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Toxicology in Vitro 21 (2007) 32–40 www.elsevier.com/locate/toxinvit 0887-2333/$ - see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2006.07.016 Experimental exposure of arsenic in cultured rat intestinal epithelial cells and cell line: Toxicological consequences Raj K. Upreti ¤ , A. Kannan, A.B. Pant Industrial Toxicology Research Centre, Biomembrane Toxicology Division, Mahatma Gandhi Marg, P.O. Box 80, Lucknow 226 001, India Received 16 May 2006; accepted 25 July 2006 Available online 25 August 2006 Abstract Arsenic is a naturally occurring metalloid and the drinking water contamination by inorganic arsenic remains a major public health problem. The trivalent arsenic (arsenite) is more toxic than the pentavalent form (arsenate), and is known to cause gastrointestinal toxic- ity. SpeciWc immortal cell lines are considered to be suitable for toxicity screening and testing of chemicals as they are easy to handle and possess most of the biochemical pathways present in the corresponding cells present in vivo. The present study was designed to evaluate and compare the in vitro toxicity of arsenite on rat intestinal epithelial cell line (IEC-6) and primary cultures of rat intestinal epithelial cells (IEC). To evaluate in vitro toxicity, cultures of IEC and IEC-6 cells were assessed for viability, morphometric analysis, membrane transport enzymes and structural constituents for membrane damage, dehydrogenase activity test for respiratory and energy producing processes and esterase activity test for intra and extra cellular degradation, following the post exposures to arsenite (0–20 ppm). SigniW- cantly similar concentration-dependent changes in these toxicity-screening parameters in IEC and IEC-6 were observed. Highest tested concentration of arsenite (20ppm) was found to be detrimental in both IEC and IEC-6. Furthermore, to evaluate arsenite toxicity in epi- thelial cells of rat intestine, intestinal loops were Wlled with arsenite solutions and incubated for 30 min in situ. In situ studies also showed a signiWcant arsenite concentration-dependent decline in epithelial cell membrane transport enzyme activities and total hexose and sialic acid contents. Concomitant release of membrane enzymes, hexose and sialic acid in the intestinal luminal Xuid following higher arsenite exposures further indicated partial membrane damage. Similar morphological changes in IEC and IEC-6 were also evident. These Wnd- ings also suggest that IEC-6 cell lines are suitable for initial screening of gastrointestinal cellular toxicity caused by arsenite. © 2006 Elsevier Ltd. All rights reserved. Keywords: Arsenic; Intestine; Epithelial cells; IEC-6; Gastrointestinal toxicity 1. Introduction The drinking water contamination by arsenic is a major health problem. Most cases of human toxicity from arsenic have been associated with exposure to inorganic arsenic. Inorganic arsenic (i-As) comprises two valence states, As(III) and As(V) and the trivalent arsenite form is more toxic than the pentavalent arsenate. Acute and chronic arsenic exposure via drinking water has been reported in many countries (Toxicological ProWle for Arsenic, 2005). Inorganic arsenic is known to cause irritation of stomach and intestine, with symptoms such as stomachache, nausea, vomiting and diarrhea. Prolonged ingestion can lead to car- diovascular disorders, liver and kidney injuries, neurologi- cal and skin disorders (Abernathy et al., 2003; Rossman, 2003; Bashir et al., 2006). Chronic ingestion in drinking water has been associated with increased incidence of human cancer (Jager and Wegman, 1997; Thomas et al., 2001). The main paths for human exposure to As are drink- ing water and foods; consequently, the intestinal epithelium is the Wrst physiological barrier to As metabolism and dis- tribution towards the tissues through the bloodstream. The literature is lacking in studies on i-As bio-availability in foods, and the only existing data in this sense are derived from research relating to i-As solubility in simulated gastrointestinal media (bio-accessibility) carried out by Laparra et al. (2003, 2004). * Corresponding author. Tel.: +91 522 2613786; fax: +91 522 2611547. E-mail address: upretirk@rediVmail.com (R.K. Upreti).

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Page 1: Experimental exposure of arsenic in cultured rat intestinal epithelial cells and cell line: Toxicological consequences

Toxicology in Vitro 21 (2007) 32–40www.elsevier.com/locate/toxinvit

Experimental exposure of arsenic in cultured rat intestinal epithelialcells and cell line: Toxicological consequences

Raj K. Upreti ¤, A. Kannan, A.B. Pant

Industrial Toxicology Research Centre, Biomembrane Toxicology Division, Mahatma Gandhi Marg, P.O. Box 80, Lucknow 226 001, India

