Research Article Lymphocyte Oxidative Stress/Genotoxic...

Research ArticleLymphocyte Oxidative StressGenotoxic Effects Are Related toSerum IgG and IgA Levels in Coke Oven Workers

Meili Gao1 Yongfei Li2 Aqun Zheng3 Xiaochang Xue4 Lan Chen5 and Yu Kong1

1 Department of Biological Science and Engineering Institute ofMitochondrial Biology andMedicineTheKey Laboratory of BiomedicalInformation Engineering of Ministry of Education School of Life Science and Technology Xirsquoan Jiaotong UniversityXianning West Road 28 Xirsquoan Shaanxi 710049 China

2 School of Materials and Chemical Engineering Xirsquoan Technological University Xirsquoan 710032 China3 School of Science Xirsquoan Jiaotong University Xirsquoan Shaanxi 710049 China4Department of Biopharmaceutics School of Pharmacy State Key Laboratory of Cancer Biology Fourth Military Medical UniversityXirsquoan Shaanxi 710032 China

5 Center of Shared Experimental Facilities The Key Laboratory of Biomedical Information Engineering of Ministry of EducationSchool of Life Science and Technology Xirsquoan Jiaotong University Xirsquoan Shaanxi 710049 China

Correspondence should be addressed to Yu Kong kynksinacom

Received 17 January 2014 Revised 7 June 2014 Accepted 9 June 2014 Published 20 July 2014

Academic Editor Raimo Pohjanvirta

Copyright copy 2014 Meili Gao et alThis is an open access article distributed under theCreativeCommonsAttributionLicensewhichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

We investigated oxidative stressgenotoxic effects levels immunoglobulin levels polycyclic aromatic hydrocarbons (PAHs) levelsexposed in 126 coke oven workers and in 78 control subjects and evaluated the association between oxidative stressgenotoxiceffects levels and immunoglobulin levels Significant differences were observed in biomarkers including 1-hydroxypyrene levelsemployment time percentages of alcohol drinkers MDA 8-OHdG levels CTL levels and CTM MN CA frequency and IgG IgAlevels between the control and exposed groups Slightly higher 1-OHP levels in smoking users were observed For the dose-responserelationship of IgG IgA IgM and IgE by 1-OHP each one percentage increase in urinary 1-OHP generates a 0109 0472 0051and 0067 decrease in control group and generates a 0312 0538 0062 and 0071 decrease in exposed group respectivelyExcept for age alcohol and smoking status IgM and IgE a significant correlation in urinary 1-OHP and other biomarkers in thetotal population was observed Additionally a significant negative correlation in genotoxicoxidative damage biomarkers of MDA8-OH-dG CTL levels and immunoglobins of IgG and IgA levels especially in coke oven workers was found These data suggestthat oxidative stressDNA damage induced by PAHs may play a role in toxic responses for PAHs in immunological functions

1 Introduction

Coke oven workers are constantly exposed to coke ovenemissions which are toxic chemicals especially polycyclicaromatic hydrocarbons (PAHs) PAHs are formed duringcombustion of fossil fuels and typified by the 1-hydroxypyrenelevels The 1-hydroxypyrene level has been shown to be agood marker for total PAHs exposure [1 2] Some of PAHsare carcinogenic due to their metabolites and their ability togenerate genetic damage and further oxidative DNA damagethrough the production of reactive oxygen species duringmetabolism [3 4]

Of many indicators for oxidative DNA damage 8-hydroxy-21015840-deoxyguanosine (8-OHdG) represents an

important product from oxidative damage to DNA8-OHdG is formed in a promutagenic DNA lesion inducedby the reaction of hydroxyl radicals with guanosine atthe C8 site in DNA A growing number of surveys andoccupational studies indicated that elevated levels of 8-OHdG in DNA from leukocytes or excretion in urine havebeen observed in PAHs exposure of smokers and workers[5ndash8] Malondialdehyde (MDA) which is an end productof the oxidation of polyunsaturated fatty acids and candetermine the degree of lipid peroxidation has been used asa marker for oxidative stress [9]

It has been reported that low level PAH exposure causesDNA single strand breakage the formation of DNA damages

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 801346 10 pageshttpdxdoiorg1011552014801346

2 The Scientific World Journal

and immunotoxicity [10ndash12] Immunotoxicity can changelymphocytic subpopulation in peripheral blood and serumimmunoglobulin levels in coke oven workers exposed toPAHs [12 13] As for genotoxic risk factors the comet assaymicronucleus (MN) assay and chromosomal aberrations(CA) assay have been used to evaluate the biomarkers ofearly biological effects [14] The comet assay has been foundto be a very sensitive method for measuring DNA damageThe MN test was found to provide a cytogenetic parameterand allowed the detection of both clastogenic and aneugenicagents [14] Chromosomal damage has also been found toprovide CA such as chromosome breakage chromosomedeletion and chromosome polyploid [13 14]

In the present study we investigated if there was anyrelation between the levels of MDA 8-OHdG and genotoxicdamages and immunoglobulin levels in serum and lympho-cytes of workers exposed to coke oven emission

2 Materials and Methods

21 Study Subjects The 126 coke oven workers and 78 non-coke-oven workers who were all males and worked in thesame steel company in northern China were studied in thispaper These 126 coke oven workers were in active serviceat the time of the study were employed for at least 6months and were recruited as the exposed group The 78non-coke-oven workers were staff members of the officesand hospitals of the same steel company and served as thecontrol group The workers exposed to known mutagenicagents such as radiotherapy and chemotherapy in the last 3months were excludedQuestionnaires were administered bytrained interviewers to collect information on demographicinformation including age length of employment smokingand alcohol habits Individuals who had smoked for 3monthswere considered as smokers Those who drank more thantwice a week in the last six months were classified as drinkersBlood samples were collected at the end of these days In themorning 5mL fasting venous blood and 10mL urine sampleswere collected from each subject for further analysis Thestudywas approved by the Ethics Committee of Xirsquoan JiaotongMedical College and was performed in accordance with theHelsinki Declaration (1964)

22 1-Hydroxypyrene in Urine Assay Urine 1-hydroxypyrene(1-OHP) was measured by the method described by Jon-geneelen and Anzion [15] and Siwinska et al [16] Brieflyaliquots of 10mL urine samples were enzymatically decon-jugated and transferred to primed C18 octadecyl cartridges(Beckman USA) Then the samples were washed with 10mLof water and eluted with 9mL of methanol The compo-nents of the elutae was subjected to a high pressure liquidchromatography of the Waters Alliance 2695 (Waters USA)with the XAqua C18 150 times 41mm column A fluorescencedetector of F1000 (Hitachi Japan) was quantitatively assayed1-hydroxypyrene concentration The wavelengths of excita-tion and emission were 229 nm and 400 nm respectivelyThe concentrations of 1-OHP were normalised to urinarycreatinine Urinary creatinine concentrationswere assayed by

a standard colorimetric method with the picric acid reactionand absorption at 520 nm [17]

23 Serum Biomarkers Assay Blood samples were obtainedafter an overnight fast Samples (3mL) of venous blood wereincubated in a water bath for 30 minutes at 37∘C and cen-trifuged at 4500 rpm for 10 minutes The supernatants werestored at minus70∘C The levels of serum MDA represented lipidperoxidation product weremeasured spectrophotometricallyby amodification of themethod described by Buege andAust[18] The spectrophotometric measurements were done withShimadzu UV-1208 spectrophotometer (Japan) The MDAconcentration was calculated using its extinction coefficientof 156times105 Lmol cm at 535 nm Enzyme linked immunosor-bent assay (ELISA)was used tomeasure the concentrations of8-OHdG in accordance with the procedures described in theassay kit (Sigma USA) Serum IgA IgE IgG and IgM levelswere measured by a nephelometric method [19]

