1 3

Arch Toxicol (2014) 88:585–596DOI 10.1007/s00204-013-1151-0

INORGANIC COMPOUNDS

Ogg1 genetic background determines the genotoxic potential of environmentally relevant arsenic exposures

Jordi Bach · Adriana Sampayo‑Reyes · Ricard Marcos · Alba Hernández

Received: 7 August 2013 / Accepted: 22 October 2013 / Published online: 5 November 2013 © Springer-Verlag Berlin Heidelberg 2013

during the complete course of the exposure suggests a rel-evant role in arsenic-associated carcinogenic risk in turn.

Keywords Arsenic · 8-OH-dG · Ogg1 · Oxidative DNA damage · Genotoxic · Comet assay

Introduction

Inorganic arsenic (i-As) is a widely spread environmental contaminant known to be cytotoxic, genotoxic and carcino-gen in humans (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans 2004). Epidemiological data clearly show an increase in the incidence of cancers of the skin, lung, urinary bladder, liver, prostate and kidney in human populations living in arsenic-rich areas, although the associated carcinogenic mechanism(s) remain incom-pletely characterized (IARC Working Group on the Evalu-ation of Carcinogenic Risks to Humans 2004; Straif et al. 2009).

Several studies have detected increases in DNA dam-age, sister-chromatid exchanges, micronuclei or chromo-somal aberrations in populations exposed to arsenic in drinking water (Marchiset-Ferlay et al. 2012; Basu et al. 2001; Sampayo-Reyes et al. 2010; Rossman 2003). It has been described that arsenic genotoxicity can be, at least in part, attributed to the reactive oxygen species (ROS) that are generated during i-As biotransformation (Flora 2011; Kitchin and Ahmad 2003; Liu et al. 2003; Ruiz-Ramos et al. 2009; Hei and Filipic 2004a; Liu et al. 2001). In this context, several studies have shown that i-As is able to induce the generation of 7,8-dihydro-2′-deoxyguanosine (8-OH-dG) both in vitro and in vivo (Sampayo-Reyes et al. 2010; Gomez et al. 2005; Hei and Filipic 2004b; Eblin et al. 2006; Yamauchi et al. 2004; Fujino et al. 2005;

Abstract Inorganic arsenic (i-As) is a well-established human carcinogen to which millions of people are exposed worldwide. It is generally accepted that the genotoxic effects of i-As after an acute exposure are partially linked to the i-As-induced production of reactive oxygen species, but it is necessary to better determine whether chronic sub-toxic i-As doses are able to induce biologically significant levels of oxidative DNA damage (ODD). To fill in this gap, we have tested the genotoxic and oxidative effects of environmentally relevant arsenic exposures using mouse embryonic fibroblast MEF mutant Ogg1 cells and their wild-type counterparts. Effects were examined by using the comet assay complemented with the use of FPG enzyme. Our findings indicate that MEF Ogg1−/− cells are more sensitive to arsenite-induced acute toxicity, genotoxicity and ODD. Long-term exposure to sub-toxic doses of arsen-ite generates a detectable increase in ODD and genotoxic DNA damage only in MEF Ogg1-deficient cells. Alto-gether, the data presented here point out the relevance of ODD and Ogg1 genetic background on the genotoxic risk of i-As at environmentally plausible doses. The persistent accumulation of DNA 8-OH-dG lesions in Ogg1−/− cells

J. Bach · R. Marcos · A. Hernández (*) Grup de Mutagènesi, Departament de Genètica i de Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Edifici Cn, Campus de Bellaterra, 08193 Cerdanyola del Vallès, Spaine-mail: alba.hernandez@uab.es

A. Sampayo-Reyes Centro de Investigación Biomédica del Noreste, Instituto Mexicano del Seguro Social (IMSS), Monterrey, Mexico

R. Marcos · A. Hernández CIBER Epidemiología y Salud Pública, ISCIII, Madrid, Spain

586 Arch Toxicol (2014) 88:585–596

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Matsui et al. 1999; Engstrom et al. 2010). Widely used as a biomarker of oxidative DNA damage (ODD) induction, 8-OH-dG has also been used as an indicator of the geno-toxic effects induced by many chemicals, including arsenic (Hirano 2008). DNA damage is considered to be essential for the initiation of carcinogenesis and other age-related changes in response to exogenous and endogenous carcino-gens. Indeed, lesions in genomic DNA caused by reactive oxygen species (ROS) are closely associated with aging (Ames and Shigenaga 1992; Ames 1989) and various dis-eases, including cancer (Marnett 2000; Tsuzuki et al. 2007; Clayson et al. 1994; Cerutti 1994). Notably, 8-OH-dG has stimulated the most interest as a potential pre-carcinogenic lesion, because this oxidized base can miscode with ade-nine producing G → T transversions (Shibutani et al. 1991; Wallace 1998). This and other point mutations generated via oxidative DNA damage are known to be involved in cancer development as they are common feature of human cancers.

8-OH-dG is efficiently removed from DNA via the short-patch base excision repair (BER) pathway. The func-tional mechanism of 8-OH-dG repair is a multistep process in which 8-oxo-dGuanine DNA glycosylase 1 (OGG1) first recognizes the lesion and then catalyzes its removal by cleaving the oxidized base, producing an AP site (Boi-teux and Radicella 2000; Shinmura and Yokota 2001). As OGG1 is fundamental in maintaining DNA stability, it is assumable that those human polymorphisms that alter the protein function, and thus the individual’s capacity to repair damaged DNA, may lead to genetic instability and carcino-genesis. Indeed, a common non-synonymous genetic poly-morphism at codon 326, Ser326Cys, in the OGG1 gene is a strong candidate as a genetic factor for cancer risk (Yamane et al. 2004), and case–control studies have been able to found association with an increased incidence of lung, prostate and esophageal cancers (Hirano 2008; Karahalil et al. 2012).

