective in children and adolescents with moderate to severepsoriasis [9]

The use of GT has recently decreased for severalreasons including suspected mutagenicitycarcinogenicityof CCT [11ndash13] The mutagenicitycarcinogenicity of CCTis probably caused by the presence of polycyclic aro-matic hydrocarbons (PAHs) Typical representative of muta-geniccarcinogenic PAHs benzo[a]pyrene (BaP) is metab-olized into a highly reactive metabolite benzo[a]pyrene-78-diol-910-epoxide (BPDE) that is able to bind to the structureof DNA RNA and proteins [14 15] The complex of reactivemetabolite and DNA structure is known as a DNA adduct Inthe case of BPDE it is a BPDE-DNA adduct [16] An alter-native PAHs carcinogenic pathway includes the inductionof oxidative stress caused by redox cycling between PAHsdihydrodiols and PAH-o-chinons with subsequent formationof various oxidized forms of nucleobases and nucleosides[17 18]

Children are more susceptible to the harm effects ofenvironmental exposure and medical treatments than adultsas their organism is developing [19 20] Recent trends inchildhood cancers in the USA and Europe seem to confirmchildrenrsquos increased exposures to genotoxic substances [21]

Elevated levels of biomarkers confirm deleterious effectsof carcinogenic PAHs on human health and underline theimportance of biomarker analysis as a preventive measure inchildren Suitable biomarkers include among others PAH-DNA adducts markers of oxidative stress and chromosomalaberration [19]

Presented study used biomarkers of oxidative stress andgenotoxicity for description of adverse (side) effects asso-ciated with application of GT (combined exposure of 3coal tar ointment and UV radiation) in children Mentionedeffects can damage cells accelerate aging induce diseasesand shorten life expectancy

2 Materials and Methods

21 Study Group The group was formed of children withchronic stable plaque psoriasis treated by GT at the Clinic ofDermal and Venereal Diseases University Hospital HradecKralove (Czech Republic) Over the period of two yearswe collected data of 19 children The group consisted of 12girls and 7 boys (average age of 12 years age range 5ndash17years) Exposure history of the patients was checked by thequestionnaire Regardless of the young age of respondentswe also assessed their smoking habitsThose patientswhohadsignificant prior exposure to PAHs andor artificialUVRwereexcluded from the monitored groupThe study was approvedby the Ethics Committee of theUniversity Hospital inHradec

Table 1 Detailed characteristic of the treatment

Number ofpatients

Total durationof GT (days)

Time of UVRexposure(days)

Daily topicalapplication ofCCT ointment( of bodysurface)

1 22 18 322 17 10 193 18 13 374 22 22 385 22 19 316 18 15 207 14 9 408 14 10 289 17 17 3110 14 13 4011 19 15 2812 21 21 3713 22 22 3714 14 9 3015 14 10 1716 17 17 3817 22 23 2818 17 17 3619 18 18 28

Kralove Czech Republic Informed written consent wasobtained from parents of each patient

22 Goeckerman Therapy (GT) Within the therapy derma-tological ointment containing 3 of CCT was administereddaily overnight on psoriatic lesions According to the extentof lesions 17ndash40 of the total body surface was covered byCCT ointment Each morning the residues of CCT ointmentwere removed from the body (using oil bath) and the patientswere whole-body irradiated by UVR

UVR exposure was dosed according to skin conditionand in cases of adverse reactions has been temporarilysuspended For this reason the total duration of GT (days)may not be equivalent to the time of UVR exposure(days) Irradiation time was individual according to thedisease status (1ndash15minday) The density of used radiationwas 24975120583Wcm2 of UV-B and 1318 120583Wcm2 of UV-A(controlled by Sola-Scope 2000 spectrometer Solatell UK)Duration of the treatment was modified according to thetreatment effectiveness (average duration of 18 days rangeof 14ndash22 days) Detailed individual characteristic of GTis summarized in Table 1 The effectiveness of the therapywas calculated from basic characteristics of actual diseasestatus (erythema desquamation and skin infiltration) andexpressed as the PASI score (Psoriasis Area and SeverityIndex) [22] Calculation of the efficiency of the treatment wasperformed according the formula (100minus [PASI after GTPASIbefore GT] times 100)

Oxidative Medicine and Cellular Longevity 3

Table 2 The content of selected PAHs in pharmaceutical grade crude coal tar (CCT)

PAHs IARCa mgg PAHs IARCa mggBenzo(a)pyrene 1 0008 Acenaphthene 3 0104Dibenz(ah)anthracene 2A 0 Anthracene 3 2494Benz(a)anthracene 2B 0 Benzo(ghi)perylene 3 0Benzo(b)fluoranthene 2B 0 Fluoranthene 3 0413Benzo(k)fluoranthene 2B 0 Fluorene 3 0299Chrysene 2B 0028 Phenanthrene 3 2520Indeno(123-cd)pyrene 2B 0 Pyrene 3 0241Naphthalene 2B 3286 Acenaphthylene mdash 0153aClassification according to WHO (IARC Monographs on the Evaluation of Carcinogenic Risk to Humans Volumes 1ndash109 2014)Group 1 the agent is carcinogenic to humansGroup 2A the agent is probably carcinogenic to humansGroup 2B the agent is possibly carcinogenic to humansGroup 3 the agent is not classifiable as to its carcinogenicity to humans

23 Selected PAHs in Pharmaceutical Grade Crude Coal Tar(CCT) The pooled sample of CCT (01973 g) was soni-cated (30min) with 25mL of n-hexane and subsequentlywas diluted fifty times Instrumental analysis of the extractwas performed by gas-chromatography-mass detection GCAgilent HP 6890MS HP 5975 column HP-5ms Agilent30m025mm025 120583m

24 Oxidative Damage of DNA and RNA Samples of bloodwere collected from the cubital vein a day before thefirst treatment and immediately after the last procedureof GT (BD Vacutainer sampling tubes) Blood serum wasisolated by centrifugation and stored under minus70∘C untilanalysis Repeated thawing and freezing were avoided Theextent of DNARNA oxidative damage was evaluated byEIA Kit (Enzyme Immunoassay Cayman Chemical Com-pany Michigan USA) Oxidative damage of DNA andRNA was presented as the sum of three oxidized guaninespecies in serum 8-hydroxy-21015840-deoxyguanosine from DNA8-hydroxyguanosine fromRNA and 8-hydroxyguanine fromeither DNA or RNA The level of DNARNA oxidativedamage was expressed in picograms of all guanine species permilliliter of serumwith detection limit of 33 pg of speciesmLof serum

25 Benzo[a]pyrene-78-diol-910-epoxide-DNAAdducts (BPDE-DNA Adducts) EDTA-treated peripheral blood sampleswere collected a day before the first treatment and imme-diately after the last procedure of GT DNA isolation fromlymphocytes was performed and samples were subsequentlydiluted to a concentration of 2120583g of DNA in 1mL The levelof BPDE-DNA adducts was determined using the standardmethod OxiSelect BPDE-DNA Adduct ELISA Kit (Cell Bio-labs Inc San Diego USA) The results were expressed asnanograms of BPDE-DNA adducts per microgram of DNAwith detection limit of 39 ng BPDE-DNA120583g of DNA

26 Chromosomal Aberration in Peripheral LymphocytesHeparin-treated peripheral blood samples were collected aday before the first treatment and immediately after the lastprocedure of GT and the level of chromosomal aberration

in peripheral lymphocytes was evaluated by standardizedmethod [23]Themethod is based onmicroscopic analysis oflymphocytic chromosomes undergoing mitotic metaphaseIn each blood sample 100 of mitotic sets were evaluatedWe counted structurally aberrated cells (SAC) numericallyaberrated cells (NAC) and total number of aberrated cells(ABC)

