Toxicology in Vitro 26 (2012) 369–372

Contents lists available at SciVerse ScienceDirect

Toxicology in Vitro

journal homepage: www.elsevier .com/locate / toxinvi t

Comet assay analysis of DNA damage in T- and B-lymphocytes separatedby MACS for human biomonitoring studies

So-Young Park a,1, Eunkyung Cho b,1, Eunha Oh c, Donggeun Sul b,d,⇑a Pharmacognosy Lab., College of Pharmacy, Dankook University, San#29, Anseo-Dong, Dongnam-Gu, Cheonan-Si, Chungnam 330-714, Republic of Koreab Graduate School of Medicine, Korea University, 126-1 Anam-Dong 5 Ka, Sungbuk-Ku, Seoul 136-701, Republic of Koreac Center for Life & Environmental Science, Neodin Medical Institute, 2-3 Youngdab-Dong, Sungdong-Ku, Seoul 133-847, Republic of Koread Environmental Toxico-Genomic & Proteomic Center, College of Medicine, Korea University, 126-1 Anam-Dong 5 Ka, Sungbuk-Ku, Seoul 136-701, Republic of Korea

a r t i c l e i n f o

Article history:Received 5 February 2009Accepted 17 November 2011Available online 26 November 2011

Keywords:B-lymphocytesComet assayDNA damageHydrogen peroxideMethyl methane sulfonateT-lymphocytes

0887-2333/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.tiv.2011.11.014

⇑ Corresponding author at: Graduate School of MedAnam-Dong 5 Ka, Sungbuk-Ku, Seoul 136-701, Repub6170; fax: +82 2 927 7220.

E-mail address: dsul@korea.ac.kr (D. Sul).1 These authors equally contributed to this work.

a b s t r a c t

In this study, we developed and tested a method for human biomonitoring using Comet assays withhuman T- and B-lymphocytes obtained by magnetic cell sorting (MACS). We evaluated DNA damageinduced by treatment with hydrogen peroxide (H2O2; 5, 25 and 50 lM) and methyl methane sulfonate(MMS; 5, 25 and 50 lM) in both human B- and T-lymphocytes obtained by MACS, and compared theirDNA damage levels. Significant, dose-dependent levels of DNA damage were found in T-lymphocytesand B-lymphocytes. Furthermore, the level of DNA damage was significantly greater in B-lymphocytesthan in T-lymphocytes, suggesting that human B-lymphocytes may be a more sensitive target than T-lymphocytes for the evaluation of DNA damage. In addition, we compared these in vitro exposure datawith previous studies that showed DNA damage in B- and T-lymphocyte and granulocytes of control sub-jects and industrial workers exposed in vivo to environmental toxicants. The use of single types of humanperipheral blood mononuclear cells obtained by MACS, for Comet assays gave sensitive and reliable datafor human biomonitoring for environmental toxicants.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

A progressive reduction in the levels of exposure of industrialworkers to environmental genetic toxicants has led to a shift inthe focus of biological monitoring to lower occupational and envi-ronmental exposures. For this reason, the use of single cell gel elec-trophoresis (SCGE), or the Comet assay, to investigate DNA damagehas increased in recent years (Moller et al., 2000). In animal stud-ies, the Comet assay has been carried out in lymphocytes and mul-tiple organs to determine DNA damage induced by toxicants(McNamee et al., 2000). For human biological monitoring, theComet assay is commonly used in leukocytes or freshly isolatedlymphocytes because of its sensitivity (Kassie et al., 2000).

Using the Comet assay, we previously reported that mononu-clear and polynuclear lymphocytes from automobile emissioninspectors showed higher levels of DNA damage compared tocontrols (Im et al., 2006). In another study, waste incinerationworkers showed significantly higher levels of DNA damage thanthe control group (Oh et al., 2005). In addition, Sul and colleagues

ll rights reserved.

icine, Korea University, 126-1lic of Korea. Tel.: +82 2 920

found that DNA damage was significantly increased in T-lympho-cytes, B-lymphocytes, and granulocytes from automobile emissioninspectors and waste incineration workers (Sul et al., 2003). Themost sensitive cell type for evaluating DNA damage induced bytoxicants was the B-lymphocyte. Giovannelli (2003) have alsoreported that isolated mononuclear lymphocytes were more sensi-tive to DNA damage induced by H2O2 than non-isolated mononu-clear lymphocytes.