Studies on the relationship between environmental factors and 8-OH-dG generation and its repair capacity pointed out that OGG1 down-regulation or inhibition of the enzyme activity appears as a common mechanism of action (Hirano 2008). For arsenic compounds, their abil-ity of inhibiting the 8-OH-dG repair activity has been already reported, at least within the acute range of expo-sure (Hirano 2008; Ebert et al. 2011; Hartwig et al. 2003; Osmond et al. 2010).

Despite of the attributed role of ODD on arsenic-related toxic, genotoxic and carcinogenic effects and the docu-mented indispensable role of the wild-type OGG1 in the maintenance of DNA stability, few studies exist exploring the effect of arsenic exposure within different Ogg1 genetic backgrounds. Therefore, to cast light on the relationship between the Ogg1 gene and the arsenic-induced genotoxic

DNA damage after an environmentally relevant type of exposure, the genotoxic and oxidative effects of arsenic were examined by the comet assay in MEF wild-type and mutant Ogg1 cells after long-term exposures to sub-toxic arsenite concentrations. These Ogg1 knockout cells, sharing the same genetic background than their parental wild-type cells, lack the nicking activity for substrate DNA containing 8-OH-dG, exhibiting an approximately fourfold increased sensitivity toward ROS inducing agents such as potas-sium bromate (KBrO3) or hydrogen peroxide (H2O2) (Cas-tillo et al. 2011). Organisms with an intact function of the BER pathway exhibit an efficient ability to eliminate low induced 8-OH-dG levels, and this has restricted researchers to the measure of only the remaining non-repaired ODD. Since Ogg1−/− cells are unable to eliminate the 8-OG-dG from DNA, we have used our MEF Ogg1 knockout system to clearly determine the level of ODD that arsenic is able to generate at the sub-toxic range.

Taken together, the data presented here point out the relevance of ODD and Ogg1 genetic background on the genotoxic potential of inorganic arsenic at environmentally plausible doses. The findings encourage for the develop-ment of association studies in human populations chroni-cally exposed to arsenic to unravel the role of common OGG1 polymorphisms, especially Ser326Cys, on arsenic-induced cancer and disease.

Materials and methods

Culture conditions and in vitro arsenic exposure

Wild-type mouse embryonic fibroblasts (MEF) and their derived Ogg1−/− MEF were kindly shared by Drs. Deb-orah Barnes. The MEF wt and MEF Ogg1−/− cell lines were maintained in DMEM:F12 medium (Life Technolo-gies, NY, USA) supplemented with 10 % fetal bovine serum (FBS) and 2.5 μg/mL plasmocin (InvivoGen, CA, USA) in a humidified atmosphere of 5 % CO2 and 95 % air at 37 °C.

For the short-term in vitro studies, MEF cells were exposed to sub-toxic doses of arsenic in the form of sodium arsenite (SA, NaAsO2; up to 20 μM; pH = 7.4; Sigma-Aldrich, MO, USA) for 24 and 48 h. MEF cells (5 × 105 per 100-mm dishes) were plated in triplicate for each treat-ment in complete medium, incubated overnight and placed in fresh medium (control) or in fresh medium containing arsenic.

For the long-term in vitro analyses, MEF cells were continuously exposed to sub-toxic doses of SA (0.5, 1 and 2 μM) for up to 17 weeks. The arsenic-containing medium was changed every 48 h, and sub-confluent cells were pas-saged weekly. Three separate flasks for each treatment

587Arch Toxicol (2014) 88:585–596

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were maintained, and arsenic-exposed cells were in all cases compared with unexposed passage-matched controls.

Cell viability

MEF cells were plated in triplicates (0.3 × 106 cells per well, 6-well plates), incubated overnight in complete medium and placed in fresh medium with increasing con-centrations of SA for 24 or 48 h. Cell viability was then assessed by the Beckman counter method with a Z™ Series coultercounter (Beckman coulter, CA, USA). The IC50 was derived from averaging three independent survival curves.

Total RNA extraction and RT-PCR

Total RNA was extracted using TRIzol Reagent (Invit-rogen, USA), and RNase-free DNase I (DNA-free™ kit; Ambion, UK) was used to remove DNA contamination. The first-strand cDNA synthesis was performed using 1 μg of total RNA and the Omniscript Reverse Transcrip-tion Kit (Qiagen, CA, USA) following the manufacturer instructions. The resulting cDNA was subjected to PCR using DFS-Taq DNA Polymerase (Bioron, Ludwigshafen, Germany) to qualitatively evaluate the expression of Ogg1 and As3mt genes. Each 25 μL of reaction volume con-tained 0.5 μL of cDNA, 3 U of DSF-Taq DNA polymerase, 2.5 μL of 10× DSF-Taq Buffer, 1.5 mM MgCl2 (Bioron, Ludwigshafen, Germany) and 200 nM of each primer pairs. The cycling parameters began with 95 °C for 5 min, then 30 cycles of 95 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s. The primer pairs used were: Ogg1 fwd 5′-CAAGGTGT-GAGACTGCTGAG-3′, Ogg1 rev 5′-CCAACTTCCTCAG-GTGAGTC-3′, As3mt fwd 5′-GCATCGAGAAGTTGGCA-GAG-3′, As3mt rev 5′-ATCCTTCCAGTACAGAGCGC-3′.

Arsenic speciation analysis

MEF cells were plated into 25-mm culture dishes and cultured at an approximate cell density of 70 % conflu-ence. Culture medium was then replaced with fresh FBS-free medium containing 0, 1, 2 and 5 μM SA or 0.5 μM MMAIII. After 24 or 48 h of incubation, medium was col-lected, and each cell monolayer was washed three times with PBS and lysed on ice for 10 min with a cell-lysing buffer containing 10 mM Tris (pH 7.4), 10 mM EDTA (pH 8.0) and 0.5 % Triton X-100.