27 Statistical Analysis Obtained data were analyzed byusing MATLAB rel 2013b software (MathWorks Inc Mas-sachusetts USA) Because the Lilliefors test of normalityhad rejected the hypothesis of normal distribution the datawere analyzed by theWilcoxon signed rank test The possibleassociations between monitored biomarkers and importantfactors of exposure were evaluated by using Spearman RankOrder Correlations

3 Results

For analysis of selected PAHs we used a pooled sampleof CCT (from five different samples of CCT collected indifferent time of the study) The analyses were done for16 selected PAHs reported by the US EPA standards (USEnvironmental Protection Agency) The results are listed inTable 2The total content of 16 selected PAHs was 9546mggCCT and used dermatological ointment contains 3 ofCCT This means that 100 g of 3 dermatological ointmentcontained altogether 286mg of 16 selected PAHs As resultedfrom analysis of CCT (Table 2) one PAH is sorted intothe group of proven carcinogens one PAH in the group ofprobably carcinogens and six PAHs into the group of pos-sibly carcinogens Remaining PAHs are registered by IARChowever they are not classifiable as to their carcinogenicity tohumans

Table 3 summarizes the results of biomonitoring ofoxidative stress and genotoxic effects associated with theapplication of GT in observed group After GT (3 of CCT)we found significantly increased serum levels of oxidativedamage of DNA and RNA (119875 lt 005) BPDE-DNA adducts(119875 lt 001) and total number of aberrated cells (119875 lt0001) Increased levels of biomarkers are clearly visible in

4 Oxidative Medicine and Cellular Longevity

1000

1500

2000

2500

3000

3500

4000

1000 1500 2000 2500 3000 3500 4000

Oxidative damage DNARNA before GT (pgmL)

Oxi

dativ

e dam

age D

NA

RN

A aft

er G

T (p

gm

L)

0

5

10

0 5 10

Nonsmokers P = 0078 n = 14

Smokers P = NA n = 5

n (mdash)

n(mdash

)

Figure 1 Distribution of values of oxidative damage of DNA andRNAThe oxidative damage of DNARNA is presented as the sum ofthree oxidized guanine species per mL of serum Scatter plot depictsanti-BPDE-DNAvalues before and after theGT therapyAll together19 dots represent 38 measurements each dot belongs to a onepatientThe top histogram shows data distribution before treatmentthe right side histogram corresponds to the posttreatment valuesdistribution The white zones represent nonsmokers data and theblack zones depict smokersrsquo ones NA statistically not available

the attached scatter plots (Figures 1 2 and 3) which depictsdistribution of values of oxidative damage of DNA andRNA BPDE-DNA adducts and chromosomal aberrationin peripheral lymphocytes (total number of aberrated cells)before and after the GT Each dot belongs to at least onepatient (number of patients belonging to one dot is depictedin histograms) Values above the dotted diagonal line reflectthe increase of the oxidative and genotoxic damage after thetherapy Probability of difference between the pre- and theposttreatment medians was evaluated by Wilcoxon signedrank test The top histogram shows distribution of followedparameters before treatment the right side histogram cor-responds to the posttreatment values distribution On thesehistograms the development of changes in the distribution ofvalues can be well observed

The effectiveness of GT was high and the values ofPASI score significantly decreased after GT (119875 lt 0001)Before the therapy we found 204128ndash223 (medianlower-upper quartile of PASI) and after the therapy 9453ndash102(medianlower-upper quartile of PASI) Figure 4 depicts thePASI score with a similar layout as Figures 1ndash3 The valuesrepresenting positive treatment effect are below the diagonaldotted line The Wilcoxonrsquos test for smokers for such smallgroup did not have sufficient power to detect differences

Smokers P = NA n = 4

4

6

8

10

12

14

16

18

20

22

4 6 8 10 12 14 16 18 20 22

BPDE-DNA before GT (ngmL)

BPD

E-D

NA

after

GT

(ng

mL)

0

2

4

6

0 2 4

Nonsmokers P = 0014 n = 10

n(mdash

)

n (mdash)

Figure 2 Distribution of values of BPDE-DNA adducts The levelsare expressed as ng of BPDE-DNA adducts per 120583g of DNA Scatterplot depicts BPDE-DNA values before and after the GT therapy Alltogether 14 dots represent 28 measurements each dot belongs to aone patient The top histogram shows data distribution before treat-ment the right side histogram corresponds to the posttreatmentvalues distributionThewhite zones represent nonsmokers data andthe black zones depict smokersrsquo ones NA statistically not available

between pre- and posttreatment values and therefore 119875 isreported as not available (NA)

Table 4 presents the analysis of potential associationsbetween monitored biomarkers (values after GT) and impor-tant characteristics of exposure We found significant rela-tionship between (1) time of UV exposure and total numberof aberrated cells (119875 lt 005) (2) daily topical application ofCCT ointment ( of body surface) and BPDE-DNA adducts(119875 lt 005) and (3) daily topical application of CCT ointment( of body surface) and PASI score (119875 lt 001)

Cigarette smoking was reported by 5 patients Numberof smoked cigarettes was overall low and irregular andranged from one to four cigarettes per ldquoordinaryrdquo day (duringhospitalization they almost did not smoke at all) Groupof respondents who admitted smoking was not statisticallyevaluable because of low number of the patients (4-5) Forthe reasons above the statistical evaluations in Tables 3 and 4were performed for the whole group of respondents and thevalues of potential smokers were marked only in Figures 1ndash4

4 Discussion

The carcinogenicity of individual PAHs and PAHs containingmixtures has been studied in experimental animals Vir-tually no data exist on the carcinogenicity of individualPAHs in humans and only a limited amount of data on

Oxidative Medicine and Cellular Longevity 5

0

02

04

06

08

1

12

14

16

18

2

0 02 04 06 08 1 12 14 16 18 2 20100

20

10

0

ABC (total number of aberrant cells) before GT (mdash)

ABC

(tot

al n

umbe

r of a

berr

ant c

ells)

after

GT

(mdash)

Nonsmokers P = 0002 n = 14

Smokers P = NA n = 5

n (mdash)

n(mdash

)

Figure 3 Distribution of values of chromosomal aberration inperipheral lymphocytes (total number of aberrated cells) The levelsare expressed as percentage of total number of aberrated cells (ABC)Scatter plot depicts ABC values before and after the GT therapy Alltogether 5 dots represent 19 subjects and 38 measurements numberof patients belonging to one dot is shown in histograms The tophistogram shows data distribution before treatment the right sidehistogram corresponds to the posttreatment values distributionThewhite zones represent nonsmokers data and the black zones depictsmokersrsquo ones NA statistically not available

the carcinogenicity of PAHs containing mixtures is availablefor humans There is evidence that a number of individualPAHs are carcinogenic in experimental animals while othershave been found to be noncarcinogenic Because of thevarying carcinogenic potency of individual PAHs it is notpossible to provide a single weight of evidence carcinogenic-ity assessment for PAHs as a class As it was shown inTable 2 the carcinogenicity of each PAHs should be assessedseparately [24]

While it is not possible to provide a carcinogenicityassessment for PAHs as a class it may be possible to evaluatethe carcinogenicity of PAHs containing complex mixturesThere is good evidence that some PAHs containing mixturesare carcinogenic to humans (eg bitumen carbon black coaltars creosote and soot) Coal tars are classified accordingto IARC (International Agency for Research on Cancer) ingroup 1 the agent is carcinogenic to humans [24]

