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Mutation Research 744 (2012) 172– 183

Contents lists available at SciVerse ScienceDirect

Mutation Research/Genetic Toxicology andEnvironmental Mutagenesis

j o ur nal homep ag e: www.elsev ier .com/ locate /gentoxCo mm uni t y add re ss : www.elsev ier .com/ locate /mutres

extran sulfate sodium-induced ulcerative colitis leads to increasedematopoiesis and induces both local as well as systemic genotoxicity in mice

.P. Trivedi, G.B. Jena ∗

acility for Risk Assessment & Intervention Studies, Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Sector-67, S.A.S.agar, Punjab 160062, India

r t i c l e i n f o

rticle history:eceived 17 August 2011eceived in revised form 23 February 2012ccepted 3 March 2012vailable online 10 March 2012

eywords:nflammationenotoxicityell proliferationextran sulfate sodium

a b s t r a c t

Ulcerative colitis is a chronic gastrointestinal disorder eliciting the risk of colorectal cancer, the thirdmost common malignancy in humans. The present study was aimed to characterize dextran sulfatesodium-induced ulcerative colitis and to elucidate its influence on the bone marrow cell prolifera-tion and the subsequent stimulation of the systemic genotoxicity in mice. Experimental colitis wasinduced in Swiss mice using 3% (w/v) dextran sulfate sodium in drinking water. The severity of col-itis was assessed on the basis of clinical signs, colon length, oxidative stress parameters, variouspro-inflammatory markers, histopathological evaluation and immunohistochemical staining of 8-oxo-7,8-dihydro-2′-deoxyguanosine in the colon of dextran sulfate sodium treated mice. Further, assessmentof genotoxicity was carried out using alkaline and modified comet assays in the colon and lymphocytesand micronucleus assay in the peripheral blood of mice. For the evaluation of inflammation-induced cell

ice proliferation in the bone marrow, proliferating cell nuclear antigen immunostaining was carried out inthe bone marrow of mice. Dextran sulfate sodium induced severe colitis as evident from the elevateddisease activity index, reduced colon length, increased oxidative stress, histological abnormalities andoxidative DNA damage in the colon of mice. Moreover, colitis-induced elevated prostaglandin-E2 level inthe plasma of dextran sulfate sodium treated mice stimulated the cell proliferation in the bone marrow,which further triggered colitis-induced DNA damage in the peripheral blood of mice.

. Introduction

The prevalence of inflammatory bowel diseases, such as ulcer-tive colitis and Crohn’s disease is generally more in westernountries, but is becoming common in rest of the world due tohe adoption of western lifestyle [1]. According to the report ofccess Economics Pty Limited as a proposal for Australian Crohn’s

nd Colitis Association (ACCA), inflammatory bowel disease is moreommon than epilepsy or road accidents, and its prevalence isomparable with type 1 diabetes or schizophrenia [2]. Imbalance

Abbreviations: DSS, dextran sulfate sodium; IL-6, interleukin-6; TNF-�, tumorecrosis factor-alpha; NF�B, nuclear factor kappa B; COX-2, cyclooxygenase-2; PG-2, prostaglandin-E2; End-III, endonuclease-III; FPG, formamidopyrimidine DNAlycosylase; H&E, hematoxylin and eosin; DMSO, dimethylsulfoxide; NMPA, normalelting point agarose; LMPA, low melting point agarose; EDTA, ethylenediamine

etraacetic acid; HBSS, Hank’s balanced salt solution; DAI, disease activity index;DA, malondialdehyde; GSH, reduced glutathione; MPO, myeloperoxidase; TL, tail

ength; TM, tail moment; OTM, olive tail moment; % DNA, % DNA in comet tail;-oxo-dG, 8-oxo-7,8-dihydro-2′-deoxyguanosine; PCNA, proliferating cell nuclearntigen; DAB, 3,3′-diaminobenzidinetetrahydrochloride.∗ Corresponding author. Tel.: +91 172 2214682 87x2152; fax: +91 172 2214692.

E-mail addresses: priyankatrvd2@gmail.com (P.P. Trivedi), gbjena@gmail.com,bjena@niper.ac.in (G.B. Jena).

383-5718/$ – see front matter © 2012 Elsevier B.V. All rights reserved.oi:10.1016/j.mrgentox.2012.03.001

© 2012 Elsevier B.V. All rights reserved.

in the regulatory immune mechanisms that control the intestinalcellular and the bacterial homeostasis may lead to the inductionof inflammatory bowel disease [3]. Ulcerative colitis is a chronicgastrointestinal disorder associated with the inflammation of thesuperficial layer of the colon mucosa [4]. It generally affects a partof colon or the entire colon in an uninterrupted manner and theinflammation is typically confined to the mucosa [5]. Patients withulcerative colitis have a higher risk of developing colorectal cancer,which is the third most common malignancy in humans [6].

There are several reports stating that the risk of colorectal can-cer is greater in the patients suffering from extensive colitis dueto inappropriate inflammatory response to a luminal pathogen,abnormal immune response to intestinal bacterial flora, role ofcytokines and oxidative DNA damage at the local sites of inflamma-tion in the colon [7,8]. Interestingly Westbrook et al. [8,9] reportedthat intestinal mucosal inflammation leads to systemic genotox-icity in ulcerative colitis-induced mice and emphasized that theglobal effect of genotoxicity in the peripheral blood is mainly dueto oxidative stress. However, report existed in the literature that

Asian dust and titanium dioxide-induced pulmonary inflammationin mice as well as periodontitis in patients lead to peripheral bloodDNA damage [10,11]. Ulcerative colitis leads to an elevation ofvarious inflammatory markers such as interleukin-6 (IL-6), tumor

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ecrosis factor-alpha (TNF-�), nuclear factor kappa B (NF�B) andyclooxygenase-2 (COX-2) [7].

The correlation between the local inflammation and the sys-emic genotoxicity is still far from clearly understood, and moretudies are required to validate the same using various experimen-al models. In a research article published in the journal Nature,t has been reported that prostaglandin-E2 (PG-E2), synthesizedrom COX-2, leads to an increase in the number of hematopoietictem cells in the zebrafish aorta, gonad and mesonephros regions12]. Several reports claimed that erythropoiesis per se can triggerhe induction of micronuclei in response to different stress fac-ors [13–17]. Moreover, it has been well reported that the ratef cell proliferation per se triggers the rate of DNA damage inhe cells [18,19]. In the present experiment our observations wellorrelated with the findings made by Westbrook et al., that ‘intesti-al inflammation induces systemic DNA damage in experimentalolitis model’. Further we have explored the possibility that thencrease in PG-E2 level can have an influence on the bone mar-ow cell proliferation as evident from increased RETs-to-ERTs ratio,

BC count and proliferating cell nuclear antigen (PCNA) positiveells in the bone marrow with an increase in the extent of colitisnd the subsequent induction of systemic genotoxicity. Further anffort has been made to establish a correlation, if any, between theocal inflammation and the systemic genotoxicity.