Received 16 May 2006; accepted 25 July 2006Available online 25 August 2006

Abstract

Arsenic is a naturally occurring metalloid and the drinking water contamination by inorganic arsenic remains a major public healthproblem. The trivalent arsenic (arsenite) is more toxic than the pentavalent form (arsenate), and is known to cause gastrointestinal toxic-ity. SpeciWc immortal cell lines are considered to be suitable for toxicity screening and testing of chemicals as they are easy to handle andpossess most of the biochemical pathways present in the corresponding cells present in vivo. The present study was designed to evaluateand compare the in vitro toxicity of arsenite on rat intestinal epithelial cell line (IEC-6) and primary cultures of rat intestinal epithelialcells (IEC). To evaluate in vitro toxicity, cultures of IEC and IEC-6 cells were assessed for viability, morphometric analysis, membranetransport enzymes and structural constituents for membrane damage, dehydrogenase activity test for respiratory and energy producingprocesses and esterase activity test for intra and extra cellular degradation, following the post exposures to arsenite (0–20 ppm). SigniW-cantly similar concentration-dependent changes in these toxicity-screening parameters in IEC and IEC-6 were observed. Highest testedconcentration of arsenite (20 ppm) was found to be detrimental in both IEC and IEC-6. Furthermore, to evaluate arsenite toxicity in epi-thelial cells of rat intestine, intestinal loops were Wlled with arsenite solutions and incubated for 30 min in situ. In situ studies also showeda signiWcant arsenite concentration-dependent decline in epithelial cell membrane transport enzyme activities and total hexose and sialicacid contents. Concomitant release of membrane enzymes, hexose and sialic acid in the intestinal luminal Xuid following higher arseniteexposures further indicated partial membrane damage. Similar morphological changes in IEC and IEC-6 were also evident. These Wnd-ings also suggest that IEC-6 cell lines are suitable for initial screening of gastrointestinal cellular toxicity caused by arsenite.© 2006 Elsevier Ltd. All rights reserved.

Keywords: Arsenic; Intestine; Epithelial cells; IEC-6; Gastrointestinal toxicity

1. Introduction

The drinking water contamination by arsenic is a majorhealth problem. Most cases of human toxicity from arsenichave been associated with exposure to inorganic arsenic.Inorganic arsenic (i-As) comprises two valence states,As(III) and As(V) and the trivalent arsenite form is moretoxic than the pentavalent arsenate. Acute and chronicarsenic exposure via drinking water has been reported inmany countries (Toxicological ProWle for Arsenic, 2005).Inorganic arsenic is known to cause irritation of stomachand intestine, with symptoms such as stomachache, nausea,

* Corresponding author. Tel.: +91 522 2613786; fax: +91 522 2611547.E-mail address: [email protected] (R.K. Upreti).

0887-2333/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.doi:10.1016/j.tiv.2006.07.016

vomiting and diarrhea. Prolonged ingestion can lead to car-diovascular disorders, liver and kidney injuries, neurologi-cal and skin disorders (Abernathy et al., 2003; Rossman,2003; Bashir et al., 2006). Chronic ingestion in drinkingwater has been associated with increased incidence ofhuman cancer (Jager and Wegman, 1997; Thomas et al.,2001). The main paths for human exposure to As are drink-ing water and foods; consequently, the intestinal epitheliumis the Wrst physiological barrier to As metabolism and dis-tribution towards the tissues through the bloodstream. Theliterature is lacking in studies on i-As bio-availability infoods, and the only existing data in this sense are derivedfrom research relating to i-As solubility in simulatedgastrointestinal media (bio-accessibility) carried out byLaparra et al. (2003, 2004).

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R.K. Upreti et al. / Toxicology in Vitro 21 (2007) 32–40 33

Gastrointestinal tract represents the major site of expo-sure to chemicals through food and water ingestion andautomatically becomes important target organ as well as asite of access of toxicants into the organism. Althoughintestinal epithelium is equipped to metabolize most of thetoxicants and excrete them, but acute and/or chronic expo-sure of these chemicals may cause cellular damage and leadto a number of biological consequences. Oral uptake is oneof the major routes of exposure to toxic heavy metals suchas chromium and arsenic, which in turn can cause gastroin-testinal cellular toxicity (Shrivastava et al., 2002, 2003;Upreti et al., 2004). Much interest has now been focused ondeveloping in vitro toxicity test models evaluating their use-fulness in predicting toxicities. Recently, we have shownthat resident facultative bacteria isolated from rat gut canbe used as an alternate to animals for the preliminaryscreening of gastrointestinal tract cellular toxicity causedby heavy metals (Upreti et al., 2005a,b).

