Experimental Parasitology 125 (2010) 230–235

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Experimental Parasitology

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Rejoining of gamma-ray-induced DNA damage in Cryptosporidium parvum measuredby the comet assay

Soo-Ung Lee a, Mikyo Joung a, Taekyoung Nam a, Woo-Yoon Park b, Jae-Ran Yu a,*

a Department of Environmental and Tropical Medicine, Konkuk University School of Medicine, Seoul 143-701, Republic of Koreab Department of Radiation Oncology, College of Medicine, Chungbuk National University, Cheongju 361-763, Republic of Korea

a r t i c l e i n f o

Article history:Received 11 November 2009Received in revised form 18 January 2010Accepted 22 January 2010Available online 1 February 2010

Keywords:Cryptosporidium parvumRadiationDNA damageDouble-strand break

0014-4894/$ - see front matter � 2010 Elsevier Inc. Adoi:10.1016/j.exppara.2010.01.021

* Corresponding author. Fax: +82 2 2049 6192.E-mail address: maria205@kku.ac.kr (J.-R. Yu).

a b s t r a c t

Cryptosporidium parvum is a well-known waterborne intracellular protozoan that causes severe diarrhealillness in immunocompromised individuals. This organism is highly resistant to harsh environmentalconditions and various disinfectants, and it exhibits one of the highest known resistances to gamma irra-diation. We investigated rejoining of gamma-ray-induced DNA damage in C. parvum by neutral cometassay. Oocysts were gamma irradiated at various doses (1, 5, 10, and 25 kGy) and were incubated for var-ious periods (6–96 h) after exposure to 10 kGy. The comet tail moment showed that the number of DNAdouble-strand breaks increased concomitantly with the gamma irradiation dose. When investigatingrejoining after irradiation at 10 kGy, double-strand breaks peaked at 6 h postirradiation, and rejoiningwas highest at 72 h postirradiation. The observed rejoining pattern suggests that repair process occursslowly even when complex DNA double-strand breaks in C. parvum were induced by high dose irradia-tion, 10 kGy.

� 2010 Elsevier Inc. All rights reserved.

1. Introduction

Cryptosporidium parvum is an obligate intracellular protozoanparasite that infects the gastrointestinal epithelium in a wide rangeof vertebrates, including humans and other mammals (O’Donog-hue, 1995). This parasite can cause watery diarrhea in infectedindividuals and can be fatal to those who are immunocompro-mised, such as patients with acquired immune deficiencysyndrome (O’Donoghue, 1995). Three features of the life cycle ofC. parvum enhance its waterborne transmission to humans: (i)C. parvum is transmitted zoonotically; (ii) it is monoxenous, whichfacilitates the maintenance of its entire life cycle, and autoinfectionensures large numbers of oocysts are excreted in feces; (iii) oocystsare environmentally robust (Smith, 1990). Previous studies foundthat C. parvum exhibited the highest known resistance to gammairradiation among parasites (Kato et al., 2001; Yu and Park,2003). It has been suggested that gamma irradiation at 50 kGy isnecessary for the complete elimination of C. parvum infectivity inmice (Yu and Park, 2003). However, there has been no report onthe mechanism underlying the high radioresistance of C. parvumto gamma irradiation.

Ionizing radiation induces cell death by DNA damage, with DNAdouble-strand breaks (DSB) representing the principal lesion thatcan lead to cell death via the generation of lethal chromosomal

ll rights reserved.

aberrations or the direct induction of apoptosis (Radford, 1985;Willers et al., 2004). The comet assay is a single-cell gel electropho-resis-based method that is useful for measuring DNA damage inindividual eukaryotic cells, yeast, protozoa, plants, and inverte-brates, and it is especially useful for measuring radiation-inducedDNA damage (Kumaravel and Jha, 2006; Olive, 2009; Olive andBanath, 2006; Ostling and Johanson, 1984). In particular, thealkaline comet assay is a sensitive method for detecting mostlysingle-strand break in cells, whereas the neutral comet assay de-tects mainly DSB and can be useful for assessing DNA fragmentsassociated with apoptosis (Olive and Banath, 2006). In the presentstudy, we used the neutral comet assay to investigate rejoining ofgamma-ray-induced DNA DSB in C. parvum.