As can be seen from the results the amount of 100 g of3 CCT ointment contains 0024mg of proved carcinogensand 9942mg of possible carcinogensThe average amount of3 CCT ointment applied to treated skin was approximately4mgcm2 Extent of psoriatic lesions ranged approximatelyfrom 10 to 80 of the body surface and thus correspondingtotal dose of applied CCT ointment ranged from units to tens

Nonsmokers P = 0000 n = 14

Smokers P = NA n = 5

0

5

10

15

20

25

30

0 5 10 15 20 25 30

PASI before GT (mdash)

PASI

after

GT

(mdash)

0

5

10

0 5 10

n(mdash

)

n (mdash)

Figure 4 Distribution of PASI score The levels are dimensionlessScatter plot depicts PASI score before and after the GT therapy Alltogether 18 dots represent 38measurements each dot belongs at leastto a one patient The top histogram shows data distribution beforetreatment the right side histogram corresponds to the posttreat-ment values distribution The white zones represent nonsmokersdata and the black zones depict smokersrsquo ones NA statistically notavailable

of grams The total amount of carcinogenic PAHs applied tothe skin of given patient (and related hazard) can be assumedon the base of these data As an example we can describe thehazards of some patient with a body surface area of about14000 cm2 and with the 50 of skin affected by psoriaticlesions (7000 cm2) Then the theoretical total daily dose of16 selected PAHs will be 80mg and of carcinogenic PAHs27mg (PAHs from the group of 16) However although thegroup of 16 selected PAHs is toxicologically significant andrepresentative it should be noted that the group of selected 16PAHs represents only a small fraction of the total content ofPAHs in the CCT

It must be noted that the mixture of UV-A and UV-Bradiation (part of GT) is also ranked by IARC into Group1 with definition ultraviolet radiation wavelengths 100ndash400 nm encompassing UVA UVB and UVC It is evidentthat both components of the therapy (CCT and UVR)present a relatively high carcinogenic potential Moreover insituation of combined exposure to PAHs and UV radiationeven their synergistic effects on generation of ROS lipidperoxidation and DNA damage can be expected [25 26]

PAHsmay exert their mutagenic genotoxic and carcino-genic properties by two major mechanisms One pathwayincludes induction of oxidative stress [17 18 27] secondpathway includes formation of specific PAHs-DNA adducts[16 28 29]

4 Oxidative Medicine and Cellular Longevity

1000

1500

2000

2500

3000

3500

4000

1000 1500 2000 2500 3000 3500 4000

Oxidative damage DNARNA before GT (pgmL)

Oxi

dativ

e dam

age D

NA

RN

A aft

er G

T (p

gm

L)

0

5

10

0 5 10

Nonsmokers P = 0078 n = 14

Smokers P = NA n = 5

n (mdash)

n(mdash

)

Figure 1 Distribution of values of oxidative damage of DNA andRNAThe oxidative damage of DNARNA is presented as the sum ofthree oxidized guanine species per mL of serum Scatter plot depictsanti-BPDE-DNAvalues before and after theGT therapyAll together19 dots represent 38 measurements each dot belongs to a onepatientThe top histogram shows data distribution before treatmentthe right side histogram corresponds to the posttreatment valuesdistribution The white zones represent nonsmokers data and theblack zones depict smokersrsquo ones NA statistically not available

the attached scatter plots (Figures 1 2 and 3) which depictsdistribution of values of oxidative damage of DNA andRNA BPDE-DNA adducts and chromosomal aberrationin peripheral lymphocytes (total number of aberrated cells)before and after the GT Each dot belongs to at least onepatient (number of patients belonging to one dot is depictedin histograms) Values above the dotted diagonal line reflectthe increase of the oxidative and genotoxic damage after thetherapy Probability of difference between the pre- and theposttreatment medians was evaluated by Wilcoxon signedrank test The top histogram shows distribution of followedparameters before treatment the right side histogram cor-responds to the posttreatment values distribution On thesehistograms the development of changes in the distribution ofvalues can be well observed

The effectiveness of GT was high and the values ofPASI score significantly decreased after GT (119875 lt 0001)Before the therapy we found 204128ndash223 (medianlower-upper quartile of PASI) and after the therapy 9453ndash102(medianlower-upper quartile of PASI) Figure 4 depicts thePASI score with a similar layout as Figures 1ndash3 The valuesrepresenting positive treatment effect are below the diagonaldotted line The Wilcoxonrsquos test for smokers for such smallgroup did not have sufficient power to detect differences

Smokers P = NA n = 4

4

6

8

10

12

14

16

18

20

22

4 6 8 10 12 14 16 18 20 22

BPDE-DNA before GT (ngmL)

BPD

E-D

NA

after

GT

(ng

mL)

0

2

4

6

0 2 4

Nonsmokers P = 0014 n = 10

n(mdash

)

n (mdash)

Figure 2 Distribution of values of BPDE-DNA adducts The levelsare expressed as ng of BPDE-DNA adducts per 120583g of DNA Scatterplot depicts BPDE-DNA values before and after the GT therapy Alltogether 14 dots represent 28 measurements each dot belongs to aone patient The top histogram shows data distribution before treat-ment the right side histogram corresponds to the posttreatmentvalues distributionThewhite zones represent nonsmokers data andthe black zones depict smokersrsquo ones NA statistically not available

between pre- and posttreatment values and therefore 119875 isreported as not available (NA)

Table 4 presents the analysis of potential associationsbetween monitored biomarkers (values after GT) and impor-tant characteristics of exposure We found significant rela-tionship between (1) time of UV exposure and total numberof aberrated cells (119875 lt 005) (2) daily topical application ofCCT ointment ( of body surface) and BPDE-DNA adducts(119875 lt 005) and (3) daily topical application of CCT ointment( of body surface) and PASI score (119875 lt 001)

Cigarette smoking was reported by 5 patients Numberof smoked cigarettes was overall low and irregular andranged from one to four cigarettes per ldquoordinaryrdquo day (duringhospitalization they almost did not smoke at all) Groupof respondents who admitted smoking was not statisticallyevaluable because of low number of the patients (4-5) Forthe reasons above the statistical evaluations in Tables 3 and 4were performed for the whole group of respondents and thevalues of potential smokers were marked only in Figures 1ndash4

4 Discussion

The carcinogenicity of individual PAHs and PAHs containingmixtures has been studied in experimental animals Vir-tually no data exist on the carcinogenicity of individualPAHs in humans and only a limited amount of data on

Oxidative Medicine and Cellular Longevity 5

0

02

04

06

08

1

12

14

16

18

2

0 02 04 06 08 1 12 14 16 18 2 20100

20

10

0

ABC (total number of aberrant cells) before GT (mdash)

ABC

(tot

al n

umbe

r of a

berr

ant c

ells)

after

GT

(mdash)

Nonsmokers P = 0002 n = 14

Smokers P = NA n = 5

n (mdash)

n(mdash

)

Figure 3 Distribution of values of chromosomal aberration inperipheral lymphocytes (total number of aberrated cells) The levelsare expressed as percentage of total number of aberrated cells (ABC)Scatter plot depicts ABC values before and after the GT therapy Alltogether 5 dots represent 19 subjects and 38 measurements numberof patients belonging to one dot is shown in histograms The tophistogram shows data distribution before treatment the right sidehistogram corresponds to the posttreatment values distributionThewhite zones represent nonsmokers data and the black zones depictsmokersrsquo ones NA statistically not available

the carcinogenicity of PAHs containing mixtures is availablefor humans There is evidence that a number of individualPAHs are carcinogenic in experimental animals while othershave been found to be noncarcinogenic Because of thevarying carcinogenic potency of individual PAHs it is notpossible to provide a single weight of evidence carcinogenic-ity assessment for PAHs as a class As it was shown inTable 2 the carcinogenicity of each PAHs should be assessedseparately [24]