24 Comet Assay Micronuclei Test and Chromosomal Aber-rations Assay Venous blood was collected from all subjectsusing heparinized syringes Lymphocyte cultures were set upby adding 05mL of heparinised blood to 45mL of chromo-some medium (RPMI 1640 Gibco) supplemented with 20heat inactivated fetal bovine serum (Gibco) antibiotics (peni-cillin and streptomycin) andL-glutamine Lymphocyteswerestimulated by 1 phytohaemagglutinin (Gibco) The cometassay micronuclei test and CA assay were performed toassay the genotoxic damage of lymphocytes in workers DNAdamage to lymphocytes was assayed by the alkaline single-cell gel technique [20 21] Images of 25 randomly selectedlymphocytes were analyzed for each sample The slides wereexamined using a Comet 40 image analysis systemfittedwithanOlympus BX50 fluorescencemicroscope For the collectedcell sample of each subject images of 50 randomly selectedcells were analyzed for each sample The mean level of comettail length (CTL) and comet tail moment (CTM) of the 50cells was as the value of the sample The determination ofmicronuclei in lymphocytes was performed as described byFenech andMorley [22] The cultures were incubated at 37∘Cfor 72 hours and 44 hours after the initiation of culturescytochalasin-B (Sigma) at a concentration of 6mgmL wasadded to arrest cytokinesis MN slides were stained with10 Giemsa in phosphate buffer A total of 1000 binucleatescells with well-preserved cytoplasmwere examined by a well-trained research assistant under double blindness for eachsubject on coded slides throughout the study The data arereported as the occurrence rate of permil micronucleated cellsfor each subject The CA assay was according to the methoddescribed by Carrano and Natarajan [23] Briefly colcemidwas added to arrest the cells in metaphase after incubatingthe cultures for 48 hours at 37∘C then cells were collectedby centrifugation resuspended in a prewarmed hypotonicsolution (0075M KCl) for 25 minutes and fixed in aceticacid methanol (1 3 vv) Air dried preparations were madeand the slides were stained with Giemsa A total of 100well spread metaphases containing 46 plusmn 1 chromosomes wasexamined for each subject on coded slides otherwise the

The Scientific World Journal 3

Table 1 Demographic characteristics of workers in the control and exposed groups

Variables Control group (78) Exposed group (126) 119875 value1-OHP (120583molmol creatine) 055 plusmn 028 936 plusmn 214lowastlowastlowast lt0001a

Age (year) 324 plusmn 49 336 plusmn 52 0103a

Employment time (year) 127 plusmn 25 141 plusmn 27lowastlowastlowast lt0001a

Current smokers yes () 41 (526) 79 (627) 0153b

Alcohol users yes () 18 (231) 57 (452) 0001b

MDA (nmolmL) 123 plusmn 037 197 plusmn 061lowastlowastlowast lt0001a

8-OH-dG (nmolmol creatinine) 129 plusmn 015 220 plusmn 019lowastlowastlowast lt0001a

CTL (120583m) 316 plusmn 058 749 plusmn 089lowastlowastlowast lt0001a

CTM 085 plusmn 023 1048 plusmn 352lowastlowastlowast lt0001a

MN (permil) 183 plusmn 051 278 plusmn 063lowastlowastlowast lt0001a

CA (N AR) 10 128 36 286 0009b

IgG (gL) 036 plusmn 011 012 plusmn 003lowastlowastlowast lt0001a

IgA (gL) 209 plusmn 044 166 plusmn 044lowastlowastlowast lt0001a

IgM (gL) 176 plusmn 069 162 plusmn 072 0172a

IgE (IUmL) 17390 plusmn 3024 16776 plusmn 4316 0273a

1-OHP 1-hydroxypyrene CTL comet tail length CTM comet tail moment MN micronucleus CA chromosomal aberrations Values are shown mean plusmn SDexcept where indicated awhen compared with control by Mann-Whitney test bwhen compared with control by chi-square testlowastlowastlowast119875 lt 0001

Table 2 1-OHP levels by smoking use status in control and exposed group

Control group Exposed groupSmoking (No) Smoking (yes) Smoking (No) Smoking (yes)

119899 37 41 47 791-OHP level 048 plusmn 021 057 plusmn 031 862 plusmn 212 958 plusmn 365119875 valuea 0142 0096awhen compared between nonsmoking and smoking users control and exposed group by chi-square test

data were discarded The data are reported as the occurrencenumber and occurrence rate of CA for each group

25 Statistical Analysis All analyses were carried out usingthe Statistical Package for Social Sciences (SPSS130)We usedMann-Whitney and Pearson chi-square tests to compare thedemographics and lifestyle variables between the exposureand control groups We used the Mann-Whitney test tocompare values of biomarkers between the exposure andcontrol groups Spearmanrsquos rank correlation coefficient wascalculated to evaluate the relations between MDA CTL IgGand the impact of independent variables (occupational PAHsexposure age length of employment smoking and alcoholdrinking) on dependent variables Pearson correlation wascalculated to evaluate the relations betweenMDA and 8-OH-dG level 8-OH-dG and CTL level MDA and IgG IgA levelAll statistical tests were two-sided with a significant level of119875 lt 005

3 Results

31 Demographic Characteristics of Study Subjects Table 1shows the characteristics of study subjects by work site

Exposed workers were 1 year older (mean age) than controlsubjects Significant differences were observed in 1-OHP lev-els employment time percentages of alcohol drinkers MDA8-OHdG levels CTL level andCTMMNCA frequency IgGand IgA between the control and exposed groups but nosignificance for age current smokers distribution and IgMand IgE levels

Table 2 shows the results of urinary 1-OHP relative tosmoking status in exposed workers and controls after adjust-ment for exposure concentration A slight higher of 1-OHPlevels in smoking users were observed both in control andexposed groups but this was not significant (119875 = 0142 incontrol and 119875 = 0096 in exposed group resp)

32 Dose-Response Relationships of Urinary 1-OHP withImmunoglobins and Correlation between 1-OHP and OtherBiomarkers The dose-response relationship of IgG IgAIgM and IgE by 1-OHP was analyzed as in Table 3 Thevalues of IgG IgA IgM and IgE and 1-OHP were all ln-transformed in the multiple linear regression models For thecontrol group each one percentage increase in urinary 1-OHPgenerates a 0109 (119875 = 0353) 0472 (119875 lt 0001) 0051(119875 = 0658) and 0067 (119875 = 0565) decrease in IgG IgA


Table 3 Dose-response relationship with the levels of 1-OHP with immunoglobins

1-OHP level

IgG (gL) IgA (gL) IgM (gL) IgE (IUmL)120573 120573 120573 120573

(95 CI) (95 CI) (95 CI) (95 CI)119875 119875 119875 119875

Control groupminus0109 minus0472 minus0051 minus0067

(minus0359ndash0130) (minus0438ndash0296) (minus0445ndash0283) (minus0941ndash0518)0353 lt0001 0658 0565

Exposed groupminus0312 minus0538 minus0062 minus0071

(minus03455ndash0176) (minus0463ndash0211) (minus0903ndash0465) (minus0233ndash0098)lt0001 lt0001 0592 0420

Table 4 Correlations of 1-OHP levels and the other studiedvariables in total subjects