2. Materials and methods

2.1. Animal care and C. parvum oocyst preparation

Specific pathogen-free C57BL6/J female mice (8–9 weeks old)were purchased from Daehan BioLink Co. (Eumsung, Republic ofKorea) and housed under conditions of constant temperature andcontrolled illumination. Mice were orally infected with C. parvumoocysts (KKU isolate) after inducing immunosuppression by pro-viding dexamethasone phosphate disodium salt (Sigma, St. Louis,MO, USA) ad libitum in drinking water at a dose of 10 mg/ml (Yangand Healey, 1993). Mice feces were collected from the wire bottomcages, and the oocysts were purified according to the method

S.-U. Lee et al. / Experimental Parasitology 125 (2010) 230–235 231

described by Petry et al. (1995). Purified oocysts were maintainedat 4 �C for less than 2 weeks in filtered (0.22 lm) distilled water.The animal study was approved by the Animal Care and Use Com-mittee of the Konkuk University.

2.2. Gamma irradiation of C. parvum oocysts

A 1.5-ml microcentrifuge tube containing 2 � 107 purified C.parvum oocysts in 1 ml of filtered (0.22 lm) distilled water wasimmersed in a 50-ml tube filled with distilled water to inducebackscattering and to reduce the temperature increase caused bythe absorption of high-dose radiation energy. Irradiation was per-formed at room temperature (20 �C) for 2 h with a 60Co IR221 HighPerformance Tote Irradiator (MDS Nordion, Ottawa, Canada). C.parvum oocysts were gamma irradiated at various doses (1, 5, 10,and 25 kGy), and they were incubated for various incubation times(6, 12, 24, 48, 72, and 96 h) after exposure to 10 kGy gammairradiation. Oocysts in the control group were maintained at roomtemperature during irradiation. The temperature of the control andirradiated sample tubes were the same before and immediatelyafter irradiation (20.2 ± 0.3 �C versus 20.4 ± 0.3 �C, respectively).

2.3. Comet assay

The neutral comet assay was applied to C. parvum using thecommercial CometAssay kit (Trevigen, Gaithersburg, MD, USA),which is based on a modified method of Ostling and Johanson(1984). C. parvum oocysts (2 � 107) exposed to gamma rays weremixed with molten low-melting-point agarose (Trevigen) at a ratioof 1:10 (v/v), and 75 ll of this mixture was immediately embeddedin a CometSlide (Trevigen) that was placed flat at 4 �C in the darkfor 10 min to improve adherence of the sample in a humiditybox. The slide was immersed for 30 min in the pre-chilled (4 �C) ly-sis solution provided with the CometAssay kit. After removing ex-cess buffer from the slide, it was placed twice in electrophoresisbuffer (90 mM Tris base, 90 mM boric acid, and 2 mM ethylenedi-aminetetraacetic acid) for 5 min at room temperature to removethe lysis solution. The slide was then placed in a horizontal electro-phoresis tank (Mupid, Advance, Tokyo, Japan), and electrophoresiswas conducted for 20 min at 28 V (1 V/cm, 400 mA). After remov-ing excess electrophoresis buffer from the slide, it was rinsedbriefly in distilled water, and then the gel was fixed by immersionin 70% ethanol for 5 min and air dried at room temperature for15 min. The sample slide was stained for 1 h with the green fluo-rescent DNA-binding dye, SYBR Green I, provided with the Comet-Assay kit. The migrated DNA (comet) was observed under anepifluorescence microscope at 600� magnification (BX61, Olym-pus, Tokyo, Japan). As a positive control, oxidative DNA damage

Fig. 1. Cryptosporidium parvum oocysts lysed by the lysis buffer provided with the C

of C. parvum was induced by treatment with 25 lM KMnO4 for20 min at 4 �C as described at CometAssay kit (Trevigen).

2.4. Analysis of comet assay

DNA damage was assessed using an automatic image analysissystem (CometAssay IV software, Perceptive Instruments, Suffolk,UK) to measure the tail moment, which is defined as the productof the amount of DNA in the tail and the displacement betweenthe center of mass of the comet head and the center of mass ofthe tail (tail length) (Olive, 1999). Duplicate samples were pre-pared for each experimental group, and 30 cells in each samplewere randomly selected and analyzed. Statistical significance wasdetermined by analysis of variance.

