acrylonitrile-induced toxicity and oxidative stress in isolated rat colonocytes

Environmental Toxicology and Pharmacology 19 (2005) 371–377

Acrylonitrile-induced toxicity and oxidative stress inisolated rat colonocytes

Ahmed M. Mohamadina, Ebtehal El-Demerdashb, Hesham A. El-Beshbishya,Ashraf B. Abdel-Naimb,∗

a Department of Biochemistry and Tumor Marker Oncology Research unit, Faculty of Pharmacy, Al-Azhar University, Cairo, Egyptb Department of Pharmacology and Toxicology, Faculty of Pharmacy, Ain Shams University, Abbasia, Cairo, Egypt

Received 4 May 2004; accepted 3 September 2004Available online 2 November 2004

Abstract

Acrylonitrile (ACN), an environmental toxic pollutant, has been detected in drinking water, food products and occupational environment.T culturedr tes werei ility andl roxidation asi ed nearlya nt decreasei pendent onb (p entd eated cells.A ited LDHl DFO), ani xic effecti ortant rolea©

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he objective of the present work was to investigate the cytotoxic effects as well as the oxidative stress induced by ACN inat colonocytes. Colonocytes were exposed in vitro to different concentrations of ACN (0.1–2.0 mM) for 60 min. Also, colonocyncubated with ACN (1.0 mM) for different time intervals extending to 180 min. Cytotoxicity was determined by assessing cell viabactate dehydrogenase (LDH) release. Oxidative stress was assessed by determining reduced glutathione (GSH) level and lipid pendicated by thiobarbituric acid reactive substances (TBARS) production. Exposure of colonocytes to ACN (1.0 mM) for 60 min caus

50% decrease in cell viability and induced a 2.5-fold increase of LDH leakage. In the same experiment, ACN caused a significan cellular GSH content as well as a significant enhancement of TBARS accumulation. These toxic responses to ACN were deoth concentration and duration of exposure to ACN. There was a good correlation between LDH release and TBARS formationr2 = 0.97,< 0.05). Treatment of colonocytes with GSH,N-acetyl-l-cysteine (NAC) or dithiothreitol (DDT) prior to exposure to ACN afforded differegrees of protection as indicated by significant decrease in the LDH leakage and TBARS formation as compared to ACN alone-trlso, pretreatment of colonocytes with the antioxidant enzyme superoxide dismutase (SOD) or catalase (CAT) significantly inhib

eakage and TBARS production. Preincubation with dimethyl sulfoxide (DMSO), a hydroxyl radical scavenger or desferroxiamine (ron chelator, diminished ACN-induced LDH leakage and TBARS generation. Our results suggest that ACN has a potential cytoton rat colonocytes; and thiol group-donors, antioxidant enzymes, hydroxyl radical scavengers and iron chelators can play an impgainst ACN-induced colonotoxicity.2004 Elsevier B.V. All rights reserved.

eywords:Acrylonitrile; Colonocytes; Oxidative stress

. Introduction

Acrylonitrile (ACN) is an extensively produced aliphaticitrile that is used in the synthesis of acrylic fibers, resinsnd plastics (IARC, 1979). It is also used in the manufacturef soft prosthesis material (Parker and Bradern, 1990), highermeable dialysis tubing (Ward et al., 1993) and medicalloves (Walsh et al., 2004). ACN has been detected in drink-

∗ Corresponding author. Tel.: +20 10 5182741; fax: +20 2 6831492.E-mail address:[email protected] (A.B. Abdel-Naim).

ing water (Rubio et al., 1990), food products (Ventura et al.2004), occupational environment (Ochiai et al., 2003) andcigarette smoke (Nazaroff and Singer, 2004). Animal studiesperformed on male and female rats indicated that ACNcarcinogen (Maltoni et al., 1988). Epidemiological studieconducted on workers exposed to ACN indicated increincidence of colon cancer (O’Berg, 1980) and stomach canc(Delzell and Monson, 1982).

