γ-H2AX as a biomarker for DNA double-strand breaks in ecotoxicology

Marko Geric,́ Goran Gajski, Vera Garaj-Vrhovac n

Institute for Medical Research and Occupational Health, Ksaverska cesta 2, 10000 Zagreb, Croatia

a r t i c l e i n f o

Article history:Received 27 September 2013Received in revised form27 March 2014Accepted 29 March 2014

Keywords:γ-H2AX foci assayGenotoxicityDouble-strand breaksCytostatic pharmaceuticalsEnvironmental concentrations

a b s t r a c t

The visualisation of DNA damage response proteins enables the indirect measurement of DNA damage.Soon after the occurrence of a DNA double-strand break (DSB), the formation of γ-H2AX histone variantsis to be expected. This review is focused on the potential use of the γ-H2AX foci assay in assessing thegenotoxicity of environmental contaminants including cytostatic pharmaceuticals, since standardmethods may not be sensitive enough to detect the damaging effect of low environmental concentra-tions of such drugs. These compounds are constantly released into the environment, potentiallyrepresenting a threat to water quality, aquatic organisms, and, ultimately, human health. Our reviewof the literature revealed that this method could be used in the biomonitoring and risk assessment ofaquatic systems affected by wastewater from the production, usage, and disposal of cytostaticpharmaceuticals.

& 2014 Elsevier Inc. All rights reserved.

1. Introduction

DNA double-strand breaks (DSBs) are one of the most criticalevents affecting DNA when a cell or an organism is exposed toionizing radiation or, chemical or environmental stress. If notadequately repaired, DSBs can have severe consequences on cellsleading to cell death or induction of genomic instability, which canin turn trigger carcinogenesis (Redon et al., 2010a; Vandersickel etal., 2010). Soon after a DSB is formed, the cell's complex machineryinitiates a DNA damage response, which is frequently character-ized by changes in signalization, transcription, and cell cycle thusleading to DNA repair. The proteins involved into DNA repair canroughly be divided into three major groups: (a) proteins thatdetect DSBs and trigger signalling due to DNA damage;(b) proteins that amplify the DNA damage signal; (c) proteins

involved in the DNA repair processes (Bekker-Jensen and Main-land, 2010).

Although the precise mechanisms of DSB detection are stillunknown, it has been proposed that, soon after the occurrence of aDSB, the NBS1 protein is recruited to the site of the DSB, which theninitiates the formation of the NBS1/MRE11/RAD50 (NMR) complex(Kobayashi, 2004). Subsequently, the ATM protein is activated throughthe auto-phosphorylation of serine 1981. ATM is one of the mostpotent histone H2AX activators, which phosphorylates serine 139residue in highly conserved region (Kinner et al., 2008). As for γ-H2AXphosphorylation kinetics, it is important to highlight that this event iscompleted within just a fewminutes after a DSB occurrence, while thehighest concentration of the γ-H2AX is approximately 30 min after aDSB occurrence (Banáth et al., 2010). This phenomenon can beexplained by γ-H2AX's dual role in this signalling pathway: it recruitsmore MRN complexes to the DSB site thus increasing the signalizationvia a positive feedback loop and binding the proteins responsible forDNA damage repair (BRCA1, 53BP1, TOPBP1, MDC1, etc.) (Minter-Dykhouse et al., 2008; Paull et al., 2000). Depending on the efficacyof damage response proteins, this signal loop can result in DNA repair,cell cycle arrest, or initiation of apoptosis (Kobayashi, 2004; Kobayashiet al., 2008; Srivastava et al., 2009, Yuan et al., 2010). It has beenestablished that H2AX phosphorylation mediated by the ATM proteinoccurs when DSBs are induced by radiation, while phosphorylationmediated by ATR protein or DNA-dependent protein kinases is to beexpected when DSBs are induced by replication stress and hyperto-nical stress, respectively (Shiloh, 2003; Rakiman et al., 2008).

Among the fields where visualisation of the γ-H2AX could be usedto quantify the amount of DSBs (reviewed by Redon et al., 2011a), thismethod still predominantly measures radiation-induced DSBs. Still,

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Ecotoxicology and Environmental Safety

http://dx.doi.org/10.1016/j.ecoenv.2014.03.0350147-6513/& 2014 Elsevier Inc. All rights reserved.

Abbreviations: 5-FU, 5-fluorouracil; 53BP1, p53 binding protein 1; API, aphidico-lin; ARA-C, cytarabine; ATM, ataxia telangiectasia mutated; ATR, ataxia telangiec-tasia related; BLAP, ß-lapachone; BLM, bleomycin; BRCA1, breast cancer type1 susceptibility protein; cDDP, cisplatin; CFGE, constant field gel electrophoresis;COL, colcemid; DOX, doxorubicin; DSB, DNA double-strand break; EGFR, epidermalgrowth factor receptor; ERL, erlotinib; ETO, etoposide; FITC, fluorescein isothio-cyanate; GEF, gefitinib; GEM, gemcitabine; HPBLs, human peripheral blood lym-phocytes; IMB, imatinib mesylate; IRI, irinotecan; MDC1, mediator of DNA-damagecheckpoint 1; MMC, mitomycin C; MRE11, meiotic recombination 11 homolog A;MTX, methotrexate; MTXT, mitoxantrone; NBS1, neijmegen breakage syndrome1 protein; NPRL2, nitrogen permease regulator-like 2; PT, paclitaxel; RABiT, rapidautomated biodosimetry tool; RAD50, DNA repair protein RAD50; TOPBP1, topoi-somerase (DNA) II binding protein 1; TOPO, topoisomerase; TPT, topotecan; VCR,vincristine; ZID, zidovudine.

n Corresponding author. Fax: þ385 1 4673 303.E-mail address: vgaraj@imi.hr (V. Garaj-Vrhovac).