. Materials and methods

.1. Animals

All the animal experiment protocols were approved by the Institutional Ani-al Ethics Committee (IAEC) and the experiments on animals were performed in

ccordance with the Committee for the Purpose of Control and Supervision of Exper-mentation on Animals (CPCSEA) guidelines. Experiments were performed on malewiss mice (25–28 g) procured from the Central Animal Facility of the institute. Allhe animals were kept under controlled environmental conditions at room tem-erature (22 ± 2 ◦C) with 50 ± 10% humidity and controlled cycle of 12 h light and2 h dark. Standard laboratory animal feed (purchased from commercial supplier)nd water were provided ad libitum. Animals were acclimatized to the experimentalonditions for a period of 1 week prior to the commencement of the experiment.

.2. Chemicals

DSS (MW 36,000–40,000 Da, CAS no. 9011-18-1) was purchased from MPiomedicals. Acridine orange (CAS no 10127-02-3), endonuclease-III (End-III),

ormamidopyrimidine DNA glycosylase (FPG), SYBR Green I (CAS no. 163795-5-3), hexadecyltrimethylammonium bromide (CAS no. 57-09-0), o-dianisidineihydrochloride (CAS no. 20325-40-0), 1,1,3,3-tetramethoxy propane (CAS no. 102-2-3), 2-thiobarbituric acid (CAS no. 504-17-6), bovine serum albumin (CAS no.048-46-8), hematoxylin and eosin (H&E) and trizma (CAS no. 77-86-1) were pur-hased from Sigma–Aldrich Chemicals, Saint Louis, MO, USA. DimethylsulfoxideDMSO), normal melting point agarose (NMPA), low melting point agarose (LMPA),riton X-100, ethylenediamine-tetraacetic acid (EDTA) and Hank’s balanced saltolution (HBSS) were obtained from HiMedia Laboratories Ltd., Mumbai.

.3. Induction of colitis

For the induction of colitis, mice were administered 3% (w/v) DSS dissolvedn drinking water for various time periods. One cycle consisted of 7 days of DSSreated water followed by 14 days of normal drinking water. Animals were dividednto various groups, each group consisting of 8 animals; and mild, moderate andevere colitis were induced in them. Group 1 received normal drinking water anderved as control. Group 2 (mild colitis) received DSS (3%, w/v) for 7 days and thenimals were sacrificed on the 8th day. Group 3 (moderate colitis) received DSS (3%,/v) from days 1 to 7 and 22 to 28 and the animals were sacrificed on the 29th day.uring the remission period (days 8–21) mice were administered normal drinkingater. Similarly, group 4 (severe colitis) received DSS (3%, w/v) from days 1 to 7,

2 to 28 and 43 to 49 and the animals were sacrificed on the 50th day. During theemission period (days 8–21 and 29–42) mice were administered normal drinking

ater. Detailed experimental design is explained in Fig. 1. To assess the extent of

olitis, weight loss, stool consistency and rectal bleeding were monitored daily inrder to calculate the disease activity index (DAI). DAI is referred to as the averageombined score of weight loss (0–4), stool consistency (0–4) and rectal bleeding0–4) used to score clinical symptoms [20].

search 744 (2012) 172– 183 173

2.4. Measurement of malondialdehyde (MDA) level

Oxidative stress involves the formation of lipid peroxides from polyunsaturatedfatty acids, which further react to form reactive substances like MDA. MDA on reac-tion with thiobarbituric acid forms thiobarbituric acid reactive substances (TBARS),which show maximum absorbance at 532 nm and can be easily detected spectropho-tometrically. Measurement of MDA level is used for the assessment of oxidativestress. MDA level in tissue homogenate was measured according to the methodpreviously described [21] with some modifications. The colon was homogenizedin ice cold phosphate buffer (pH 7.4) for the determination of lipid peroxidationlevel. After homogenization and centrifugation, the supernatant was collected forthe determination of MDA level in tissue samples. MDA level was estimated spec-trophotometrically as an end product of lipid peroxidation using thiobarbituricacid reactive substance method. Lipid peroxidation was calculated from the stan-dard curve generated using 1,1,3,3-tetramethoxy propane and expressed as nmolMDA/mg protein.

2.5. Measurement of glutathione (GSH) content

Elevated oxidative stress may lead to a decrease in the GSH content, a naturalantioxidant present in the body. GSH when reacts with Ellman’s reagent produces5-thio nitro benzoic acid, which is a yellow colored complex showing maximumabsorbance at 412 nm and can be measured spectrophotometrically. GSH content intissue homogenate was measured according to the method previously described[22] with some modifications. For the determination of GSH content, an equalvolume of 5% sulfosalicylic acid was added to tissue homogenate and mixed. Themixture was kept for 30 min in ice bath. After centrifugation for 10 min, the super-natant was collected carefully without disturbing the sediment. GSH content wasmeasured using Ellmann’s reagent (5,5′-dithiobis-2-nitrobenzoic acid solution).GSH levels were calculated using a standard reference curve using reduced glu-tathione as a standard, and expressed as �mol GSH/mg protein.

2.6. Determination of protein content

Protein concentration in tissue homogenate was determined as described [23]with bovine serum albumin as the standard protein.

2.7. Measurement of myeloperoxidase (MPO) activity

MPO is a peroxidase enzyme, most abundantly present in the neutrophils. Itexhibits peroxidase activity that catalyzes oxidation of various substrates such aso-dianisidine by H2O2, which results into the formation of brown colored substanceshowing maximum absorbance at 460 nm and can be measured spectrophotometri-cally. MPO activity was determined as an indicator of polymorphonuclear leucocyteaccumulation as described [24] with some modifications. The colon tissue washomogenized in ice cold 50 mmol/l potassium phosphate buffer (pH 6.0) contain-ing 0.5% hexadecyltrimethylammonium bromide. The homogenate was frozen andthawed three times, centrifuged at 4000 × g for 20 min at 4 ◦C and the level of MPO insupernatant was measured using the o-dianisidine. The rate of change in absorbancewas measured spectrophotometrically at 460 nm, and MPO activity was expressedin units per 100 mg of protein.

2.8. Measurement of IL-6, TNF- ̨ and PG-E2 levels

Plasma levels of IL-6 (Ray Bio ELISA Kit Mouse IL-6, Norcross, GA), TNF-� (RayBio ELISA Kit Mouse TNF-alpha, Norcross, GA) and PG-E2 (Cusabio Biotech Co. LtdMouse PG-E2 ELISA Kit, Newark, DE) were evaluated using commercially availablekits according to the manufacturer’s instructions.