Arsenic species were measure by HPLC-ICP-MS. To measure pentavalent species, the HPLC system consisted of an Agilent 1100 HPLC (Agilent Technologies, Inc.) with Hamilton PRP-X100 column (10 μm, 250 × 4.1 mm) and guard cartridge. The mobile phase was 50 mM ammo-nium carbonate (pH 9) with 4 % methanol at a flow rate of 1.0 mL/min at 30 °C. To measure trivalent species, a reverse

phase C18 column (Gemini 5u C18 110A, 150 × 4.60 mm, Phenomenex, Torrance, CA) and guard cartridge was used. The mobile phase (pH 5.85) contained 4.7 mM tetrabutyl-ammonium hydroxide, 2 mM malonic acid and 4 % (v/v) methanol at a flow rate of 1.2 mL/min at 50 °C. The oper-ating parameters were as follows: Rf power 1,500 watts, plasma gas flow 15 L/min, carrier flow ~0.9 L/min, 0.15 L/min makeup and arsenic measured at 75 m/z. An Agilent 7500ce ICP-MS with a Conikal nebulizer (Glass Expan-sion) was used as the detector.

Comet assay

The ODD and the genotoxic DNA damage for MEF wt and MEF Ogg1−/− cells acutely or chronically exposed to arsenite were evaluated by the alkaline comet assay with and without the use of formamidopyrimidine DNA glyco-sylase (FPG) enzyme. Sheet films of the type Gelbond® to process a large number of samples (McNamee et al. 2000) were used.

The comet assay detects single- and double-stranded DNA breaks in naked supercoiled DNA. Strand breaks that are present cause the supercoiled DNA to relax, allowing loops of DNA to migrate toward the anode upon electro-phoresis, forming a ‘comet tail’. The use of lesion-specific enzymes in the comet assay, specifically formamidopy-rimidine DNA glycosylase (FPG), allows for the detection of 8-OH-dG, in addition to certain imidazole ring-opened purines and also causes breaks at apurinic/apyrimidinic sites (AP sites) (Collins 2004).

To perform this assay, complete media was aspirated from the cell monolayer, and ice-cold 0.2 % ethylenedi-aminetetraacetic acid (EDTA) in PBS was placed onto the cells for 5 min. This wash solution was then aspirated, and the cells were detached from culture dishes using trypsin. Cells were then centrifuged at 130 G for 8 min, and the pel-let was washed twice in RPMI medium and resuspended in PBS to obtain about 17,500 cells/25 μL. Cells were mixed with 0.75 % LMP at 37 °C (1:10) and dropped onto Gel-bond® film 10.5 × 7.5 cm. Forty-eight drops (7 μL each) were placed in each Gelbond® sheet, and 16 samples were run simultaneously, so each sample was represented by 3 drops. The use of hydrophilic films facilitates the rapid pro-cessing of numerous samples, increasing the efficiency of the alkaline comet technique, without sacrificing the reli-ability or sensitivity of the assay. Three identical Gelbond® films with the same type of samples were processed simul-taneously in each experiment. Gels were then lysed over-night by immersion in ice-cold lysis buffer at 4 °C (2.5 M NaCl, 0.1 M Na2EDTA, 0.1 M Tris base, 1 % Triton X-100, 1 % lauroyl sarcosinate, 10 % DMSO), at pH 10. Two Gel-bond® films replicates were gently washed twice in enzyme buffer at pH 8.0 (10 mM HEPES; 0.1 M KCl; 0.5 mM

588 Arch Toxicol (2014) 88:585–596

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EDTA; 0.2 mg/mL BSA) 5-50 min at 4 °C and then incu-bated for 30 min at 37 °C in only enzyme buffer (negative control) or in enzyme buffer containing FPG. The other Gelbond® film was kept in the lysis buffer. The three Gel-bond® films were then washed with electrophoresis buffer (5 min) and placed into a horizontal gel electrophoresis tank, and DNA was allowed to unwind for 35 min in 0.3 M NaOH and 1 mM Na2EDTA, pH = 13.2, before the electro-phoresis, which was carried out for 20 min at 0.8 V/cm and 300 mA at 4 °C. After electrophoresis, the Gelbond® films were rinsed with cold PBS for 15 min and fixed in abso-lute ethanol for 2 h, before air-drying overnight at room temperature.

After drying, Gelbond® films were stained for 20 min with SYBR Gold, 1/10,000 dilution of stock solution in TE buffer (10 mM Tris; 1 mM EDTA pH 7.5), supplied by Molecular Probes (495 nm excitation, 537 nm emis-sion). SYBR Gold stain appears to possess ideal proper-ties for automated scoring of comets for tail and comet length parameters due to its intense staining and greater resistance to fading. Each stained gel was appropriately labeled and was cut in half and mounted on an acrylic slide of 52.5 × 75 × 3 mm. A coverslip of 74 × 49 mm (IZASA, Barcelona, Spain) was applied and gently pressed onto the drops forming a tight seal. Finally, the gels were visualized for comets using an epifluorescent microscope at 20× magnification. The image for each individual cell was acquired employing the Komet 5.5 Image analysis sys-tem (Kinetic Imaging Ltd, Liverpool, UK). Cells were ana-lyzed according to their percentage of DNA in tail, as an adequate measure of DNA damage. One hundred randomly selected comet images were analyzed per sample. Three different samples (triplicates) were processed for each i-As treatment.

Statistical analysis

Once demonstrated the normal distribution of the parame-ters tested, an unpaired Student’s t test was used to compare arsenic-treated cells with untreated time-matched controls at individual time points. For the comet assay, the median ‘DNA in tail’ value was calculated for each triplicate from one hundred cells, and the mean of the three obtained val-ues was then generated. In all cases, a two-sided P < 0.05 was considered statistically significant.