Variables 119903a

119875-valueb

Age (year) 0157 0233a

Employment time (year) 0455 lt0001a

Current smokers yes () 0329 0073b

Alcohol users yes () 0165 0209b

MDA (nmolmL) 0623 lt0001a

8-OH-dG (nmolmol creatinine) 0668 lt0001a

CTL (120583m) 0571 lt0001a

CTM 0682 lt0001a

MN (permil) 0459 lt0001a

CA (N AR) 0422 0009b

IgG (gL) minus0732 lt0001a

IgA (gL) minus0677 lt0001a

IgM (gL) minus0209 0127a

IgE (IUmL) minus0187 0211aaSpearman rank correlation coefficient bSpearman rank correlation coeffi-cient for the comparisons between each of the studied variables

IgM and IgE respectively For the exposed group each onepercentage increase in urinary 1-OHPgenerates a 0312 (119875 lt0001) 0538 (119875 lt 0001) 0062 (119875 = 0592) and 0071(119875 = 0420) decrease in IgG IgA IgM and IgE respectivelyCorrelation between 1-OHP and other indices was assayed asin Table 4 Except for age alcohol status current smokersIgM and IgE a significant correlation in urinary 1-OHPand other biomarkers in the total population (exposed andcontrols) (119875 lt 0001 for other biomarkers 119875 = 0009 for CAresp) was observed in the studied subjects

33 Correlation between OxidativeGenotoxic DamageBiomarkers and Immunoglobin Levels We observed asignificant correlation in MDA and 8-OH-dG levels 8-OH-dG and CTL levels in the control group (119903 = 0556119875 lt 0001 119903 = 0644 119875 lt 0001 resp) (Figures 1(a) and1(c)) and in the exposed group (119903 = 0640 119875 lt 0001119903 = 0794 119875 lt 0001 resp) (Figures 1(b) and 1(d)) Alsowe examined the correlations in genotoxicoxidative damagebiomarkers and immunoglobins of exposed workers andcontrol workers next to further evaluate a possible role

and a potent biomedical significance of immunoglobinlevels in genotoxicoxidative damage The correlationbetween MDA 8-OH-dG CTL and IgG IgA of workersexposed to coke oven emission is plotted in Figures 2(b)2(d) 2(f) 2(h) 2(j) and 2(l) As can be seen there is asignificant negative correlation between MDA 8-OH-dGCTL and IgG IgA levels in exposed workers (119875 lt 0001)A similar significant negative correlation between MDAand IgG IgA level 8-OH-dG and IgG in control workers(Figure 2(a) 119903 = minus0498 119875 lt 0001 Figure 2(c) 119903 = minus0363119875 = 0001 Figure 2(e) 119903 = minus0229 119875 = 0004 resp)was observed However in the control group 8-OH-dGlevels were not significantly correlated with IgA levels(Figure 2(g) 119903 = minus0080 119875 = 0486) Also CTL levels werenot significantly correlated with IgG and IgA in controlworkers (Figure 2(i) 119903 = minus0126 119875 = 0270 Figure 2(k)119903 = minus0110 119875 = 0339 resp) Further PAH exposureenhances this negative correlation Interestingly althoughmost individuals with low IgG IgA levels tended to havemore MDA 8-OH-dG and CTL levels some with high IgGIgA also had more MDA 8-OH-dG and CTL levels as canbe seen particularly in Figures 2(b) 2(f) 2(h) 2(j) and 2(l)respectively

4 Discussion

The working environment at a coke plant can negativelyaffect the employed workers who are exposed to coke ovenemissions containing PAHs [24] PAHs are introduced tothe environment in numerous ways and are ubiquitouslydistributed They are taken up by humans at the workplaceby smoking by breathing polluted air and by consumingfood and medicines They are potent oxidativegenotoxicdamage and immunotoxic compounds Current occupationalexposure to PAHs was investigated in China coke oven plantsin order to establish which resulted in oxidativegenotoxicDNA damages and immunotoxic effects which are consid-ered to be indicators for long term adverse health effectsBiological monitoring of the internal dose was based onthe analysis of urinary 1-hydroxypyrene Biological effectmonitoring includingMDA 8-OH-dG CTL CTMMN CAand IgA IgE IgG and IgM levels as well as the correlationamong these markers were evaluated


04 06 08 10 12 14 16 18 2010

11

12

13

14

15

168-

OH

-dG

(nm

olm

ol cr

eatin

ine)

MDA (nmolmL)

(a)

05 10 15 20 25 30

18

19

20

21

22

23

24

25

26

27

8-O

H-d

G (n

mol

mol

crea

tinin

e)

MDA (nmolmL)

(b)

10 11 12 13 14 15 16

40

45

50

55

60

65

70

75

CTL

(120583m

)

8-OH-dG (nmolmol creatinine)

(c)

18 19 20 21 22 23 24 25 26 27

55

60

65

70

75

80

85

90

95


CTL

(120583m

)

(d)

Figure 1 Correlation between MDA level and 8-OH-dG level in lymphocytes of workers in control group ((a) 119903 = 0556 119875 lt 0001) andexposed group ((b) 119903 = 0640 119875 lt 0001) and correlation between 8-OH-dG and CTL level in lymphocytes of workers in control group ((c)119903 = 0644 119875 lt 0001) and exposed group ((d) 119903 = 0794 119875 lt 0001)

In the present study the coke oven workers seemed tohave been exposed to a significant high level of PAHs basedon the urinary 1-OHP The PAH exposure of coke ovenworkers in the present study was similar with that in Siwinskaet alrsquos [16] and in Zhang et alrsquos [25] study conducted incoke oven workers and much higher than that reported byPavanello et al [26] in CMBN assay in coke oven workersThough the length of exposure is not therefore the onlyrisk factor Yang et al reported that the length of time spentoutdoors had marginal positive associations with urinary 1-OHP levels in nonoccupationally exposed Koreans [27] Sosignificantly higher length of employment timemay promotea synergistic effect on excretion of 1-OHP in PAH exposedcoke oven workers Significant distributions for alcohol usersand no significant distributions for smoking status in exposedcoke oven workers were observed in the present study Onthe contrary no significant difference was found in alcoholusers between control and coke oven workers in Leng et alrsquos[28] and Zhang et alrsquos studies [25] Other studies [29 30]

indicated that the smoking of cigarettes significantly increasesthe urinary 1-OHP concentration while alcohol consump-tion does not affect the urinary 1-OHP concentration Wefurther analyzed the smokers and nonsmokers in controland exposed group The findings indicated the smoking wasactually increase 1-OHP levels but not significance Thismay be partially due to the relatively higher exposure toPAHs which may hide the effect of smoking on 1-OHPconcentrations [25] Hence the smoking life style may notsignificantly affect the urinary 1-OHP concentration in thisstudy

Metabolic transformation of PAHs generates reactiveelectrophilic metabolites causing DNA damage and trigger-ing the production of reactive oxygen species that lead tooxidative stress [31] The analysis of urinary excretion of 8-OH-dG is a useful approach to assess individual cancer riskdue to oxidative stress [32] Data from the present study alsoshowed that levels of MDA 8-OHdG was associated withexposure to carcinogenic PAHs among coke oven workers


04 06 08 10 12 14 16 18 20

01

02

03

04

05

06

07

IgG

(gL

)

MDA (nmolmL)

(a)

05 10 15 20 25 30004

006

008

010

012

014

016

018

020

IgG

(gL

)

MDA (nmolmL)

(b)

04 06 08 10 12 14 16 18 201012141618202224262830

IgA

(gL

)