2.9. Western blot analysis

Western blot analysis is used to identify proteins, which have been separatedfrom one another according to their size by gel electrophoresis, using specific anti-bodies. Colon was homogenized in RIPA buffer (50 mM Tris/HCl, 150 mM sodiumchloride, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, pH 7.6), sonicated, cen-trifuged, and the supernatant was collected. Protein concentration was determinedin supernatant as described previously [23]. For western blot analysis, proteins weretransferred onto nitrocellulose membrane, and immunoblot analysis was performedby using the anti NF�B (rabbit polyclonal 1:500; Santa Cruz, CA, USA), anti COX-2(rabbit polyclonal 1:500; Santa Cruz, CA, USA), anti-actin (rabbit polyclonal 1:2500;Santa Cruz, CA, USA) and HRP-conjugated secondary antibodies (anti-rabbit; SantaCruz, CA, USA). Proteins were detected using enhanced chemiluminescence, andquantified with the help of software Imagequant TL (Imagequant 350, GE Healthcare,Hongkong, China).

2.10. Histological evaluation

Histological evaluation was carried out to study the microscopic anatomy ofcells and tissues using light microscopy. Histological slides were prepared as pre-viously standardized in our laboratory [25]. The sections were stained using H&E,

174 P.P. Trivedi, G.B. Jena / Mutation Research 744 (2012) 172– 183

Fig. 1. Schematic diagram illustrates the experimental design. Group 1 received normal drinking water and served as control. Group 2 received DSS (3%, w/v) for 7 days andthe animals were sacrificed on the 8th day. Group 3 received DSS (3%, w/v) from days 1 to 7 and 22 to 28 and the animals were sacrificed on the 29th day. During the remissionperiod (days 8–21) mice were administered normal drinking water. Similarly, group 4 received DSS (3%, w/v) from days 1 to 7, 22 to 28 and 43 to 49 and the animals weres e werew

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acrificed on the 50th day. During the remission period (days 8–21 and 29–42) micater followed by 14 days of normal drinking water.

ounted with DPX mounting media, and examined under the microscope (Olym-us BX51 microscope, Tokyo, Japan). Microscopic scoring was done quantitativelyy measuring the submucosal thickness (in �m) in the colonic sections. Furtheristological score was assigned according to the criteria described [26]. Collagenstimation was carried out by Weigert’s hematoxylin and van Geinson’s stainings described [27] with some modifications. The slides were stained with Weigert’sematoxylin (composed of 0.5% (w/v) hematoxylin, 50% (v/v) alcohol, 2% (v/v) fer-ic chloride solution, 0.5% (v/v) conc. HCl and 47.5% (v/v) distilled water) and vaneinson’s solution (composed of 13.1% (v/v) of 1% acid fuchsin solution and 86.9%

v/v) saturated picric acid). After dehydration in absolute alcohol and cleaning withylene, sections were mounted with DPX mounting media and examined under anlympus BX51 microscope (Tokyo, Japan). Evaluation of collagen content is basedn the principle of selective binding of acid aniline dye (acid fuchsin, a componentf van Geinson’s solution) to the collagen. The sections were counterstained withematoxylin and picric acid. Under Weigert’s-van Gieson staining nucleus takes vio-

et color with hematoxylin, muscles and cytoplasm take yellow color with picric acidnd collagen takes bright red color with acid fuchsin.

.11. Estimation of DNA damage by single cell gel electrophoresis (SCGE) assay

.11.1. Alkaline comet assayComet assay is a reliable and a sensitive method for the detection of DNA dam-

ge using gel electrophoresis. The alkaline comet assay was performed as described28,29] with some modifications. A small piece of colon was placed in 1 ml coldank’s balanced salt solution containing 20 mM EDTA and 10% DMSO and minced

nto fine pieces. 10 �l of this was mixed with 90 �l of low melting point agarose andayered over the surface of a frosted slide [pre-coated with 1% normal melting pointgarose] to form a microgel and allowed to set at 4 ◦C for 5 min. For the isolation

f lymphocytes, blood was collected from the retro-orbital sinus and the lympho-ytes were isolated using Ficoll histopaque solution. A final cell-agarose suspension100 �l) was prepared containing ≈1 × 104 lymphocytes/ml in 0.5% LMPA and lay-red over the surface of a frosted slide [pre-coated with 1% NMPA] to form a microgelnd allowed to set at 4 ◦C for 5 min. A second layer of 1% LMPA was added and allowed

ig. 2. Effect of DSS treatment on (A) % change in body weight and (B) disease activity*P < 0.01 vs. control. (C) Representative photomicrograph showing the induction of coliti

administered normal drinking water. One cycle consisted of 7 days of DSS treated

to set at 4 ◦C for 5–10 min. The slides were then immersed in lysis solution (2.5 MNaCl, 100 mM EDTA, 10 mM Tris–HCl buffer (pH 10.0), 1% Triton X-100 and 10%DMSO) at 4 ◦C for 24 h. After 24 h, the slides were washed with chilled water, thencoded and placed in a specifically designed horizontal electrophoresis tank and DNAwas allowed to unwind for 20 min in alkaline solution containing 300 mM NaOHand 1 mM EDTA (pH >13.0). Electrophoresis was conducted at 0.6 V/cm, 300 mA for30 min in a horizontal electrophoresis unit (SCIE-PLAS Ltd., UK, Max volts 1000 V,Max current 500 mA). After neutralization the slides were washed with chilled waterand stained with SYBR Green I (1:10,000 dilutions). The fluorescent labeled DNA wasvisualized using an AXIO Imager M1 fluorescence microscope (Carl Zeiss, Germany).The parameters for the DNA damage analysis include: tail length (TL, in �m), tailmoment (TM), olive tail moment (OTM) and % DNA (% DNA) in comet tail. Fifty cellswere randomly counted from each slide and a total of hundred cells were counted foreach animal and the resulting images were captured on a computer and processedwith image analysis software (Metasystem software, Comet Imager V.2.0.0).

2.11.2. Modified comet assayThe alkaline comet assay using lesion-specific enzymes was used to detect

oxidized purines and pyrimidines (that are generated as a result of oxidative stress-induced DNA damage) as described [30] with some modifications. The cell-agarosesuspension slides were prepared as described above for the normal comet assay.After lysing, the slides were washed three times with the enzyme buffer (40 mMHEPES, 100 mM KCl, 0.5 mM EDTA and 0.2 mg/ml BSA) at room temperature andwere incubated at 37 ◦C for 30 min with: (i) Endo-III (1:1000), (ii) FPG (1:1000) and(iii) with enzyme buffer (control). End-III recognizes oxidized pyrimidines while FPGrecognizes oxidized purines. Slides were coded and placed in a specifically designed

horizontal electrophoresis tank and DNA was allowed to unwind for 20 min in alka-line solution containing 300 mM NaOH and 1 mM EDTA (pH >13). The DNA waselectrophoresed at 0.6 V/cm, 300 mA for 30 min. After neutralization, the slides werestained with SYBR Green I (1:10,000 dilutions) for 1 h and covered with coverslips.Image analysis and data scoring was same as mentioned for normal comet assay.

index (DAI). All the values are expressed as mean ± SEM (n = 8), ***P < 0.001 ands in mice after 3 cycles of DSS.