Target organs for ACN toxicity in animals include tgastrointestinal tact, zymbal gland and brain (Maltoni etal., 1977). Quast et al. (1980)reported that rats chronica

382-6689/$ – see front matter © 2004 Elsevier B.V. All rights reserved.oi:10.1016/j.etap.2004.09.004

372 A.M. Mohamadin et al. / Environmental Toxicology and Pharmacology 19 (2005) 371–377

exposed to ACN developed gastrointestinal tumors. Kineticstudies byFarooqui and Ahmed (1983)indicated that ACNmolecules irreversibly bind to macromolecules of varioustissues in rats and the gastrointestinal tract was a major targetfor ACN molecular interaction. The gastrointestinal tractis well endowed with the enzymatic machinery necessaryto form large amounts of reactive oxygen species (ROS)(Grisham and Granger, 1988). Rat colon is particularlysubject to oxidative stress induced by several factorsincluding polymorphonuclear leukocytes infiltration andischemia/reperfusion (Weiss, 1986; Bhaskar et al., 1995).In addition, sources of ROS in the colon include mucosaloxidases such as xanthine oxidase and aldehyde oxidase(Krenitsky et al., 1974). Generation of free radicals has beenimplicated in the activation of ACN and other nitriles tothe very toxic metabolite cyanide (Mohamadin et al., 1996;Abdel-Naim and Mohamadin, 2004). Therefore, it is hypoth-esized that ACN-induced oxidative stress and generation ofROS would activate the chemical to cyanide. ACN is knownto induce GSH depletion and the consequences of oxidativestress in different organs in the rat (Kedderis et al., 1995).GSH depletion was reported to potentiate ACN-inducedgastric DNA damage in rats (Ahmed et al., 1996).

Although, the colon is a potential target for ACN toxicity(Farooqui and Ahmed, 1983; Jacob and Ahmed, 2003), littlei ob-j ntialc d byA

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contents with saline (37◦C). Then, the lumen was filled withDPBS containing 0.25% BSA and 5 mmol/L EDTA-Na2 andboth ends were ligated. The ligated colon was placed in a flaskcontaining 100 ml-oxygenated DPBS for 45 min at 37◦C ina water bath with shaking at 60 cycles/min. The luminal fluidwas then drained into a polystyrene tube and the colon was ev-erted onto a glass rod. Additional cells were isolated from themucosa into the tube with the luminal fluid by gently stirringfor a period of 30 s. The cells were then counted and viabilitywas determined by using trypan blue exclusion method. Cellsyield was more than 90% viable.

2.3. Treatments

Cytotoxic effect of ACN was evaluated in a dose–responseas well as a time–course experiment. In the dose–responseexperiment, colonocytes were exposed to different concen-trations of ACN (0.1, 0.5, 1, 1.5 and 2 mM) for 60 min. ACNwas diluted in DPBS to the required concentrations. In thetime–course experiment, colonocytes were incubated withACN (1 mM) for different time intervals (0, 30, 60, 120,180 min).

Cytotoxicity was determined by assessing cell viabilityand lactate dehydrogenase release (LDH). Oxidative stressinduced by ACN was determined by assessing GSH level andl Int rein-c ),D T( thea tentw ectsw pro-d

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s known about the ACN colonotoxicity. Therefore, theective of the present study was to investigate the poteytotoxic effects as well as the oxidative stress induceCN in cultured rat colonocytes.

. Materials and methods

.1. Animals and chemicals

Sprague–Dawley rats of either sex (130–150 g) were uCN, bovine serum albumin (BSA), catalase from bov

iver (CAT), cholral hydrate, dithiothreitol (DTT), desferriomine (DFO), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNBimethyl sulfoxide (DMSO), Dulbecco’s phosphate buffealine (DPBS), ethylene diamine tetra-acetic acid disodalt (EDTA-Na2), N-acetyl-l-cysteine (NAC), reduced glathione (GSH), superoxide dismutase from bovine eryytes (SOD), 1,1,3,3-tetramethoxypropane, thiobarbicid and Triton X-100 were obtained from Sigma–Aldrhemical Co. (St. Louis, MO, USA). All other chemicere of analytical grade.

.2. Isolation of colonocytes

Rat colonocytes (colonic epithelial cells) were isolatedng a modification of the procedure originally describedoediger and Truelove (1979). Briefly, over night fasted ran= 3–4) were anesthetized with cholral hydrate (300 mg.p.), abdomen was opened and the colons were rapidlyected out (cecum to rectum) and flushed clear of lum

ipid peroxidation as indicated by formation of TBARS.he mechanistic part of our study, colonocytes were pubated with GSH (1 mM), NAC (1 mM), DTT (1 mMMSO (80 mM), DFO (10 mM), SOD (500 U/ml) or CA

1500 U/ml). All pretreatments were done 30 min beforeddition of ACN and their concentrations were consisith those in the literature. The potential protective effere evaluated by assessing LDH leakage and TBARSuction by colonocytes after 180 min of incubation.