MDA (nmolmL)

(c)

05 10 15 20 25 300608101214161820222426

IgA

(gL

)

MDA (nmolmL)

(d)

10 11 12 13 14 15 16

01

02

03

04

05

06

07

IgG

(gL

)


(e)

18 19 20 21 22 23 24 25 26 27004

006

008

010

012

014

016

018

020

IgG

(gL

)


(f)

10 11 12 13 14 15 161012141618202224262830

IgA

(gL

)


(g)

18 19 20 21 22 23 24 25 26 270608101214161820222426

IgA

(gL

)


(h)

Figure 2 Continued


40 45 50 55 60 65 70 75

01

02

03

04

05

06

07

IgG

(gL

)

CTL (120583m)

(i)

55 60 65 70 75 80 85 90 95004

006

008

010

012

014

016

018

020

IgG

(gL

)

CTL (120583m)

(j)

40 45 50 55 60 65 70 751012141618202224262830

IgA

(gL

)

CTL (120583m)

(k)

55 60 65 70 75 80 85 90 950608101214161820222426

IgA

(gL

)

CTL (120583m)

(l)

Figure 2 Correlation between MDA and IgG IgA level in lymphocytes of workers in control group ((a) 119903 = minus0498 119875 lt 0001 (c)119903 = minus0363 119875 = 0001) and exposed group ((b) 119903 = minus0606 119875 lt 0001 (d) 119903 = minus0814 119875 lt 0001) respectively correlation between8-OH-dG and IgG IgA level in lymphocytes of workers in control group ((e) 119903 = minus0229 119875 = 0004 (g) 119903 = minus0080 119875 = 0486) and exposedgroup ((f) 119903 = minus0405 119875 lt 0001 (h) 119903 = minus0523 119875 lt 0001) respectively correlation between CTL and IgG IgA level in lymphocytesof workers in control group ((i) 119903 = minus0126 119875 = 0270 (k) 119903 = minus0110 119875 = 0339) and exposed group ((j) 119903 = minus0312 119875 lt 0001 (l)119903 = minus0350 119875 lt 0001) respectively

Significant differences were observed for CTL levels MNand CA frequency of PAH exposed workers In agreementwith our results Siwinska et al reported that the level ofurinary 1-OHP was correlated with genotoxic effects in cokeoven workers as determined by a number of assays includingmicronuclei frequency and DNA damagemeasured by sister-chromatid exchange and comet assays [16] Significant cor-relation in MDA and 8-OH-dG levels 8-OH-dG and CTLlevels in all subjects was observed in the present study It iswell known that reactive oxygen species generated by PAHsmay induce damage to DNA It is implied that oxidative DNAmodification and oxidative DNA damage generated by PAHsexposed in coke oven workers [33] High levels of MDA8-OHdG and DNA damage were also indicated by severaloccupational studies [3 8 34] Additionally accumulatingevidence suggested that ethanol can induce oxidative DNAdamage in humans [3 35] The significant distributions foralcohol users in exposed coke oven workers indicates thatalcohol drinking may synergetically increase levels of MDA8-OHdG and DNA damage in alcohol users of coke ovenworkers

A general observation has been that the immune systemof humans is the most sensitive to modulation by PAHs Fourkinds of immunoglobulins were lower in coke oven workersbut this was significant only for IgG and IgA (119875 lt 0001)The results of this study regarding immunoglobulin levelsalso show consistent with the data of Szczeklik et al [36]who demonstrated biosynthesis of IgG and IgAwasmarkedlydepressed immunoglobulins in serum of coke oven workersOh et al [13] found that the four kinds of immunoglobulintypes were lower in automobile emission inspectors but thiswas significant only for IgG (119875 = 0047) On the otherhand Winker et al [37] did not find any alterations ofthe serum immunoglobulin levels in PAH-exposed workersSimilarly the four kinds of immunoglobulin typeswere foundin lower amounts in the waste incineration workers but thisdisparity was not significant one [38] By contrast there wasalso a significant enhancement in serum IgG levels and thepercentage ofmonocytes in the PAHexposed asphalt workerscompared to the control group [12] In addition Szczekliket al also found that serum IgE had a trend toward increasedvalues in serum of coke oven workers [36]These may be due


to the fact that the place of adaptive responses or changesmaycome to plays in situation of chronic exposure [39] GenerallyIgG plays a major role in humoral immune response throughcomplement fixation and neutralization and reduction inIgG may lead to downregulation of antibody-mediated hostresistance which could enhance influence of infection ortumor progression [40 41] The decrease in IgA productionsuggests that PAHs may compromise protection of mucosalsurfaces in the respiratory tract [42]There has been reportedthat many PAHs are known to be potent suppressors ofthe immune response Additionally the immunosuppressivepotential of PAHs is linked to their carcinogenic potency[43] Accordingly in some extent the PAH exposure incoke oven workers showed the immune depression andthe increase in potential carcinogenic effect through thesignificant reduction of IgG and IgA levels Further exceptage alcohol status smoking status IgM IgE and othermarkers were significantly associated with PAH exposureas indicated by 1-OHP levels As previous descriptions themarkers of employment time MDA 8-OH-dG CTL CTMMN (permil) CA IgG and IgA may be more important markersin coke oven workers

Further a significant negative correlation in geno-toxicoxidative damage biomarkers of MDA 8-OH-dG CTLlevels and immunoglobins of IgG IgA levels especiallyin coke oven workers was found in the present studyStudies have suggested that oxidative stress-induced lipidperoxidation may play a role in toxic responses to PAHin immunological systems For example Zhigacheva et alfound that the increased lipid peroxidation products werelinked to modification of leucocyte membranes and reducedactivity of lymphocyte enzymes in animals after 6 months ofPAH inhalation as compared with controls [44] In additionmoderate activation of lipid peroxidation and oxidative dam-age as determined by plasma MDA and 8-OHdG levels weredetected simultaneously with significant alterations in IgAand IgE levels described by Jeng et al [42] Also a significantdifference was found regarding the levels of IgE and the levelsof DNA damage in polynuclear cells were found in wasteincineration workers described by Oh et al [38] FurtherPAH exposure enhances this negative correlation (Figure 2)Generally speaking PAHs are cytotoxic at high doseswhile lower doses of PAHs frequently result in alterationof immunological responses It is likely that heavier PAHspecies increased redox activity and subsequently inducedgeneration of ROS [42] which altered IgE and IgA levels inworkers exposed to PAHs Further research should clarify theeffects of high molecular weights of PAHs on immunologicalfunction

The study also has some limitations First our samplesize is relatively small and this is one of the most commonreasons for the failure to replicate reported associations acrossstudies [45] This may be due to no significance for 1-OHPlevels in smokers and nonsmokers IgM IgE levels betweenthe control and exposed group In particular the numberof subjects in CA assay was only 10 for control and 36 inexposed group So further investigation still recommendslarge sample population in the future On the other hand

though alcohol consumption does not affect the urinary 1-OHP concentration [29 30] excess alcohol consumptionmay result in the enhancement of PAH metabolites [25] andthemechanism is needed further study Finally staffmembersof the offices and hospitals of the same steel company servedas the control group There may be differences in economicsand nutrition status between control and coke oven workersThis may be affected by food intakes which are also knownas an important route of exposure to PAHs [9] To betterunderstand the factors related to the increase of 1-OHPthe food consumption social activities and nutrition statusshould be included in the questionnaire and further analyzedin the future