P.P. Trivedi, G.B. Jena / Mutation Research 744 (2012) 172– 183 175

Fig. 3. Effect of DSS treatment on (A) colon length (in cm) of mice. All the values are expressed as mean ± SEM (n = 8), ***P < 0.001 vs. control. (B) Representative photomi-crograph depicting the reduction in the colon length of DSS treated mice after 1, 2 and 3 cycles of DSS.

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Fig. 4. Effect of DSS treatment on (A) MDA and (B) GSH levels in mice co

.12. Detection of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) in colon androliferating cell nuclear antigen in bone marrow by immunohistochemicalnalysis

Immunohistochemistry is used to detect antigens in cells of a tissue section bytilizing the principle of antibodies binding specifically to the antigens in biologicalissues. Paraffin embedded tissues were cut into 5 �m thick sections, deparaffinized

n xylene and rehydrated with graded alcohol. Tissue sections were incubated initrate buffer at 95–100 ◦C for 20 min for antigen retrieval. Sections were then incu-ated in 1% H2O2 and in 5% normal goat serum blocking solution. The colon and theone sections were then incubated overnight at 4 ◦C in the mouse anti-8-oxo-dG

ig. 5. (A) Effect of DSS treatment on MPO level in mice colon. (B) (a) Representative phob) the histogram showing the effect of DSS treatment on the expression of transcription fxpression and are mean ± SEM of three individual experiments. (C) Effect of DSS treatmxpressed as mean ± SEM (n = 8), ***P < 0.001, **P < 0.01 and *P < 0.05 vs. control.

ll the values are expressed as mean ± SEM (n = 8), ***P < 0.001 vs. control.

(1:100) and rabbit anti-PCNA (1:100) primary antibodies respectively. Sections werewashed with PBS, and then incubated with peroxidase-marked secondary antibody(Santa Cruz, CA, USA) for 2 h at room temperature. Reaction product was detectedusing 3,3′-diaminobenzidinetetrahydrochloride (DAB, Novocastra). Sections weredehydrated through graded alcohols, cleared in xylene and coverslipped in per-manent mounting medium. Sections were examined with a microscope (OlympusBX51, Japan). For immunostaining of bone marrow smear with PCNA, samples were

processed on poly-l-lysine coated slides as described with some modifications [31].Bone marrow cells were isolated from the femur bone, suspended in fetal bovineserum and centrifuged. The pellet was suspended in the residual fetal bovine serumand a drop of it was smeared on a slide, fixed with 4% paraformaldehyde and

tomicrograph showing the expression of transcription factor NF�B and COX-2 andactor NF�B (i) and COX-2 (ii) in mice colon. The data were normalized with �-actinent on (a) IL-6, (b) TNF-� and (c) PG-E2 levels in mice plasma. All the values are

176 P.P. Trivedi, G.B. Jena / Mutation Research 744 (2012) 172– 183

Fig. 6. (A) Representative photomicrographs of mice colon sections stained with hematoxylin and eosin (H&E) indicating increase in the thickness of the submucosal layerdue to DSS treatment. (i) Control, (ii) 1 cycle of DSS treatment, (iii) 2 cycles of DSS treatment and (iv) 3 cycles of DSS treatment and representative photomicrographs of micec cating( cyclesc ice. A

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olon sections stained with Weigert’s hematoxylin and van Geinson’s solution indiv) Control, (vi) 1 cycle of DSS treatment, (vii) 2 cycles of DSS treatment and (viii) 3

olon in mice. (C) Effect of DSS treatment on the histological score of the colon in m

ermeabilized using 0.5% Triton X-100 in PBS. Rest procedure was as mentionedbove.

.13. Estimation of DNA damage by peripheral blood micronucleus assay

The peripheral blood micronucleus assay is used to detect the damage inducedo the chromosomes or the mitotic apparatus of erythroblasts by analysis of erythro-ytes as sampled in peripheral blood cells of animals. The peripheral blood smearsere prepared as described [32] with some modifications. The blood was collected

rom tail tip and the smears were prepared on pre-cleaned slides. The smear wasllowed to dry at room temperature and fixed in absolute methanol for 5 min. Afterxation, the slides were stained with acridine orange and were washed thrice withhosphate buffer (pH 6.8). The slides were observed under a microscope and scored.ata were expressed as RET-to-ERT ratio and MN ERTs/1000 ERTs.

.14. Hematology

Blood samples were collected from the mice at the time of sacrifice forematological analysis. The hematology parameters were analyzed using a MELETCHLOESING LABORATORIES (MS9-5 France) hematology analyzer.

.15. Statistical analysis

Results were shown as mean ± standard error of mean (SEM) for each group.tatistical analysis was performed using Jandel Sigma Stat (Version 3.5) statisti-al software. For multiple comparisons, one-way analysis of variance (ANOVA) wassed. In case ANOVA showed significant differences, post hoc analysis was performedith Tukey’s test. P < 0.05 was considered to be statistically significant. Linear

increase in the collagen deposition in the submucosal layer due to DSS treatment. of DSS treatment. (B) Effect of DSS treatment on the thickness of submucosa of thell the values are expressed as mean ± SEM (n = 8), ***P < 0.001 vs. control.

regression analysis was performed in order to observe the correlation between thelocal inflammation and the systemic genotoxicity.

3. Results

3.1. Effect on body weight and DAI

DSS treatment led to a significant decrease in the body weightand increase in the DAI of the animals as compared to the controlgroup. Severe rectal bleeding was observed during the second andthe third cycles of treatment (Fig. 2).

3.2. Effect on colon length

DSS treatment led to a significant decrease in the colon length ofthe animals as compared to the control group depicting its severetoxicity on the colon (Fig. 3).

3.3. Effect on oxidative stress

DSS treatment led to a significant increase in the MDA anddecrease in the GSH levels in the colon as compared to the con-trol group (Fig. 4), thus indicating that elevated oxidative stress isinvolved in the DSS-mediated colitis in mice.

P.P. Trivedi, G.B. Jena / Mutation Research 744 (2012) 172– 183 177

Fig. 7. (A) Representative photomicrographs showing the DNA migration pattern in mice colon nuclei stained with SYBR Green I. (A) Control, (B) 3 cycles of DSS treatment.The symbols “−” and “+” represent cathode and anode respectively during electrophoresis of negatively charged DNA. (B) Effect of DSS treatment on the oxidative DNAdamage in (i) colon and (ii) lymphocytes of mice by alkaline and modified comet assays using lesion-specific enzymes, FPG and End-III. All the values are expressed asmean ± SEM (n = 8), ***P < 0.001, **P < 0.01 and *P < 0.05 vs. control.

178 P.P. Trivedi, G.B. Jena / Mutation Research 744 (2012) 172– 183

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ig. 8. Representative photomicrographs illustrating the immunohistochemical staSS treatment and (D) 3 cycles of DSS treatment. Inset shows the magnified images oositive cells.

.4. Effect on inflammatory markers

There was a significant increase in various inflammatory mark-rs such as MPO level (Fig. 5A), NF�B and COX-2 expression (Fig. 5B)n the colon and IL-6, TNF-� and PG-E2 levels in the plasma (Fig. 5C)f the DSS treated animals as compared to the control group.