3. Results

Excystation of the C. parvum oocyst wall was confirmed by lightmicroscopy (Olympus, Tokyo, Japan) after exposure to lysis bufferprovided with the CometAssay kit. For the comet assay to besuccessful, the C. parvum oocyst wall should be disrupted becausethe thick oocyst wall can inhibit penetration of dye into thesporozoite nucleus, which is located inside the oocyst wall. Thegamma-irradiated C. parvum oocysts achieved about 90% lysis afterexposure to the lysis solution for 30 min at 4 �C (Fig. 1).

The SYBR Green I-stained DNA of non-irradiated control C. par-vum sporozoites was round and had a well-defined nucleoid mar-gin, whereas DNA of C. parvum sporozoites damaged by radiationappeared as a smear (Fig. 2). Increasing the irradiation dose re-sulted in the SYBR Green I-stained DNA appearing enlarged andwith a longer migrated tail (Fig. 2A). After irradiation at 10 kGy,the SYBR Green I-stained comet tail moment was highest at 6 hpostirradiation, and DNA tail migration was observed up to 48 hpostirradiation (Fig. 2B). However, the amount of DNA stained withSYBR Green I became much smaller at 72–96 h postirradiation,eventually appearing not to differ in size from the non-irradiatedsporozoites (Fig. 2B).

The comet tail moment was calculated as the percentage ofDNA in the tail multiplied by the distance between the means ofthe head and tail distributions (tail length) according to Oliveand Banath (2006). In the neutral comet assay, the comet tail mo-ment increased significantly in all of the gamma-irradiated groups(1–25 kGy) (Fig. 3A). Oxidative damage induced by KMnO4 as a po-sitive control also showed significant (P < 0.05) DNA damage (datanot shown).

Irradiation at 1, 5, 10, and 25 kGy resulted in 4.0-, 4.9-, 5.6-, and6.0-fold increases in the comet tail moment relative to non-irradi-

ometAssay kit. Bar, 5 lm; arrows, sporozoites from the C. parvum oocyst wall.

Fig. 2. Changes in nuclear morphology of Cryptosporidium parvum sporozoites as detected by the comet assay for different doses of gamma irradiation (A) and differentincubation times after irradiation at 10 kGy (B). DNA was stained with SYBR Green I and observed by epifluorescence microscopy. PC, C. parvum oocysts treated with 25 lMKMnO4 for 20 min at 4 �C as a positive control. Bar, 2 lm.

Table 1Parameters of the comet assay, tail length, and percentage tail DNA determined usingan image analysis system, showing DNA damage of C. parvum oocysts after differentdoses of gamma irradiation. Data are mean ± SD values (n = 60). Superscript ‘‘a”denotes a significant difference from control (P < 0.05). PC, C. parvum oocysts treatedwith 25 lM KMnO4 for 20 min at 4 �C as a positive control.

Irradiation dose (kGy) Tail length Tail DNA (%)

0 16.5 ± 6.1 9.3 ± 10.11 48.4 ± 13.1a 20.3 ± 9.2a

5 51.0 ± 7.6a 25.5 ± 6.2a

10 60.4 ± 17.9a 24.1 ± 8.5a

25 67.0 ± 20.8a 23.1 ± 7.1a

PC (KMnO4) 45.4 ± 11.4a 25.0 ± 10.5a

232 S.-U. Lee et al. / Experimental Parasitology 125 (2010) 230–235

ated C. parvum, respectively (Fig. 3A). The comet tail momentshowed that DNA damage reached a maximum 6 h after 10-kGyirradiation, decreasing gradually thereafter back down to the samelevel of DNA damage present in the non-irradiated control group at72 h postirradiation (Fig. 3B). However, the comet tail moment sig-nificantly increased again at 96 h postirradiation (P < 0.05) com-pared to the non-irradiated controls (Fig. 3B). The comet tailmoments increased 7.4-, 6.3-, 6.1-, 4.5-, 1.2-, and 2.5-fold relativeto the non-irradiated controls at 6, 12, 24, 48, 72, and 96 h postir-radiation, respectively (Fig. 3B). The tail length and tail DNA(percentage) increased significantly (P < 0.05) compared to thenon-irradiated controls in all irradiated groups (Table 1). Boththe tail length and tail DNA increased significantly up to 48 h post-irradiation (Table 2). However, the tail DNA was not significantlyincreased at 72 h after 10-kGy irradiation (Table 2). The distribu-tion of changes in the DNA tail length showed that non-irradiated