In this study, we developed and tested a method for human bio-monitoring using Comet assays with human T- and B-lymphocytesobtained by MACS. We evaluated DNA damage induced by treat-ment with H2O2 and MMS, and compared their DNA damage levels.Finally, we evaluated these in vitro exposure data in comparison toprevious studies showing DNA damage in B- and T-lymphocytesand granulocytes of control subjects and industrial workersexposed in vivo to environmental toxicants. The use of individualhuman peripheral blood mononuclear cells gave sensitive andreliable data for human biomonitoring of environmental toxicants.

2. Materials and methods

2.1. Subjects

The subjects (n = 10) were healthy male volunteers recruitedbetween June 5 and October 10, 2007. Their mean age was 25 years

370 S.-Y. Park et al. / Toxicology in Vitro 26 (2012) 369–372

(ranges 24–28). All subjects completed a questionnaire that in-cluded questions regarding smoking, drinking, and medication.All the subjects were non-smokers with no history of medication.This study was approved by the IRB at the Hospital of KoreaUniversity.

2.2. Sample preparation of B- and T-lymphocytes

Peripheral blood samples were collected by heparinized veni-puncture from each human subject and delivered immediately tothe laboratory. The Comet assay was carried out within 3 h accord-ing to the method previously described (Sul et al., 2002).

Peripheral blood mononuclear cells (PBMCs) were isolated byFicoll-Paque density gradient centrifugation, after which PBMCswere further separated to using MACS (Fig. 1) (Sul et al., 2002).

2.3. Chemical treatments

Chemical treatments were performed according to Andreoli,(1999) with minor modifications. Briefly, T- and B-lymphocytes(1 � 106 ml�1) were exposed to two concentrations of H2O2 (5,25 and 50 lM) or MMS (5, 25 and 50 lM) for 5 min on ice. Follow-ing the exposure period, cells were washed with PBS and used forthe Comet assay.

2.4. Comet assay

The Comet assays were performed as described previously (Parket al., 2008). Briefly, cover slips coated as previously describedwere submersed in lysis solution for 1 h, then placed in unwindingbuffer for 20 min and followed by electrophoresis for 20 min at25 V and 300 mA (0.8 V/cm). After electrophoresis, the slides wereneutralized and stained overnight with 50 ll of 10 lg/ml ethidiumbromide. The slides were examined using a Komet 4.0 image anal-ysis system (Kinetic Imaging, Liverpool, UK) fitted with an Olym-pus BX50 fluorescence microscope equipped with an excitationfilter of 515–560 nm and a barrier filter of 590 nm. For each treat-ment group, two slides were prepared and 50 randomly chosennuclei per slide (100 cells total) were scored manually. The OliveTailmoment parameter [= (Tail.mean � Head.mean) � Tail%DNA/100], was calculated automatically using a Komet 4.0 image anal-ysis system, which was used for global Comet description.

Fig. 1. Experimental scheme to prepare PBMCs, T-lymphocytes and B-lymphocytesfrom whole blood.

2.5. Statistical analysis

Statistical analyses were performed using Wilcoxon signed ranktest to determine whether differences between the exposed andcontrol groups were significant. The level of statistical significanceemployed in all cases was p < 0.05.

3. Results

3.1. Isolation of B- and T-lymphocytes using MACS

As shown in Fig. 1, T- and B-lymphocytes were isolated fromwhole blood for Comet assays. As the result, one ml of whole bloodcontained 5.4 � 106 mononuclear lymphocytes. Of these, 70%, or3.8 � 106, were T-lymphocytes, while 8.3%, or 4.5 � 105, were B-lymphocytes, collected using MACS. After MACS separation, theT- and B-lymphocytes were over 95% pure, determined by flowcytometry using anti-CD3 and anti-CD19 antibodies.