In last couple of decades, in vitro systems using cell linesand primary cultures have been accepted as screening toolsto understand the pharmacological and toxicological con-sequences in a rapid and reliable manner, since most of thebiochemical pathways are well expressing in them (Zucco,1993; Wils et al., 1994; Rossi et al., 1996; Sambruy et al.,2001). Although reports are available on arsenic cytotoxic-ity in cell lines (Bode and Dong, 2002), however the cellularkinetics comparing primary cultures of intestinal cells andimmortal intestinal cell line (IEC-6) is lacking. Thus, in thepresent investigation, experiments were carried out to studythe modulatory eVect of trivalent i-As on cellular kineticsand morphometric changes in primary cultures of rat intes-tinal epithelial cells and immortal rat intestinal cell line(IEC-6) along with a response comparison following in situexperimentation in rat intestinal epithelial cells.

2. Materials and methods

2.1. Chemicals and reagents

All the chemicals and reagents were purchased eitherfrom Sigma–Aldrich, E. Merck, Gibco, BRL, Hi-Media,India, otherwise stated. These were of analytical grade withhighest purity available. Sodium meta-arsenite (Sigma) wasused in experiments and arsenite solution containing vari-ous concentrations of As(III) was prepared either in dis-tilled water or in incomplete Dulbecco’s ModiWed Eagle’sMedium (DMEM).

2.2. Animals

Healthy adult male albino Wistar rats procured from theAnimal Breeding Facility of Industrial ToxicologyResearch Centre, Lucknow, were used for the isolation ofintestinal epithelial cells and for in situ studies. The animalswere housed individually under standard animal houseconditions with natural light/dark cycle and a temperatureof 25§2 °C. The standard animal food pellets and water

were given ad libitum. Clearance from the Animal EthicalCommittee of the Institute was obtained for the use of ani-mals.

2.3. Cell line (IEC-6)

IEC-6, normal rat small intestine cell line (ATCC CRL1592), was initially procured from National Centre for CellSciences, Pune, India and since then has been maintained atour Institute. Monolayers of cells were grown in DMEMsupplemented with 5% fetal bovine serum (FBS), 10�g/mlinsulin, 100 mg/l penicillin, 100 mg/l streptomycin and2.5 mg/l fungizone (Invitrogen, Groningen, The Nether-lands) at 37 °C in a humidiWed atmosphere of 5%CO2–95%air. Cells were screened for viability assessment usingtrypan blue dye exclusion staining method under a phase-contrast microscopy, prior to the start of every experiment.The batches showing more then 95% cell viability were usedin the study. All experiments were done on cells betweenpassages 10 and 25.

2.4. Isolation and culture of rat intestinal epithelial cells (IEC)

Intestinal epithelial cells were prepared by the method ofWeiser (1973). In brief, the small intestines were Xushedgently with normal saline containing 1.0 mM dithiothreitol.The cecal end of the intestine was ligated and solution ‘A’containing 1.5 mmol/l KCl, 96 mmol/l NaCl, 27 mmol/lsodium citrate, 8 mmol/l KH2PO4, 5.6 mmol/l Na2HPO4(pH 7.3) was Wlled after clamping the other end with arteryforceps. The intestine was immersed in solution ‘A’ andincubated at 37 °C for 15 min in a constant temperatureshaker bath. The intestine was emptied; Xuid discarded andwas Wlled with solution ‘B’ containing 1.5 mmol/l EDTAand 0.5 mmol/l dithiothreitol in PBS (pH 7.2) and immersedin solution ‘A’ for incubation. After incubation, the con-tents were emptied into a plastic centrifuge tube to recoverthe Wrst epithelial cell population. The process of Wllingwith solution ‘B’ and collecting the washing was repeated atdiVerent time periods of incubation (2, 2, 3, and 4 min,respectively) and fractions were pooled. Intestinal epithelialcells were collected by centrifugation (3000 rpm, 15 min at4 °C). Cells were washed twice with incomplete DMEM andthen re-suspended in DMEM supplemented with 5% fetalbovine serum (FBS), 10 �g/l insulin, 100 mg/l penicillin,100 mg/l streptomycin and 2.5 mg/l fungizone. Cells wereseeded in poly-L-lysine pre-coated Xasks and grown at37 °C in a humidiWed atmosphere of 5%CO2–95%air. AtconXuence, cells were expanded by passaging them to sub-sequent Xasks by trypsinization. Cells of 3–6 passages wereused in the experiments after ascertaining the cell viability.

2.5. Experimental protocol

Pre-conXuent growth were allowed for both rat intesti-nal epithelial cells and IEC-6 cell line cultures and then the

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34 R.K. Upreti et al. / Toxicology in Vitro 21 (2007) 32–40

medium was switched to a serum-free, phenol-red freemedium for 24 h. The medium was changed again andreplaced with fresh serum-free medium containing variousconcentrations of arsenite (1–20 ppm). Parallel-untreatedsets were run under identical conditions and served as con-trol. The cultures were analyzed for cytotoxicity, biochemi-cal marker enzymes and morphometric changes.