Results

Wild-type mouse embryonic fibroblasts (MEF wt) and its isogenic Ogg1 knockout (MEF Ogg1−/−) cell lines (see Fig. 1) were used to test the hypothesis that Ogg1 genetic background and oxidative DNA damage play an important

role in arsenic genotoxic potential at environmentally rel-evant doses.

We first examined the MEF wt and MEF Ogg1−/− arsenic biotransformation ability due to its outstanding role in arsenic toxic, genotoxic and carcinogenic poten-tial (Hernandez and Marcos 2008). The expression of the key enzyme for arsenic methylation As3mt was verified by conventional RT-PCR (Fig. 1). Also, an analysis of arsenic metabolism by HPLC-ICP-MS revealed that our MEF wt and MEF Ogg1−/− cells were able to biotransform arse-nic, as indicated by the presence of all organic species resulting from i-As metabolism in the cell lysates and in the medium after exposure to AsIII and MMAIII for 24 and 48 h (Tables 1 and 2). The rate of biotransformation was found to be similar in both MEF wt and Ogg1−/− cell lines.

We next evaluated the role of Ogg1 on the arsenic acute toxic and genotoxic potential. The viability of MEF cells after acute arsenite exposure appeared to be signifi-cantly different between both studied genetic backgrounds (Fig. 2). After 24 h of arsenite exposure (Fig. 2a), the IC50 value found in MEF wt was 21.33 ± 1.00 μM, whereas the IC50 value found in MEF Ogg1−/− was 12.60 ± 0.70 μM (P < 0.001). The same pattern was found after 48 h of arsenite exposure (Fig. 2b), where the IC50 values were 17.85 ± 1.25 and 8.17 ± 0.53 μM, respectively (P < 0.001). Therefore, Ogg1 background showed as rel-evant for arsenic-induced toxic effects under an acute sce-nario of exposure.

The oxidative DNA damage (ODD) measured by the comet assay after 1.5, 3 and 24 h of arsenite exposure in MEF wt and MEF Ogg1−/− cells is shown in Fig. 3. Interestingly, we were able to detect significant levels of ODD in MEF Ogg1−/− cells. In these Ogg1-deficient cells, the ODD was found after 1.5, 3 and 24 h of arsen-ite exposure at all examined doses and in a dose-depend-ent manner (Fig. 3b). After 24 h of exposure, i.e., the level of ODD after 5 μM arsenite was increased 3.94-fold and rose up to 4.85-fold and 6.01-fold after 10 and 20 μM of

Fig. 1 mRNA expression of Ogg1 and As3mt genes in MEF cell lines. Conventional RT-PCR with specific primers confirms the lack of Ogg1 in MEF Ogg1−/− cells. The same experimental technique demonstrates that both MEF wt and MEF Ogg1−/− cells express the key enzyme As3mt necessary for arsenic biotransformation

589Arch Toxicol (2014) 88:585–596

1 3

Tabl

e 1

Ars

enic

spe

ciat

ion

anal

ysis

of

ME

F w

t cel

ls a

fter

MM

Am

and

As11

1 exp

osur

e

Mea

n va

lue

± S

D o

f th

ree

inde

pend

ent e

xper

imen

tsAsm

/ng

AsV

/ng

MM

Am

/ng

MM

AV

/ng

DM

AV

/ng

Sum

of

sp/n

gTo

tal s

peci

ated

/ng

% o

f re

cove

ry

MM

AII

I

24

h18

7 ng

0.5

nMC

ell l

ysat

e1.

42 ±

0.0

41.

89 ±

0.0

45.

16 ±

0.3

22.

80 ±

0.0

90.

80 ±

0.0

612

.31

± 0

.37

150.

34 ±

3.0

880

Med

ium

2.18

± 0

.03

6.22

± 0

.08

81.3

3 ±

3.0

244

.50

± 2

.17

4.00

± 0

.10

138.

33 ±

3.0

8

48

h18

7 ng

0.5

nMC

ell l

ysat

e1.

52 ±

0.1

22.

39 ±

0.0

74.

54 ±

0.4

92.

96 ±

0.1

50.

80 ±

0.0

312

.21

± 0

.37

153.

33 ±

5.5

982

Med

ium

2.63

± 0

.16

5.00

± 0

.26

84.3

6 ±

4.3

845

.70

± 2

.08

3.45

± 0

.05

141.

12 ±

5.2

3

AsII

I

24

h75

0 ng

2 nM

Cel

l lys

ate

10.5

3 ±

2.8

43.

57 ±

0.2

80.

23 ±

0.0

20.

75 ±

0.0

90.

94 ±

0.0

416

.48

± 1

.84

544.

44 ±

12.

6473

Med

ium

513.

35 ±

10.

207.

44 ±

0.3

04.

33 ±

0.6

72.

56 ±

0.5

00.

25 ±

0.0

952

7.93

± 1

1.19

1,87

5 ng

5 nM

Cel

l lys

ate

15.0

1 ±

4.1

39.

56 ±

0.4

10.

64 ±

0.0

31.

57 ±

0.0

91.

63 ±

0.1

328

.42

± 4

.36

1,43

4.01

± 4

3.03

77

Med

ium

1,36

8.66

± 3

9.14

20.5

0 ±

0.4

611

.50

± 0

.95

4.38

± 0

.43

0.56

± 0

.09

1,40

5.60

± 3

8.79

3,00

0 ng

8 nM

Cel

l lys

ate

29.1

2 ±

1.6

215

.87

± 0

.61

0.80

± 0

.08

1.96

± 0

.22

2.05

± 0

.16

49.8

0 ±

2.4

42,

375.

42 ±

26.