.5. Effect on histology

DSS treatment led to a significant increase in the cellular infiltra-ion into the lamina propria and submucosa, extensive distortion ofhe crypt morphology and increased epithelial cell layer erosions asompared to the control group. The extent of inflammation and cel-ular toxicity in the colon increased after the 2nd and the 3rd cyclesf DSS treatment. Further, DSS treatment resulted into the collagen

eposition in the submucosal layer as observed from the Weigert’sematoxylin and van Geinson’s staining (Fig. 6A). Moreover, it ledo a significant increase in the thickness of the submucosa as wells histological score as compared to the control group (Fig. 6A–C).

ig. 9. (A) Representative photomicrographs depicting the effect of DSS treatment on

reatment. (B) Effect of DSS treatment on (i) RETs-to-ERTs ratio and (ii) MN ERTs/1000 ERs mean ± SEM (n = 8), ***P < 0.001, **P < 0.01 and *P < 0.05 vs. control.

of 8-oxo-dG in mice colon. (A) Control, (B) 1 cycle of DSS treatment, (C) 2 cycles ofrea highlighted in the box showing differences between the staining of cytoplasmic

3.6. Effect on DNA damage

Comet assay is a sensitive and reliable assay for the detection ofDNA damage (Fig. 7A). DSS treatment led to a significant increase inthe DNA damage in colon cells as well as lymphocytes as comparedto the control group (Fig. 7B). Treatment of the cells with End-IIIand FPG further increased the sensitivity of the comet assay, andDSS treated animals depicted a significant increase in the oxidativestress-induced DNA damage in colon cells as well as in lymphocytesas compared to the control group.

3.7. Effect on 8-oxo-dG expression in colon

Higher intensity of immunostaining for 8-oxo-dG in the colonwas observed in the animals treated with DSS as compared to thecontrol group indicating oxidative stress-induced DNA damage inthe colon of DSS treated animals (Fig. 8).

cell proliferation and micronucleus formation, (i) control and (ii) 3 cycles of DSSTs. The blood smear was stained with acridine orange. All the values are expressed

P.P. Trivedi, G.B. Jena / Mutation Research 744 (2012) 172– 183 179

Fig. 10. Representative photomicrographs illustrating the immunohistochemical staining of PCNA in (A) bone section (i) control, (ii) 3 cycles of DSS treatment, (B) bonemarrow smear (i) control, (ii) 3 cycles of DSS treatment at 40× magnification and (iii) control, (iv) 3 cycles of DSS treatment at 100× magnification. Arrows indicate PCNApositive cells. (C) Effect of DSS treatment on % PCNA positive cells in the bone marrow smear. All the values are expressed as mean ± SEM (n = 8), ***P < 0.001 and *P < 0.05 vs.control.

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.8. Effect on micronucleus formation

DSS treatment led to a significant increase in the frequency oficronuclei formation in the peripheral blood of mice after the 2nd

nd the 3rd cycles as compared to the control group. Moreover,here was a significant increase in the number of reticulocytes asvident from a siginificant increase in the RETs-to-ERTs ratio in theeripheral blood of DSS treated mice as compared to the controlroup, indicating increased cell proliferation due to inflammationFig. 9).

.9. Effect on PCNA expression in bone marrow

In order to confirm increased cellular proliferation in the bonearrow of DSS treated animals, immunostaining of PCNA was

arried out. Higher intensity of immunostaining for PCNA wasbserved in the bone section as well as bone marrow smear of thenimals treated with DSS as compared to the control group indicat-

ng increased cell proliferation in the bone marrow of DSS treatednimals. Further, quantitative estimation of PCNA positive cells inhe bone marrow smear depicted a significant increase in the %CNA positive cells due to DSS treatment (Fig. 10).

3.10. Effect on hematological parameters

There was a statistically significant increase in the total WBCcount and differential WBC count (% Lymphocytes and % Neu-trophils) due to DSS treatment as compared to the control group.DSS treatment did not lead to a statistically significant change inother hematological parameters (Table 1).

3.11. Correlation between the local inflammation and thesystemic genotoxicity

A positive correlation was observed between the local inflam-mation and the systemic genotoxicity in animals treated with DSS.Strong correlations were observed between NF�B level in colonand TL (R2 = 0.72), TM (R2 = 0.698), OTM (R2 = 0.924) and % DNAin tail (R2 = 0.975) in lymphocytes, COX-2 level in colon and TL

(R2 = 0.900), TM (R2 = 0.886), OTM (R2 = 0.998) and % DNA in tail(R2 = 0.993) in lymphocytes as well as MPO level in colon and TL(R2 = 0.980), TM (R2 = 0.973), OTM (R2 = 0.980) and % DNA in tail(R2 = 0.933) in lymphocytes of DSS treated animals (Fig. 11).

180 P.P. Trivedi, G.B. Jena / Mutation Research 744 (2012) 172– 183

Table 1Effect of DSS treatment on the hematological parameters in mice. All the values are expressed as mean ± SEM (n = 8), ***P < 0.001, **P < 0.01 and *P < 0.05 vs. control.

Parameters Groups

Control DSS-7 DSS-14 DSS-21

WBC (×103/mm3) 2.88 ± 0.06 3.09 ± 0.06 3.67 ± 0.10*** 4.27 ± 0.04***RBC (×106/mm3) 9.35 ± 0.04 9.58 ± 0.08 9.75 ± 0.23 9.77 ± 0.23MCV (fl) 47.63 ± 0.76 47.96 ± 1.21 46.23 ± 0.93 47.33 ± 0.58Hct (%) 46.03 ± 1.87 49.36 ± 1.64 43.06 ± 1.84 43.86 ± 0.55Hb (g/dl) 15.06 ± 0.65 15.16 ± 0.38 14.30 ± 0.20 13.56 ± 0.13PLT (×103/mm3) 1166.66 ± 17.61 1170.00 ± 35.72 1159.66 ± 32.66 1133.00 ± 33.00Lym (%) 75.26 ± 0.76 76.30 ± 1.34 80.93 ± 1.27** 85.26 ± 1.43***Mon (%) 6.53 ± 0.70 6.20 ± 0.20 6.33 ± 0.06 6.23 ± 0.17Nut (%) 7.46 ± 0.35 8.70 ± 0.96 8.96 ± 0.17* 9.26 ± 0.46*Eos (%) 9.86 ± 1.16 8.26 ± 0.52 8.20 ± 0.50 8.73 ± 0.43Bas (%) 0.06 ± 0.03 0.06 ± 0.06 0.03 ± 0.03 0.06 ± 0.03Unid (%) 0.90 ± 0.35 0.83 ± 0.16 0.06 ± 0.16 0.02 ± 0.10

Fig. 11. Linear regression analysis showing the correlation of NF�B level in colon with different comet assay parameters such as tail length (a) tail moment (b) olive tailmoment (c) and % DNA in tail (d) in lymphocytes of mice. Similar correlation between COX-2 level and different comet assay parameters [(e), (f), (g) and (h)] as well asbetween MPO level and different comet assay parameters [(i), (j), (k) and (l)] has been shown indicating a correlation between the local inflammation and the systemicgenotoxicity in mice treated with DSS.