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Fig. 3. Comet tail moment of Cryptosporidium parvum after gamma irradiationusing different doses (A) and for different incubation times after gamma irradiationat 10 kGy (B). Asterisks denote a significant difference from control (P < 0.05). Dataare mean and SD values for 30 comets (each from duplicate experiments).

controls had a mean tail length of 10–20 (arbitrary units) in theneutral comet assay (Fig. 4). Increasing the radiation dose shiftedthe high-frequency fraction between 50 and 60 (Fig. 4A). Also,the fraction with a tail length of 50–70 increased up to 48 h post-irradiation, and the fraction with a short tail length (20–40) in-creased again at 72 h postirradiation (Fig. 4B). The highestfrequency fraction shifted to 50 at 96 h postirradiation (Fig. 4B).

4. Discussion

Kato et al. (2001) reported that C. parvum oocysts that received2-kGy irradiation showed the same excystation rates as non-irradi-ated oocysts, and irradiation at 20 and 50 kGy induced excystationrates of 50% and 0%, respectively. Using nucleic acid staining, wefound that about 90% and 50% of C. parvum oocysts were viable fol-lowing irradiation at 10 and 50 kGy, respectively (Yu and Park,2003). These studies demonstrate that C. parvum is resistant toradiation at doses more than 5–20 times higher than those for par-asitic helminthes (Bickle et al., 1979; Chai et al., 1995; Lee et al.,1989; Verster et al., 1976) and coccidian protozoans, such as Toxo-

Table 2Parameters of the comet assay, tail length, and percentage tail DNA determined by animage analysis system, showing DNA damage of C. parvum oocysts for differentincubation time after gamma irradiation at 10 kGy. Data are mean ± SD values(n = 60). Superscript ‘‘a” denotes a significant difference from control (P < 0.05). PC, C.parvum oocysts treated with 25 lM KMnO4 for 20 min at 4 �C as a positive control.

Time after irradiation (h) Tail length Tail DNA (%)

0 23.9 ± 5.3 10.1 ± 9.66 75.3 ± 17.6a 27.2 ± 13.2a

12 66.9 ± 20.6a 25.4 ± 10.1a

24 65.4 ± 19.9a 26.8 ± 6.1a

48 55.1 ± 15.0a 22.4 ± 7.7a

72 35.5 ± 11.1a 12.6 ± 9.496 40.8 ± 9.0a 16.1 ± 8.4a

PC (KMnO4) 62.6 ± 13.6a 21.2 ± 7.2a

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plasma gondii and Eimeria necatrix, (Dubey et al., 1996; Singh andGill, 1975; Song et al., 1993) which can be controlled by gammairradiation at 0.1–0.6 and 0.6–2 kGy, respectively.

There is a strong correlation between cellular radiosensitivityand DNA damage (Wada et al., 2005). The comet assay measuresvarious types of DNA damage and repair in individual cells, includ-ing cells from invertebrates and plants (Olive, 1999). This methodis advantageous in that it requires only a small number of cells, is

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Fig. 4. Frequency distributions of DNA damage of Cryptosporidium parvum quantified as thof gamma irradiation (A) and different incubation times after irradiation at 10 kGy (B).parvum sporozoites in each assay.

highly sensitive to detecting DNA damage, and is applicable to anytype of eukaryotic cell (Olive, 1999). A previous study using the co-met assay found a linear dose–response relationship for DSB withradiation doses ranging from 5 to 300 Gy (Olive, 1995). In the pres-ent study, the comet tail moment showed that the DNA DSB of C.parvum induced by high dose (1–25 kGy) gamma irradiation alsoincreased linearly with the dose. The tail length indicates the dis-tance that the DNA has migrated out of the cell, and smaller DNA

B

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e comet tail length (arbitrary units) in the neutral comet assay using different dosesData are mean and SEM values (n = 3). Comet tail length was measured using 30 C.