4579

Med

ium

2,27

0.00

± 2

6.45

28.4

3 ±

1.6

318

.60

± 0

.80

7.87

± 0

.65

0.71

± 0

.26

2,32

5.62

± 2

7.94

48

h75

0 ng

2 nM

Cel

l lys

ate

8.82

± 0

.45

2.98

± 0

.47

0.16

± 0

.01

0.55

± 0

.04

0.82

± 0

.17

13.3

2 ±

0.8

554

0.25

± 5

.25

72

Med

ium

511.

75 ±

3.3

58.

53 ±

0.3

64.

87 ±

0.2

61.

62 ±

0.8

40.

15 ±

0.0

351

6.85

± 5

.73

1,87

5 ng

5 nM

Cel

l lys

ate

13.0

9 ±

0.7

27.

66 ±

0.2

50.

60 ±

0.1

80.

80 ±

0.1

71.

43 ±

0.1

123

.58

± 0

.99

1,58

2.59

± 1

0.47

84

Med

ium

1,49

5.63

± 9

.34

41.5

5 ±

2.3

317

.75

± 0

.25

3.04

± 0

.07

10.4

± 0

.29

1,55

9.01

± 1

0.26

3,00

0 ng

8 nM

Cel

l lys

ate

27.4

2 ±

0.4

613

.29

± 0

.80

0.85

± 0

.18

1.02

± 0

.08

1.93

± 0

.21

44.5

2 ±

1.0

72,

571.

31 ±

25.

9786

Med

ium

2,44

3.33

± 2

3.75

55.7

6 ±

2.8

722

.24

± 4

.54

4.56

± 0

.09

0.89

± 0

.08

2,52

6.79

± 2

5.16

590 Arch Toxicol (2014) 88:585–596

1 3

Tabl

e 2

Ars

enic

spe

ciat

ion

anal

ysis

of

ME

F O

ggl−

/− c

ells

aft

er M

MA

and

As

expo

sure

Mea

n va

lue

± S

D o

f th

ree

inde

pend

ent e

xper

imen

tsAsm

/ng

AsV

/ng

MM

Am

/ng

MM

AV

/ng

DM

AV

/ng

Sum

of

sp/n

gTo

tal s

peci

ated

/ng

% o

f re

cove

ry

MM

AII

I

24

h18

7 ng

0.5

nMC

ell l

ysat

e2.

15 ±

0.0

21.

45 ±

0.0

53.

25 ±

0.2

52.

77 ±

0.1

10.

84 ±

014

10.4

6 ±

0.4

515

4.97

± 8

.09

83

Med

ium

3.10

± 0

.10

6.87

± 0

.13

79.1

1 ±

6.4

653

.07

± 2

.70

2.35

± 0

.13

144.

51 ±

7.7

9

48

h18

7 ng

0.5

nMC

ell l

ysat

e0.

78 ±

0.1

32.

35 ±

0.0

52.

95 ±

10

2.40

± 0

.46

0.95

± 0

.05

9.49

± 0

.51

162.

32 ±

11.

0687

Med

ium

2.23

± 0

.25

4.46

± 0

.12

93.8

7 ±

5.2

249

.37

± 5

.50

3.12

± 0

.17

153.

05 ±

10.

71

AsII

I

24

h75

0 ng

2 nM

Cel

l lys

ate

8.71

± 0

.44

2.92

± 0

.51

0.15

± 0

.01

0.54

± 0

.04

0.75

± 0

.08

13.0

8 ±

0.9

453

4.51

± 9

.77

71

Med

ium

497

± 9

.29

8.40

± 0

.29

4.47

± 0

.72

2.01

± 0

.18

0.15

± 0

.03

512.

36 ±

10.

37

1,87

5 ng

5 nM

Cel

l lys

ate

12.9

± 0

.70

7.49

± 0

.45

0.60

± 0

.02

0.77

± 0

.12

1.29

± 0

.11

23.0

5 ±

0.9

4 4

1,57

1.65

± 1

3.37

84

Med

ium

1,48

6.67

± 9

.60

40.6

0 ±

2.5

014

.53

± 0

.25

2.81

± 0

.18

0.99

± 0

.13

1,54

8.60

± 1

2.4

3,00

0 ng

8 nM

Cel

l lys

ate

27.0

0 ±

0.3

912

.63

± 0

.80

0.80

± 9

.16

0.99

± 0

.10

1.84

± 0

.17

43.2

7 ±

0.9

32,

556.

01 ±

22.

4185

Med

ium

2,42

9.33

± 2

0.03

56.2

0 ±

2.0

321

.73

± 3

.64

4.64

± 0

.15

0.83

± 0

.06

2,51

2.73

± 2

1.48

48

h75

0 ng

2 nM

Cel

l lys

ate

12.8

6 ±

1.5

54.

07 ±

0.1

20.

29 ±

0.0

10.

87 ±

0.0

41.

30 ±

0.2

619

.39

± 1

.61

536.

3 ±

53.

3672

Med

ium

499.

10 ±

56.

118.

58 ±

0.3

84.

95 ±

0.0

93.

95 ±

0.8

20.

33 ±

0.0

851

6.90

± 5

4.89

1,87

5 ng

5 nM

Cel

l lys

ate

17.8

7 ±

2.0

710

.10

± 0

.34

0.97

± 0

.03

1.85

± 0

.15

1.81

± 0

.16

32.3

2 ±

1.8

41,

481.

77 ±

83.

7479

Med

ium

1,40

6.00

± 8

0.54

23.5

3 ±

0.9

514

.45

± 0

.83

4.69

± 0

.56

0.77

± 0

.10

1,44

9.45

± 8

2.4

3,00

0 ng

8 nM

Cel

l lys

ate

31.1

2 ±

3.0

217

.48

± 0

.89

0.91

± 0

.07

2.47

± 0

.49

2.17

± 0

.26

54.1

4 ±

4.5

22,

530.

07 ±

32.