While it is not possible to provide a carcinogenicityassessment for PAHs as a class it may be possible to evaluatethe carcinogenicity of PAHs containing complex mixturesThere is good evidence that some PAHs containing mixturesare carcinogenic to humans (eg bitumen carbon black coaltars creosote and soot) Coal tars are classified accordingto IARC (International Agency for Research on Cancer) ingroup 1 the agent is carcinogenic to humans [24]

As can be seen from the results the amount of 100 g of3 CCT ointment contains 0024mg of proved carcinogensand 9942mg of possible carcinogensThe average amount of3 CCT ointment applied to treated skin was approximately4mgcm2 Extent of psoriatic lesions ranged approximatelyfrom 10 to 80 of the body surface and thus correspondingtotal dose of applied CCT ointment ranged from units to tens

Nonsmokers P = 0000 n = 14

Smokers P = NA n = 5

0

5

10

15

20

25

30

0 5 10 15 20 25 30

PASI before GT (mdash)

PASI

after

GT

(mdash)

0

5

10

0 5 10

n(mdash

)

n (mdash)

Figure 4 Distribution of PASI score The levels are dimensionlessScatter plot depicts PASI score before and after the GT therapy Alltogether 18 dots represent 38measurements each dot belongs at leastto a one patient The top histogram shows data distribution beforetreatment the right side histogram corresponds to the posttreat-ment values distribution The white zones represent nonsmokersdata and the black zones depict smokersrsquo ones NA statistically notavailable

of grams The total amount of carcinogenic PAHs applied tothe skin of given patient (and related hazard) can be assumedon the base of these data As an example we can describe thehazards of some patient with a body surface area of about14000 cm2 and with the 50 of skin affected by psoriaticlesions (7000 cm2) Then the theoretical total daily dose of16 selected PAHs will be 80mg and of carcinogenic PAHs27mg (PAHs from the group of 16) However although thegroup of 16 selected PAHs is toxicologically significant andrepresentative it should be noted that the group of selected 16PAHs represents only a small fraction of the total content ofPAHs in the CCT

It must be noted that the mixture of UV-A and UV-Bradiation (part of GT) is also ranked by IARC into Group1 with definition ultraviolet radiation wavelengths 100ndash400 nm encompassing UVA UVB and UVC It is evidentthat both components of the therapy (CCT and UVR)present a relatively high carcinogenic potential Moreover insituation of combined exposure to PAHs and UV radiationeven their synergistic effects on generation of ROS lipidperoxidation and DNA damage can be expected [25 26]

PAHsmay exert their mutagenic genotoxic and carcino-genic properties by two major mechanisms One pathwayincludes induction of oxidative stress [17 18 27] secondpathway includes formation of specific PAHs-DNA adducts[16 28 29]

Oxidative Medicine and Cellular Longevity 5

0

02

04

06

08

1

12

14

16

18

2

0 02 04 06 08 1 12 14 16 18 2 20100

20

10

0

ABC (total number of aberrant cells) before GT (mdash)

ABC

(tot

al n

umbe

r of a

berr

ant c

ells)

after

GT

(mdash)

Nonsmokers P = 0002 n = 14

Smokers P = NA n = 5

n (mdash)

n(mdash

)

Figure 3 Distribution of values of chromosomal aberration inperipheral lymphocytes (total number of aberrated cells) The levelsare expressed as percentage of total number of aberrated cells (ABC)Scatter plot depicts ABC values before and after the GT therapy Alltogether 5 dots represent 19 subjects and 38 measurements numberof patients belonging to one dot is shown in histograms The tophistogram shows data distribution before treatment the right sidehistogram corresponds to the posttreatment values distributionThewhite zones represent nonsmokers data and the black zones depictsmokersrsquo ones NA statistically not available

the carcinogenicity of PAHs containing mixtures is availablefor humans There is evidence that a number of individualPAHs are carcinogenic in experimental animals while othershave been found to be noncarcinogenic Because of thevarying carcinogenic potency of individual PAHs it is notpossible to provide a single weight of evidence carcinogenic-ity assessment for PAHs as a class As it was shown inTable 2 the carcinogenicity of each PAHs should be assessedseparately [24]

While it is not possible to provide a carcinogenicityassessment for PAHs as a class it may be possible to evaluatethe carcinogenicity of PAHs containing complex mixturesThere is good evidence that some PAHs containing mixturesare carcinogenic to humans (eg bitumen carbon black coaltars creosote and soot) Coal tars are classified accordingto IARC (International Agency for Research on Cancer) ingroup 1 the agent is carcinogenic to humans [24]

As can be seen from the results the amount of 100 g of3 CCT ointment contains 0024mg of proved carcinogensand 9942mg of possible carcinogensThe average amount of3 CCT ointment applied to treated skin was approximately4mgcm2 Extent of psoriatic lesions ranged approximatelyfrom 10 to 80 of the body surface and thus correspondingtotal dose of applied CCT ointment ranged from units to tens

Nonsmokers P = 0000 n = 14

Smokers P = NA n = 5

0

5

10

15

20

25

30

0 5 10 15 20 25 30

PASI before GT (mdash)

PASI

after

GT

(mdash)

0

5

10

0 5 10

n(mdash

)

n (mdash)

Figure 4 Distribution of PASI score The levels are dimensionlessScatter plot depicts PASI score before and after the GT therapy Alltogether 18 dots represent 38measurements each dot belongs at leastto a one patient The top histogram shows data distribution beforetreatment the right side histogram corresponds to the posttreat-ment values distribution The white zones represent nonsmokersdata and the black zones depict smokersrsquo ones NA statistically notavailable

of grams The total amount of carcinogenic PAHs applied tothe skin of given patient (and related hazard) can be assumedon the base of these data As an example we can describe thehazards of some patient with a body surface area of about14000 cm2 and with the 50 of skin affected by psoriaticlesions (7000 cm2) Then the theoretical total daily dose of16 selected PAHs will be 80mg and of carcinogenic PAHs27mg (PAHs from the group of 16) However although thegroup of 16 selected PAHs is toxicologically significant andrepresentative it should be noted that the group of selected 16PAHs represents only a small fraction of the total content ofPAHs in the CCT

It must be noted that the mixture of UV-A and UV-Bradiation (part of GT) is also ranked by IARC into Group1 with definition ultraviolet radiation wavelengths 100ndash400 nm encompassing UVA UVB and UVC It is evidentthat both components of the therapy (CCT and UVR)present a relatively high carcinogenic potential Moreover insituation of combined exposure to PAHs and UV radiationeven their synergistic effects on generation of ROS lipidperoxidation and DNA damage can be expected [25 26]

PAHsmay exert their mutagenic genotoxic and carcino-genic properties by two major mechanisms One pathwayincludes induction of oxidative stress [17 18 27] secondpathway includes formation of specific PAHs-DNA adducts[16 28 29]

6 Oxidative Medicine and Cellular Longevity

Table 3 Oxidative damage of DNARNA BPDE-DNA adducts and chromosomal aberrations in children treated by GT

Oxidative damage of DNA and RNAa

Significance of differencesBefore GT After GT(119899 = 19) (119899 = 19)

2557 2687119875 lt 005

(2008ndash2789) (2146ndash3238)BPDE-DNA adductsb

Significance of differencesBefore GT After GT(119899 = 14) (119899 = 14)

565 72119875 lt 001

(497ndash73) (535ndash79)Chromosomal aberration in peripheral lymphocytesc

Significance of differencesBefore GT After GT(119899 = 19) (119899 = 19)

Structurally aberrated cells00 10

119875 lt 001(00-00) (00ndash10)