2.6. MTT assay

MTT (3-4,5-dimethyl thiazol-2-yl) 2,5-diphenyl tetrazo-lium bromide), a pale yellow substrate is converted into far-mazone, a violet compound by the activity of succinatedehydrogenase of mitochondria. Since the conversion takesplace in living cells, the amount of formazone produced isdirectly correlated with the number of viable cells present.The MTT assay was done following the method of Mos-mann (1983) with slight modiWcation. In brief, cells(1£104/well in 100�l medium) were seeded in 96 well plateand allowed to adhere for 24 h at 37 °C in 5%CO2–95%atmosphere. Medium was aspirated and replaced withmedium containing arsenite ranging from 1.0 ppm to20.0 ppm and incubated for 19 h at 37 °C. Then, 10 �l ofMTT (5 mg/ml stock solution) in phosphate buVered saline(PBS) was added to each well containing 100 �l of cell sus-pension and re-incubated for another 5 h at 37 °C. The reac-tion mixture was carefully taken out and 200�l of DMSOwas added to each well and mixed thoroughly. After 10 min,the color was read at 530 nm, using Multiwell MicroplateReader (Biotek, USA). The untreated sets were also runparallel under the identical conditions and served as con-trol. The data presented are the mean§SD from threeindependent experiments.

2.7. Morphometric analysis

Following the exposure of arsenite, live cells were exam-ined under the phase-contrast inverted microscope (Leica,Germany) to observe the exposure induced morphologicalchanges as well as the alteration in cell growth pattern, ifany. The changes were quantiWed using automatic imageanalysis software Lieca Q Win 500, hooked up with theinverted phase-contrast microscope and expressed in per-cent area covered under the microscopic Welds.

2.8. Dehydrogenase activity (DHA) test

Dehydrogenase activity was tested by slightly modiWedmethod as described by Liu (1985). In brief, cell suspensionsin 0.025 M isotonic phosphate buVer containing 1.6£106 cells/ml were prepared. Two milliliters cell suspension,1.0 ml resazurin (50 mg/l in phosphate buVer), and arsenitesolution to the Wnal concentrations of 0–10 ppm and steril-ized deionized water to a Wnal volume of 6.0 ml. Tubes with-out arsenite were considered as control. All the tubes wereincubated for 5 h at 20 °C. The test was stopped after 5 h byadding 0.25 ml 1.0% HgCl2 solution, followed by centrifu-

gation at 2500g for 25 min. DHA activity was determinedspectrophotometrically as the absorbance of resazurin inthe supernatant at 601 nm before incubation minus theabsorbance after incubation. Percent inhibition was calcu-lated from the photometric readings of samples, controlsand blanks.

2.9. Esterase activity (EA) test

Esterase activity test was determined following slightlymodiWed method described by Obst and Holzapfel-Pschorn(1988). Three milliliters cell suspension (1.6£ 106 cells/ml)was incubated with 0.01 ml Xuorescin diacetate (FDA) solu-tion (10 mg FDA/ml acetone), without or with diVerentconcentrations of arsenite and sterile deionized water to aWnal volume of 6.0 ml at 20 °C for 5 h under constant agita-tion. Reaction was stopped after 5 h by adding 3.0 ml ace-tone, followed by centrifugation at 2500g for 25 min.Esterase activity in supernatant was determined by the for-mation of free Xuorescin, which was measured spectropho-tometrically as the absorbance at 490 nm. A 1:1 mixture ofwater and acetone was used as a photometric blank. Per-cent inhibition was calculated from the photometric read-ings of samples, controls and blanks.

2.10. In vitro cell membrane studies

IEC and IEC-6 cultures were exposed to diVerent con-centrations of As(III) (0–10 ppm) at 37 °C for 24 h with con-stant shaking. Following incubation, cells were harvested,washed with 30 mmol/l tris-buVer containing 2.5 mmol/lEDTA, pH 8.1 and re-suspended in the same buVer undercold condition. All subsequent steps were also carried out at4 °C. Cells were disrupted by four 15 s bursts with ultrasonicprocessor and centrifuged at 450g for 10 min. To obtain themembrane fraction, the sub-fractionation was carried outaccording to Forstner et al. (1968). The membrane fractionwas suspended in a convenient volume of 2.5 mmol/l EDTA-buVer and used for estimation of biochemical endpoints.