234 S.-U. Lee et al. / Experimental Parasitology 125 (2010) 230–235

fragments move the farthest (Kumaravel and Jha, 2006), whichstrongly suggests that increasing the gamma radiation dose frag-ments the DSB-containing DNA into smaller pieces.

The bacterium Deinococcus radiodurans is also highly resistantto ionizing radiation, and it possesses various mechanisms forrepairing damage induced by ionizing radiation (Battista, 1997;Daly et al., 2004; Hansen, 1978; Servinsky and Julin, 2007; Zah-radka et al., 2006). Irradiation at 3 kGy induces remarkable DNADSB in D. radiodurans, but chromosomes reform 3 h after radiationexposure without loss of viability or mutation (Battista, 1997). Therepair of DSB in mammalian cells reportedly involves fast and slowcomponents, with the fast component of total rejoining, which cor-responds to repair of simple DSBs, occurring predominantly duringthe first 2 h (Belli et al., 2000; Lobrich, 1998; Nunez et al., 1996).During this fast component, correct rejoining occurs during thefirst 2 h, whereas the slow component corresponds to more com-plex DSB, which require more time for rejoining and may have highchance of misrejoining (Belli et al., 2000; Lobrich, 1998). In thepresent study, the comet tail moment showed that DNA DSB werehighest at 6 h postirradiation but thereafter rejoined rapidly until72 h, with a significant number of the comet tail moments restoredto the normal non-irradiated levels. We note that the time requiredto transport our irradiated samples from the radiation facility toour research institute prohibited the determination of DSB rejoin-ing at time points earlier than 6 h postirradiation. Although the co-met tail moment showed that complete rejoining occurred after72 h postirradiation, there was a high possibility of incorrectrejoining of DNA DSB in a major portion of C. parvum oocysts be-cause 10 kGy-irradiated oocysts showed effective reduction ofin vitro infectivity on HCT-8 cells (Lee et al., 2009) and becausethe rejoining with the lowest tail moment occurred at 72 h postir-radiation. C. parvum remains in a dormant state in the environmentand hence does not undergo cell cycling or proliferation; thus thereis a limitation to know that DNA DSB repair can be evaluated. In aprevious study, the in vitro infectivity of HCT-8 cells with C. par-vum sporozoites that had received 10-kGy irradiation was reducedeffectively (more than 2 log10), and the infectivity was not recov-ered until 72 h postirradiation (Lee et al., 2009). A study of thein vivo infectivity using mice, however, found that the infectivityby C. parvum was still remained even after irradiation up to10 kGy (Yu and Park, 2003). These studies demonstrate that thereis a discrepancy between the in vitro and in vivo infectivity of C.parvum after 10 kGy irradiation, and they suggest that a substantialnumber of irradiated oocysts retain their infectivity in mice.Although infectivity is not completely recovered after 10 kGy irra-diation, it is very interesting that DNA DSB rejoining occurred at72 h postirradiation.

Radiation damage is largely mediated by reactive oxygen spe-cies (ROS) production (Riley, 1994). Therefore, ROS scavengingantioxidant enzymes may be important factors for radiation resis-tance. Anisakis simplex third-stage larvae are radioresistant, andsuperoxide dismutase may play a role in this radioresistance (Seoet al., 2006). However, the basal level of antioxidant enzymes, suchas superoxide dismutase, catalase, glutathione transferase, gluta-thione reductase and glutathione peroxidase activity was verylow in C. parvum oocysts (Entrala et al., 1997). Thus, another anti-oxidant enzymes or repair systems may be involved in radioresis-tance of C. parvum. Future molecular and genetic studies should beconducted to uncover more details about the mechanism underly-ing the high radioresistance and DNA damage repair of C. parvum.

Acknowledgments

This research was supported by the program of the Basic AtomicEnergy Research Institute, which forms part of the Nuclear R&DPrograms funded by the Ministry of Science & Technology of Korea,

and by the Second-Phase of the BK (Brain Korea) 21 Project in2009. This study complied with the current laws of the Republicof Korea (where the experiments were performed).

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