Numerically aberrated cells00 00 NS(00-00) (00ndash10)

Total number of aberrated cells00 10

119875 lt 0001(00-00) (00ndash10)aOxidative damage of DNA and RNA is presented as the sum of three oxidized guanine species in serum 8-hydroxy-21015840-deoxyguanosine from DNA 8-hydroxyguanosine from RNA and 8-hydroxyguanine from either DNA or RNAThe levels are expressed as pg of the sum of oxidized guanine species per mLof serumThe average concentration is presented as median and lower-upper quartile because of nonnormal data distribution 119875 Wilcoxonmatched-pairs testbThe levels are expressed as ng of BPDE-DNA adducts per 120583g of DNA and the average concentration is presented as median and lower-upper quartile becauseof nonnormal data distribution 119875 Wilcoxon matched-pairs testcThe levels are expressed as percentage of structurally aberrated cells (SAC) numerically aberrated cells (NAC) and total number of aberrated cells (ABC)The average levels of aberration are presented as median and lower-upper quartile because of nonnormal data distribution 119875 Wilcoxon matched-pairs testNS nonsignificant difference

Table 4The associations between monitored biomarkers (levels after GT) and important characteristics of exposure (Spearman Rank OrderCorrelations)

Total durationof GT(days)

Time of UVR exposure(days)

Daily topical application ofCCT ointment ( of body

surface)Oxidative damage of DNAand RNAa NS NS NS

BPDE-DNA adductsb NS NS 119903 = 062

119875 lt 005

Total number of aberratedcellsc NS 119903 = 055

119875 lt 005NS

PASI scored NS NS 119903 = 065

119875 lt 001aUnit pg of the sum of three oxidized guanine species per ml of serum

bUnit ng of BPDE-DNA adducts per 120583g of DNAcUnit percentage of total number of aberrated cells (ABC)dDimensionless

Oxidative stress represents a deregulation of the home-ostasis between the reactive oxygen species and the mecha-nisms of detoxification and repair [27] The currently avail-able epidemiological and laboratory data indicate that oxida-tive stress plays a central role in carcinogenic potential effectof PAHs [30] PAHs can be metabolized by the cytochrome

P450 enzymes to active semiquinones which are knownfree radicals intermediates and can go through redox cycling(between PAHs dihydrodiols and PAH-o-chinons) and gen-erate reactive oxygen species (ROS) [28] Subsequently theROS can then cause oxidativemodification ofDNA and lipidsin the body [30] Oxidative damage to nucleic acids has been

Oxidative Medicine and Cellular Longevity 7

found to be associated with a variety of diseases includingcancer and aging Guanine is the base that is the most proneto oxidationThe 8-hydroxy-21015840-deoxyguanosine (fromDNA)is the form of oxidized guanine that is the most commonlystudied critical biomarker of oxidativeDNAdamage in adultsand children exposed to PAHs [31ndash33]

In recent years it has become increasingly clear thatboth DNA and RNA are damaged by oxidation in dis-ease states and that the repair processes that are initiatedto correct this damage release multiple oxidized guanineresidues including the ribose free base (8-oxo-guanineor 8-hydroxyguanine) the nucleoside from RNA (8-oxo-guanosine or 8-hydroxyguanosine) and the deoxynucleo-side from DNA (8-oxo-deoxyguanosine or 8-hydroxy-21015840-deoxyguanosine) [2 18] These residues are released into thebody fluid including cerebrospinal fluid plasmaserum andurine under the condition of oxidative stress [1 2]

While 8-hydroxy-21015840-deoxyguanosine is the form mostresearches are familiar with other published studies havereported that the RNA residue 8-hydroxyguanosine is a betterbiomarker of age-related oxidant damage [17] and that thebase (8-hydroxyguanine) is a better biomarker in some can-cer patients [3 27 34] From the reasons described above it isrecommended to analyze more than one oxidation (damage)product since if just one damage product is measured thereis a risk that the specific damage (product) may not be a goodmarker of the oxidative stress and thus not give a true pictureof the amount of oxidative stress the subject in question isexposed to [35]

It was shown in Table 3 that the level of oxidative damageofDNAandRNAwas significantly increased after the therapy(119875 lt 005) This exposure scenario indicates (in given groupof children) increased hazard of oxidative stress related to thetreatment However it must be noted that although higherlevel of oxidized guanine residues indicates elevated level ofoxidative stress it can also reflect a high level of efficiencyof the processes that work to repair this damage (oxidativestress can be high and the repair processes eliminate itseffects) Therefore the interpretation of this bioindicator forthe purpose of health risk assessment requires a certaindegree of caution

In the presented study the oxidative damage of DNA andRNA was presented as the sum of three oxidized guaninespecies in serum 8-hydroxy-21015840-deoxyguanosine from DNA8-hydroxyguanosine fromRNA and 8-hydroxyguanine fromeither DNA or RNA (pgmL of serum) To the best ofour knowledge the state of oxidative stress (expressed asdifferent serum oxidized guanine species) in children der-mally exposed to CCT and UVR has not been yet reportedPreviously published studies were focused mostly on rela-tionship between inhalation exposure to PAHs and differentguanine species in urine (not in serum) For example thestudy of Rossnerova et al presented inhalation exposureto benzo[a]pyrene and related urinary 8-oxo-78-dihydro-21015840-deoxyguanosine in children living in highly polluted areaThe level of urinary 8-oxo-78-dihydro-21015840-deoxyguanosine ofthese children ranged from 596 to 673 nmolmmol creati-nine [32] Other authors described impact of inhalation expo-sure of children to PAHs (traffic pollution polluted versus

nonpolluted area) on urinary 8-hydroxy-21015840-deoxyguanosine[31] They did not find significant difference for urinary8-hydroxy-21015840-deoxyguanosine concentration between thegroups from polluted and nonpolluted areas (2087 versus1678 120583molmol creatinine) It can be seen that urinary 8-hydroxy-21015840-deoxyguanosine concentration was only slightlyhigher in the group from polluted area The authors assumethat the potential of coexposure of the children to other pol-lutants affects 8-hydroxy-21015840-deoxyguanosine concentrationbesides the PAHs However as stated previously it cannotbe excluded that the used oxidation product (8-hydroxy-21015840-deoxyguanosine) was not the most suitable marker of theoxidative stress and thus did not give a real picture of theamount of oxidative stress [35] On the other hand a clear linkbetween heavy occupational exposure to PAHs (coke ovenworkers) and the levels of urinary 8-hydroxydeoxyguanosinewas found in the work of Kuang et al [30] The levels of uri-nary 8-hydroxydeoxyguanosine ranged from 817 (control) to1157 (heavily exposed group) nmolmmol creatinine

Benzo(a)pyrene is notable for being the first chemical car-cinogen to be discovered BaP is a five-ring PAH known to beprocarcinogen Itsmechanism of carcinogenesis is dependenton a three-step enzymatic metabolism to final mutagenThe steps include generation of benzo(a)pyrene-78-epoxidebenzo(a)pyrene-78-diol and finally benzo(a)pyrene-78-diol-910-epoxide (BPDE) Very reactive BPDE bindscovalently to proteins lipids and DNA (guanine residues)to produce BPDE-DNA adducts If left unrepaired DNAadducts may lead to permanent mutations resulting incell transformation and ultimately to tumor development[15] Biological monitoring includes indicators of exposurebiological effect and susceptibility BPDE-DNA adductscan be indicative of both exposure and genotoxic effect ofBaP The level of BPDE-DNA adducts is determined by thecharacter of BaP metabolism (metabolic enzyme activities)and by the degree of reparation mechanisms