9784

Med

ium

2,41

4.33

± 3

3.26

31.3

6 ±

1.8

520

.20

± 1

.42

9.13

± 0

.70

0.90

± 0

.10

2,47

5.93

± 3

4.60

591Arch Toxicol (2014) 88:585–596

1 3

arsenite, respectively. MEF wt cells, however, presented significant detectable levels of ODD only after 20 μM of arsenite exposure (Fig. 3a), indicating that MEF cells hav-ing the wild-type Ogg1 glycosidase enzyme are able to rapidly excise the arsenic-induced 8-OH-dG lesions from their DNA, making the ODD undetectable, at least by the experimental technique used here. The data presented in Fig. 3 also show that the highest ODD induced by arsenite in MEF Ogg1−/− cells occurs 1.5 h after the exposure and that it diminishes progressively after 3 and 24 h of expo-sure regardless of the dose, indicating that the generation of ROS after acute i-As exposure begins soon after the exposure.

The genotoxic DNA damage measured by the comet assay after 1.5, 3 and 24 h of arsenite exposure in MEF wt and MEF Ogg1−/− cells is shown in Fig. 4. The geno-toxic DNA damage detected in MEF wt cells (Fig. 4a) cor-responds to DNA strand breaks and AP sites induced by arsenic, but also to the transient alkali-labile sites generated during the repair of 8-OH-dG by BER mechanisms. In fact, the genotoxic damage found in MEF wt after 20 μM of arsenite was higher than in MEF Ogg1−/− cells, probably due to their higher 8-OH-dG repair rate. Similar to what we found for the ODD damage, these lesions are gener-ated soon after the exposure and diminish the longer is the time of exposure. As some of the detected lesions such as SSB are known to be rapidly repaired by the cells, we can

consider the time-point of 24 h the one that better reflects the genotoxic DNA damage able to persist and therefore able to attain harmful consequences for the cell. After 24 h of arsenite exposure, MEF wt cells show significant increases in genotoxic DNA damage only at the doses of 20 and 30 μM. MEF Ogg1−/−, however, show significant increases in genotoxic DNA damage at all the experimen-tal doses analyzed. Taken together, MEF wt cells present detectable levels of ODD and increased levels of persisting genotoxic DNA damage only at 20 μM of arsenite expo-sure (Figs. 3a, 4a). On the other hand, MEF Ogg1−/− cells show detectable levels of ODD and persisting genotoxic DNA damage at 5, 10 and 20 μM of arsenite (Figs. 3b, 4b). Again, Ogg1 background showed to be relevant for arsenic-induced effects under an acute scenario of exposure, in this case in relation with genotoxic effects.

As human populations are chronically exposed to low doses of inorganic arsenic, the main goal of this work

Fig. 2 Cell viability curves for MEF cells in response to various con-centrations of arsenite for 24 h (a) or 48 h (b). Data are presented as mean values of independent experiments (n = 3); error bars cor-respond to 95 % confidence intervals. P values are two-sided (Stu-dent’s t test). **P < 0.005 compared with time-matched controls. ***P < 0.001 compared with time-matched controls

Fig. 3 Oxidative DNA damage (ODD) for MEF wt (a) and MEF Ogg1−/− deficient cells (b) in response to various concentrations of arsenite for 1.5, 3 and 24 h. Alkaline comet assay with FPG enzyme reveals the ODD induced by arsenite under an acute scenario of expo-sure. MEF wt cells show detectable ODD only at the highest dose, whereas MEF Ogg1−/− cells show increased levels of ODD at all doses analyzed. Data are presented as mean values of independent experiments (n = 3); error bars correspond to 95 % confidence inter-vals. ***P < 0.001 compared with time-matched controls

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was to measure how a deficient Ogg1−/− genetic back-ground affects on the ODD and the genotoxic DNA dam-age induced by arsenite after an environmentally relevant type of exposure. Thus, MEF cells were exposed to sub-toxic arsenite doses (according to Fig. 2) for 17 weeks, and the alkaline comet assay, with and without the addition of FPG enzyme, was performed at different time points along the exposure period. Figure 5a shows the ODD found in MEF wt cells chronically exposed to 0.5, 1 and 2 μM of arsenite. Significant detectable levels of ODD were found

only after 17 weeks of exposure to 1 and 2 μM of arsenite. Figure 5b shows the ODD found in MEF Ogg1−/− chroni-cally exposed to 0.5, 1 and 2 μM of arsenite. In contrast to what was found for the wild-type Ogg1 genetic back-ground, the lack of a functional Ogg1 enzyme reveals an scenario of arsenic-induced chronic ODD (Fig. 5b) where MEF Ogg1−/− cells present detectable levels of ODD during the complete course of the exposure (4, 10 and 17 weeks of arsenite exposure). This data evidence that inorganic arsenic is able to induce ODD also at low envi-ronmentally relevant doses to an extent that wt cells are able to eliminate up to the week 17 of exposure. Ogg1-deficient cells, however, are unable to eliminate the arsenic-induced ODD. The genotoxic DNA damage found in MEF cells after chronic arsenite exposure is shown in Fig. 6. MEF wt cells did not show increasing sensitivity toward

Fig. 4 Genotoxice DNA damage for MEF wt (a) and MEF Ogg1−/− deficient cells (b) in response to various concentrations of arsenite for 1.5, 3 and 24 h. Alkaline comet assay reveals the genotoxic DNA damage induced by arsenite under an acute scenario of exposure. In all cases, the genotoxic DNA damage induced by arsenite occurs pre-dominantly at the first hour of exposure. After 24 h of arsenite expo-sure, MEF wt cells do not show increased levels of genotoxic DNA damage up to 20 μM, whereas MEF Ogg1−/− cells show increased levels of genotoxic DNA damage at 5 μM dose. Data are presented as mean values of independent experiments (n = 3); error bars cor-respond to 95 % confidence intervals. *P < 0.05 compared with time-matched controls. **P < 0.005 compared with time-matched controls. ***P < 0.001 compared with time-matched controls