2.11. In situ studies

Laparotomy on each rat was performed by midline inci-sion under light ether anesthesia. The intestine was exterior-ized, washed with normal saline, using a syringe and a bluntneedle, through two small cuts. One cut was made slightlydistal to the duodeno–jejunal junction and another at thedistal end of the ileum. After washing, the opening wasligated and 30-cm-length loop was prepared from the upperend of the intestine using sterile threads. Intestinal loopswere Wlled with diVerent concentrations of arsenite solutions(1–10 ppm) through the proximal opening, which was thenimmediately ligated. Control loops contained normal physi-ologic saline solution. The whole intestine was kept in situand the abdomen was closed. Proper breathing and anesthe-sia of the animal were maintained throughout the experi-ment. Intestinal loops were removed after 30 min in situ

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R.K. Upreti et al. / Toxicology in Vitro 21 (2007) 32–40 35

incubation. The luminal Xuid was collected and intestine wasplaced in ice-cold normal saline. After inverting the intestine,epithelial layer was scrapped oV gently with the help of aglass slide. The scraping was weighed, placed in 75 volumesof 5.0 mmol/l EDTA, adjusted to pH 7.4 with sodiumhydroxide, and homogenized. The brush border membrane(BBM) was prepared according to Forstner et al. (1968).

2.12. Enzyme assays and biochemical estimations

Alkaline phosphatase was determined according toWeiser (1973) and Ca2+–Mg2+-ATPase as described byHidalgo et al. (1983). Enzyme units were deWned as micro-moles of product formed or liberated per minute under theassay conditions. SpeciWc activity was expressed as units permilligram of protein. Protein was determined according toLowry et al. (1951) using bovine serum albumin as stan-dard. Carbohydrates (total hexoses) were estimated by theanthrone reagent method (Roe, 1955). Total lipid wasextracted according to Folch et al. (1957). Phospholipidswere quantiWed following digestion with 70% perchloricacid and estimated according to the method of Wagneret al. (1962). Cholesterol was estimated according to Zlatkiset al. (1953). The estimation of sialic acid was carried outaccording to the method of Warren (1959) using N-acetylnuraminic acid as standard.

2.13. Statistical analysis

The results were expressed as mean§SD and compari-sons were made with appropriate controls employingStudent’s ‘t’ test. Probability values of less than 0.05 wereconsidered signiWcant. The comparison of the dose-dependent eVects on diVerent endpoints between IEC andIEC-6 were also compared using the ANOVA statisticalapproach.

3. Results

3.1. EVects of arsenite on cell viability

Cell viability of IEC and IEC-6 following 24 h in vitroexposure to arsenite revealed a concentration-dependentreduction. Arsenite concentration at 10 ppm showed 45–50% non-viable cells in both types of cell cultures, whereas,20 ppm concentration caused more than 85% cell death(Fig. 1).

3.2. EVects of arsenite on DHA and EA activity

DHA and EA tests revealed an arsenite concentration-dependent inhibition in both IEC and IEC-6 as comparedto their respective controls. Inhibition of dehydrogenaseand esterase activities was found statistically insigniWcantat 1 ppm whereas, higher concentrations revealed signiW-cant inhibitions. In case of IEC, the decrease in DHA at 5and 10 ppm arsenite concentrations were 41% and 69% and

for IEC-6 it was 52% and 73%, respectively. A more or lesssimilar pattern of decrease in EA was observed in IEC andIEC-6 (Fig. 2).

3.3. In vitro eVect of arsenite on membrane enzymes and constituents

Cultures of IEC and IEC-6 cells grown in media without orwith arsenite for 24 h, revealed a concentration-dependent

Fig. 1. Cell viability of intestinal epithelial cells (IEC) and intestinal epi-thelial cell line (IEC-6) following 24 h in vitro exposure to arsenite. Valuesare mean § SD from three set of experiments.

Fig. 2. Dehydrogenase and esterase activity of intestinal epithelial cells(IEC) and intestinal epithelial cell line (IEC-6) following in vitro exposureof arsenite. Values are mean § SD from three set of experiments.*P < 0.05.

EA-test

0

20

40

60

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100

1 2 5 10As-concentration (ppm)

Per

cent

inhi

bitio

n

IEC

IEC-6

DHA-test

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1 2 5 10As-concentration (ppm)

Per

cent

inhi

bitio

n

IEC

IEC-6

**

**

**

**

*

**

*

Page 5: Experimental exposure of arsenic in cultured rat intestinal epithelial cells and cell line: Toxicological consequences

36 R.K. Upreti et al. / Toxicology in Vitro 21 (2007) 32–40

decrease in membrane enzyme alkaline phosphatase andCa2+–Mg2+-ATPase activities as compared to their respec-tive controls (Fig. 3). SigniWcant decrease of 28.5% and43.2% in alkaline phosphatase activity of IEC with 5 and10 ppm arsenite concentrations were evident. The respectivedecline were 36% and 65% in case of IEC-6 cell lines.Although the trend of the dose–response was similarbetween the IEC and IEC-6 for these endpoints but thecomparative decline were statistically higher in case of IEC-6. The inhibition of Ca2+–Mg2+-ATPase activity at 2, 5 and10 ppm concentrations were 26%, 41.3% and 66.2% for IEC;and 19.4%, 50.4% and 59.4% for IEC-6, respectively. Com-parisons made at each endpoint revealed statistically sig-niWcant similarity between IEC and IEC-6.