3.2. DNA damages in T-lymphocytes

T-lymphocytes were exposed to 5, 25 or 50 lM of either H2O2 orMMS, and DNA damage was evaluated using Comet assays(Fig. 2A). The mean Olive Tailmoment of the control T-lymphocyteswas 1.438 ± 0.054. Five lM of H2O2 and MMS did not significantlyalter the Olive Tailmoments compared to the controls. However,the mean Olive Tailmoments of the T-lymphocytes were signifi-cantly higher than the control [2.408 ± 0.057 (p = 0.001) and2.966 ± 0.0191 (p = 0.001), respectively] after being exposed to25 lM or 50 lM of H2O2, indicating that significant DNA damageoccurred from both treatments with H2O2. After the cells were ex-posed to 25 lM or 50 lM of MMS, the Olive Tailmoments were2.088 ± 0.078 (p = 0.001) and 3.190 ± 0.214 (p = 0.001), respec-tively, indicating that significant DNA damage occurred from bothMMS treatments.

3.3. DNA damages in B-lymphocytes

B-lymphocytes were exposed to 5, 25 or 50 lM of either H2O2

or MMS, and DNA damage was evaluated using Comet assays(Fig. 2B). The mean value of the Olive Tailmoment of the controlB-lymphocytes was 1.364 ± 0.065, and those exposed to 5 lMH2O2 and MMS was not significantly different from the controls.However, the exposure of B-lymphocytes to 25 lM or 50 lM ofH2O2 significantly increased the Olive Tailmoments to2.790 ± 0.069 (p = 0.001) and 3.562 ± 0.139 (p = 0.001), respec-tively, indicating that significant DNA damage occurred at bothtreatment levels. Additionally, the exposure of B-lymphocytes to25 lM or 50 lM of MMS significantly increased the Olive Tailmo-ments to 2.758 ± 0.122 (p = 0.001) and 3.668 ± 0.264 (p = 0.001),respectively, indicating that both levels of MMS caused significantDNA damage in B-lymphocytes.

3.4. Comparison of DNA damage in T-lymphocytes and B-lymphocytesinduced by H2O2 or MMS

The levels of DNA damage were compared between T- and B-lymphocytes exposed to 5, 25 or 50 lM of either H2O2 or MMS.The Olive Tailmoments of the control cells were 1.438 ± 0.054and 1.364 ± 0.065 in T-lymphocytes and B-lymphocytes, respec-tively, suggesting that there were no significant differences in thelevels of DNA damage between different cell types (Table 1). TheDNA damage induced by the treatment of 5 lM H2O2 or MMS werenot significantly different from the controls, and the levels of DNAdamage on T- and B-lymphocytes were not, either. However, the

Fig. 2. The Olive tailmoments of T- or B-lymphocytes exposed to H2O2 or MMS. (A)T-lymphocytes separated from PBMCs by MACS were exposed to 5, 25 or 50 lM ofH2O2 or MMS for 5 min. (B) B-lymphocytes isolated from PBMCs by MACS wereexposed to 5, 25 or 50 lM of H2O2 or MMS for 5 min. DNA damage was determinedusing Comet assays and the Olive Tailmoments were calculated. Data represent themeans ± SD (n = 5). ⁄: Significantly different from controls (p < 0.001).

Table 1DNA damage in T-lymphocyte and B-lymphocytes induced by H2O2 and MMS.

Treatment T-lymphocytes B-lymphycytesMean ± SD Mean ± SD

H2O2 (lM)Control 1.438 ± 0.054c,d 1.364 ± 0.065b,c,d

5 lM 1.632 ± 0.081c,d 1.692 ± 0.110b,c,d

25 l-M 2.408 ± 0.057a,b,d 2.790 ± 0.069a,b,d,�

50 lM 2.966 ± 0.191a,d,c 3.562 ± 0.139a,b,c,�

MMS (lM)Control 1.438 ± 0.054c,d 1.364 ± 0.065c,d

5 lM 1.536 ± 0.092c,d 1.650 ± 0.173c,d

25 lM 2.088 ± 0.078a,b,d 2.758 ± 0.122a,b,d,�

50 lM 3.190 ± 0.214a,b,c 3.668 ± 0.264a,b,c,�

a Significantly different with control by Wilcoxon signed rank test (p < 0.001).b Significantly different with 5 lM by Wilcoxon signed rank test (p < 0.001).c Significantly different with 25 lM by Wilcoxon signed rank test (p < 0.001).d Significantly different with 50 lM by Wilcoxon signed rank test (p < 0.001).