5 Conclusion

In conclusion the present study provided yet more evidencethat PAHs cause significant oxidative stressDNA damagethrough the MDA 8-OHdG levels and CTL level CTMMN and CA frequency in coke oven workers Moreoverour immunoglobulin study showed that IgG and IgA weresignificantly reduced by PAHs In addition our investigationof the association between MDA and 8-OH-dG levels 8-OH-dG and CTL levels MDA 8-OH-dG CTL levels andimmunoglobins of IgG IgA levels was proven to be ameaningful or relevant association It is implied that oxidativestressDNA damage induced by PAHsmay play a role in toxicresponses for PAH in immunological systems

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors extend specials thanks to Dr Fei Yan of theFourthMilitaryMedicalUniversity for his assistance in statis-tical analysis The research was supported by the Fundamen-tal Research Funds for the Central Universities (2012jdhz48)and by the Shaanxi Postdoctoral Science Foundation fundedproject (18420024)

References

[1] H Wang W Chen H Zheng et al ldquoAssociation betweenplasma BPDE-Alb adduct concentrations and DNA damageof peripheral blood lymphocytes among coke oven workersrdquoOccupational and Environmental Medicine vol 64 no 11 pp753ndash758 2007

[2] G Castano-Vinyals A DrsquoErrico N Malats and M KogevinasldquoBiomarkers of exposure to polycyclic aromatic hydrocarbonsfrom environmental air pollutionrdquo Occupational and Environ-mental Medicine vol 61 no 4 article e12 2004

[3] J Zhang M Ichiba T Hanaoka et al ldquoLeukocyte 8-hydroxydeoxyguanosine and aromatic DNA adduct in coke-oven workers with polycyclic aromatic hydrocarbon expo-surerdquo International Archives of Occupational and EnvironmentalHealth vol 76 no 7 pp 499ndash504 2003


[4] M Gao Y Li Y Sun et al ldquoA common carcinogenbenzo[a]pyrene causes p53 overexpression in mouse cervixvia DNA damagerdquo Mutation Research Genetic Toxicology andEnvironmental Mutagenesis vol 724 no 1-2 pp 69ndash75 2011

[5] M Lodovici C Casalini R Cariaggi L Michelucci and PDolara ldquoLevels of 8-hydroxydeoxyguanosine as a marker ofDNA damage in human leukocytesrdquo Free Radical Biology andMedicine vol 28 no 1 pp 13ndash17 2000

[6] B Marczynski H P Rihs B Rossbach et al ldquoAnalysis of 8-oxo-78-dihydro-21015840-deoxyguanosine and DNA strand breaks inwhite blood cells of occupationally exposed workers compari-son with ambient monitoring urinary metabolites and enzymepolymorphismsrdquo Carcinogenesis vol 23 no 2 pp 273ndash2812002

[7] 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 amp Technology vol 47 no 13 pp 7446ndash7456 2013

[8] A L Liu W Q Lu Z Z Wang et al ldquoElevated level of urinary8-hydroxy-21015840-deoxyguanosine lymphocytic micronuclei andserum glutathione S-transferase in workers exposed to cokeoven emissionsrdquo Environmental Health Perspectives vol 114 no5 pp 673ndash677 2006

[9] S Bae X Pan S Kim et al ldquoExposures to particulate matterand polycyclic aromatic hydrocarbons and oxidative stress inschoolchildrenrdquo Environmental Health Perspectives vol 118 no4 pp 579ndash583 2010

[10] R Crebelli P Carta C Andreoli et al ldquoBiomonitoring ofprimary aluminium industry workers detection of micronucleiand repairable DNA lesions by alkaline SCGErdquo MutationResearchmdashGenetic Toxicology and Environmental Mutagenesisvol 516 no 1-2 pp 63ndash70 2002

[11] S J Kwack and BM Lee ldquoCorrelation betweenDNAor proteinadducts and benzo[a]pyrene diol epoxide I-triglyceride adductdetected in vitro and in vivordquo Carcinogenesis vol 21 no 4 pp629ndash632 2000

[12] A Karakaya B Yucesoy A TurhanO Erdem S Burgaz andAE Karakaya ldquoInvestigation of some immunological functionsin a group of asphalt workers exposed to polycyclic aromatichydrocarbonsrdquo Toxicology vol 135 no 1 pp 43ndash47 1999

[13] E Oh H Im H-S Kang et al ldquoComparison of immunnolog-ical and genotoxicological parameters in automobile emissioninspectors exposed to polycyclic aromatic hydrocarbonsrdquo Envi-ronmental Toxicology and Pharmacology vol 21 no 1 pp 108ndash117 2006

[14] S Sellappa B Mani and K S Keyan ldquoCytogenetic biomon-itoring of road paving workers occupationally exposed topolycyclic aromatic hydrocarbonsrdquo Asian Pacific Journal ofCancer Prevention vol 12 no 3 pp 713ndash717 2011

[15] F J Jongeneelen and R B M Anzion ldquoAnalyses of hazardoussubstances in biological materialsrdquoDeutsche Forschungsgemein-schaft vol 3 pp 151ndash169 1990

[16] E Siwinska DMielzynska and L Kapka ldquoAssociation betweenurinary 1-hydroxypyrene and genotoxic effects in coke ovenworkersrdquoOccupational and environmental medicine vol 61 no3 article e10 2004

[17] R C Baselt Biological MonitoringMethods for Industrial Chem-icals Biomedical Publications Davis Calif USA 1980

[18] J A Buege and S D Aust ldquoMicrosomal lipid peroxidationrdquoMethods in Enzymology vol 52 pp 302ndash310 1978

[19] J Brostoff G K Scadding D Male and I M Roitt ClinicalImmunology Gower Medical Publishing London UK 1991

[20] N P Singh M T McCoy R R Tice and E L Schneider ldquoAsimple technique for quantitation of low levels of DNA damagein individual cellsrdquo Experimental Cell Research vol 175 no 1pp 184ndash191 1988

[21] V J McKelvey-Martin M H L Green P Schmezer B LPool-Zobel M P de Meo and A Collins ldquoThe single cellgel electrophoresis assay (comet assay) a European reviewrdquoMutation ResearchmdashFundamental and Molecular Mechanismsof Mutagenesis vol 288 no 1 pp 47ndash63 1993

[22] M Fenech and A A Morley ldquoMeasurement of micronucleiin lymphocytesrdquo Mutation ResearchmdashGenetic Toxicology andEnvironmental Mutagenesis vol 147 no 1-2 pp 29ndash36 1985

[23] A V Carrano and A T Natarajan ldquoConsideration for pop-ulation monitoring using cytogenetic techniquesrdquo MutationResearch vol 204 no 3 pp 379ndash406 1988

[24] M T Wu I-F Mao C K Ho et al ldquoUrinary 1-hydroxypyreneconcentrations in coke oven workersrdquo Occupational and Envi-ronmental Medicine vol 55 no 7 pp 461ndash467 1998

[25] J Zhang M Ichiba K Hara et al ldquoUrinary 1-hydroxypyrenein coke oven workers relative to exposure alcohol consump-tion and metabolic enzymesrdquoOccupational and EnvironmentalMedicine vol 58 no 11 pp 716ndash721 2001

[26] S Pavanello L Kapka E SiwinskaDMielzynska C Bolognesiand E Clonfero ldquoMicronuclei related to Anti-B[a]PDE-DNAadduct in peripheral blood lymphocytes of heavily polycyclicaromatic hydrocarbon-exposed nonsmoking coke-oven work-ers and controlsrdquo Cancer Epidemiology Biomarkers and Preven-tion vol 17 no 10 pp 2795ndash2799 2008