.4. Assay of LDH leakage

The activity of the cytosolic enzyme LDH was esated according to the method described byWroblewski andadue (1955)by assessing the rate of conversion of NA1.5 mmol/L) to NAD+ in the presence ofl(+)-lactic acid50 mmol/L) in culture supernatants (S), and in the remng cells (C) after lysis with Triton X-100. The percentageDH leakage was calculated as follows:

leakage= S

S + C× 100

.5. Assay for cellular GSH

Reduced GSH levels in colonocytes were determineeasuring total soluble-reduced sulfhydryl content. A

ncubation, cells were washed three times with phospuffered saline (PBS), and then, 0.7 ml of 0.2% Triton00 and 2.5% sulfosalicylic acid in PBS was added. S

ions were centrifuged at 3000×g for 5 min. A 0.5 ml-aliquo

A.M. Mohamadin et al. / Environmental Toxicology and Pharmacology 19 (2005) 371–377 373

of the acid-soluble supernatant medium was then added to1.0 ml of 0.3 M Na2HPO4 solution. Spectrophotometric de-terminations were performed at 412 nm immediately after theaddition of 0.125 ml of 5,5′-dithiobis (2-nitrobenzoic acid)(40 mg/100 ml in 1% sodium citrate) (Beutler et al., 1963).

2.6. Lipid peroxidation assay

Lipid peroxidation was assessed by determining TBARSby the method ofUchiyama and Mihara (1978). Test solutionsobtained from cultured cells were cooled to 4◦C and cen-trifuged for 10 min at 1000×g. Aliquots (750�l) of the su-pernatant were combined with an equal volume of cold 12%trichloroacetic acid and centrifuged for 10 min at 1000×gat 4◦C to remove precipitated protein. One ml of supernatantwas added to 1 ml thiobarbituric acid reagent (0.6% thio-barbituric acid in 0.1N NaOH), and the mixture was heatedat 100◦C for 20 min. The mixture was allowed to cool andTBARS were extracted with 3 ml of 1-butanol. Appropriateblanks were concurrently made. A 1.0 mM stock solution of1,1,3,3-tetraethoxypropane in water was diluted in variousamounts of 0.01 N HCl to produce malondialdehyde (MDA);these solutions were used as TBARS standards. The 1-butanolfractions and MDA standards were determined at wavelengthof 553 nm. TBARS content was always expressed as nmol/mgpa

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Fig. 1. (A) Dose–response effect of ACN on colonocytes viability after a60 min incubation time; * significantly different from control atp< 0.05. (B)Time–course effect of ACN (1 mM) on colonocytes viability; *significantlydifferent from corresponding control atp< 0.05.

ACN. The chemical caused a significant and time-dependentdecrease in cell viability starting from 60 min after beginningof incubation and showed maximal cytotoxicity at 180 min(28%).

Plasma membrane damage was assessed by monitoringLDH leakage from colonocytes exposed to ACN. Incuba-tion of colonocytes with different concentrations of ACN(0.1–2.0 mM) for 60 min showed a significant increase inLDH leakage compared to the control incubations. This ef-fect was concentration-dependent and in comparison to thebasal LDH leakage observed in the control incubations, theenzyme leakage was progressively elevated to 2.5-folds (for1.0 mM ACN) and finally to five-folds (for 2.0 mM ACN)(Fig. 2A). The time–course effects of 1.0 mM ACN on LDHrelease from colonocytes are shown inFig. 2B. LDH leakagewas significantly elevated at all time points. At 180 min afterACN addition to incubations, the percentage of LDH releasewas maximal and mounted to approximately four-folds thatof the corresponding control value.

3.2. Assessment of oxidative stress-induced by ACN

Incubating colonocytes in the presence of different ACNconcentrations (0.1–2.0 mM) resulted in an observable lossof cellular GSH (Fig. 3A). However, the decrease in GSHl han0 ono-c SH

rotein. Protein was determined by the method ofLowry etl. (1951)with bovine serum albumin as the standard.

.7. Data analysis

The GraphPad Prism® (version 1.02) computer prograGraphPad Software Incorporated) was used to conduression analysis and to plot collected data. Data arressed as means± S.E.M. Individual group comparisoere conducted using the two-tailed unpaired Student’st-test.ultiple group comparisons were carried out using onenalysis of variance (ANOVA) followed by Dunnet’s testost-hoc analysis. Statistical analyses were performedoftware program SPSS® for Windows (version 8). The 0.0

evel of probability was used as the criterion for significan

. Results

.1. Assessment of ACN cytotoxicity

Cell survival was assessed by trypan blue excluethod after exposing rat colonocytes to ACN (0.1–2.0 m

ncubation of colonocytes for 60 min with ACN (at concrations higher than 0.1 mM) showed significant decreasell viability in a concentration-related manner. Also, An a concentration of 1.0 mM reduced the viability to apprmately 50% (Fig. 1A). Therefore, subsequent time–couxperiments and mechanistic experiments were perfosing this concentration. The data inFig. 1B illustrate the

ime course of viability for colonocytes exposed to 1.0 m

evel was significant only at ACN concentrations higher t.5 mM. In the time–course experiment, exposure of colytes to 1.0 mM ACN significantly reduced cellular G