� Significantly different from T-lymphocyte with the same condition (p < 0.05).

Table 2DNA damages in lymphocytes, T- and B-lymphocytes, and granulocytes in humanbiomonitoring.

Lymphocytes B-lymphocytes T-lymphocytes Granulocytes

Control – 1.40 ± 0.27 1.42 ± 0.22 2.72 ± 0.59Automobilea – 2.44 ± 0.32* 1.41 ± 0.22 3.32 ± 0.38*

Wasteb – 2.36 ± 0.37* 1.76 ± 0.27* 2.85 ± 0.49Control 1.22 ± 0.26 1.23 ± 0.37 1.12 ± 0.19 2.29 ± 0.36Exposurec 1.27 ± 0.56 2.11 ± 0.37* 1.17 ± 0.30 2.70 ± 0.53*

All values expressed as the mean ± S.D.* Significantly different from control subjects (p < 0.05).

a Automobile emission inspection workers.b Waste incineration workers (Sul et al., 2003).c Workers exposed to benzene, PAHs or dioxin (Lee et al., 2004).

S.-Y. Park et al. / Toxicology in Vitro 26 (2012) 369–372 371

exposure with H2O2 and MMS with 25 or 50 lM significantly in-creased the levels of DNA damage in T-lymphocytes and B-lym-phocytes compared to the controls. Particularly, the OliveTailmoments of B-lymphocytes by the exposure of both toxicantsat 25 and 50 lM significantly increased compared to those of T-lymphocytes (p < 0.05).

4. Discussion

DNA damage plays a significant role in the development ofhuman degenerative diseases and aging (Ames, 1983). In addition,environmental toxicants can induce DNA damage. For example,benzene acts as a mutagen by causing oxidative DNA damagethrough the formation of hydroxyl radicals (Andreoli et al.,1997). It is therefore critical to develop sensitive and accuratemethods to measure DNA damage in human.

The Comet assay has been widely used to detect DNA damagesuch as strand breaks, alkali-labile sites, DNA cross-linking, andincomplete excision repair sites. This technique is a very sensitiveand useful tool to detect genotoxic damage at the individual celllevel and in human biomonitoring (Kassie et al., 2000). In animalstudies, this assay has been carried out in lymphocytes and multi-ple organs to quantify DNA damage and to characterize mutagens(McNamee et al., 2000). The Comet assay also has applications inhuman biomonitoring for occupational or environmental exposureto genotoxic agents (Dusinska and Collins, 2008). Most biomoni-toring using the Comet assay has utilized peripheral blood lympho-cytes (Al Zabadi et al., 2008). However, several studies have usedindividual lymphocytes, including T-and B-lymphocytes and gran-ulocytes. For example, DNA damage was determined using a Cometassay in T-and B-lymphocytes and granulocytes from automobileemission inspectors and water incineration workers (Sul et al.,2003). In addition, workers exposed to various chemicals includingbenzene, PAHs or dioxins were also evaluated for the DNA damagein lymphocytes, T-and B-lymphocytes and granulocytes (Lee et al.,2004) using a Comet assay.

The purification of human lymphocyte subpopulations, such asT-lymphocytes and B-lymphocytes, allows better insight into theirfunction and regulation, and we employed the MACS system, inwhich the magnetically labeled cells are separated over a MACSColumn placed in a MACS Separator. Combining the MACS systemand the Comet assay offers an efficient method for evaluating DNAdamage induced by toxicants in each cell type. To evaluate theeffectiveness of this method, we determined DNA damage in T-and B-lymphocytes, which were exposed to various concentrationsof H2O2 and MMS. Previously, the general toxicity and the geneticdamage caused by H2O2 were investigated in human lymphocytes(Andreoli et al., 1999), and significant DNA damage was observedto various concentrations of H2O2. In addition, an alkylating agent,MMS, significantly increased DNA damage in human lymphocytesby inducing DNA lesions that led to strand breaks in the alkaline