It was shown in Table 3 that the level of BPDE-DNAadducts was significantly increased after the therapy (565versus 72 ng BPDE-DNA adducts120583g DNA 119875 lt 001) andit indicates increased hazard of genotoxic effects related tothe treatment However similar to the scenariowith oxidativestress it must be noted that the final level of BPDE-DNAadducts reflects combined effect of intensity of exposure toBaP metabolic activity (production of BPDE-DNA adducts)and efficiency of DNARNA repair mechanisms The worstscenario should include high level of exposure high pro-duction of BPDE-DNA adducts (high metabolic activity ofresponsible CYP enzymes) and limited activity of repairsystem This indicates (analogously to the biomarkers ofoxidative stress) that the interpretation of the level of BPDE-DNA adducts for purposes of health risk assessment requiresa certain degree of caution

The number of epidemiological studies related to BPDE-DNA adducts in children is limited [15 29 36] In addi-tion these studies are different in used methods unitsand analyzed biological materials Thus the comparison ofresults is a little bit complicated For example Pauk et alcompared the levels of total DNA adducts in lymphocytesbetween different groups of respondents [15] These groups

8 Oxidative Medicine and Cellular Longevity

included healthy persons patients with chronic obstructivepulmonary disease (noncancerous lung disease which occursmainly due to smoking and other long-term respiratoryexposure to chemical substances) and patients with lungcancer The level of total DNA adducts was determinedusing P32-postlabeling method and ranged between 0710and 1329AU (arbitrary units) The patients with diseaseslargely associated with smoking (PAHs) showed increasedlevels of total DNA adducts in comparison to nonsmokingpatients The influence of tobacco smoke (PAHs) on the levelof total DNA adducts and B(a)P-like adducts in newbornsand mothers was studied by Topinka et al [36] PAH-DNA adducts and B(a)P-like adducts were determined usingP32-postlabeling method In subjects unexposed to tobaccosmoke the level of total DNA adducts in lymphocytes rangedfrom 106 (newborns) to 113 (mothers) and the level of B(a)P-like adducts ranged from 016 (newborns) to 019 (mothers)DNA adducts108 nucleotides In subjects exposed to tobaccosmoke the level of total DNA adducts in lymphocytes rangedfrom092 (newborns) to 118 (mothers) and the level of B(a)P-like adducts ranged from 015 (newborns) to 022 (mothers)DNA adducts108 nucleotides

Genotoxic exposures occurring during childhood maycontinue for several years become chronic and eventuallyplay a relevant role in the etiology of childhood as wellas adulthood cancers Chromosomal aberrations in humanperipheral lymphocytes represent well-established biomarkerof genotoxicity probably the only one which has beeninternationally standardized and validated for children [1921 37]

In our study we found significantly increased levels ofABC (119875 lt 001) and SAC (119875 lt 005) after GT and thesefindings support assumption of elevated genotoxic hazard(Table 3) Before GT we found only two aberrations (ABC)in the total number of 1900 cells (010) One aberration wasclassified as a structural abnormality (SAC) and one cell as anumerical abnormality (NAC) After GT we found seventeenaberrations (ABC) in the total number of 1900 cells (080)Eleven aberrations were classified as structural abnormalities(SAC) and six as numerical abnormalities (NAC) Declaredreference value for the total number of aberrated cells (ABC)of healthy Czech children ranges from 0 to 185 This is arange of values for a group of children (119899 = 20) [23 38] Fromthe above it is evident that treatment increased the level ofaberrated cells in lymphocytes (genotoxic hazard) Under thiscondition we can consider that a similar situation of elevatedgenotoxic hazard was obvious also in other tissuesorgansthat are well perfused with blood In conclusion it must beemphasized that the level of abnormalities which was foundafter the therapy (080) is still below the upper level of thereference range for healthy Czech children (185 119875 lt 001)This means that although there was an increase of genotoxicrisk this risk is within the acceptable values

The results of the present study agree well with the resultsof our previous work [8] In that work we studied a group ofchildren treated with GT using 5 coal tar ointment Afterthe treatment there were found significantly increased levelsof aberrations (ABC median 20 lower-upper quartile

20-30) As it can be seen the median value of ABC in thecase of foregoing study (5 of CCT) was greater thanmedianvalue presented in this paper (3 CCT) and exceeded theupper level of the reference range for healthy Czech children(185) The results from both studies suggest possible dosedependency

Nowadays it is generally accepted that the high frequencyof cytogenetic biomarkers chromosomal aberrations canpredict an increased risk of genotoxic effect in children afterexposure to PAHs Exposure situations can be describedeither by simply increasing the levels from initial state (ifknown) or by comparison with the reference value(s) It isknown that cytogenetic biomarkers may vary with genderand age in children (adults) The age extent of children inour group ranged from 5 to 17 years and the group includesboys and girls It may therefore be the question whether thisage range and gender differences can lead to some shiftsof reference values of chromosomal aberrations Merlo etal analyzed data from 16 published epidemiologic studiesperformed on pediatric population and from a large sampleof Czech children aged 7ndash16 years For the whole referentpopulation (age range 0ndash19 years) the mean frequency ofchromosomal aberration was 124 (95 CI = 105ndash147)Similar baseline levels were found for chromosome breaksfrequency in boys and girls 122 and 121 respectively Inconclusion based on the reviewed studies baseline levels forchromosomal aberrations were similar in boys and girls andfailed to show any increase with age [37]

While it appears that most of genotoxic effects of GT canbe attributed to PAHs it is likely that UVR as integral part ofGT increases the risk for mutagenicity and carcinogenicityUVR is known to increase the toxicity of PAHs throughphotoactivation and photomodification [39] Skin damagecaused by exposure to BaP is further increased by exposureto both UV-A and UV-B radiation The effects related toUV-A radiation seem to be more significant [40] It hasbeen shown that BaP in combination with UV-A radiationsynergistically induced oxidative DNA damage These factsindicate that UV-A radiation is able to transform BaP intomore harmful compounds [39] Another research demon-strates that BaP in combination with UV-A radiation cansubstantially increase oxidative damage of DNA via a ROSmechanism (primarily represented as elevated levels of 8-hydroxy-21015840-deoxyguanosine) [16 25 41] In the presentedwork (Table 4) we found significant relationship betweentime of UV exposure and total number of aberrated cells(119875 lt 005)

The effectiveness of the therapy was expressed as the PASIscore In the presented study the PASI score was significantlydecreased after GT (119875 lt 0001) and confirmed generallyassumed high effectiveness of the GT for children [8 9]We found significant relationship between the PASI scoreand daily topical application of CCT ointment ( of bodysurface)

5 Conclusion

We found significantly increased markers of oxidativestress (8-hydroxy-21015840-deoxyguanosine 8-hydroxyguanosine

Oxidative Medicine and Cellular Longevity 9

and 8-hydroxyguanine) significantly increased levels ofBPDE-DNA adducts and significantly increased levels oftotal number of chromosomal aberration in peripheral lym-phocytes of children with psoriasis treated by GT Thesefindings can indicate increased hazard of oxidative stressand genotoxic effects related to the treatment However itmust be acknowledged that the oxidized guanine species andBPDE-DNA adducts also reflect the individual variations inmetabolic enzyme activity (different extent of bioactivation ofBaP to BPDE) as well as the overall efficiency of DNARNArepair systemThe study confirmed good clinical effect of theGT (significantly decreased PASI score)

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgments

This research was supported by Charles University in PragueFaculty of Medicine in Hradec Kralove Czech RepublicProjects PRVOUK P3709 and PRVOUK P3710The authorsthank Dana Fialova for proofreading