Ecotoxicology and Environmental Safety 105 (2014) 13–21

there is huge potential for this method to be used in environmentalgenotoxicology where it could be used to detect the negative impactsof xenobiotics. Accordingly, the present review aimed to highlight thecapacity of the γ-H2AX histone to represent a sensitive biomarker ofeffect during exposure to certain compounds. Also, special attentionwas given to cytostatic pharmaceuticals, since these drugs are usuallyhighly toxic and constantly released into aquatic ecosystems fromhealthcare facilities, patient homes, wastewater treatment planteffluents, landfill leakages, and pharmaceutical industrial plants.Consequently, their presence can be detected in waste-, surface-, andground waters (Besse et al., 2012; Negreira et al., 2013a). Apart fromtheir constant release into the environment, their stability in aquaticsolutions (Negreira et al., 2013b) qualifies them as “pseudo persistentpollutants” that could affect water quality and non-target organismsfrom all trophic levels, including humans (Filipič et al., 2013; Hernandoet al., 2006). This review emphasizes the ability of the γ-H2AX fociassay to serve as a more sensitive method than standard genotoxicitybioassays in the detection of genome changes at low exposureconcentrations.

2. γ-H2AX foci assay as a reliable method for DSBs detection

During the last 50 years, many methods have been developedto detect DNA damage (Garcia-Sagredo, 2008). Some of the mostpopular genotoxicity assays do not measure DSBs exclusively suchas the comet assay or micronucleus test, although some modifica-tions can be made in order to draw attention to DSBs (Fenech,2006; Suspiro and Prista, 2011; Tice et al., 2000). Comet assayelectrophoresis under neutral pH conditions could be used todetect DNA fragments that occur due to DSBs, whereas themicronucleus test with centromere labelling can suggest thatmicronucleus (MN) originated following a DSB if its labellingproves negative (Collins et al., 2008; Fenech, 2007). Some of theparameters of chromosome aberrations and sister chromatidexchange may also indicate that a DSB occurred, but thesemethods sometimes have low sensitivity and/or specificity (Obeet al., 2002; Schvartzman and Goyanes, 1980). The specificity canbe satisfactory if methods like the G-2 assay (Brzozowska et al.,2012), pulsed- or constant-field gel electrophoresis (Lob̈rich et al.,2010; Rydberg, 2000) are used, but their sensitivity is probablysomewhat lower than of the γ-H2AX foci assay (Chaudhry, 2008;Valdiglesias et al., 2013). Based on a study on six differentgenotoxicants conducted by Smart et al. (2011), the γ-H2AX fociassay responded impressively with an average sensitivity of91 percent and average specificity of 89 percent. In the last decade,the γ-H2AX foci assay became a powerful biodosimetry toolbecause of its specificity, high reproducibility, sensitivity, andrapidness, which are also important characteristics in other fieldsof research. Therefore we have attempted to support these state-ments with corresponding data from the literature.

Flow cytometry measurement of the γ-H2AX after irradiationwith different X-ray doses (0–8 Gy) was conducted by Hamasaki etal., 2007, who observed a dose-responsive increase in DSBs in bothcultured and uncultured human T lymphocytes. The applicabilityof this method in monitoring DSB repair and inter-individualvariability of γ-H2AX formation was also demonstrated. A similarresponse of the γ-H2AX formation was observed in a study by Jostet al., 2009 indicating that this parameter is a reliable DSBbiomarker for doses exceeding 1 Gy where the frequency ofdicentrics in human peripheral blood lymphocytes (HPBLs) alsorose, and they represent a good biomarker of radiation exposure(IAEA, 1986). When discussing doses lower than 1 Gy (0.025–0.1 Gy) γ-H2AX did not produce a dose-responsive increase ofsignal, although for certain doses it managed to reach a significantincrease in γ-H2AX foci formation. On the other hand, a paper by

Rothkamm et al. (2013a) demonstrated that laboratories withexperienced scientists can detect increase of γ-H2AX foci evenafter a 0.1 and 0.7 Gy exposure dose. Human MRC-5 lung fibro-blasts also lead to dose-dependent increase in the number of fociper cell following synchrotron microbeam radiotherapy 0.5–2 Gy(Rothkamm et al., 2012).

Except from its use in the detection of radiation-induced DSBs,the γ-H2AX foci assay proved useful for monitoring DNA repairkinetics. In a study by Avondoglio et al. (2009) on U251 cellsirradiated with X-rays, 2–8 Gy induced a dose-dependent increasein γ-H2AX signal intensity and an increase in the number of fociper cells was also detected. When the cells were irradiated with asingle dose of 2 Gy, the γ-H2AX signal increased in the first hourafter irradiation increases thus reflecting cell recruitment of DNArepair mechanisms to DSB foci. After 6 and 24 h, that signaldecreased dramatically, which was proven by the presence ofefficient DNA repair mechanisms. A similar pattern in repairkinetics was detected on human MRC-5 lung fibroblasts where0.2 and 2 Gy resulted with a progression in repair after 3 min,15 min, 2 h and 24 h (Rothkamm and Lob̈rich, 2003).

