Toxicology in Vitro 28 (2014) 485–491

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Toxicology in Vitro

journal homepage: www.elsevier .com/locate / toxinvi t

Oxidative damage in keratinocytes exposed to cigarette smokeand aldehydes

http://dx.doi.org/10.1016/j.tiv.2014.01.0040887-2333/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Address: Department of Anatomy and Cell Biology,Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, P.O.Box 9649, Haifa, Israel. Tel.: +972 48295388; fax: +972 48295403.

E-mail address: Reznick@technion.ac.il (A.Z. Reznick).

Katia Avezov a,b, Abraham Z. Reznick a,⇑, Dror Aizenbud a,b

a Department of Anatomy and Cell Biology, Rappaport Faculty of Medicine, Technion – Israel Institute of Technology, Haifa, Israelb Orthodontic and Craniofacial Department, School of Graduate Dentistry Rambam Health Care Campus, Haifa, Israel

a r t i c l e i n f o a b s t r a c t

Article history:Received 26 August 2013Accepted 8 January 2014Available online 18 January 2014

Keywords:Cigarette smokea,b-Unsaturated aldehydesKeratinocytesOxidative stressGlutathioneProtein carbonylation

Cigarette smoke (CS) is a significant environmental source of human exposure to chemically activesaturated (acetaldehyde) and a,b-unsaturated aldehydes (acrolein) inducing protein carbonylation anddysfunction. The exposure of oral tissues to environmental hazards is immense, especially in smokers.The objectives of the current study were to examine the effect of aldehydes originating from CS onintracellular proteins of oral keratinocytes and to observe the antioxidant response in these cells.

Intracellular protein carbonyl modification under CS, acrolein and acetaldehyde exposure in the HaCaTkeratinocyte cell line, representing oral keratinocytes was examined by Western blot. Possibleintracellular enzymatic dysfunction under the above conditions was examined by lactate dehydrogenase(LDH) activity assay. Oxidative stress response was investigated, by DCF (2,7-dichlorodihydrofluorescein)assay and GSH (glutathione) oxidation.

Intracellular protein carbonyls increased 5.2 times after CS exposure and 2.7 times after exposure to1 lmol of acrolein. DCF assay revealed an increase of fluorescence intensity 3.2 and 3.1 times after CSand acrolein exposure, respectively. CS caused a 72.5% decrease in intracellular GSH levels comparedto controls. Activity of intracellular LDH was preserved.

a,b-Unsaturated aldehydes from CS are capable of intracellular protein carbonylation and have a role inintracellular oxidative stress elevation in keratinocytes, probably due to the reduction in GSH levels.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction periodontal disease (Valko et al., 2007; Lavie and Lavie, 2009;

Cigarette smoke (CS) is a multipotent mixture of numerouscomponents associated with disorders in different organs, andinvolved in many pathological processes (Kuper et al., 2002). Themolecular mechanisms involved in CS damage to the organismare diverse, and have not yet been fully disclosed. One of themechanisms of CS damage is oxidative stress (OS), defined as animbalance between the load of chemically reactive oxidants suchas reactive oxygen and nitrogen species (ROS and RNS) and theability of a biological system to detoxify them or to repairthe resulting damage (Valko et al., 2007). Disturbances in thenormal redox state of cells can cause toxic effects to all cellularcomponents, including proteins, lipids, carbohydrates and DNA.OS is thought to be involved in the development of over 100seemingly unrelated diseases including cancer, Parkinson’s,Alzheimer’s, atherosclerosis, myocardial infarction as well as

Reibel, 2003; D’Aiuto et al., 2010).There are endogenous as well as exogenous ROS/RNS sources.

Endogenously ROS are a natural byproduct of mitochondrialoxygen metabolism. ROS/RNS are also produced by leukocytes inorder to destroy pathogens. Exogenously free radicals originatefrom sources such as ionizing radiation and environmental pollu-tants such as CS. The gas phase of CS contains more than 1015 freeradicals per puff while the particulate phase contains more than1017 free radicals per gram (Swan and Lessov-Schlaggar, 2007).ROS contained in the CS gas phase include hydrogen peroxide(H2O2), superoxide (O�2 ) and hydroxyl radical (OH�). Additionally,cigarette smoke is a great source of RNS such as nitric oxide(NO). NO and superoxide may react and form peroxynitrite(ONOO–), a potent oxidizing and nitrating compound which alsohas been linked to a variety of pathological conditions (Hasniset al., 2007).