A concentration-dependent decline in membrane con-stituents viz. hexose and sialic acid was evident in both thetypes of cells as compared to their respective controls(Fig. 4). Hexose content at 5 ppm arsenite concentrationshowed signiWcant decline of 27.5% and 19.4% in IEC andIEC-6, respectively. At the highest tested concentration of10 ppm, the decrease in IEC and IEC-6 were 40.3% and51%, respectively. In case of sialic acid content, respectivesigniWcant decline of 28.3% and 36%, were evident only at10 ppm arsenite concentration. Similarly, cholesterol and

Fig. 3. In vitro eVect of arsenite on membrane enzymes of intestinal epithe-lial cells (IEC) and intestinal epithelial cell line (IEC-6). Cells wereexposed to various concentrations of arsenite at 37 °C for 24 h. Values aremean § SD from three set of experiments. *P < 0.05.

Alkaline phosphatase

0

1

2

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4

0 2 5 10As-concentration (ppm)

Sp.

act

ivity

(U

nits

/mg

prot

ein)

IEC

IEC-6

*

**

*

Ca-Mg-ATPase

0

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0.2

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0 2 5 10As-concentration (ppm)

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(U

nits

/mg

prot

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IEC

IEC-6

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*

phospholipids, the major structural constituents of mem-brane, also revealed concentration-dependent decreases inthe IEC and the IEC-6 cell lines. SigniWcant decline of cho-lesterol and phospholipids in both types of cells at 5 ppmarsenite concentration was in the range of 22–27% and at10 ppm concentration, it was in the range of 37–47%. How-ever, in both types of cells the C:P ratio did not change inall the tested concentrations of arsenite (Fig. 5). Statisticalanalysis between IEC and IEC-6 at each endpoint of indi-vidual parameter revealed that the respective declines weresigniWcantly similar.

Fig. 4. In vitro eVect of arsenite on membrane hexose and sialic acid con-tents of intestinal epithelial cells (IEC) and intestinal epithelial cell line(IEC-6). Values are mean § SD from three set of experiments. *P < 0.05.

Hexose

0

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Control 2 ppm 5 ppm 10 ppm

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*

Sialic acid

0

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60

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IEC IEC-6ug

/ m

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otei

nControl 2 ppm 5 ppm 10 ppm

**

Fig. 5. In vitro eVect of arsenite on membrane cholesterol and phospho-lipid contents of intestinal epithelial cells (IEC) and intestinal epithelialcell line (IEC-6). Values are mean § SD from three set of experiments.*P < 0.05.

0

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40

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Control 2 ppm 5 ppm 10 ppm

Chol. Phos. Chol. Phos.IEC IEC-6

*

*

** *

* **

Page 6: Experimental exposure of arsenic in cultured rat intestinal epithelial cells and cell line: Toxicological consequences

R.K. Upreti et al. / Toxicology in Vitro 21 (2007) 32–40 37

A quantitative comparison done by calculating theEC50 of each endpoint between the two types of cells issummarized in Table 1. The calculated arsenite EC50 for alltested endpoints were in the range of 4.97–5.55 ppm. Therewas no signiWcant diVerence in EC50 between the two typesof cells.

3.4. In situ eVect of arsenite on IEC

A concentration-dependent decrease of 20%, 35%, 50%and 62% in BBM alkaline phosphatase activity wasobserved with arsenite concentrations of 1, 2, 5, and10 ppm, respectively. Similarly, the decline in Ca2+–Mg2+-ATPase activity was 13%, 21%, 34% and 49%, respectively(Fig. 6). BBM hexose and sialic acid contents also revealedarsenite concentration-dependent decline, which were sta-tistically signiWcant at 5 and 10 ppm concentrations. At10 ppm concentration, the decline in hexose and sialic acidcontents was 62% and 42%, respectively (Fig. 7). Con-versely, the cholesterol and phospholipids content of BBMdid not reveal signiWcant changes.

3.5. Morphometric analysis

Highlights of the morphometric analysis are summarizedin Fig. 8. The adverse eVects on cell physiology were appar-

Table 1A comparative proWle of EC50 for IEC and IEC-6 membrane enzymesand constituents following in vitro exposure of arsenite

Values are mean § SD from three experiments.