3. Detection of cytostatics-induced DSBs with γ-H2AXfoci assay

The H2AX histone variant represents 2–20 percent of the H2Ahistone family and has a role in the recruitment of proteins,remodelling of chromatin and signal amplification and transduc-tion when a cell's genome is disturbed by DSBs (Rothkamm andHorn, 2009). Chemotherapy, along with radiotherapy and surgery,is one of the most common choices in cancer treatment (Enger etal., 2007). Usually, the negative effects of cancer treating agentsare monitored in vitro and in later stages of drug development inin vivo population studies to evaluate the efficacy of candidatedrugs and their negative effects on healthy cells and tissues (Kanget al., 2008). There is still a lack of studies dealing with the toxicityof wastewaters containing pharmaceuticals. Accordingly, in astudy by Ismail et al. (2007), when the genotoxic effects of theantibiotic calicheamicin γ1 on human blood white cells wereexamined, the relative FITC signal elevated logarithmically con-trary to the comet's tail intensity which increased linearly, there-fore demonstrating the greater sensitivity of the γ-H2AX foci assayto detect early produced DSBs. In order to evaluate the efficacy ofthe γ-H2AX foci assay in regard to pharmaceutical exposure tocytostatic drugs (antimetabolites, crosslinkers, topoisomeraseinhibitors, microtubules inhibitors, and tyrosine kinase inhibitors),a review of the currently available literature was done and theresults are summarized in Table 1. Cytostatics are reviewed basedon their modes of action according to their occurrence in hospitaleffluents, wastewaters treatment plant effluents or freshwater asreviewed by Zhang et al. (2013).

3.1. Antimetabolites

Drugs 5-fluorouracil (5-FU) and methotrexate (MTX) are well-known antimetabolites widely used in cancer therapy. Their mainmode of action is to inhibit thymidylate synthase and dihydrofo-late reductase, respectively. These enzymes are both involved inthe thymine synthesis pathway and therefore inhibit its de novoproduction (Showalter et al., 2008). Using the γ-H2AX, the abilityof 5-FU to cause DSBs was noticed by Ikeda et al. (2010) onHEC-1A, HEC-1B, KLE and Ishikawa cell lines, as well as byMatsuzaki et al. (2010.) on CHL, V79, and CHO cell lines. Exceptfor 5-FU, Matsuzaki et al. (2010) also used MTX as a DSB-inducingagent in their study. In the study by Ikeda et al. (2010), a modestincrease in γ-H2AX formation was observed in concentrations

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Table 1Summary of the results from the studies that tested induction of γ-H2AX using different treating agents at given concentrations and exposure periods on various study models.

Reference Treating agents Study model Concentration Exposure period Result (γ-H2AX detection)

Clingen et al. (2008) cDDP AGO human fibroblasts,AA8, CHO-K1, UV96, irs1SF, xrs5 cells cDDP 5–10 μM 1 h cDDP: þþ (AGO fibroblasts and in CHO sublines whereUV96 were the most sensitive)

Ewald et al. (2007) GEM Human adult myelogenus leukemia cell lines ML-1 and OCI-AML3 GEM 10 nM 0–24 h GEM: þþEwald et al. (2008) GEM Human adult myelogenus leukemia cell lines OCI-AML3 GEM 0.1 μmol/L 2 h GEM: þþ

ARA-C ARA-C 0.5 μmol/L ARA-C: þþGalluzzi et al. (2010) cDDP Non-small cell lung cancer: A549 cells cDDP 50 μM 3, 6, 9 h CDDP: þþ (after 3 h of exposure)Garcia-Canton et al. (2013) ETO BEAS-2B bronchial epithelial cell lines ETO 0–1000 μM 3, 24 h ETO: þþ

API API 0–100 μM API: þþBLM BLM 0–10 μM BLM: þþZID ZID 0–1000 μM ZID: þþ

Huang et al. (2004) cDDP Promyelocytic leukemic: HL-60 cells cDDP 0.5, 1, 2 μM cDDP 1, 3 h cDDP: þþTPT, TPT 0.15 μM TPT 1, 1.5, 3 h TPT: þþMTXT MTXT 0.15 μM MTXT 2, 3 h MTXT: þþ

Ikeda et al. (2010) 5-FU Endometroid 5-FU 3 μg/mL 3, 6, 12, 5-FU:þ(Ishikawa cells were the most sensitive)cDDP Adenocarcinoma: HEC-1A, HEC-1B, Ishikawa cells, KLE cDDP 10 μg/mL 24, 48, 72, 96, 120 h cDDP: þþ (Ishikawa cells were the most sensitive after

72 h of exposure)DOX DOX 0.1 μg/mL DOX: þþ (HEC-1A were the most sensitive after 3 h of exposure)

Jayachandran et al. (2010) cDDP Non-small cell lung cancer cells: H1299, H322 cDDP 3 μM 24, 48, 72 h cDDP:þ(H1299 cells), þþ (when stimulated with NPRL2)Kim et al. (2011) BLM U20S osteosarcoma cells BLM 2–50 μg/mL 1 h BLM: þþ

cDDP cDDP 2–50 μM cDDP: -ETO ETO 2–50 μM ETO: þþDOX DOX 0.5–2 μM DOX: þþ5-FU 5-FU 2–50 mg/mL 5-FU: -

Li et al. (2008) ERL MCF7DRGFP human breast cancer cells ERL 1 μM 16 h ERL: þþLiu et al., 2007 IMB GIST882 cell line, IMR90 (lung fibroblasts) IMB 1 μM 1–72 h IMB: þþMatsuzaki et al. (2010) cDDP CHL/IU cells cDDP 0–25 μg/mL 24 h cDDP: þþ (although CHO cells presented þ)

ETO CHO-K1 cells ETO 0–50 μg/mL ETO: þþ5-FU V79 cells 5-FU 0–200 μg/mL 5-FU: þþ (except CHO cells)MMC MMC 0–100 μg/mL MMC: þþIRI IRI 0–200 μg/mL IRI: þMTX MTX 0–0.5 μg/mL MTX: þþ (although CHO cells were less sensitive than