[27] M Yang S Kim E Lee et al ldquoSources of polycyclic aromatichydrocarbon exposure in non-occupationally exposed kore-ansrdquo Environmental and Molecular Mutagenesis vol 42 no 4pp 250ndash257 2003

[28] S Leng Y Dai Y Niu et al ldquoEffects of genetic polymorphismsof metabolic enzymes on cytokinesis-block micronucleus inperipheral blood lymphocyte among coke-oven workersrdquo Can-cer Epidemiology Biomarkers and Prevention vol 13 no 10 pp1631ndash1639 2004

[29] M Yang J Jang S Kim et al ldquoGenetic effects on urinary 1-hydroxypyrene levels in a Korean populationrdquo Carcinogenesisvol 24 no 6 pp 1085ndash1089 2003

[30] H Nan H Kim H Lim et al ldquoEffects of occupation lifestyleand genetic polymorphisms of CYP1A1 CYP2E1 GSTM1 andGSTT1 on urinary 1-hydroxypyrene and 2-naphthol concentra-tionsrdquo Carcinogenesis vol 22 no 5 pp 787ndash793 2001

[31] J Wang E H Weyand and R G Harvey ldquoSynthesis ofsuspected carcinogenic metabolites of 7H-benzo[c]fluorene acoal tar component implicated in causation of lung tumorsrdquoJournal of Organic Chemistry vol 67 no 17 pp 6216ndash6219 2002

[32] B Halliwell ldquoFree radicals antioxidants and human diseasecuriosity cause or consequencerdquoTheLancet vol 344 no 8924pp 721ndash724 1994

[33] M S Cooke M D Evans R Dove et al ldquoDNA repair isresponsible for the presence of oxidatively damaged DNAlesions in urinerdquoMutation ResearchmdashFundamental and Molec-ular Mechanisms of Mutagenesis vol 574 no 1-2 pp 58ndash662005

[34] X Yang J Zheng Y Bai et al ldquoUsing lymphocyte and plasmaHsp70 as biomarkers for assessing coke oven exposure amongsteel workersrdquoEnvironmental Health Perspectives vol 115 no 11pp 1573ndash1577 2007


[35] R Guo and J Ren ldquoAlcohol and acetaldehyde in public healthfrommarvel tomenacerdquo International Journal of EnvironmentalResearch and Public Health vol 7 no 4 pp 1285ndash1301 2010

[36] A Szczeklik J Szczeklik Z Galuszka J Musial E KolarzykandD Targosz ldquoHumoral immunosuppression inmen exposedto polycyclic aromatic hydrocarbons and related carcinogens inpolluted environmentsrdquo Environmental Health Perspectives vol102 no 3 pp 302ndash304 1994

[37] NWinker H Tuschl R Kovac and E Weber ldquoImmunologicalinvestigations in a group of workers exposed to various levels ofpolycyclic aromatic hydrocarbonsrdquo Journal of Applied Toxicol-ogy vol 17 pp 23ndash29 1996

[38] E Oh E Lee H Im et al ldquoEvaluation of immuno- andreproductive toxicities and association between immunotoxico-logical and genotoxicological parameters in waste incinerationworkersrdquo Toxicology vol 210 no 1 pp 65ndash80 2005

[39] A O Odewabi O A Ogundahunsi and M Ekor ldquoEffect ofexposure to solid wastes in relation to employment durationon some important markers of health and disease in wastemanagement workers of Ogun State in southwest NigeriardquoHuman and Experimental Toxicology vol 32 no 12 pp 1231ndash1244 2013

[40] H A Kim E M Kim Y C Park et al ldquoImmunotoxicologicaleffects of agent orange exposure to the Vietnam war Koreanveteransrdquo Industrial Health vol 41 no 3 pp 158ndash166 2003

[41] R Jefferis and D S Kumararatne ldquoSelective IgG subclassdeficiency quantification and clinical relevancerdquo Clinical andExperimental Immunology vol 81 no 3 pp 357ndash367 1990

[42] H A Jeng C Pan N Diawara et al ldquoPolycyclic aromatichydrocarbon-induced oxidative stress and lipid peroxidationin relation to immunological alterationrdquo Occupational andEnvironmental Medicine vol 68 no 9 pp 653ndash658 2011

[43] P Rajendran T Jayakumar I Nishigaki et al ldquoImmunomod-ulatory effect of mangiferin in experimental animals withBenzo(a)pyrene-induced lung carcinogenesisrdquo InternationalJournal of Biomedical Science vol 9 no 2 pp 68ndash74 2013

[44] I V Zhigacheva E B Burlakova L S Evseenko et alldquoHumoral immunity polycyclic aromatic hydrocarbons andnitrosaminesrdquo Doklady Biological Sciences vol 383 no 1ndash6 pp120ndash122 2002

[45] K Fagerstrom ldquoTime to first cigarette the best single indicatorof tobacco dependencerdquo Monaldi Archives for Chest Diseasevol 59 no 1 pp 91ndash94 2003

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


and immunotoxicity [10ndash12] Immunotoxicity can changelymphocytic subpopulation in peripheral blood and serumimmunoglobulin levels in coke oven workers exposed toPAHs [12 13] As for genotoxic risk factors the comet assaymicronucleus (MN) assay and chromosomal aberrations(CA) assay have been used to evaluate the biomarkers ofearly biological effects [14] The comet assay has been foundto be a very sensitive method for measuring DNA damageThe MN test was found to provide a cytogenetic parameterand allowed the detection of both clastogenic and aneugenicagents [14] Chromosomal damage has also been found toprovide CA such as chromosome breakage chromosomedeletion and chromosome polyploid [13 14]

In the present study we investigated if there was anyrelation between the levels of MDA 8-OHdG and genotoxicdamages and immunoglobulin levels in serum and lympho-cytes of workers exposed to coke oven emission

2 Materials and Methods

21 Study Subjects The 126 coke oven workers and 78 non-coke-oven workers who were all males and worked in thesame steel company in northern China were studied in thispaper These 126 coke oven workers were in active serviceat the time of the study were employed for at least 6months and were recruited as the exposed group The 78non-coke-oven workers were staff members of the officesand hospitals of the same steel company and served as thecontrol group The workers exposed to known mutagenicagents such as radiotherapy and chemotherapy in the last 3months were excludedQuestionnaires were administered bytrained interviewers to collect information on demographicinformation including age length of employment smokingand alcohol habits Individuals who had smoked for 3monthswere considered as smokers Those who drank more thantwice a week in the last six months were classified as drinkersBlood samples were collected at the end of these days In themorning 5mL fasting venous blood and 10mL urine sampleswere collected from each subject for further analysis Thestudywas approved by the Ethics Committee of Xirsquoan JiaotongMedical College and was performed in accordance with theHelsinki Declaration (1964)

22 1-Hydroxypyrene in Urine Assay Urine 1-hydroxypyrene(1-OHP) was measured by the method described by Jon-geneelen and Anzion [15] and Siwinska et al [16] Brieflyaliquots of 10mL urine samples were enzymatically decon-jugated and transferred to primed C18 octadecyl cartridges(Beckman USA) Then the samples were washed with 10mLof water and eluted with 9mL of methanol The compo-nents of the elutae was subjected to a high pressure liquidchromatography of the Waters Alliance 2695 (Waters USA)with the XAqua C18 150 times 41mm column A fluorescencedetector of F1000 (Hitachi Japan) was quantitatively assayed1-hydroxypyrene concentration The wavelengths of excita-tion and emission were 229 nm and 400 nm respectivelyThe concentrations of 1-OHP were normalised to urinarycreatinine Urinary creatinine concentrationswere assayed by

a standard colorimetric method with the picric acid reactionand absorption at 520 nm [17]