Furthermore, cigarette smoke constitutes one of the largestenvironmental sources of human exposure to another group ofhighly chemically active substances: volatile aldehydes. Theseinclude saturated (mainly acetaldehyde) and a,b-unsaturatedaldehydes (mainly acrolein and crotonaldehyde) capable of protein

486 K. Avezov et al. / Toxicology in Vitro 28 (2014) 485–491

carbonylation leading to protein dysfunction, an increase in oxida-tive stress and disease onset, including cancerous transformationprocesses (Nystrom, 2005; Dalle-Donne et al., 2003; Colomboet al., 2010; Kehrer and Biswal, 2000). Smoke from a single ciga-rette contains twenty times less unsaturated aldehydes (0.21 lmolcrotonaldehyde and 0.8 lmol acrolein) than saturated aldehydes(19.3–22.7 lmol acetaldehyde) (Reznick et al., 1992). However,the former are a major source of reactive double bonds reactingwith -SH (thiol) groups of proteins in a Michael addition reaction(Nagler et al., 2000). In this reaction aldehydic carbonyls are at-tached to a protein and induce structural alterations. This is partic-ularly critical in enzymes’ activity, since carbonylation can lead totheir dysfunction.

Oral cavity tissues are the first to encounter cigarette smokeand its toxic constituents entering the body. Moreover, oral cellsare uniquely susceptible to free radical damage because the oralmucus membranes allow rapid absorption of substances acrosstheir surface. All oral tissues are affected by CS: teeth, mucosa,salivary glands and the saliva. The effects of cigarette smoke onthe oral cavity range from simple tooth staining, inflammatoryconditions to oral cancer (Reibel, 2003). In addition, macromole-cules such as enzymes found in the saliva may lose their activity(Zappacosta et al., 2002). For instance, lactate dehydrogenase(LDH) activity was shown to decrease after exposure to CS (Nagleret al., 2001). Although the effect of CS on saliva is transient as it iscontinuously secreted, CS components such as aldehydes, whichare readily dissolved in saliva, can potentially penetrate into oralkeratinocytes and inactivate intracellular as well as extracellularproteins (Lambert et al., 2005). This specifically applies to heavysmokers suffering from an additive effect. Furthermore, theincrease in oral oxidative stress from free radical formationthrough periodontal infection, alcohol, dental procedures andsubstances leads to further breakdown of cell walls of oral tissuesand exacerbates inflammation (Ismahil et al., 2011). Recent studieshave shown that this is a major contributing factor to systemicinflammatory diseases, including rheumatoid arthritis, vascularand cardiovascular diseases (D’Aiuto et al., 2010; Lee and Park,2013).

Biological systems have developed the ability to detoxify bothendogenously generated ROS and environmental oxidative andchemically active agents (Tomitori et al., 2012). One of the imme-diate antioxidant agents is glutathione (GSH), a tripeptide contain-ing a thiol group acting as a reducing agent. GSH reduces disulfidebonds in cytoplasmatic proteins by serving as an electron donor. Inthis process, GSH is converted to its oxidized form, glutathionedisulfide (GSSG). Once oxidized, GSSG can be reduced to GSH bythe glutathione reductase enzyme (GSH-RD), using NADPH as anelectron donor. The ratio between GSH and GSSG is used as a mea-sure of the cellular oxidative status.

Despite constant exposure of oral cavity tissue in smokers to CSand its chemically active constituents such as aldehydes, the effectof aldehydes on oral cells and antioxidant defense systems was notextensively studied. The objectives of the current study were toexamine the effect of aldehydes originating from CS on intracellu-lar proteins of keratinocytes and to observe the antioxidant re-sponse in these cells.

Fig. 1. Exposure of cell cultures to CS. 1 I: CS exposure system. Reproduced withpermission of the copyright owner. From: Rom O, Mech Ageing Dev (2012). 1 II Thecorrelation between the extent of lowering the pressure in the reservoir and thedetected nitrite concentrations in PBS samples.