CHL and V79 cell lines)VCR VCR 0–1 μg/mL VCR, COL and PT: -COL COL 0–1 μg/mLPT PT 0–100 μg/mL

Muslimović et al. (2009) ETO SV40 human fibroblasts ETO 62.5–500 μM 40 min ETO: -Olive and Banáth (2009) cDDP Cervical carcinoma cells: SiHa, glioma cells: U87, ovarian cancer cells:

SKOV3, melanoma cells: HT144, colon cancer cells: WiDr0.5, 1, 2, 5, 10 μg/mL 1, 2 h cDDP: þþ (time- and concentration-dependent increase

of foci, UV41 cells were the most sensitive)Chinese hamster cells: UV41, AA8, irs3, V79–4

Smart et al. (2008) ETO V79 hamster ETO 0.001–10 μg/mL 4 h ETO: þþMTXT Fibroblasts MTXT 0.001–10 μg/mL MTXT: þþ

Srivastava et al. (2011) ETO MCF-7, HeLa, breast carcinoma tissue ETO 10 μM (Positive control) ETO: þþTanaka et al., 2008 GEF Non-small cell lung cancer: A549 and 1299cells GEF 1–2 μM 24 h GEF: – (irradiated, GEF pre-treated: þ)Terai et al. (2009) cDDP FSaII fibrosarcoma mice Cells cDDP 5 μM 1 h cDDP: þþ; BLPC: þþ;

BLPC BLPC 5 μM cDDPþBLPC: þþ (synergistic effect)Watters et al. (2009) ETO Mouse: embryonic fibroblasts (MEF) ETO 0.006–60 μg/mL 4 h ETO: þþ

BLM lymphoma cells (L5178Y) BLM 0.1–10 μg/mL BLM: þþYu et al. (2013) GEM Human ovarian cell line (UACC-1598) GEM 20 nM 2 w GEM: þþ

Abbreviations: 5-FU – 5-fluorouracil; AMA – amasacrine; API – aphidicolin; ARA-C – cytarabine; BLM – bleomycin; BLPC – ß-lapachone; cDDP – cisplatin; COL – colcemid; DOX – doxorubicin; ERL – erlotinib; ETO – etoposide; GEF –

gefitinib; GEM – gemcitabine; IMB – imatinib mesylate; MMC – mitomycin C; MTX – methotrexate; MTXT – mitoxantrone; IRI – irinotecan hydrochlorate; PT – paclitaxel; TPT – topotecan; VCR – vincristine; ZID, zidovudine.“þþ” – increase in signal; “þ” – moderate increase in signal, “-” – no increase in signal.

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within pharmacological range (5-FU 3 μg/mL) in HEC-1A, HEC-1B,and KLE cells, whereas the induction of apoptosis was notobserved. Ishikawa cell lines proved to be more sensitive to5-FU, which resulted with an increase in γ-H2AX formation after24 h of exposure while apoptosis in Ishikawa cells was observedafter 72 h. Also after an exposure period of 24 h, Matsuzaki et al.(2010) found an increase in γ-H2AX formation in CHL and V79cells at a concentration range within 0.39–0.78 μg/mL and 12.5–25 μg/mL, respectively. A decrease in the proliferation of all threecell lines was also observed. In the same study, a similar effect wasobserved in MTX-induced DSBs, where CHL and V79 cells wereagain more sensitive than CHO cells. The γ-H2AX formation wasdetected at concentration ranges 0.008–0.012 μg/mL, 0.003–0.008 μg/mL, and 0.195–0.206 μg/mL for each cell line, respec-tively. On the contrary, Kim et al. (2011) reported an absence ofDSB formation in U20S osteosarcoma cells even after a 1 h 5-FU(50 mg/mL) treatment.

Two other cytostatics also have similar modes of action have:gemcitabine (GEM) and cytarabine (ARA-C), both of which aredeoxycytidine analogues. They have the ability to incorporate intoa DNA strand instead of cytosine thus causing the collapse of thereplication fork as well as the inhibition of ribonucleotide reduc-tase and therefore de novo dNTP synthesis. Three different studieshave shown that low concentrations of GEM (10 nM, 20 nM, and0.1 μM) are able to induce γ-H2AX foci after a short exposureperiod (2 h), but also after a prolonged exposed period (twoweeks), in ovarian cancer cell lines UACC-1598, ML-1, and OCI-AML3 human adult myelogenus leukaemia cell lines (Ewald et al.,2007; Ewald et al., 2008; Yu et al., 2013). Additionally, ARA-C wasalso a potent γ-H2AX inducer in OCI-AML3 cells at a concentrationof 0.5 μM. Interestingly, neither PFGE, neutral, nor alkaline cometassay were able to detect DNA damage caused by such lowconcentrations of GEM and ARA-C (Ewald et al., 2008), suggestingthus the higher sensitivity of γ-H2AX foci assay.

Aphidicolin (API) and zidovudine (ZID) are also cytostatic drugscapable of nucleotide or DNA synthesis inhibition. Their impact onDNA was assessed and the increased γ-H2AX intensity wasobserved in BEAS-2B bronchial epithelic cell lines at concentra-tions of 1.56 μM (API) and 125 μM (ZID) after 24 h exposure whereboth drugs decreased cell viability significantly (Garcia-Cantonet al., 2013).