Parameter EC50 (ppm)

IEC IEC-6

Alk. Phosphatase 5.00 § 0.41 5.25 § 0.43Ca2+–Mg2+-ATPase 5.16 § 0.37 5.23 § 0.52Hexose 5.55 § 0.40 5.18 § 0.47Sialic acid 5.05 § 0.50 5.23 § 0.86Cholesterol 5.20 § 0.51 4.97 § 0.53Phospholipid 5.03 § 0.37 5.00 § 0.43

Fig. 6. In situ eVect of arsenite on rat intestinal epithelial cell membraneenzymes. Values are mean § SD from three rats. *P < 0.05.

0

1

2

3

4

5

Control 1 2 5 10

As-concentration (ppm)

Alk

. Pta

se, S

p. a

ct. (

Uni

ts/m

g pr

otei

n)

0

0.1

0.2

0.3

0.4

Ca-M

g-AT

Pase

Alk.Ptase

Ca-Mg-ATPase

*

*

*

*

*

*

ent at 5 ppm arsenite concentration in both IEC-6 and IECcells, respectively (81% and 74% area of control). This wasfurther intense with the increase in arsenite concentrationsviz., 64% and 56% of control at 10 ppm and 31% and 23% ofcontrol at 20 ppm in IEC-6 and IEC cells, respectively. Thetotal covered area analysis indicated that the exposures at5 ppm and higher concentrations were able to pose the sta-tistically signiWcant reduction in cell size and mitotic indexas evidenced by the formation of protuberances and spindleshaped structures. The concentrations of arsenite at 1 and2 ppm did not show signiWcant cytotoxic eVects (Fig. 9).

4. Discussion

The signiWcant Wnding of the present study is the similari-ties in the pattern of majority of in vitro toxic eVects of arse-nite on the IEC-6 cell lines and the intestinal epithelial cellsof rats. The MTT assay, which reXects both growth and via-bility of cell population revealed a similar concentration-dependent cell viability pattern in primary cultures of ratintestinal epithelial cells and IEC-6 cell lines following arse-nite incubation for 24 h. Susceptibility to the cytotoxic eVectsof arsenicals and their metabolism in cultured rat andhuman cells and cell lines of diVerent origin have been stud-ied and a concentration-dependent decrease in the rate ofMTT conversion have been shown (Yamauchi and Fowler,1994; Huang and Lee, 1996; Styblo et al., 2000). In the pres-ent study, the exposure to lower concentrations of arsenitein IEC and IEC-6 caused only a transient inhibition of for-mazan formation by mitochondrial dehydrogenases withoutapparent changes in cell morphology. However, in bothtypes of cells exposed to high concentrations of arsenite,MTT assay and microscopic analysis showed signiWcantchanges that likely reXect the decrease in cell viability. Expo-sure to 10 ppm arsenite decreased the rate of MTT conver-sion by 40% and 45% in IEC and IEC-6, respectively.Concomitantly, changes in cell morphology such as rounding

Fig. 7. In situ eVect of arsenite on rat intestinal epithelial cell membranehexose and sialic acid contents. Values are mean § SD from three rats.*P < 0.05.

0

100

200

300

400

Control 1 2 5 10

As-concentration (ppm)

Hex

ose

ug /

mg

prot

ein

0

20

40

60

80

Sialic acid

Hexose

Sialic acid

* *

**

Page 7: Experimental exposure of arsenic in cultured rat intestinal epithelial cells and cell line: Toxicological consequences

38 R.K. Upreti et al. / Toxicology in Vitro 21 (2007) 32–40

and shrinking of cells and granulation of cell cytoplasm alsooccurred. Similar cell viability and proliferation changes inboth types of cells also indicate that methylation of arsenite,

which aVects the clearance of arsenite from cells, could beacting in similar manner. Although the methylation capacitydoes not protect cells against the acute cytotoxic eVects of

Fig. 8. Representative microphotographs of intestinal epithelial cells (IEC) and intestinal epithelial cell line (IEC-6) demonstrating the morphologicalalterations and growth pattern following the exposure of arsenite. Original magniWcation is 400£.

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R.K. Upreti et al. / Toxicology in Vitro 21 (2007) 32–40 39

arsenite, it may provide partial protection in cells exposed torelatively low concentrations (Styblo et al., 2000).

Dehydrogenase activity, in general, is linked to the respi-ratory and energy producing processes in the cell. Whereas,esterase activity is involved in intra- and extra cellular deg-radation of organic substances and considered as indicatorof general heterotrophic activity of the cell. An inhibition ofDHA and EA represents intracellular toxicity. Theseparameters have been used to study the toxicity of chemi-cals (Torslov, 1993; Upreti et al., 2005b). We observed thatarsenite concentration of 2 ppm and more, inhibited DHAand EA signiWcantly in both cell types. The dose–responsesfor the endpoints were also found to be statistically similarbetween the IEC and IEC-6. These Wndings suggest thatmode of arsenicals intracellular interaction; metabolismand toxic insult responses may be similar in both types ofcells. It has also been shown and discussed that methylatedarsenicals generated during the metabolism of arsenic in thecell, interact with cytosolic proteins and produces some ofthe toxic eVects (Thomas et al., 2001). However, a separatestudy to explore the role of methylated arsenicals in theinduction of oxidative stress and the glutathione pathwaysin IEC and IEC-6 cell line is required.