23 Serum Biomarkers Assay Blood samples were obtainedafter an overnight fast Samples (3mL) of venous blood wereincubated in a water bath for 30 minutes at 37∘C and cen-trifuged at 4500 rpm for 10 minutes The supernatants werestored at minus70∘C The levels of serum MDA represented lipidperoxidation product weremeasured spectrophotometricallyby amodification of themethod described by Buege andAust[18] The spectrophotometric measurements were done withShimadzu UV-1208 spectrophotometer (Japan) The MDAconcentration was calculated using its extinction coefficientof 156times105 Lmol cm at 535 nm Enzyme linked immunosor-bent assay (ELISA)was used tomeasure the concentrations of8-OHdG in accordance with the procedures described in theassay kit (Sigma USA) Serum IgA IgE IgG and IgM levelswere measured by a nephelometric method [19]

24 Comet Assay Micronuclei Test and Chromosomal Aber-rations Assay Venous blood was collected from all subjectsusing heparinized syringes Lymphocyte cultures were set upby adding 05mL of heparinised blood to 45mL of chromo-some medium (RPMI 1640 Gibco) supplemented with 20heat inactivated fetal bovine serum (Gibco) antibiotics (peni-cillin and streptomycin) andL-glutamine Lymphocyteswerestimulated by 1 phytohaemagglutinin (Gibco) The cometassay micronuclei test and CA assay were performed toassay the genotoxic damage of lymphocytes in workers DNAdamage to lymphocytes was assayed by the alkaline single-cell gel technique [20 21] Images of 25 randomly selectedlymphocytes were analyzed for each sample The slides wereexamined using a Comet 40 image analysis systemfittedwithanOlympus BX50 fluorescencemicroscope For the collectedcell sample of each subject images of 50 randomly selectedcells were analyzed for each sample The mean level of comettail length (CTL) and comet tail moment (CTM) of the 50cells was as the value of the sample The determination ofmicronuclei in lymphocytes was performed as described byFenech andMorley [22] The cultures were incubated at 37∘Cfor 72 hours and 44 hours after the initiation of culturescytochalasin-B (Sigma) at a concentration of 6mgmL wasadded to arrest cytokinesis MN slides were stained with10 Giemsa in phosphate buffer A total of 1000 binucleatescells with well-preserved cytoplasmwere examined by a well-trained research assistant under double blindness for eachsubject on coded slides throughout the study The data arereported as the occurrence rate of permil micronucleated cellsfor each subject The CA assay was according to the methoddescribed by Carrano and Natarajan [23] Briefly colcemidwas added to arrest the cells in metaphase after incubatingthe cultures for 48 hours at 37∘C then cells were collectedby centrifugation resuspended in a prewarmed hypotonicsolution (0075M KCl) for 25 minutes and fixed in aceticacid methanol (1 3 vv) Air dried preparations were madeand the slides were stained with Giemsa A total of 100well spread metaphases containing 46 plusmn 1 chromosomes wasexamined for each subject on coded slides otherwise the













CA (N AR) 10 128 36 286 0009b











3 Results







1-OHP level


(95 CI) (95 CI) (95 CI) (95 CI)119875 119875 119875 119875






Variables 119903a

119875-valueb

Age (year) 0157 0233a






CTL (120583m) 0571 lt0001a

CTM 0682 lt0001a


CA (N AR) 0422 0009b








4 Discussion



04 06 08 10 12 14 16 18 2010

11

12

13

14

15

168-

OH

-dG

(nm

olm

ol cr

eatin

ine)

MDA (nmolmL)

(a)

05 10 15 20 25 30

18

19

20

21

22

23

24

25

26

27

8-O

H-d

G (n

mol

mol

crea

tinin

e)

MDA (nmolmL)

(b)

10 11 12 13 14 15 16

40

45

50

55

60

65

70

75

CTL

(120583m

)


(c)

18 19 20 21 22 23 24 25 26 27

55

60

65

70

75

80

85

90

95


CTL

(120583m

)

(d)






04 06 08 10 12 14 16 18 20

01

02

03

04

05

06

07

IgG

(gL

)

MDA (nmolmL)

(a)

05 10 15 20 25 30004

006

008

010

012

014

016

018

020

IgG

(gL

)

MDA (nmolmL)

(b)

04 06 08 10 12 14 16 18 201012141618202224262830

IgA

(gL

)

MDA (nmolmL)

(c)

05 10 15 20 25 300608101214161820222426

IgA

(gL

)

MDA (nmolmL)

(d)

10 11 12 13 14 15 16

01

02

03

04

05

06

07

IgG

(gL

)


(e)

18 19 20 21 22 23 24 25 26 27004

006

008

010

012

014

016

018

020

IgG

(gL

)


(f)

10 11 12 13 14 15 161012141618202224262830

IgA

(gL

)


(g)

18 19 20 21 22 23 24 25 26 270608101214161820222426

IgA

(gL

)


(h)

Figure 2 Continued


40 45 50 55 60 65 70 75

01

02

03

04

05

06

07

IgG

(gL

)

CTL (120583m)

(i)

55 60 65 70 75 80 85 90 95004

006

008

010

012

014

016

018

020

IgG

(gL

)

CTL (120583m)

(j)

40 45 50 55 60 65 70 751012141618202224262830

IgA

(gL

)

CTL (120583m)

(k)

55 60 65 70 75 80 85 90 950608101214161820222426

IgA

(gL

)

CTL (120583m)

(l)









5 Conclusion




Acknowledgments


References




















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of













CA (N AR) 10 128 36 286 0009b











3 Results







1-OHP level


(95 CI) (95 CI) (95 CI) (95 CI)119875 119875 119875 119875






Variables 119903a

119875-valueb

Age (year) 0157 0233a






CTL (120583m) 0571 lt0001a

CTM 0682 lt0001a


CA (N AR) 0422 0009b








4 Discussion



04 06 08 10 12 14 16 18 2010

11

12

13

14

15

168-

OH

-dG

(nm

olm

ol cr

eatin

ine)

MDA (nmolmL)

(a)

05 10 15 20 25 30

18

19

20

21

22

23

24

25

26

27

8-O

H-d

G (n

mol

mol

crea

tinin

e)

MDA (nmolmL)

(b)

10 11 12 13 14 15 16

40

45

50

55

60

65

70

75

CTL

(120583m

)


(c)

18 19 20 21 22 23 24 25 26 27

55

60

65

70

75

80

85

90

95


CTL

(120583m

)

(d)






04 06 08 10 12 14 16 18 20

01

02

03

04

05

06

07

IgG

(gL

)

MDA (nmolmL)

(a)

05 10 15 20 25 30004

006

008

010

012

014

016

018

020

IgG

(gL

)

MDA (nmolmL)

(b)

04 06 08 10 12 14 16 18 201012141618202224262830

IgA

(gL

)

MDA (nmolmL)

(c)

05 10 15 20 25 300608101214161820222426

IgA

(gL

)

MDA (nmolmL)

(d)

10 11 12 13 14 15 16

01

02

03

04

05

06

07

IgG

(gL

)


(e)

18 19 20 21 22 23 24 25 26 27004

006

008

010

012

014

016

018

020

IgG

(gL

)


(f)

10 11 12 13 14 15 161012141618202224262830

IgA

(gL

)