372 S.-Y. Park et al. / Toxicology in Vitro 26 (2012) 369–372

Comet assay (Andreoli et al., 1999). Our results are in agreementwith these. Furthermore, we found that the amount of DNA dam-age that occurred by treatment with H2O2 and MMS was signifi-cantly greater in B-lymphocytes than in T-lymphocytes. Wepreviously reported that DNA damage in T- and B-lymphocytesand granulocytes obtained from emission inspection and incinera-tion workers exposed to PAHs (Table 2) was similar, with B-lym-phocytes being more sensitive to low levels of benzene thanT-lymphocytes and granulocytes (Sul et al., 2003). In addition,B-lymphocytes were more susceptible to DNA damage than T-lym-phocytes in workers exposed to benzene, PAHs or dioxins (Leeet al., 2004). Additionally, apoptotic response to ionizing radiationin human white blood cells was greater in B-lymphocytes (Wilkinset al., 2002). Our data are in agreement with these results, suggest-ing that B-lymphocytes are more sensitive to radiation than T-lym-phocytes (Anderson et al., 1977; Durum and Gengozian, 1978).

We speculate that the differences in DNA damage between B-and T-lymphocytes may be partially due to the different life spanof each cell type. B-lymphocytes have very short life spans of 3–4 days, whereas T-lymphocytes persist for about 4–10 years(Hunter, 1993; Carson, 1995), suggesting that B-lymphocytesmay be easily damaged. The differences between B- and T-lympho-cytes may also be partially due to different DNA repair capacities.Most DNA damage is rapidly recognized and repaired, but incom-plete repair can lead to cell death. T cells have a relatively highercapacity for DNA repair than B cells, causing DNA damage to disap-pear more quickly from T cells (Boerrigter and Vijg, 1991; Choet al., 2008). Taken together, these studies suggest that B-lympho-cytes are more sensitive to DNA damage than T-lymphocytes, mak-ing them a useful target for the biomonitoring of human exposureto low levels of genotoxic compounds.

In this study, we applied the MACS system to isolate individuallymphocytes, including T-lymphocytes and B-lymphocytes, andcompared their DNA damage induced by treatment with H2O2 orMMS. The Olive Tailmoments of T-lymphocytes and B-lympho-cytes with 25 or 50 lM were significantly higher in H2O2 andMMS treated groups compared to the controls. Furthermore, theOlive Tailmoments of B-lymphocytes exposed to 25 or 50 lMH2O2 or MMS were significantly higher than those of T-lympho-cytes. These results suggest that human B-lymphocytes are a moresensitive target than T-lymphocytes to DNA damage induced byH2O2 and MMS. This method of using isolated human peripheralblood mononuclear cells is likely to give more sensitive and reli-able data than previous methods of human biomonitoring for envi-ronmental toxicants.

Conflict of interest statement

None declared.

Acknowledgements

This work was supported by ACE program through the NationalResearch Foundation of Korea (NRF) grant funded by the KoreanMinistry of Education, Science and Technology (MEST) (No.20100028416).

References

Al Zabadi, H., Ferrari, L., Laurent, A.M., Tiberguent, A., Paris, C., Zmirou-Navier, D.,2008. Biomonitoring of complex occupational exposures to carcinogens: thecase of sewage workers in Paris. BMC Cancer 8, 67.

Ames, B.N., 1983. Dietary carcinogens and anticarcinogens. Oxygen radicals anddegenerative diseases. Science 221, 1256–1264.

Anderson, R.E., Standefer, J.C., Scaletti, J.V., 1977. Radiosensitivity of definedpopulations of lymphocytes. VI. Functional, structural, and biochemicalconsequences of in vitro irradiation. Cellular Immunology 33, 45–61.

Andreoli, C., Leopardi, P., Crebelli, R., 1997. Detection of DNA damage in humanlymphocytes by alkaline single cell gel electrophoresis after exposure tobenzene or benzene metabolites. Mutation Research 377, 95–104.

Andreoli, C., Leopardi, P., Rossi, S., Crebelli, R., 1999. Processing of DNA damageinduced by hydrogen peroxide and methyl methanesulfonate in humanlymphocytes: analysis by alkaline single cell gel electrophoresis andcytogenetic methods. Mutagenesis 14, 497–504.

Boerrigter, M.E., Vijg, J., 1991. Induction and disappearance of DNA single-strandbreaks in human B and T lymphocytes after exposure to ethylnitrosourea.Mutation Research 255, 49–55.