Intestine forms the initial site of exposure to arsenic andthe epithelial cells are the Wrst sites of its interaction. Epi-thelial cell membrane enzymes viz. alkaline phosphataseand Ca2+–Mg2+-ATPase are chieXy associated in uptakeand exchange of diVerent ions (Mukherjee et al., 1992).Concentration-dependent inhibition of alkaline phospha-tase and Ca2+–Mg2+-ATPase following arsenite exposureas observed in the present investigation in both types ofcells could lead to the impairment of uptake and transportof vital ions, which in turn may be responsible for arsenictoxicity. The concentration-dependent inhibition patternsof these membrane marker enzymes and calculated EC50were found to be similar in IEC and IEC-6 cells followingin vitro arsenite exposures. However, while comparing thetwo types of cells using statistical approach, it was observedthat inhibition of alkaline phosphatase at 5 and 10 ppmconcentrations were higher in IEC-6 as compared to IEC. Itis possible that alkaline phosphatase of IEC-6 is compara-tively more susceptible to higher concentrations of arsenite.

Fig. 9. Morphometric analysis of cultured intestinal epithelial cells (IEC)and intestinal epithelial cell line (IEC-6) following the exposure of variousconcentrations of arsenite. Values are presented as percent control areacovered in the microscopic Welds. Other details are as given in the text.*P < 0.05.

0204060

80100120

1 2 5 10 20

As-concentration (ppm)

Are

a (%

Con

trol

) IEC-6 IEC

**

*

**

*

Whereas, the dose–responses of Ca2+–Mg2+-ATPase weresigniWcantly similar for the both cell types.

The protein bound sialic acid and hexose are consideredas important membrane constituents of intestinal cell liningrequired for its normal functioning and the mucus glyco-protein is determined in terms of hexose content (Gotts-chalk, 1960; Murakami et al., 1988). Reduced levels ofhexose and sialic acid content following arsenite exposureas observed in IEC and IEC-6 suggests an alteration in theintegrity of membrane. The generation of reactive oxygenspecies and its role in the toxicity of inorganic arsenic iswell known (Bernstam and Nriagu, 2000; Kitchin andAhmad, 2003; Shi et al., 2004). The sugars, including sialicacid have been shown to scavenge the hydroxyl radicalsformed in the lumen, thus protecting it from the oxidativestress (Grisham et al., 1987). During the process hydroxylradical destroys the sugar to give various products, thusloosing its visco-elasticity. The decrease of sialic acid con-tent following arsenite exposure may be due to loss of elas-ticity of these epithelial cells. The concentration ofimportant structural constituents cholesterol and phospho-lipids in the membrane aVects its various physical parame-ters including the membrane Xuidity. The in vitro exposureof arsenite in the present study resulted signiWcant concen-tration-dependent decrease of cholesterol and phospholipidcontents in IEC and IEC-6 cells vis-à-vis, further suggestsan altered Xuidity of membrane. Furthermore, a quantita-tive comparison of calculated arsenite EC50 of each end-point suggested signiWcant similarities between the twotypes of cells.

The in vitro Wndings on the eVect of arsenite on IECmembrane enzymes and constituents were further validatedfollowing in situ exposures using rat intestinal loop model.A comparative analysis revealed more or less similar con-centration-dependent decline in IEC membrane alkalinephosphatase, Ca2+–Mg2+-ATPase, hexose and sialic acidcontents. In addition, a signiWcant release of these enzymesand constituents in luminal Xuid were also evident (datanot shown), which further indicates the partial damage ofmembrane.

In general, in vitro models employing speciWc cells in cul-ture including various cell lines have been used for thestudy of toxicity and metabolism of xenobiotics. Similari-ties in majority of tested responses and eVects of arsenitebetween IEC and IEC-6 cell line as observed in the presentstudy suggest that the intestinal epithelial cell line (IEC-6)can be used for initial screening of gastrointestinal cellulartoxicity caused by arsenicals. Whether the IEC-6 can beused as for other metals need to be examined.

Acknowledgements

The authors are grateful to The Director, ITRC, Luc-know, for his keen interest in the study. We thank Mr.Neeraj Mathur, Scientist, ITRC, Lucknow for statisticalanalysis. This work was supported by Net Work project ofCSIR, New Delhi, India.

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40 R.K. Upreti et al. / Toxicology in Vitro 21 (2007) 32–40

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