Fig. 5 Long-term oxidative DNA damage (ODD) for MEF wt (a) and MEF Ogg1−/− deficient cells (b) in response to an environmen-tally relevant arsenite exposure. MEF wt cells present detectable lev-els of ODD only after 17 weeks of sub-toxic arsenite exposure. MEF Ogg1−/− cells present detectable levels of ODD during the complete course of the exposure. Data are presented as mean values of tripli-cates; error bars correspond to 95 % confidence intervals. *P < 0.05 compared with time-matched controls. **P < 0.005 compared with time-matched controls. ***P < 0.001 compared with time-matched controls

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the genotoxic effects of arsenite through time (Fig. 6a), whereas MEF Ogg1−/− cells started showing increased levels of genotoxic DNA damage after 10 weeks of arsenite exposure. Taken together, our data indicate that low arsenic exposure is able to induce chronic oxidative DNA damage that cells with deficiencies in the Ogg1 enzyme are unable to eliminate. As a consequence of the persisting ODD and/or the attempt of the deficient cells of eliminating the oxi-dized bases using less efficient repair pathways, the gen-otoxic DNA damage present in Ogg1−/− cells begins to increase. Our data clearly indicate that Ogg1 is crucial for arsenic-induced genotoxic effects under a chronic scenario of exposure.

Discussion

For decades, arsenic was considered to be a non-mutagenic carcinogen because it is only weakly active in bacterial and

mammalian cell mutation assays (Basu et al. 2001). How-ever, when assayed using model systems in which both intragenic and multilocus mutations can readily be detected arsenic is, indeed, found to be a strong dose-dependent mutagen which induces mostly multilocus deletions (Hei and Filipic 2004a). Furthermore, results obtained using micronucleus, sister-chromatid exchanges or chromosome aberrations assays indicate that some arsenic compounds are potent clastogens in both human and rodent cells in culture (Marchiset-Ferlay et al. 2012). Furthermore, many studies have detected increases in DNA damage and/or the above-mentioned cytogenetic end points in human popu-lations exposed to arsenic via drinking water (Marchiset-Ferlay et al. 2012; Basu et al. 2001; Sampayo-Reyes et al. 2010,Rossman 2003). The role of reactive oxygen species in mediating the genotoxic response to arsenic exposure has been long discussed, and many authors are lately accepting a relevant role of arsenic-induced oxidative DNA damage in arsenic-mediated genotoxicity (Hei and Filipic 2004a). In line with this hypothesis, many authors also believe that oxi-dative mechanisms are of prime importance in i-As carci-nogenicity (Kitchin 2001), although this is certainly consid-ered a multifactorial process. Despite of the evidences, other authors have been unable to detect significant increases in 8-OH-dG levels after exposures to sub-toxic, but environ-mentally relevant doses of inorganic arsenic, creating major questions about the relevance of small chemically induced increases in DNA damage. This lack of DNA damage detec-tion can probably be attributed to the methodological dif-ficulties of the standard chromatographic technologies used for measuring low levels of ODD, due to the high back-ground readings (Collins et al. 2004). Thus, the choice of the methodology for studying the i-As-induced generation of ODD is therefore not a trivial question. In this context, the alkaline comet assay has proved to be a reliable and sen-sitive technique for the study of low genotoxic DNA dam-age such as DNA strand breaks and alkali-labile AP sites induced by chemicals and/or environmental contaminants (Dusinska and Collins 2008; Moller et al. 2000). The gen-eration of 8-OH-dG lesions induced by these compounds can be easily measured by the use of formamidopyrimidine DNA glycosylase (FPG) enzyme, as they are FPG-sensitive sites (Collins 2004). FPG-based methods seem to be less prone to the artifact of additional oxidation than standard chromatographic techniques (Collins et al. 2004). Also, measuring 8-OH-dG levels has often proven to be difficult due to the fact that most cell lines and model organisms are able to rapid and efficiently eliminate the chemically induced low levels of ODD via the short-patch BER path-way, limiting the investigators to measure only the remain-ing non-repaired DNA lesions.

The use of a suitable technique for measuring low lev-els of DNA damage such as the alkaline comet assay,

Fig. 6 Long-term genotoxic DNA damage for MEF wt (a) and MEF Ogg1−/− deficient cells (b) in response to an environmentally rel-evant arsenite exposure. Data are presented as mean values of tripli-cates; error bars correspond to 95 % confidence intervals. *P < 0.05 compared with time-matched controls. **P < 0.005 compared with time-matched controls. ***P < 0.001 compared with time-matched controls

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together with the use of a cell system deficient in detect-ing/eliminating the low-i-As-induced 8-OH-dG lesions from DNA, has allowed us to demonstrate that environ-mentally relevant i-As exposures generate persisting levels of ODD with a relevant role in arsenic-mediated chronic genotoxicity. MEF Ogg1−/− cells present an approxi-mately fourfold and fivefold increase in ODD after 24 h of 5 and 10 μM of arsenite exposure, respectively, whereas their wild-type counterparts showed no increases in ODD (see Fig. 3). When the genotoxic DNA damage was ana-lyzed at the above-mentioned doses, MEF Ogg1−/− cells showed around a twofold increase, and again no increases were detected for wild-type cells (see Fig. 4). It is likely that wild-type cells are able to efficiently eliminate/repair the ODD generated after low and medium doses of arsen-ite, and it is only after high doses of arsenite, such as that of 20 μM, when significant increases in ODD and geno-toxic DNA damage are detected. The ODD found in MEF Ogg1−/− at low and medium arsenite doses, however, triggers the appearance of significant amounts of geno-toxic DNA damage at considerable lower doses. When it comes to sub-toxic (up to 2 μM), long-term arsenite exposures, MEF wild-type cells, show only a small but significant increase in ODD after 17 weeks of exposure. MEF Ogg1−/− cells, however, showed a time-dependent increase in ODD from week 4 of exposure and at all ana-lyzed doses. Thus, for example, after chronic exposure to 1 μM of arsenite, MEF Ogg1−/− cells presented a 2.00-, 3.40- and 3.90-fold increase in ODD at weeks 4, 10 and 17 of exposure, respectively. Such a chronic ODD triggered the appearance of detectable increases in i-As-induced gen-otoxic DNA damage at weeks 10 and 17 of exposure (see Figs. 5, 6). The Ogg1 gene appeared therefore relevant for arsenic-induced toxicity and genotoxicity. Our findings are consistent with other reports indicating that ODD is mean-ingful for arsenic-induced cancer and disease. It has been described, i.e., that 8-OH-dG occurs at a higher frequency in arsenic-related skin neoplasms (Matsui et al. 1999) and in lymphocytes from arsenic-exposed individuals (Basu et al. 2005), as well as with the fact that trivalent arseni-cals induce oxidative DNA damage in cultured human cells at pathologically relevant concentrations (Schwerdtle et al. 2003).