2. Materials and methods

2.1. Cell culture and cell viability assay

HaCaT keratinocyte cell line acquired from CLS Cell LinesService (Eppelheim, Germany) was used in the experiments. HaCaTare in vitro spontaneously transformed keratinocytes from histo-logically normal human skin. The line is referred to as immortal

(>140 passages), maintains full differentiation capacity and is non-tumorogenic (Boukamp et al., 1988). These cells are widely used(Ge et al., 2012) as a model for epithelial tissue studies, includingoral epithelium investigations, due to their high proliferation rate.The cells were cultured in 100 mm Nunclon cell culturing dishesand incubated in Dulbecco’s Modified Eagle’s Medium (DMEM)supplemented with 2 mM L-glutamine, 10% fetal calf serum,100 U/ml penicillin and 0.1 mg/ml streptomycin at 37 �C humidi-fied atmosphere containing 5% CO2. Results of tests for myco-plasma, bacteria and fungi were negative. Before each set ofexperiments, cells from a frozen stock were thawed and passagedfor 6–10 times. After each passage, the cells were grown for 4 daysuntil 90% confluency was reached, then the experiments were per-formed. All the experiments were executed in 100 mm Nuncloncell culturing dishes submerged in 10 ml of cell culturing medium.For the DCF assay, the cells were cultured on 0.17 mm coverslipsplaced in 100 mm petri dishes. Viability was assessed by the trypanblue exclusion method. Immediately after the experiments, thecells were lysed, centrifuged to remove cell debris and preservedin an ultra-low temperature freezer (�80 �c) for carbonylationand GSH/GSSG assays for up to 15 days. Fresh cell lysates wereused for the LDH enzyme activity assay.

2.2. Exposure of cell cultures to CS and aldehydes

The study was carried out using cigarettes (filtered ‘‘Time’’ cig-arettes, Dubek, Israel, containing 14 mg of tar and 0.9 mg of nico-tine per cigarette) combined with a source of lowered pressuresystem as previously described (Rom et al., 2013). The CS exposuresystem is schematically shown in Fig. 1I. In short: an open 100 mmPetri dishes with cell cultures submerged in 10 ml of culture med-ium (as mentioned before) were placed in a sealed reservoir with asidearm to which a cigarette was attached. A reproducible lowpressure was created in the reservoir by a vacuum pump and valveA was closed. When the attached cigarette was lit, valve B wasopened for 10 s and the smoke from the lit cigarette was drawnin. This was considered a single ‘‘puff’’. According to previousworks in our laboratory, a single cigarette smoked in the aboveapparatus statistically contains nine ‘‘puffs’’ (Weiner et al., 2008).

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After each ‘‘puff’’ the reservoirs were sealed with the smoke insideand incubated. The number of ‘‘puffs’’ and time of incubation dif-fered in each experiment. Samples subjected to air puffs insteadof CS were used as controls. The amount of CS drawn into the res-ervoir was regulated by decreasing the pressure inside the reser-voir using a vacuum pump. Thus, the dose of CS entering thereservoir equated the level of lowered pressure created insidethe reservoir. In order to calibrate the system and to determinethe reproducibility of smoking intensity, equal amounts of phos-phate-buffered saline (PBS) (with no nitrites, NO�2 ) were exposedto different CS volumes by lowering the pressure of the smokingsystem. When the pressure was lowered, higher concentrationsof nitrites (NO�2 ), originating from CS exposure, were detected inPBS by means of the Griess reagent (from Sigma–Aldrich), whichserved as an indicator of the smoking intensity. Fig. 1II demon-strates the direct correlation between the extent of lowering thepressure in the reservoir and the detected nitrite concentrations.

In separate experiments, cell cultures were incubated withpurified aldehydes in order to mimic exposure to aldehydic con-tent of a single cigarette reported to be present in a mainstreamsmoke of one 2R1 University of Kentucky reference cigarette(O’Neill et al., 1994). To mimic a cumulative effect, a tenfold alde-hyde content was applied. Accordingly, separate cell cultures weretreated with 1 lmol and 10 lmol of acrolein (Sigma–Aldrich), and20 lmol and 200 lmol of acetaldehyde (Sigma–Aldrich). Incuba-tion conditions were similar to the cultures exposed to CS.