Carson, D.A., 1995. Williams Hematology. In: Beutler, E., Lichtman, M.A., Coller, B.S.,Kipps, T.J. (Eds.), Composition and Biochemistry of Lymphocytes and Plasma.McGraw-Hill, New York, pp. 916–921.

Cho, E., Park, S.Y., Sul, D., 2008. New Research on DNA damage. In: Kimura, H.,Suzuki, A. (Eds.), Comparison of DNA damage and the expression of repairrelated molecules, including DNA polymerase b, APE/ref-1, PCNA, and GADD45,in human T and B lymphocytes exposed to hydrogen peroxide and methylmethanesulfonate. Nova Science Publisher, Hauppauge, New York, pp. 305–318.

Durum, S.K., Gengozian, N., 1978. The comparative radiosensitivity of T and Blymphocytes. International Journal of Radiation Biology and Related Studies inPhysics, Chemistry, and Medicine 34, 1–15.

Dusinska, M., Collins, A.R., 2008. The comet assay in human biomonitoring: gene-environment interactions. Mutagenesis 23, 191–205.

Giovannelli, L., Pitozzi, V., Riolo, S., Dolara, P., 2003. Measurement of DNA breaksand oxidative damage in polymorphonuclear and mononuclear white bloodcells: a novel approach using the comet assay. Mutation Research 538, 71–80.

Hunter, R.C., 1993. Hematopoiesis. In: Brown, B.A. (Ed.), Hematology. Lea & Febiger,pp. 36–82.

Im, H., Oh, E., Mun, J., Khim, J.Y., Lee, E., Kang, H.S., Kim, E., Kim, H., Won, N.H., Kim,Y.H., Jung, W.W., Sul, D., 2006. Evaluation of toxicological monitoring markersusing proteomic analysis in rats exposed to formaldehyde. Journal of ProteomeResearch 5, 1354–1366.

Kassie, F., Parzefall, W., Knasmuller, S., 2000. Single cell gel electrophoresis assay: anew technique for human biomonitoring studies. Mutation Research 463, 13–31.

Lee, E., Oh, E., Lee, J., Sul, D., Lee, J., 2004. Use of the tail moment of the lymphocytesto evaluate DNA damage in human biomonitoring studies. Toxicological Science81, 121–132.

McNamee, J.P., McLean, J.R., Ferrarotto, C.L., Bellier, P.V., 2000. Comet assay: rapidprocessing of multiple samples. Mutation Research 466, 63–69.

Moller, P., Knudsen, L.E., Loft, S., Wallin, H., 2000. The comet assay as a rapid test inbiomonitoring occupational exposure to DNA-damaging agents and effect ofconfounding factors. Cancer Epidemiology, Biomarkers & Prevention 9, 1005–1015.

Oh, E., Lee, E., Im, H., Kang, H.S., Jung, W.W., Won, N.H., Kim, E.M., Sul, D., 2005.Evaluation of immuno- and reproductive toxicities and association betweenimmunotoxicological and genotoxicological parameters in waste incinerationworkers. Toxicology 210, 65–80.

Park, S.Y., Kim, H.S., Cho, E.K., Kwon, B.Y., Phark, S., Hwang, K.W., Sul, D., 2008.Curcumin protected PC12 cells against beta-amyloid-induced toxicity throughthe inhibition of oxidative damage and tau hyperphosphorylation. Food andChemical Toxicology 46, 2881–2887.

Sul, D., Lee, D., Im, H., Oh, E., Kim, J., Lee, E., 2002. Single strand DNA breaks in T- andB-lymphocytes and granulocytes in workers exposed to benzene. ToxicologyLetters 134, 87–95.

Sul, D., Oh, E., Im, H., Yang, M., Kim, C.W., Lee, E., 2003. DNA damage in T- and B-lymphocytes and granulocytes in emission inspection and incineration workersexposed to polycyclic aromatic hydrocarbons. Mutation Research 538, 109–119.

Wilkins, R.C., Wilkinson, D., Maharaj, H.P., Bellier, P.V., Cybulski, M.B., McLean, J.R.,2002. Differential apoptotic response to ionizing radiation in subpopulations ofhuman white blood cells. Mutation Research 513, 27–36.