P.P. Trivedi, G.B. Jena / Mutation Research 744 (2012) 172– 183 181

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. Discussion

In the present investigation, DSS induced colitis in mice whendministered for 7 days in drinking water, and the extent of col-tis increased when it was administered for 14 and 21 days afterhe remission period as evident from the DAI. The induction ofnflammation in the colonic tissue was confirmed by the signifi-ant increase in various inflammatory markers, such as MPO, NF�Bnd COX-2 as compared to the control animals. It has been reportedhat DSS treatment in mice elevated the phospho-I�B�, COX-2, sev-ral pro-inflammatory cytokines as well as plasma PG-E2 level [33].oreover, NF�B has been known to be activated in the colonicucosa from the patients with ulcerative colitis [34]. Further, it

as been reported that NF�B is a critical activator gene, which up-egulates the expression of COX-2 [35]. Histological examinationlearly depicted the infiltration of inflammatory cells into the lam-na propria and submucosa as well as extensive disruption of therypt morphology in the colonic tissue sections. DSS treatment alsoed to collagen deposition in the submucosa of the colon as evidentrom the Weigert’s hematoxylin and van Geinson’s staining. Suchbnormality in the colon histology has already been reported dueo DSS treatment in mice [4,26].

It has been known that inflammation plays a pivotal role inhe generation of oxidative stress, which ultimately leads to DNAamage at the local site of inflammation [36]. Further, it has beeneported that in ulcerative colitis, shorter telomeres have beenssociated with chromosomal instability and tumor progressionn colon [37]. In the present study, inflammation in the colon ledo the generation of oxidative stress as depicted from a signifi-ant increase in the MDA and decrease in the GSH levels as wells DNA damage as observed from a significant increase in various

omet assay parameters in the colonic tissue. Comet assay (nor-al and modified) generally detects the extent and nature of DNA

amage, but not the cell types from which the DNA damage origi-ated [38]. In order to discriminate in between the involvement of

ved in the ulcerative colitis-induced systemic genotoxicity.

epithelial and inflammatory cells in the quantification of DNA dam-age in comet assay, the methodology developed by Gontijo et al.[39] must be validated using the present experimental model. Inthe present study, oxidative stress-induced DNA damage in colonwas confirmed with modified comet assay using lesion specificenzymes, End-III and FPG, as well as immunostaining of 8-oxo-dGwas carried out. It was clearly evident from these assays that ulcer-ative colitis led to the generation of oxidative stress-induced DNAdamage in colon. It has been reported that an increased level ofan oxidative DNA damage biomarker, 8-oxo-dG, was found in thecolonic mucosa of rat fed with 3% and 6% DSS in drinking water for2 days [40]. Interestingly in the present experiment, apart from thenuclei, cytoplasmic staining was observed for 8-oxo-dG immuno-histochemical staining, which might be due to the accumulation of8-oxo-dG in the mitochondrial DNA. This observation can be wellcorroborated with the findings reported by Tsuruya et al., whichstate that the level of cytoplasmic staining represents the 8-oxo-dGexpression in the mitochondrial DNA in an ischemic-reperfusioninjury in the rat kidney [41].

In the present investigation, colitis led to a significant increasein the pro-inflammatory markers in the plasma of mice as apparentfrom increased levels of IL-6, TNF-� and PG-E2 in the plasma of DSStreated animals as compared to the control animals. Further, to sub-stantiate the finding that intestinal inflammation leads to systemicgenotoxicity in mice, alkaline and modified comet assays as wellas micronucleus assay in the peripheral blood of mice were carriedout. It was evident from various comet assay parameters that col-itis led to oxidative stress-induced DNA damage in the peripheralblood lymphocytes. It has been reported that IKK� links inflam-mation and tumorigenesis via DNA damage in a mouse model ofcolitis-associated cancer [42]. Moreover, inflammatory cytokines

have been known to induce DNA damage and inhibit DNA repair incholangiocarcinoma cells by a nitric oxide-dependent mechanism[43]. Thus, there are many reports depicting that inflammation isa roadway to DNA damage. In the present study, colitis resulted in

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82 P.P. Trivedi, G.B. Jena / Mutat

he local and the systemic inflammation, which may be responsibleor the subsequent genotoxicity observed.

Further, there was a significant increase in the micronucleirequency in the peripheral blood as well as the number of reticu-ocytes as evident from a significant increase in the RETs-to-ERTsatio in DSS treated animals as compared to the control animalsndicating an increased cell proliferation. In order to ascertain thencreased cell proliferation in bone marrow, immunostaining ofCNA was carried out which revealed higher PCNA immunostain-ng in the bone marrow of DSS treated animals as compared tohe control animals. It has been reported that PCNA is the mae-tro of the replication fork, and orchestrates several functions byecruiting crucial players to the replication fork, and is involved inell proliferation [44]. The hematological analysis revealed that DSSreatment led to a significant increase in the WBC count. It has beeneported that COX-2 is an enzyme responsible for the conversionf arachidonic acid to prostaglandins, and is implicated in colorec-al tumorigenesis [45]. COX-2 is involved in the synthesis of PG-E2,hich in turn, has been reported to stimulate hematopoiesis. PG-E2as been known to induce the micronucleus formation by variousutagens in mouse bone marrow cells by triggering the erythroid

rogenitor cells into the cell cycle [46]. Moreover, PG-E2 has beeneported to increase cyclic AMP secretion, which in turn, is knowno induce erythropoiesis [47–50]. Further, it has been reported that

acrophages are the key orchestrators of inflammation, and theyre involved in the production of extrarenal erythropoietin [51,52].ur study results well corroborated with the findings made byestbrook et al., that ulcerative colitis manifests a global effect,

articularly the level of systemic genotoxicity in the peripherallood [8,9]. Apart from it, the present experiment depicts thatSS led to a significant increase in the PG-E2 level, which in turn,

nduced hematopoiesis as evident from increased RETs-to-ERTsatio, WBC count and PCNA positive cells in the bone marrow withn increase in the extent of colitis and at least in part might be ariggering influence to induce peripheral DNA damage. The possi-le mechanisms involved in the ulcerative colitis-induced systemicenotoxicity have been shown in Fig. 12. Detailed molecular mech-nisms which orchestrate events leading to systemic genotoxicityue to colitis need to be further explored.

onflict of interest

None.

cknowledgments

We wish to acknowledge the financial assistance receivedrom National Institute of Pharmaceutical Education and ResearchNIPER), Mohali to undertake the present study. The author’s arerateful to the anonymous reviewers for their critical suggestionso improve the quality as well as clarity of the manuscript. Further,he authors would like to thank Ms. Sapana Kushwaha and Mr. Sab-ir Khan for critically reading the manuscript and improving the

anguage.

eferences

[1] R.B. Sartor, Mechanisms of disease: pathogenesis of Crohn’s disease and ulcer-ative colitis, Nat. Clin. Pract. Gastroenterol. Hepatol. 3 (2006) 390–407.