The work carried out by Kojima et al. (2009) showed how i-As biomethylation is required for arsenic-induced ODD at environmentally relevant doses and that methyla-tion accompanied by detectable levels of arsenic-induced ODD exacerbate the appearance of a cancer phenotype in vitro (Kojima et al. 2009). In such work, cellular ODD was measured during arsenite exposures at approximately biweekly intervals for up to 30 weeks in a hepatic rat cell line, and authors found a remarkable, but delayed, increase in ODD in an arsenite concentration-dependent manner,

starting after 5 weeks of exposure. The reasons for the observed delay are unknown, but it correlates with what we found in our MEF wild-type cells where we are unable to see an increase in ODD until week 17 of exposure. If ODD is certainly linked to methylation of arsenic, then the more exaggerate delay observed by ours may be due to the described differences in the methylation rate between cell lines. Highly metabolically active cells such as those from liver are known to more efficiently methylate arsenic than others. When it comes to the MEF Ogg1−/− deficient cell line, however, we found significant increases in ODD start-ing from week 4 of treatment. As these cells are unable to eliminate the i-As-generated 8-OH-dG from DNA, these findings indicate, first, that i-As is able to induce 8-OH-dG at low doses of exposure and environmentally relevant type of exposure and, second, that cells with entire Ogg1 and therefore correct BER pathway are able to efficiently elimi-nate the low amount of ODD generated, at least during the first week of exposure.

In humans, genetic polymorphisms in DNA repair genes have been considered to be genetic factors underlying cancer risk by causing inter-individual differences in the capacity to prevent mutagenesis by DNA damage (Goode et al. 2002). Of the most studied BER sequence, variants are those of OGG1. The OGG1 gene encodes a protein with DNA glycosylase and AP lyase activities that removes 8-OH-dG from DNA. The OGG1 protein suppresses G:C to T:A transversions caused by 8-OH-dG in human cells in vivo (Yamane et al. 2003). Ogg1 null mice showed higher contents of 8-OH-dG and higher rates of G:C to T:A mutations in their DNA than wild-type mice and were predisposed to lung adenocarcinoma and adenoma tumors (Klungland et al. 1999; Minowa et al. 2000; Sakumi et al. 2003). A C/G polymorphism at the nucleotide position 1245 in exon 7 of the OGG1 gene (rs1052133) results in an amino acid substitution from a serine to cysteine at codon 326 (Kim et al. 2003). The repair activity of OGG1 was found to be greater with a 326Ser than a 326Cys. In this regard, the frequent OGG1 Ser326Cys polymorphism has been associated with gastric and lung cancer (Kara-halil et al. 2012). Also, the G-T transversion resulting from 8-OH-dG lesion is commonly observed in human cancers caused by mutations in the tumor suppressor p53 such as in lung cancer.

Some authors have previously shown that after a realis-tic scenario of arsenic exposure DNA repair is affected by different mechanisms and therefore very effectively, which might facilitate the carcinogenic process of inorganic arse-nic (Ebert et al. 2011), but only a few works have been published dealing with environmental arsenic and polymor-phisms in OGG1 gene. Interestingly, however, it has been described that methylated arsenicals such as DMAV exert carcinogenicity in the lungs of Ogg1−/− mutant mice, with

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a possible role for persistent accumulation of DNA oxida-tive adducts (Kinoshita et al. 2007). Also, recent epidemio-logical data indicate that subjects with both high urinary As levels and the OGG1 Cys/Cys genotype had a significantly higher risk of hypertension (Chen et al. 2012).

As a summary, this work is among the few studies showing that i-As at environmentally relevant low doses is able to induce significant increases in ODD leading to the appearance and accumulation of i-As-induced geno-toxic DNA damage. Although complex and tedious, the long-term studies using sub-toxic doses represent more accuratelly the real scenario of risk associated with envi-ronmentally exposure to inorganic arsenic. In addition, the observed effects are particularly relevant for those biologi-cal systems deficient in the BER pathway and especially for those with decreased or lacking OGG1 activity. It is likely that the polymorphisms of OGG1 are only a minor contrib-utor to human cancer in general (Hirano 2008), but it may be of great importance when it comes to chemical-induced and i-As-induced cancer in particular. In this context, the development of biomonitoring studies to characterize the role of common OGG1 polymorphisms on arsenic-induced cancer and disease is recommended.

Acknowledgments This work was supported by the ‘Generalitat de Catalunya’ [2009SGR-725], the ‘Universitat Autònoma de Bar-celona’ [APOSTA-2011 to A.H. and PIF-UAB 2010 to J.B.] and the Spanish Ministry of Education and Science [SAF2008-02933 and SAF2011-23146].

Conflict of interest The authors declare that they have no conflict of interest.

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