Fig. 2. (A) Dose–response effect of ACN on LDH leakage from colono-cytes after a 60 min incubation time; * significantly different from controlat p< 0.05. (B) Time–course effect of ACN (1 mM) on LDH leakage fromcolonocytes; * significantly different from corresponding control atp< 0.05.

Fig. 3. (A) Dose–response effect of ACN on GSH level in colonocytes aftera 60 min incubation time; * significantly different from control atp< 0.05.(B) Time–course effect of ACN (1 mM) on GSH level in colonocytes; *significantly different from corresponding control atp< 0.05.

Fig. 4. (A) Dose–response effect of ACN on lipid peroxidation in colono-cytes after a 60 min incubation time; * significantly different from controlatp< 0.05. (B) Time–course effect of ACN (1 mM) on lipid peroxidation incolonocytes; * significantly different from corresponding control atp< 0.05.

levels at all time intervals, compared to corresponding controlvalues. GSH depletion was maximal at 180 min after ACNaddition (58% of the control value) (Fig. 3B).

The effect of various concentrations of ACN (0.1–2.0 mM)on lipid peroxidation, as indicated by TBARS formation, wasestimated.Fig. 4A shows a significant and concentration-related increase of TBARS production in colonocytes as com-pared with the control value. In the time–course experiment,ACN (1.0 mM) resulted in a significant increase in the pro-duction of TBARS in colonocytes, which occurred early at30 min of incubation (339% of control value) and reachedits maximum level after 180 min (443% of control value)(Fig. 4B). In addition, the same experiment indicated that theincrease in TBARS formation significantly correlated withLDH leakage. The correlation coefficient was found to be0.97 atp< 0.05 (Fig. 5).

3.3. Assessment of potential protective effects ofdifferent antioxidants

The protective effects of different thiol-containing com-pounds on ACN-induced LDH release and lipid peroxidationin colonocytes are illustrated inTable 1. All of the testedcompounds (GSH, NAC and DTT) at a molar concentrationequivalent to that of ACN (1.0 mM) could significantly re-d ively,a ilarp o ob-s 9%

uce LDH release by about 48, 59 and 53%, respects compared with ACN alone-treated incubations. Simrotective effects offered by these compounds were alserved on ACN-induced TBARS production (43, 60 and 4


Table 1Protective effects of thiol-containing compounds on ACN-induced LDH leakage and lipid peroxidation in colonocytes

Addition LDH leakage (% of total) TBARS (nmol/mg protein)

None (control) 18.6± 1.01 0.83± 0.01ACN (1.0 mM) 70.8± 5.20a 4.56± 0.12a

ACN + GSH (1.0 mM) 36.6± 1.40a,b 2.62± 0.09a,b

ACN + NAC (1.0 mM) 29.3± 1.12a,b 1.83± 0.08a,b

ACN + DTT (1.0 mM) 33.2± 2.00a,b 2.34± 0.12a,b

Data are shown as mean± S.E.M. of six incubations. GSH, reduced glutathione; NAC,N-acetyl-l-cystiene; DTT, dithiothreitol. All thiol compounds wereadded 30 min before the addition of ACN. LDH and TBARS were determined 180 min after the addition of ACN.

a Significantly different from control group atp< 0.05.b Significantly different from ACN alone-treated group atp< 0.05.

Fig. 5. Correlation between ACN-induced LDH leakage and lipid peroxi-dation in colonocytes.

for GSH, NAC and DDT, respectively). However, pretreat-ment with thiol-containing compounds did not restore thebasal levels of LDH release or lipid peroxidation.