[2] The Economic Costs of Crohn’s Disease and Ulcerative Colitis, 2007, availablefrom: www.accesseconomics.com.au/publicationsreports (cited 09.06.2007).

[3] M. Mohamadzadeh, E.A. Pfeiler, J.B. Brown, M. Zadeh, M. Gramarossa, E. Man-aglia, P. Bere, B. Sarraj, M.W. Khan, K.C. Pakanati, M.J. Ansari, S. O’Flaherty, T.

Barrett, T.R. Klaenhammer, Regulation of induced colonic inflammation by Lac-tobacillus acidophilus deficient in lipoteichoic acid, Proc. Natl. Acad. Sci. U.S.A.108 (Suppl. 1) (2011) 4623–4630.

[4] N. Eijkelkamp, C.J. Heijnen, A. Lucas, R.T. Premont, S. Elsenbruch, M. Sched-lowski, A. Kavelaars, G protein-coupled receptor kinase 6 controls chronicity

[

search 744 (2012) 172– 183

and severity of dextran sodium sulphate-induced colitis in mice, Gut 56 (2007)847–854.

[5] C. Abraham, J. Cho, Mechanisms of disease: inflammatory bowel disease, N.Engl. J. Med. 361 (2009) 2066–2078.

[6] M. Saleh, G. Trinchieri, Innate immune mechanisms of colitis and colitis-associated colorectal cancer, Nat. Rev. Immunol. 11 (2011) 9–20.

[7] Y. Okada, Y. Tsuzuki, J. Miyazaki, K. Matsuzaki, R. Hokari, S. Komoto, S. Kato,A. Kawaguchi, S. Nagao, K. Itoh, T. Watanabe, S. Miura, Propionibacteriumfreudenreichii component 1.4-dihydroxy-2-naphthoic acid (DHNA) attenuatesdextran sodium sulphate induced colitis by modulation of bacterial flora andlymphocyte homing, Gut 55 (2006) 681–688.

[8] A.M. Westbrook, B. Wei, J. Braun, R.H. Schiestl, Intestinal mucosal inflammationleads to systemic genotoxicity in mice, Cancer Res. 69 (2009) 4827–4834.

[9] A.M. Westbrook, B. Wei, J. Braun, R.H. Schiestl, More damaging than we think:systemic effects of intestinal inflammation, Cell Cycle 8 (2009) 2482–2483.

10] Y.J. Hwang, Y.S. Jeung, M.H. Seo, J.Y. Yoon, D.Y. Kim, J.W. Park, J.H. Han, S.H.Jeong, Asian dust and titanium dioxide particles-induced inflammation andoxidative DNA damage in C57BL/6 mice, Inhal. Toxicol. 22 (2010) 1127–1133.

11] T. Konopka, K. Krol, W. Kopec, H. Gerber, Total antioxidant status and8-hydroxy-2′-deoxyguanosine levels in gingival and peripheral blood of peri-odontitis patients, Arch. Immunol. Ther. Exp. (Warsz.) 55 (2007) 417–422.

12] T.E. North, W. Goessling, C.R. Walkley, C. Lengerke, K.R. Kopani, A.M. Lord,G.J. Weber, T.V. Bowman, I.H. Jang, T. Grosser, G.A. Fitzgerald, G.Q. Daley, S.H.Orkin, L.I. Zon, Prostaglandin E2 regulates vertebrate haematopoietic stem cellhomeostasis, Nature 447 (2007) 1007–1011.

13] A. Anagnostou, S. Schade, M. Ashkinaz, J. Barone, W. Fried, Effect of proteindeprivation on erythropoiesis, Blood 50 (1977) 1093–1097.

14] J. Caro, A.J. Erslev, R. Silver, O. Miller, G. Birgegard, Erythropoietin production inresponse to anemia or hypoxia in the newborn rat, Blood 60 (1982) 984–988.

15] S.H. Cheshier, S.S. Prohaska, I.L. Weissman, The effect of bleeding on hematopoi-etic stem cell cycling and self-renewal, Stem Cells Dev. 16 (2007) 707–717.

16] G. Steinheider, R. Neth, H. Marquardt, Evaluation of nongenotoxic and geno-toxic factors modulating the frequency of micronucleated erythrocytes in theperipheral blood of mice, Cell Biol. Toxicol. 2 (1986) 197–211.

17] N. Yajima, Y. Kurata, T. Sawai, Y. Takeshita, Induction of micronucleatederythrocytes by recombinant human erythropoietin, Mutagenesis 8 (1993)221–229.

18] B.N. Ames, M.K. Shigenaga, L.S. Gold, DNA lesions, inducible DNA repair, and celldivision: three key factors in mutagenesis and carcinogenesis, Environ. HealthPerspect. 101 (Suppl. 5) (1993) 35–44.

19] A. Vikram, D.N. Tripathi, P. Ramarao, G.B. Jena, Evaluation of streptozotocingenotoxicity in rats from different ages using the micronucleus assay, Regul.Toxicol. Pharmacol. 49 (2007) 238–244.

20] S.N. Murthy, H.S. Cooper, H. Shim, R.S. Shah, S.A. Ibrahim, D.J. Sedergran,Treatment of dextran sulfate sodium-induced murine colitis by intracoloniccyclosporin, Dig. Dis. Sci. 38 (1993) 1722–1734.

21] H. Ohkawa, N. Ohishi, K. Yagi, Assay for lipid peroxides in animal tissues bythiobarbituric acid reaction, Anal. Biochem. 95 (1979) 351–358.

22] M.S. Moron, J.W. Depierre, B. Mannervik, Levels of glutathione, glutathionereductase and glutathione S-transferase activities in rat lung and liver, Biochim.Biophys. Acta 582 (1979) 67–78.

23] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement withthe Folin phenol reagent, J. Biol. Chem. 193 (1951) 265–275.

24] D. Rachmilewitz, P.L. Simon, L.W. Schwartz, D.E. Griswold, J.D. Fondacaro, M.A.Wasserman, Inflammatory mediators of experimental colitis in rats, Gastroen-terology 97 (1989) 326–337.

25] A. Vikram, D.N. Tripathi, P. Ramarao, G.B. Jena, Intervention of d-glucose ame-liorates the toxicity of streptozotocin in accessory sex organs of rat, Toxicol.Appl. Pharmacol. 226 (2008) 84–93.

26] K. Yoshihara, T. Yajima, C. Kubo, Y. Yoshikai, Role of interleukin 15 in colitisinduced by dextran sulphate sodium in mice, Gut 55 (2006) 334–341.