References

[1] Y Ma L Zhang S Rong et al ldquoRelation between gastric cancerand protein oxidation DNA damage and lipid peroxidationrdquoOxidative Medicine and Cellular Longevity vol 2013 Article ID543760 6 pages 2013

[2] B Nie W Gan F Shi et al ldquoAge-dependent accumulation of8-oxoguanine in the DNA and RNA in various rat tissuesrdquoOxidative Medicine and Cellular Longevity vol 2013 Article ID303181 9 pages 2013

[3] K Shinmura M Goto H Tao et al ldquoImpaired 8-hydroxy-guanine repair activity of MUTYH variant pArg109Trp foundin a Japanese patient with early-onset colorectal cancerrdquoOxida-tiveMedicine andCellular Longevity vol 2014Article ID61735112 pages 2014

[4] M G Lebwohl H Bachelez J Barker et al ldquoPatient per-spectives in the management of psoriasis results from thepopulation-based Multinational Assessment of Psoriasis andPsoriatic Arthritis Surveyrdquo Journal of the American Academy ofDermatology vol 70 no 5 pp 871ndash881 2014

[5] I Lara-Corrales S Ramnarine and P Lansang ldquoTreatment ofchildhood psoriasis with phototherapy and photochemother-apyrdquoClinicalMedicine Insights Pediatrics vol 7 pp 25ndash33 2013

[6] M Seyhan B K Coskun H Saglam et al ldquoPsoriasis in child-hood and adolescence evaluation of demographic and clinicalfeaturesrdquo Pediatrics International vol 48 no 6 pp 525ndash5302006

[7] P E Beattie and M S Lewis-Jones ldquoA comparative study ofimpairment of quality of life in children with skin disease andchildren with other chronic childhood diseasesrdquo British Journalof Dermatology vol 155 no 1 pp 145ndash151 2006

[8] L Borska J Smejkalova M Cerna et al ldquoUrinary mutagenicityand genotoxic risk in children with psoriasis after therapeuticexposure to polycyclic aromatic hydrocarbons and ultravioletradiationrdquoMutation Research vol 696 no 2 pp 144ndash147 2010

[9] K R KortuemMD P Davis PMWitmanM TMcEvoy andS A Farmer ldquoResults of Goeckerman treatment for psoriasis inchildren a 21-year retrospective reviewrdquo Pediatric dermatologyvol 27 no 5 pp 518ndash524 2010

[10] M L Orseth and T G Cropley ldquoWhatrsquos in a name Goecker-man therapyrdquo JAMADermatology vol 149 no 12 p 1409 2013

[11] K V Paghdal and R A Schwartz ldquoTopical tar back to thefuturerdquo Journal of the American Academy of Dermatology vol61 no 2 pp 294ndash302 2009

[12] L Borska Z Fiala and C Andrys ldquoGoeckermanrsquos regimengenotoxic pro-apoptotic anti-inflamatory and anti-angiogeniceffectrdquo in Dermatology Research Focus on Acne Melanomaand Psoriasis F Columbus Ed pp 141ndash165 Nova SciencePublishers Hauppauge NY USA 2010

[13] J H J Roelofzen K K H Aben P G M Van Der Valk J LM Van Houtum P C M Van De Kerkhof and L A L MKiemeney ldquoCoal tar in dermatologyrdquo The Journal of Dermato-logical Treatment vol 18 no 6 pp 329ndash334 2007

[14] L Borska C Andrys J Krejsek et al ldquoSerum level of antibodyagainst benzo [a]pyrene-78-diol-910-epoxide-DNA adducts inpeople dermally exposed to PAHsrdquo Journal of ImmunologyResearch vol 2014 Article ID 834389 6 pages 2014

[15] N Pauk S Klimesova J Kara J Topinka and J Labaj ldquoTherelevance of monitoring of antibodies against the polycyclicaromatic hydrocarbon (PAH) and PAH-DNA adducts in serumin relation to lung cancer and chronic obstructive pulmonarydisease (COPD)rdquo Neoplasma vol 60 no 2 pp 182ndash187 2013

[16] D Gao Y Luo D Guevara et al ldquoBenzo[a]pyrene and itsmetabolites combined with ultraviolet a synergistically induce8-hydroxy-2 1015840-deoxyguanosine via reactive oxygen speciesrdquoFree Radical Biology and Medicine vol 39 no 9 pp 1177ndash11832005

[17] W Gan B Nie F Shi et al ldquoAge-dependent increases inthe oxidative damage of DNA RNA and their metabolitesin normal and senescence-accelerated mice analyzed by LC-MSMS urinary 8-oxoguanosine as a novel biomarker of agingrdquoFree Radical Biology and Medicine vol 52 no 9 pp 1700ndash17072012

[18] F Shi B Nie W Gan et al ldquoOxidative damage of DNA RNAand theirmetabolites in leukocytes plasma and urine ofMacacamulatta 8-oxoguanosine in urine is a useful marker for agingrdquoFree Radical Research vol 46 no 9 pp 1093ndash1098 2012

[19] R J Sram B Binkova M Dostal et al ldquoHealth impact ofair pollution to childrenrdquo International Journal of Hygiene andEnvironmental Health vol 216 no 5 pp 533ndash540 2013

[20] N Holland A Fucic D F Merlo R Sram and M Kirsch-Volders ldquoMicronuclei in neonates and children effects ofenvironmental genetic demographic and disease variablesrdquoMutagenesis vol 26 no 1 pp 51ndash56 2011

[21] M Neri S Bonassi L E Knudsen et al ldquoChildrenrsquos exposure toenvironmental pollutants and biomarkers of genetic damage IOverview and critical issuesrdquoMutation Research vol 612 no 1pp 1ndash13 2006

[22] M A De Rie A Y Goedkoop and J D Bos ldquoOverview ofpsoriasisrdquo Dermatologic Therapy vol 17 no 5 pp 341ndash3492004

[23] AHEM ldquoStandardized operating approaches for biologicalmonitoring of genotoxic effects of environmental factorsrdquo ActaHygienica Epidemiologica et Microbiologica National Instituteof Health Praha vol 3 pp 6ndash17 2003

[24] IARC ldquoIARC monographs on the evaluation of carcinogenicrisks to humans Agents classified by the IARCmonographsrdquo in

10 Oxidative Medicine and Cellular Longevity

International Agency for Research on Cancer 150 Cours AlbertThomas 69372 pp 1ndash109 Lyon France 2014

[25] K E Burke and H Wei ldquoSynergistic damage by UVA radiationand pollutantsrdquo Toxicology and Industrial Health vol 25 no 4-5 pp 219ndash224 2009

[26] Y Yan J Jiang Z Huang Z Wu C Lin and F Wu ldquoAntisenseoligonucleotides targeting protein kinase C alpha inhibits theproliferation of A549 cellsrdquo Journal of SouthernMedical Univer-sity vol 29 no 6 pp 1189ndash1191 2009

[27] K Roszkowski ldquoOxidative DNA damagemdashthe possible useof biomarkers as additional prognostic factors in oncologyrdquoFrontiers in Bioscience vol 19 pp 808ndash817 2014

[28] B Marczynski M Raulf-Heimsoth A Spickenheuer et alldquoDNAadducts and strand breaks inworkers exposed to vapoursand aerosols of bitumen associations between exposure andeffectrdquo Archives of Toxicology vol 85 no 1 pp S53ndashS64 2011

[29] M K Chung J Riby H Li et al ldquoA sandwich enzyme-linkedimmunosorbent assay for adducts of polycyclic aromatic hydro-carbons with human serum albuminrdquo Analytical Biochemistryvol 400 no 1 pp 123ndash129 2010