(g)

18 19 20 21 22 23 24 25 26 270608101214161820222426

IgA

(gL

)


(h)

Figure 2 Continued


40 45 50 55 60 65 70 75

01

02

03

04

05

06

07

IgG

(gL

)

CTL (120583m)

(i)

55 60 65 70 75 80 85 90 95004

006

008

010

012

014

016

018

020

IgG

(gL

)

CTL (120583m)

(j)

40 45 50 55 60 65 70 751012141618202224262830

IgA

(gL

)

CTL (120583m)

(k)

55 60 65 70 75 80 85 90 950608101214161820222426

IgA

(gL

)

CTL (120583m)

(l)









5 Conclusion




Acknowledgments


References




















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



1-OHP level


(95 CI) (95 CI) (95 CI) (95 CI)119875 119875 119875 119875






Variables 119903a

119875-valueb

Age (year) 0157 0233a






CTL (120583m) 0571 lt0001a

CTM 0682 lt0001a


CA (N AR) 0422 0009b








4 Discussion



04 06 08 10 12 14 16 18 2010

11

12

13

14

15

168-

OH

-dG

(nm

olm

ol cr

eatin

ine)

MDA (nmolmL)

(a)

05 10 15 20 25 30

18

19

20

21

22

23

24

25

26

27

8-O

H-d

G (n

mol

mol

crea

tinin

e)

MDA (nmolmL)

(b)

10 11 12 13 14 15 16

40

45

50

55

60

65

70

75

CTL

(120583m

)


(c)

18 19 20 21 22 23 24 25 26 27

55

60

65

70

75

80

85

90

95


CTL

(120583m

)

(d)






04 06 08 10 12 14 16 18 20

01

02

03

04

05

06

07

IgG

(gL

)

MDA (nmolmL)

(a)

05 10 15 20 25 30004

006

008

010

012

014

016

018

020

IgG

(gL

)

MDA (nmolmL)

(b)

04 06 08 10 12 14 16 18 201012141618202224262830

IgA

(gL

)

MDA (nmolmL)

(c)

05 10 15 20 25 300608101214161820222426

IgA

(gL

)

MDA (nmolmL)

(d)

10 11 12 13 14 15 16

01

02

03

04

05

06

07

IgG

(gL

)


(e)

18 19 20 21 22 23 24 25 26 27004

006

008

010

012

014

016

018

020

IgG

(gL

)


(f)

10 11 12 13 14 15 161012141618202224262830

IgA

(gL

)


(g)

18 19 20 21 22 23 24 25 26 270608101214161820222426

IgA

(gL

)


(h)

Figure 2 Continued


40 45 50 55 60 65 70 75

01

02

03

04

05

06

07

IgG

(gL

)

CTL (120583m)

(i)

55 60 65 70 75 80 85 90 95004

006

008

010

012

014

016

018

020

IgG

(gL

)

CTL (120583m)

(j)

40 45 50 55 60 65 70 751012141618202224262830

IgA

(gL

)

CTL (120583m)

(k)

55 60 65 70 75 80 85 90 950608101214161820222426

IgA

(gL

)

CTL (120583m)

(l)









5 Conclusion




Acknowledgments


References




















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


04 06 08 10 12 14 16 18 2010

11

12

13

14

15

168-

OH

-dG

(nm

olm

ol cr

eatin

ine)

MDA (nmolmL)

(a)

05 10 15 20 25 30

18

19

20

21

22

23

24

25

26

27

8-O

H-d

G (n

mol

mol

crea

tinin

e)

MDA (nmolmL)

(b)

10 11 12 13 14 15 16

40

45

50

55

60

65

70

75

CTL

(120583m

)


(c)

18 19 20 21 22 23 24 25 26 27

55

60

65

70

75

80

85

90

95


CTL

(120583m

)

(d)






04 06 08 10 12 14 16 18 20

01

02

03

04

05

06

07

IgG

(gL

)

MDA (nmolmL)

(a)

05 10 15 20 25 30004

006

008

010

012

014

016

018

020

IgG

(gL

)

MDA (nmolmL)

(b)

04 06 08 10 12 14 16 18 201012141618202224262830

IgA

(gL

)

MDA (nmolmL)

(c)

05 10 15 20 25 300608101214161820222426

IgA

(gL

)

MDA (nmolmL)

(d)

10 11 12 13 14 15 16

01

02

03

04

05

06

07

IgG

(gL

)


(e)

18 19 20 21 22 23 24 25 26 27004

006

008

010

012

014

016

018

020

IgG

(gL

)


(f)

10 11 12 13 14 15 161012141618202224262830

IgA

(gL

)


(g)

18 19 20 21 22 23 24 25 26 270608101214161820222426

IgA

(gL

)


(h)

Figure 2 Continued


40 45 50 55 60 65 70 75

01

02

03

04

05

06

07

IgG

(gL

)

CTL (120583m)

(i)

55 60 65 70 75 80 85 90 95004

006

008

010

012

014

016

018

020

IgG

(gL

)

CTL (120583m)

(j)

40 45 50 55 60 65 70 751012141618202224262830

IgA

(gL

)

CTL (120583m)

(k)

55 60 65 70 75 80 85 90 950608101214161820222426

IgA

(gL

)

CTL (120583m)

(l)









5 Conclusion




Acknowledgments


References




















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


04 06 08 10 12 14 16 18 20

01

02

03

04

05

06

07

IgG

(gL

)

MDA (nmolmL)

(a)

05 10 15 20 25 30004

006

008

010

012

014

016

018

020

IgG

(gL

)

MDA (nmolmL)

(b)

04 06 08 10 12 14 16 18 201012141618202224262830

IgA

(gL

)

MDA (nmolmL)

(c)

05 10 15 20 25 300608101214161820222426

IgA

(gL

)

MDA (nmolmL)

(d)

10 11 12 13 14 15 16

01

02

03

04

05

06

07

IgG

(gL

)


(e)

18 19 20 21 22 23 24 25 26 27004

006

008

010

012

014

016

018

020

IgG

(gL

)


(f)

10 11 12 13 14 15 161012141618202224262830

IgA

(gL

)


(g)

18 19 20 21 22 23 24 25 26 270608101214161820222426

IgA

(gL

)


(h)

Figure 2 Continued


40 45 50 55 60 65 70 75

01

02

03

04

05

06

07

IgG

(gL

)

CTL (120583m)

(i)

55 60 65 70 75 80 85 90 95004

006

008

010

012

014

016

018

020

IgG

(gL

)

CTL (120583m)

(j)

40 45 50 55 60 65 70 751012141618202224262830

IgA

(gL

)

CTL (120583m)

(k)

55 60 65 70 75 80 85 90 950608101214161820222426

IgA

(gL

)

CTL (120583m)

(l)









5 Conclusion




Acknowledgments


References




















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


40 45 50 55 60 65 70 75

01

02

03

04

05

06

07

IgG

(gL

)

CTL (120583m)

(i)

55 60 65 70 75 80 85 90 95004

006

008

010

012

014

016

018

020

IgG

(gL

)

CTL (120583m)

(j)

40 45 50 55 60 65 70 751012141618202224262830

IgA

(gL

)

CTL (120583m)

(k)

55 60 65 70 75 80 85 90 950608101214161820222426

IgA

(gL

)

CTL (120583m)

(l)









5 Conclusion




Acknowledgments


References




















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of






5 Conclusion




Acknowledgments


References




















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

Research Article Lymphocyte Oxidative Stress/Genotoxic...

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