27] V.P. Dadhania, D.N. Tripathi, A. Vikram, P. Ramarao, G.B. Jena, Intervention ofalpha-lipoic acid ameliorates methotrexate-induced oxidative stress and geno-toxicity: a study in rat intestine, Chem. Biol. Interact. 183 (2010) 85–97.

28] T. Kasamatsu, K. Kohda, Y. Kawazoe, Comparison of chemically induced DNAbreakage in cellular and subcellular systems using the comet assay, Mutat. Res.369 (1996) 1–6.

29] G. Speit, A. Hartmann, The comet assay (single-cell gel test). A sensitive geno-toxicity test for the detection of DNA damage and repair, Methods Mol. Biol.113 (1999) 203–212.

30] A.R. Collins, S.J. Duthie, V.L. Dobson, Direct enzymic detection of endogenousoxidative base damage in human lymphocyte DNA, Carcinogenesis 14 (1993)1733–1735.

31] J.V. Goldstine, S. Nahas, K. Gamo, S.M. Gartler, R.S. Hansen, J.H. Roelfsema, R.A.Gatti, Y. Marahrens, Constitutive phosphorylation of ATM in lymphoblastoidcell lines from patients with ICF syndrome without downstream kinase activity,DNA Repair (Amst.) 5 (2006) 432–443.

32] H.E. Holden, J.B. Majeska, D. Studwell, A direct comparison of mouse and ratbone marrow and blood as target tissues in the micronucleus assay, Mutat. Res.391 (1997) 87–89.

33] D.C. Montrose, N.A. Horelik, J.P. Madigan, G.D. Stoner, L.S. Wang, R.S. Bruno, H.J.

Park, C. Giardina, D.W. Rosenberg, Anti-inflammatory effects of freeze-driedblack raspberry powder in ulcerative colitis, Carcinogenesis 32 (2011) 343–350.

34] L. Andresen, V.L. Jorgensen, A. Perner, A. Hansen, J. Eugen-Olsen, J. Rask-Madsen,Activation of nuclear factor kappaB in colonic mucosa from patients with col-lagenous and ulcerative colitis, Gut 54 (2005) 503–509.

ion Re

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

P.P. Trivedi, G.B. Jena / Mutat

35] A. Rossi, P. Kapahi, G. Natoli, T. Takahashi, Y. Chen, M. Karin, M.G. Santoro, Anti-inflammatory cyclopentenone prostaglandins are direct inhibitors of IkappaBkinase, Nature 403 (2000) 103–108.

36] L.R. Ferguson, Chronic inflammation and mutagenesis, Mutat. Res. 690 (2011)3–11.

37] R.A. Risques, L.A. Lai, T.A. Brentnall, L. Li, Z. Feng, J. Gallaher, M.T. Mandel-son, J.D. Potter, M.P. Bronner, P.S. Rabinovitch, Ulcerative colitis is a diseaseof accelerated colon aging: evidence from telomere attrition and DNA damage,Gastroenterology 135 (2008) 410–418.

38] A.R. Collins, V.L. Dobson, M. Dusinska, G. Kennedy, R. Stetina, The comet assay:what can it really tell us? Mutat. Res. 375 (1997) 183–193.

39] A.M. Gontijo, F.N. Elias, D.M. Salvadori, M.L. de Oliveira, L.A. Correa, J. Goldberg,J.C. Trindade, J.L. de Camargo, Single-cell gel (comet) assay detects primary DNAdamage in nonneoplastic urothelial cells of smokers and ex-smokers, CancerEpidemiol. Biomarkers Prev. 10 (2001) 987–993.

40] D. Tardieu, J.P. Jaeg, J. Cadet, E. Embvani, D.E. Corpet, C. Petit, Dextransulfate enhances the level of an oxidative DNA damage biomarker 8-oxo-7,8-dihydro-2′-deoxyguanosine, in rat colonic mucosa, Cancer Lett. 134 (1998)1–5.

41] K. Tsuruya, M. Furuichi, Y. Tominaga, M. Shinozaki, M. Tokumoto, T. Yoshim-itsu, K. Fukuda, H. Kanai, H. Hirakata, M. Iida, Y. Nakabeppu, Accumulation of8-oxoguanine in the cellular DNA and the alteration of the OGG1 expression

during ischemia-reperfusion injury in the rat kidney, DNA Repair (Amst.) 2(2003) 211–229.

42] F.R. Greten, L. Eckmann, T.F. Greten, J.M. Park, Z.W. Li, L.J. Egan, M.F. Kagnoff,M. Karin, IKKbeta links inflammation and tumorigenesis in a mouse model ofcolitis-associated cancer, Cell 118 (2004) 285–296.

[

[

search 744 (2012) 172– 183 183

43] M. Jaiswal, N.F. LaRusso, L.J. Burgart, G.J. Gores, Inflammatory cytokines induceDNA damage and inhibit DNA repair in cholangiocarcinoma cells by a nitricoxide-dependent mechanism, Cancer Res. 60 (2000) 184–190.

44] G.L. Moldovan, B. Pfander, S. Jentsch, PCNA, the maestro of the replication fork,Cell 129 (2007) 665–679.

45] R. Vadlamudi, M. Mandal, L. Adam, G. Steinbach, J. Mendelsohn, R. Kumar, Reg-ulation of cyclooxygenase-2 pathway by HER2 receptor, Oncogene 18 (1999)305.

46] Y. Suzuki, H. Shimizu, T. Ishikawa, H. Sakaba, M. Fukumoto, H. Okonogi, M.Kadokura, Effects of prostaglandin E2 on the micronucleus formation in mousebone marrow cells by various mutagens, Mutat. Res. 311 (1994) 287–293.

47] A.S. Gidari, E.D. Zanjani, A.S. Gordon, Stimulation of erythropoiesis by cyclicadenosine monophosphate, Life Sci. II 10 (1971) 895–900.

48] W. Lippmann, Inhibition of prostaglandin E2-induced cyclic amp accumu-lation in the rat anterior pituitary by 11-deoxyprostaglandin E analogs(9-ketoprostynoic acids), Prostaglandins 10 (1975) 479–491.

49] S. Pelletier, J. Dube, A. Villeneuve, F. Gobeil Jr., Q. Yang, B. Battistini, G.Guillemette, P. Sirois, Prostaglandin E(2) increases cyclic AMP and inhibitsendothelin-1 production/secretion by guinea-pig tracheal epithelial cellsthrough EP(4) receptors, Br. J. Pharmacol. 132 (2001) 999–1008.

50] Y. Suzuki, R. Takagi, I. Kawasaki, T. Matsudaira, H. Yanagisawa, H. Shimizu, Themicronucleus test and erythropoiesis: effects of cyclic adenosine monophos-

phate (cAMP) on micronucleus formation, Mutat. Res. 655 (2008) 47–51.

51] I.N. Rich, W. Heit, B. Kubanek, Extrarenal erythropoietin production bymacrophages, Blood 60 (1982) 1007–1018.

52] A. Sica, P. Allavena, A. Mantovani, Cancer related inflammation: themacrophage connection, Cancer Lett. 267 (2008) 204–215.