[30] D Kuang W Zhang Q Deng et al ldquoDose-response rela-tionships of polycyclic aromatic hydrocarbons exposure andoxidative damage to DNA and lipid in coke oven workersrdquoEnvironmental Science and Technology vol 47 no 13 pp 7446ndash7456 2013

[31] R Fan D Wang C Mao et al ldquoPreliminary study of childrensexposure to PAHs and its association with 8-hydroxy-21015840-deoxyguanosine in Guangzhou Chinardquo Environment Interna-tional vol 42 no 1 pp 53ndash58 2012

[32] A Rossnerova M Spatova P Rossner Jr Z Novakova ISolansky and R J Sram ldquoFactors affecting the frequency ofmicronuclei in asthmatic and healthy children from OstravardquoMutation Research vol 708 no 1-2 pp 44ndash49 2011

[33] V Svecova P Rossner Jr M Dostal J Topinka I Solansky andR J Sram ldquoUrinary 8-oxodeoxyguanosine levels in childrenexposed to air pollutantsrdquoMutation Research Fundamental andMolecular Mechanisms of Mutagenesis vol 662 no 1-2 pp 37ndash43 2009

[34] T B Kryston A B Georgiev P Pissis and A G GeorgakilasldquoRole of oxidative stress and DNA damage in human carcino-genesisrdquoMutation Research vol 711 no 1-2 pp 193ndash201 2011

[35] A Weimann K Broedbaek T Henriksen E S Stovgaard andH E Poulsen ldquoAssays for urinary biomarkers of oxidativelydamaged nucleic acidsrdquo Free Radical Research vol 46 no 4 pp531ndash540 2012

[36] J Topinka A Milcova H Libalova et al ldquoBiomarkers ofexposure to tobacco smoke and environmental pollutants inmothers and their transplacental transfer to the foetus Part IBulky DNA adductsrdquo Mutation Research vol 669 no 1-2 pp13ndash19 2009

[37] D F Merlo M Ceppi E Stagi V Bocchini R J Sram andP Rossner ldquoBaseline chromosome aberrations in childrenrdquoToxicology Letters vol 172 no 1-2 pp 60ndash67 2007

[38] M Cerna V Spevackova A Batariova et al ldquoHuman biomon-itoring system in the Czech Republicrdquo International Journal ofHygiene and Environmental Health vol 210 no 3-4 pp 495ndash499 2007

[39] S I N Ekunwe R D Hunter and H Hwang ldquoUltravioletradiation increases the toxicity of pyrene 1-aminopyrene and 1-hydroxypyrene to human keratinocytesrdquo International Journalof Environmental Research and Public Health vol 2 no 1 pp58ndash62 2005

[40] R A Crallan E Ingham and M N Routledge ldquoWave-length dependent responses of primary human keratinocytesto combined treatment with benzo[a]pyrene and UV lightrdquoMutagenesis vol 20 no 4 pp 305ndash310 2005

[41] R Saladi L Austin and D Gao ldquoThe combination of benzo[a]pyrene and ultraviolet A causes an in vivo time-related accu-mulation of DNA damage in mouse skinrdquo Photochemistry andPhotobiology vol 77 no 4 pp 413ndash419 2003

10 Oxidative Medicine and Cellular Longevity

International Agency for Research on Cancer 150 Cours AlbertThomas 69372 pp 1ndash109 Lyon France 2014

[25] K E Burke and H Wei ldquoSynergistic damage by UVA radiationand pollutantsrdquo Toxicology and Industrial Health vol 25 no 4-5 pp 219ndash224 2009

[26] Y Yan J Jiang Z Huang Z Wu C Lin and F Wu ldquoAntisenseoligonucleotides targeting protein kinase C alpha inhibits theproliferation of A549 cellsrdquo Journal of SouthernMedical Univer-sity vol 29 no 6 pp 1189ndash1191 2009

[27] K Roszkowski ldquoOxidative DNA damagemdashthe possible useof biomarkers as additional prognostic factors in oncologyrdquoFrontiers in Bioscience vol 19 pp 808ndash817 2014

[28] B Marczynski M Raulf-Heimsoth A Spickenheuer et alldquoDNAadducts and strand breaks inworkers exposed to vapoursand aerosols of bitumen associations between exposure andeffectrdquo Archives of Toxicology vol 85 no 1 pp S53ndashS64 2011

[29] M K Chung J Riby H Li et al ldquoA sandwich enzyme-linkedimmunosorbent assay for adducts of polycyclic aromatic hydro-carbons with human serum albuminrdquo Analytical Biochemistryvol 400 no 1 pp 123ndash129 2010

[30] D Kuang W Zhang Q Deng et al ldquoDose-response rela-tionships of polycyclic aromatic hydrocarbons exposure andoxidative damage to DNA and lipid in coke oven workersrdquoEnvironmental Science and Technology vol 47 no 13 pp 7446ndash7456 2013

[31] R Fan D Wang C Mao et al ldquoPreliminary study of childrensexposure to PAHs and its association with 8-hydroxy-21015840-deoxyguanosine in Guangzhou Chinardquo Environment Interna-tional vol 42 no 1 pp 53ndash58 2012

[32] A Rossnerova M Spatova P Rossner Jr Z Novakova ISolansky and R J Sram ldquoFactors affecting the frequency ofmicronuclei in asthmatic and healthy children from OstravardquoMutation Research vol 708 no 1-2 pp 44ndash49 2011

[33] V Svecova P Rossner Jr M Dostal J Topinka I Solansky andR J Sram ldquoUrinary 8-oxodeoxyguanosine levels in childrenexposed to air pollutantsrdquoMutation Research Fundamental andMolecular Mechanisms of Mutagenesis vol 662 no 1-2 pp 37ndash43 2009

[34] T B Kryston A B Georgiev P Pissis and A G GeorgakilasldquoRole of oxidative stress and DNA damage in human carcino-genesisrdquoMutation Research vol 711 no 1-2 pp 193ndash201 2011

[35] A Weimann K Broedbaek T Henriksen E S Stovgaard andH E Poulsen ldquoAssays for urinary biomarkers of oxidativelydamaged nucleic acidsrdquo Free Radical Research vol 46 no 4 pp531ndash540 2012

[36] J Topinka A Milcova H Libalova et al ldquoBiomarkers ofexposure to tobacco smoke and environmental pollutants inmothers and their transplacental transfer to the foetus Part IBulky DNA adductsrdquo Mutation Research vol 669 no 1-2 pp13ndash19 2009

[37] D F Merlo M Ceppi E Stagi V Bocchini R J Sram andP Rossner ldquoBaseline chromosome aberrations in childrenrdquoToxicology Letters vol 172 no 1-2 pp 60ndash67 2007

[38] M Cerna V Spevackova A Batariova et al ldquoHuman biomon-itoring system in the Czech Republicrdquo International Journal ofHygiene and Environmental Health vol 210 no 3-4 pp 495ndash499 2007

[39] S I N Ekunwe R D Hunter and H Hwang ldquoUltravioletradiation increases the toxicity of pyrene 1-aminopyrene and 1-hydroxypyrene to human keratinocytesrdquo International Journalof Environmental Research and Public Health vol 2 no 1 pp58ndash62 2005

[40] R A Crallan E Ingham and M N Routledge ldquoWave-length dependent responses of primary human keratinocytesto combined treatment with benzo[a]pyrene and UV lightrdquoMutagenesis vol 20 no 4 pp 305ndash310 2005

[41] R Saladi L Austin and D Gao ldquoThe combination of benzo[a]pyrene and ultraviolet A causes an in vivo time-related accu-mulation of DNA damage in mouse skinrdquo Photochemistry andPhotobiology vol 77 no 4 pp 413ndash419 2003