The potential protective effects of antioxidant enzymes(SOD and CAT) as well as the hydroxyl radical scavengerDMSO and the iron chelator DFO against LDH leakage andlipid peroxidation in colonocytes exposed to 1.0 mM ACN for180 min were also evaluated (Table 2). It was found that pre-treatment of colonocytes with either SOD (500 U/ml) or CAT(1500 U/ml) significantly inhibited LDH leakage by approx-imately 23 and 54%, respectively as compared with ACNalone-treated cells. Also, both of two antioxidant enzymessignificantly diminished the TBARS production-induced byCAN by 17 and 45%, respectively. Pretreatment of colono-

Table 2Protective effects of SOD, CAT, DMSO and DFO on ACN-induced LDH leak

Addition LDH leakage (% in)

None (control) 18.6± 1.01ACN (1.0 mM) 70.8± 5.20a

ACN + SOD (500 U/ml) 54.6± 3.60a,b

ACN + CAT (1500 U/ml) 32.7± 1.70a,b

ACN + DMSO (80.0 mM) 53.2± 2.60a,b

ACN + DFO (10.0 mM) 49.6± 3.10a,b

Data are shown as mean± S.E.M. of six incubations. SOD, superoxide dism ine. Allantioxidants were added 30 min before the addition of ACN. LDH and TBAR

cytes with either DMSO or DFO diminished ACN-inducedLDH leakage by 25 and 30% of total leakage, respectively,and TBARS production by 41 and 47%, respectively. How-ever, none of the used protectors could restore LDH leakageor TBARS to control values.

4. Discussion

Although the gastrointestinal tract is one of the most im-portant target organs for ACN (Farooqui and Ahmed, 1983;Jacob and Ahmed, 2003), there is a scanty of informationregarding ACN colonotoxicity. It is known that colonic ep-ithelial cells are likely to be exposed to oxidative damage byfree radicals generated within both the mucosa (from infil-trating phagocytes) and the lumen (from drugs and bacterialmetabolites) (Bhaskar et al., 1995). In the present study, thepotential cytotoxic effects as well as the oxidative stress in-duced by ACN in primary culture of rat colonocytes wereevaluated.

Our data indicated that ACN significantly decreasedcolonocytes viability and increased LDH leakage. ACN con-centration in culture media ranged from 0.1 to 2.0 mM and aconcentration of 1 mM was chosen for all subsequent stud-ies. These concentrations are relatively high compared toe rele-v andm rl ayr om-

a Significantly different from control group atp< 0.05.b Significantly different from ACN alone-treated group atp< 0.05.

age and lipid peroxidation in colonocytes

of total) TBARS (nmol/mg prote

0.83± 0.014.56± 0.12a

3.78± 0.10a,b

2.52± 0.09a,b

2.67± 0.162.44± 0.10a,b

utase; CAT, catalase; DMSO, dimethyl sulfoxide; DFO, desferrioxamS were assayed 180 min after the addition of ACN.

xpected environmental exposure. However, they areant to those used in previous studies on ACN toxicityetabolism by rat hepatocyte (Geiger et al., 1983). The uppe

imit of the used range (2 mM) was relatively high and meflect the weak bioactivation potency of colonocytes c


pared to hepatocytes. In addition, assessment of the oxida-tive stress effects of the chemical revealed that ACN inducedGSH depletion and enhanced lipid peroxidation. The toxicresponse to ACN was dependent on both concentration andduration of exposure to ACN. Indeed, the participation of ox-idative stress in the cytotoxicity of ACN has been previouslyreported in other target organs as brain (Jiang et al., 1998).Ahmed et al. (1996)reported that ACN-induced changes inthe homeostasis of tissue GSH might play a major role in theinitial processes underlying ACN toxicity. Moreover, oxida-tive stress has been implicated in the toxic insult of struc-turally related nitriles in the gastrointestinal cells and tissues.These include dibromoacetonitrile (Ahmed et al., 1991) andchloroacetonitrile (Mohamadin and Abdel-Naim, 1999). Astrong correlation (r2 = 0.97) existed between LDH leakageand TBARS production. Therefore, ACN-induced lipid per-oxidation may play a significant role in its colonotoxicity.

Lipid peroxidation and leakage of cytosolic enzymesare markers of cellular oxidative damage initiated by ROS(Farber et al., 1990). Thus, factors interfering with the genera-tion or effects of ROS are anticipated to protect against cell in-jury. The observed protective effects of GSH, DTT and NACcan be attributed to direct interaction with ROS, direct bind-ing to toxic metabolites and/or enhancement of cellular GSHsynthesis (Maxwell, 1995; Hoffer et al., 1996). Superoxidea l rad-iT t en-z nger( en-z ovet d hy-d as ar fa-c re-a ea usi ctiono roni tived fi m-a ught af-f ndD ofR pitet ibitA all stst butet

cellv ucesl astp rs,

antioxidant enzymes, hydroxyl radical scavengers and ironchelators can play an important role against ACN-inducedcolonotoxicity.

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