Bleomycin (BLM) is another potent DSBs inducer as suggestedby Dorr (1992) and Hecht (2000) that binds to CpG rich sites andproduces highly reactive free radicals with metal interactions.In a study by Watters et al. (2009), several methods with similarendpoints were compared. Bleomycin (BLM) managed to induceDSBs thus significantly increasing the γ-H2AX signal after 4 hof exposure in mouse embryonic fibroblasts at concentration of1 μg/mL. At the same time, the comet assay failed to detect adifference in DNA damage compared to accompanying control,whereas the micronucleus test proved to be more sensitive witha detection limit at 0.1 μg/mL. Similar effects were detected instudies done by Garcia-Canton et al. (2013) and Kim et al. (2011).The first group reported an increase in γ-H2AX signal in BEAS-2Bbronchial epithelial cell lines, which was observed at 0.3 μM and0.02 μM after exposure periods of 3 h and 24 h, respectively. Aftera longer exposure period BLM cell viability significantly decreased.The latter group also demonstrated a higher number of H2AX fociafter short exposure (1 h) to 2 μg/mL BLM (Kim et al., 2011).

3.2. Crosslinkers

Cisplatin (cDDP) has been used as a cancer treating agent formore than 30 years. Its main mode of action represents DNA cross-linking mainly due to interactions with nucleophilic N7-sites ofpurine bases thus leading to primarily DNA intrastrand cross-links,

but also to interstrand and DNA-protein cross-links (Siddik, 2003).Unlike cDDP, mitomycin C (MMC) is natural product isolated fromStreptomyces sp., but they both share a similar mechanism ofaction where guanine alkylation is a common occurrence (Tomasz,1995). The toxicity of both cDDP and MMC was tested in a study byMatsuzaki et al. (2010), where a decrease in CHL, CHO, and V79cell proliferation was observed. cDDP managed to induce γ-H2AXin CHL and V79 cells, while the signal increase was moderate inCHO cells. Similarly, MMC proved to be a DSB inducer in CHL andBEAS-2B cells (Garcia-Canton et al., 2013), while CHO and V79 cellshad a moderate increase in γ-H2AX signal (Matsuzaki et al. (2010).Another study on a similar model – CHO cell lines with severalsublines that were defective for some repair mechanisms provedthe cytotoxic nature of cDDP with IC50 value 44 μM after 1 htreatment. The peak of the γ-H2AX signal was observed 14 h posttreatment. Interestingly, wild type AA8 sublines with normalrepair had the least γ-H2AX formation, while UV96 (NER defec-tive) proved to be the most sensitive, since the decrease of the γ-H2AX was not observed 48 h post treatment (Clingen et al., 2008).In that study, a comparison with the comet assay was donesuggesting that the γ-H2AX foci assay is 6–10 times more sensi-tive. Another study on V79 cells showed that 1 and 2 μg/mL ofcDDP induced the γ-H2AX foci while apoptosis was not observed.The neutral comet test results were negative, while irradiatedsamples proved the existence of crosslinks using the alkalinecomet assay. UV41 cells that lacked repair mechanisms showedincrease in γ-H2AX induction and decrease in cell survival (Oliveand Banáth, 2009). cDDP also managed to induce γ-H2AX forma-tion in HL-60 cells (1 μM, 3 h treatment) without inducing apop-tosis (Huang et al. 2004); in A549 cells (50 μM, 3 h treatment)where the ATM protein was also phosphorylated (Galluzzi et al.2010); in FSaII cells (5 μM, 1 h treatment) where the percentage ofγ-H2AX positive cells increased from 43 to 66 percent 24 h posttreatment (Terai et al., 2009); and in HEC-1A, HEC-1B, KLE, andIshikawa cells (10 μg/mL, 3 h treatment) where Ishikawa cellsproved to be the most sensitive although apoptosis occurred after24 h and 96 h in HEC-1A and all other cells tested, respectively(Ikeda et al., 2010). Another interesting result regarding γ-H2AXfoci induction was done by Jayachandran et al. (2010), whereH1299 (cDDP resistant) cells were sensitized using the NPRL2tumour suppressor gene thus enabling phosphorylation of NBS1,ATM and ultimately H2AX histones. On the contrary, short termexposure (1 h) to concentrations of 50 μM cDDP failed to increasethe number of H2AX foci in U20S cells (Kim et al., 2011).

3.3. Topoisomerase inhibitors

Topoisomerases (TOPO) are enzymes that have major role inDNA replication where they are used as over- and un-winders ofDNA protecting it from over winding (Hande, 1998). Inhibitors oftopoisomerase can be divided into two major groups: TOPO I(irinotecan (IRI), topotecan (TPT), ß-lapachone (BLAP)) and TOPO IIinhibitors (etoposide (ETO), mitoxantrone (MTXT), doxorubicin(DOX)). Both types of inhibitors either form stabilized cleavagecomplexes disabling DNA re-ligation, or disrupt the catalyticturnovers of enzyme (Azarova et al., 2007). All TOPO I inhibitorsreviewed in this study proved to induce the γ-H2AX signal. IRI hadthe most intense impact on CHO cells, while CHL and V79 cellsresulted in a moderate increase of the γ-H2AX signal after 24 h ofexposure to concentrations higher than 20 μg/mL. At the sametime, cell proliferation decreased up to 100 percent when IRIconcentrations were elevated ten-fold (Matsuzaki et al., 2010).Terai et al. (2009) tested the ability of BLAP to cause DSBs and after1 h of exposure to 5 μM, the γ-H2AX signal was elevated, but thecombination of cDDP and BLAP provided the best results provingtheir synergistic effect on FSall cells. Cell proliferation decreased