2.3. Detection of protein carbonyl modification

Protein carbonyl assay was used as an indicator of proteinmodification, using the commercially available OxyBlot ProteinOxidation Detection Kit (Millipore, USA), based upon 2,4-dinitro-

Fig. 2. HaCaT keratinocytes following CS exposure. 2I: Cell viability following exposuretotal intracellular protein carbonyls. (A) Untreated control, (B) after a single puff of CS, (CAverage densitometric analyses of 3–5 different WB assays of the same experiment. TotalNAC. 2IV: Average (±SEM) cellular LDH activity following CS exposure (U/L) (⁄statistical

phenylhydrazine (DNPH) carbonyl derivation, following immuno-detection with the Western blot assay with anti-dinitrophenyl(DNP) antibodies and quantified by densitometry.

2.4. Addition of N-acetylcysteine (NAC) to cell medium

NAC 1 mM (Sigma–Aldrich), a thiol scavenger capable of tra-versing cell membranes was added to the culture medium wherenoted and incubated with the cells for one hour prior to the exper-iments. Then, the cultures were washed with saline, and an NACfree medium was added. Thereafter, the experiments were con-ducted, verifying that thiol scavenging by NAC occurred onlyintracellularly.

2.5. Measurement of intracellular GSH and GSSH

GSH levels were determined in cell lysates using the spectro-photometric evaluation method (Rahman et al., 2006) with areduction rate of 5,5-dithiobis-2-nitrobenzoic acid (DTNB, Sigma–Aldrich) to 5-thio-2-nitrobenzoic acid (TNB). Values were deter-mined by comparing the reduction rate against a standard curveof GSH. GSSG levels were determined under the same conditionsafter trapping GSH with 3-vinyl pyridine.

2.6. Detection of intracellular ROS formation

The generation of intracellular oxidants was determined by theformation of fluorescent 2070-dichlorodihydrofluorescein (DCF) onoxidation of the nonfluorescent, reduced H2DCFDA (Invitrogen).The fluorescent intensity is proportional to ROS and free radicalswithin the cytosol. HaCaT cells were grown as mentioned aboveon 0.17 mm cover slips placed in 100 mm culture plates. Prior to

to 0 (control) – 3 puffs of CS over a 1 h period. 2II: A representative WB analysis of) after 2 puffs of CS, (D) after 3 puffs of CS. 2III: Intracellular protein carbonyls ratio.carbonylated intracellular proteins exposed to CS with and w/o pre-incubation with

ly significant).

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the experiment, the cells were loaded with a medium containing10 lM of H2DCFDA followed by 60 min of incubation in 37 �C inthe dark. Then, the cell cultures were washed twice with PBS to re-move extracellular DCF, submerged in a fresh medium and ex-posed to CS and aldehydes as previously described. Immediatelyafter the experiment, the cover slips were mounted using animmu-mont mounting medium (Thermo Scientific), and cellularfluorescence was monitored by a Zeiss Axioscop 2 fluorescent up-right microscope, captured by an Olympus DP70 digital microscopecamera and quantified by densitometry.

2.7. LDH enzyme activity measurement

In order to examine CS and aldehydes effect on intracellularenzymatic activity, LDH enzyme activity in fresh cell lysates wasmeasured immediately after the exposure experiments using theUV spectrophotometric evaluation with reduced nicotinamide ade-nine dinucleotide (NADH, Sigma–Aldrich) as a coenzyme and pyru-vate (Sigma–Aldrich) as a substrate, as previously described(Recommendations of the German Society for Clinical Chemistry,1970).