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slightly after exposure to BLAP (2.5 μM) for 4 h, while at higherconcentrations the proliferation decreased exponentially. A similarimpact was observed when HL-60 cells were exposed to TPT (200–400 nM) for 3 h, where the γ-H2AX signal was highest in the S-phase of the cell cycle. This observation was to be expected,because TOPO inhibitors act mainly during DNA replication. Also,in a study by Huang et al. (2004), TPT managed to induceapoptosis. The same study dealt with DSB induction by concentra-tions of MTXT close to environmental ones (400–800 nM) andagain the γ-H2AX signal was elevated, but this time during G1,while the induction of apoptosis only occurred in the ensuing S-phase. A comparison of two TOPO II inhibitors was conducted inthe study by Smart et al. (2008). MTXT and ETO managed toproduce stabilized cleavage complexes at 0.01 and 1 μg/mL after4 h of exposure on V79 cells. On the contrary, the tail intensity ofthe neutral comet assay in this study proved DSBs at concentra-tions of 0.005 μg/mL and 0.5 μg/mL for MTXT and ETO, respec-tively. Additionally, the cytotoxicity of these two TOPO inhibitorsincreased up to 53 percent (MTXT) and 43 percent (ETO). Takentogether, the γ-H2AX signal was expected to be concentration-dependent for both of the tested cytostatics, whereas MTXTproved to be more genotoxic at lower concentrations. Similarresults were obtained in a study by Matsuzaki et al. 2010 with5 μg/mL ETO after 24 h exposure to on the same model (V79 cells),with the addition that the same pattern of genotoxicity andcytotoxicity was observed in CHO and CHL cell lines. Anotherthree studies determined the DSB-inducing potential of ETO.Srivastava et al. (2011) used 10 μg/mL ETO as a positive controlin MCF-7 and HeLa cells, Garcia-Canton et al. (2013) observedhigher γ-H2AX signal in BEAS-2B cell lines after 3 h and 24 hexposure to7.8 μM and 0.24 μM ETO, respectively, in addition thatcell viability did not drop under 75 percent, while Watters et al.(2009) detected a higher number of γ-H2AX foci when mouseembryonic fibroblast were exposed to 0.6 μg/mL ETO. Whencompared to micronucleus and comet test, the γ-H2AX foci assayreached statistical significance at same concentrations as themicronucleus test, while the comet test needed 10 times higherconcentration for the same result. Finally, the effects of DOX andETO were investigated on U20S cells where they induced DSBs atconcentrations of 0.5 μM and 2 μM, respectively, after 1 h treat-ment. Contradictory results regarding ETO were observed in astudy by Muslimović et al. (2009), where ETO (0–450 μM, 40 minexposure) failed to produce DSBs using neutral constant field gelelectrophoresis (CFGE), and at the same time alkaline CFGEdemonstrated that ETO produces only single-strand DNA breakson SV40 transformed human fibroblasts. Therefore, the inductionof γ-H2AX foci was pretty low. Interestingly, approximately threepercent of all damage were DSBs, which then correlated with theformation of γ-H2AX and apoptosis.

3.4. Microtubule inhibitors

As microtubules inhibitors, vincristine (VCR), colcemid (COL),and paclitaxel (PT) were all tested in a same study by Matsuzakiet al. (2010.) on CHL, V79, and CHO cell lines. All three drugs hadan impact on microtubules, whether by binding to tubule struc-tures or by suppressing tubules dynamics (Aardema et al., 1998).VCR, COL, and PT failed to induce γ-H2AX, as it could be expected,due to the aneugenic properties of these compounds. At the sametime, at 1 μg/mL (VCR and COL) and 100 μg/mL (PT) these com-pounds managed to decrease proliferation almost up to 100percent in all three of the cell lines tested. When using doses thatwere several-fold lower doses, the micronucleus test managed todetect an increase in the number of micronucleated cells suggest-ing that cytostasis of these compounds is not based on DNAdamage, but on the miss-segregation of chromosomes (Fenech,

2010; Matsuzaki et al., 2010). Cytostatic drugs with microtubuleinhibiting mode of action expectedly failed to induce γ-H2AX sincethey do not induce DSBs.

3.5. Tyrosine kinase inhibitors

Imatinib mesylate (IMB) is mainly used in chronic myeloidleukaemia treatment. Its mechanism of action is based on bindingto oncogenic protein tyrosine kinases thus preventing autopho-sphorylation and phosphorylation of the substrate (Maekawa etal., 2007). In a paper by Liu et al. (2007), it was observed thatGIST882 cell lines treated with 1 μM of IMB resulted in an increaseof γ-H2AX signals and apoptosis. On the contrary gefitinib (GEF),which targets epidermal growth factor receptor (EGFR), failed toinduce DSB formation in A549 and H1299 cells (Tanaka et al.,2008). In the same study, when the cells were pre-treated withGEF for 24 h and then irradiated, due to the suppression of DNArepair machinery, the number of DSBs was higher suggesting theiradditive or synergistic effect. Another cytostatic drug that alsotargets EGFR is erlotinib (ERL) and its effects on DNA wereinvestigated in a study by Li et al. (2008). Unlike GEF, 1 μM ERLmanaged to induce DSBs detected after 16 h of exposure with anincrease of γ-H2AX positive MCF7DRGFP cells. Due to altering theDNA damage response, irradiated cells pre-treated with ERLenhanced the percentage of cells positive to γ-H2AX, but radiationinduced cytotoxicity was also observed.

4. The application of γ-H2AX foci assay in environmentaltoxicology

Anticancer drugs are only one sub-group of pharmaceuticalsconstantly released into the environment (Delgado et al. 2012;WHO, 2011). Due to their potent mode of action, they represent athreat to non-target organisms from all trophic levels (Hernandoet al., 2006; Zounková et al., 2007, 2010). Modern pharmaceuticalsare designed to have a desired biological effect and be presentin target tissues in optimal concentrations. Cytotoxicity andcytostasis are the two predominant biological effects caused byanticancer agents, which can usually be characterized as highlygenotoxic, mutagenic, teratogenic or carcinogenic (Sandersonet al., 2004). Such relatively stable compounds are released intoaquatic environments a daily basis from healthcare facilities(preparing of medication, patients’ excretion), patient homes(patients’ excretion), wastewater treatment plants effluents (ifnot adequately purified), landfill leakages and pharmaceuticalplants (pharmaceutical residues) (Negreira et al., 2013b; Zhang etal., 2013).

Therefore, during the last decade, several groups of scientistexplored the occurrence of cytostatic drugs in the environmentand some were detectible in ranges from several ng/L up to 150 μg/L (Besse et al., 2012; Lenz et al., 2007; Lin and Lin, 2014; Kosjek etal., 2013; Mahnik et al., 2007; Yin et al., 2010; Zhang et al., 2013).Taking into account prediction models and actual concentrations,the aquatic environment has become a mixture of various com-pounds, where 28 anticancer drugs are present in concentrationsover 1 ng/L (Besse et al., 2012), and 13 of them exceed concentra-tions of 10 ng/L (Rowney et al., 2009). Although some of theanticancer drug concentrations reviewed in this article are severaltimes higher in laboratory-testing settings than in the environ-ment, the toxic effects of the various combinations of cytostaticsare still unknown, particularly if they include as much as 10different drugs.

On the other hand, the difference between laboratory settingsand the exposure of non-target organisms in the environment is amuch longer exposure period. Aquatic organisms are exposed to

M. Geric ́ et al. / Ecotoxicology and Environmental Safety 105 (2014) 13–21 17

low concentrations of cytostatics for days, months and, whensedentary organisms are concerned, even life-long. Althoughanticancer drugs are most commonly administered intravenouslyin clinical practice, which provides the best drug bioavailabilityand therefore the highest toxicity, non-target organisms intakexenobiotics from the environment through body surfaces andorally. Bioavailability is one of the major limitations for the oraladministration of anticancer drugs, still ETO (alone or in combina-tion with cDDP), TPT and prodrugs of 5-FU showed good bioavail-ability in cancer patients resulting with high response rates(Terwogt et al., 1999). Similarly, topical exposure can result withdifferent absorption rates depending on the body part. Vertebrateskin can serve as a reservoir capable of increasing local concentra-tions of certain cytostatics and increase their half-life (Katzung,1992). Taken together, potent anticancer drugs could penetrateinto tissues of aquatic organisms, particularly when they arechronically exposed.

This review has shown that antimetabolites and cross-linkingdrugs and TOPO and tyrosine kinase inhibitors induce DSBsdetectible by the γ-H2AX histone labelling, irrespectively of themechanism of DSB-formation. It was expected that TOPO inhibitorssuch as ETO, DOX or MTXT would produce DSBs due to their DNAcutting ability. Cytostatics with other modes of action also inducedDNA damage that γ-H2AX managed to detect, but the DSBs mighthave been formed during DNA damage responses such as NHEJ(Kuo and Yang, 2008) nonetheless showing the DNA damagingpotential of a certain drug. As Banáth et al. (2010) reported, thepeak of the H2AX signal is 30 min post irradiation and the similarcould be expected in cytostatics-treated samples. Therefore, thepossible explanation why Kim et al. (2011) failed to observe cDDP-induced DSBs could be a relatively short exposure period (1 h)compared to the same dose exposure (50 μM) after 3 h treatmentin study done by Galluzzi et al., 2010) where such an effect wasobserved. Additionally, exposure to even lower concentrations ofcDDP (5 μM) for 1 h followed by 24 h post-treatment periodallowed cells to start DNA damage response processes, thusforming H2AX foci, as seen in a paper by Terai et al. (2009).

Furthermore, the advantage of this method is its ability todetect the formation of DSBs on a broad range of different cells,such as human blood cells (Beels et al., 2009; Ismail et al., 2007;Redon et al., 2010a, Sedelinkova et al., 2008; Wu et al., 2013) orother human cells and tissues (Nagelkerke et al., 2011; Redon et al.,2011b; Simonsson et al., 2008), cultured cell lines (Ewald et al.,2007; Huang et al., 2004; Ikeda et al., 2010; Jayachandran et al.,2010; Kim et al., 2011; Matsuzaki et al., 2010; Olive and Banáth,2009; Yu et al., 2013), and animal (Fuji et al., 2013; Leopardi et al.,2010; Redon et al., 2010b) and/or plant cells (Charbonnel et al.,2011) which makes it ideal for further development and imple-mentation in environmental biomonitoring. The SQ motif at theC-terminal tail of the H2AX primary structure is highly conserved

and can thus be found in the plant Cicer arietinum, protozoanGiardia lamblia, yeast Schizosaccharomyces pombe, fish Danio rerio,monkeys Pan troglodytes, and humans (Dickey et al., 2009). Thisproperty enables the development of antibodies highly-specific tothe aquatic organisms currently used in aquatic biomonitoringsuch as various taxa of crustaceans (Cladocera, Copepoda), shells(Dressena sp., Mytilus sp., Perna sp.) and fish (Cyprinus sp., Danio sp.,Salmo sp.) (Baun et al., 2008; Bolognesi and Hayashi, 2011;Mitchelmore and Chipman, 1998; Sarkar et al., 2006).

5. Optimization of γ-H2AX foci assay

The application of the γ-H2AX foci as a biomarker of exposureand effect is currently present in many biomonitoring studies. Therange of fields covered in these studies is very broad fromexploring the effects of air pollutants (Mondal et al., 2010),polycyclic aromatic hydrocarbons (Audebert et al., 2010;Mattsson et al., 2009; Toyooka and Ibuki, 2006), cigarette smoke(Albino et al., 2004; Toyooka and Ibuki, 2009), and heavy metals(Peterson-Roth et al., 2005; Wan et al., 2012) to the effects of GSMmicrowaves on different biological models (Belyaev et al., 2009).

When interpreting the results of the γ-H2AX foci assay, itshould be kept in mind that DSBs can originate from a DNAdamaging agent but also, from caspase activity, which can thenlead to the misinterpretation of the results (Revet et al., 2011).Another important issue in evaluating genotoxicants is the appear-ance of false positive and negative results. As reviewed by Redonet al. (2011a), the γ-H2AX foci can be present without theformation of DSBs in senescent cell cultures and after tetheringsingle repair factors to chromatin (Soutoglou and Mistreli, 2008;Pospelova et al., 2009). On the other hand, the absence of theγ-H2AX foci even when DSBs occur can be expected in highosmolality conditions, which should be kept in mind whendesigning the study (Dmitrieva and Burg, 2008).

Further optimisations of the γ-H2AX foci assay can be achievedwithin the frame of scoring H2AX foci. In the studies reviewedhere, several different methods of measuring γ-H2AX signal havebeen used and these are summarized in Table 2. More precisemeasurements are done using flow cytometry due to the fact thatit analyses large numbers (thousands) of cells (Jayachandran et al.,2010; Huang et al., 2004; Muslimović et al., 2009; Smart et al.,2008), unlike microscopy analysis where only 50–280 cells arecounted per sample (Avondoglio et al., 2009; Beels et al., 2009;Redon et al., 2010a) thus giving more statistical power to theanalysis. Besides, a flow cytometric analysis is less time-consuming and provides results in less time. When using fluor-escent microscopy, there are several programs for capturingimages that help in sample analysis. Also, there is the possibilityof automating the method by using the scanning system to detect

Table 2Parameters measured in studies when using certain γ-H2AX visualization method.

Reference γ-H2AXvisualizationmethod

Measured parameter

Chu et al. (2011) Microscopy Organizing cells into classes depending on thenumber of γ-H2AX foci in the cell

Ewald et al. (2007), Li et al. (2008), Olive and Banáth (2009), Rothkamm and Lobrich (2003) Teraiet al. (2009) and Yu et al. (2013)

Microscopy Number (percentage) of γ-H2AX positive cells

Avondoglio et al. (2009), Beels et al. (2009), Clingen et al. (2008), Kim et al. (2011), Redon et al.(2010a), Rothkamm and Lobrich (2003) and Tanaka et al. (2008)

Microscopy Average number of γ-H2AX foci per cell

Avondoglio et al. (2009), Garcia-Canton et al. (2013) and Watters et al. (2009) Microscopy γ-H2AX fluorescence intensityHuang et al. (2004) and Jayachandran et al. (2010) Flow cytometry Number (percentage) of γ-H2AX positive cellsIkeda et al. (2010), Muslimović et al. (2009), Smart et al. (2008) and Watters et al. (2009) Flow cytometry γ-H2AX fluorescence intensityMatsuzaki et al. (2010) ELISA Absorbance of each sample

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the cell's fluorescence and γ-H2AX fluorescence in order to countthe foci induced by DSBs. There is no significant difference inmanual and automated scoring leading to conclusion that theautomatisation of the method would improve the rapidness andkeep its high efficacy of detection (Ivashkevich et al., 2011;Rothkamm et al., 2013b; Valente et al., 2011; Vandersickel et al.,2010). The ultimate step in automatisation of the method is theRABiT (Rapid Automated Biodosimetry Tool), a roboticallydesigned workstation responsible for the entire experiment; fromblood collection, through lymphocyte separation and slide pre-paration, up to scoring the γ-H2AX signal. In addition, this fully-automated biodosimetry tool is capable of processing approxi-mately 6000 samples per day, which makes it ideal for largerbiomonitoring studies (Garty et al., 2010, 2011; Turner et al., 2011).Another advantage of this machine is that it can prepare samplesand analyse micronuclei frequency, which are also great genotoxi-city endpoints (Fenech, 2010).

6. Conclusion and perspectives

Taken together, the γ-H2AX foci assay has already become awell-established tool for the detection of DSBs induced by radia-tion, whereas the available literature also suggests its applicabilityin measuring cytostatic drugs exposure proposing it as a newcandidate in environmental monitoring. On several occasions, thismethod had a lower detection limit than several other well-established genotoxicity tools like the comet and micronucleusassays. Hence, the γ-H2AX foci assay possesses great potential forapplication in environmental toxicology and an increase in thenumber of studies using this method as DSB marker can beexpected.

Since processes of cytostatic drug production, use, and disposalcan have a negative impact on the environment, particularlyaquatic ecosystems, the development of new tools for detectinggenome damage enables a more efficient monitoring of suchsystems. There are several steps that need to be done before theγ-H2AX foci assay would become established environmentalbiomonitoring tool.

First, it is necessary to evaluate the toxicity of mixtures ofanticancer drugs using a battery of tests and multibiomarkerapproach. Next, the introduction of aquatic organisms in γ-H2AXfoci assay analyses would be beneficial if appropriate antibodiesare to be developed.

Finally, it will be possible to apply the γ-H2AX foci assay as asensitive tool for the detection of DSBs in in vitro and in vivo testmodels induced by pharmaceuticals including cytostatic drugs inorder to provide more accurate risk assessments regarding phar-maceuticals released into the environment since they are presentin low concentrations, usually not detectable using standardgenotoxicity assays.

Conflicts of interest

None declared.

Acknowledgments

Supported by the Ministry of Science, Education and Sports ofthe Republic of Croatia (Grant no. 0022-0222148-2125) and EU FP7project CytoThreat – Fate and effects of cytostatic pharmaceuticalsin the environment and the identification of biomarkers for andimproved risk assessment on environmental exposure (Grant no.265264).

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