3. Results

3.1. Analysis of cell viability and cell morphology

The viability of cultured HaCaT cells, determined by the trypanblue exclusion analysis following exposure to CS and aldehydes

Fig. 3. HaCaT keratinocytes following aldehyde exposure. 3I: Cell viability following 1 h(C) 10 lmol of acrolein (equal to 10 cigarettes), (D) 20 lmol of acetaldehyde (equal to 1 cWB analysis of total intracellular proteins exposed to aldehydes. Average densitometric acarbonyls ratio. Average densitometric analyses of 3–5 different WB assays of the same e(U/L) (⁄statistically significant).

after 1 h of incubation is presented in Figs. 2I and 3I. Cell viabilityonly slightly decreased, dose dependently, ranging from99.3 ± 0.3% for cells exposed to 1 cigarette puff to 98.3 ± 1.4 forcells exposed to 3 cigarette puffs. After exposure to 1 lmol of acro-lein, cell viability was 98 ± 1.4%, but after 10 lmol, it decreased to83 ± 1.2. 20 and 200 lmol of acetaldehyde exposure showed97.8 ± 1.1 and 95.2 ± 3.1% of cell survival, respectively.

3.2. Analysis of intracellular protein carbonylation following CSexposure

Protein carbonylation induced by CS in whole-cell lysates wasanalyzed by Western blotting. One hour exposure of the HaCaTkeratinocyte culture to increasing volumes of CS induced a marked,dose-dependent increase in protein carbonylation (Fig. 2II and III).A single puff of CS generated an elevation of 2.6 times (p < 0.01), 2puffs induced an elevation of 3.2 times (p < 0.04), and 3 puffs of CSincreased the carbonyls content by 5.2 times (p < 0.03). Pre-incu-bation of cell cultures with 1 mM of NAC for 1 h prior to CS expo-sure, neutralized part of the carbonyl formation in the 1 and 2 CSpuff groups, but not in the 3 CS puff group (p < 0.02) (Fig. 2III).

3.3. Analysis of intracellular protein carbonylation following aldehydeexposure

Intracellular carbonyl content after exposure to saturated andunsaturated aldehydes is presented in Fig. 3. Incubation with1 lmol and 10 lmol of acrolein (equivalent to acrolein content in

of exposure to: (A) air exposed control, (B) 1 lmol of acrolein (equal to 1 cigarette),igarette), (E) 200 lmol of acetaldehyde (equal to 10 cigarettes). 3II: A representativenalyses of 3–5 different WB assays of the same experiment. 3III: Intracellular proteinxperiment. 3IV: Average (±SEM) cellular LDH activity following aldehydes exposure

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CS of 1 and 10 cigarettes, respectively) over a 1 h period caused anincrease of 2.7 times (p < 0.04) and 5 times (p < 0.01), respectively(Fig. 3II and III). Incubation with 20 and 200 lmol acetaldehyde(equivalent to acetaldehyde content in CS of 1 and 10 cigarettes,respectively) showed carbonyl accumulation similar to air exposedcontrols. In cultures pre-incubated with 1 mM of NAC for 1 h,exposure to 1 lmol of acrolein elevated intracellular carbonylsby only 1.3 times, compared to 2.7 times in the no NAC pre-incu-bated cultures. However, in cultures exposed to 10 lmol of acro-lein, no benefit was achieved from pre-incubation with NAC.

3.4. Analysis of intracellular LDH activity

The effect of CS and aldehydes on intracellular enzymaticactivity, was examined by measuring the LDH enzyme activity infresh cell lysates after exposure of the cell cultures to CS andaldehydes for 1–3 h and compared to air subjected controls. LDHintracellular enzymatic activity was preserved; one hour airexposed controls showed an average activity of 1652 ± 63 U/L,while exposure to 1, 2 and 3 puffs of CS showed activity of1701 ± 51, 1734 ± 54, and 1635 ± 98 U/L. In cell cultures incubatedfor one hour with 1 lmol of acrolein, LDH activity was 1777 ± 29,while 10 lmol of acrolein caused 1774 ± 84 U/L activity and expo-sure of cultures to 20 and 200 lmol of acetaldehyde demonstrated1667 ± 54 and 1686 ± 81 U/L LDH activity (Figs. 2IV and 3IV).

Fig. 4. DCF assay for cellular total oxidation state. Fluorescence intensity is proportionalcontrol cell culture, (B) cellular oxidative status after a single puff of CS, (C) after exposfluorescence analyses of 3–5 different DCF experiments. Total cellular fluorescence ofsignificant).

3.5. Analysis of intracellular ROS generation

HaCaT oxidative status was examined after exposure to CS,acrolein and acetaldehyde by means of the DCF assay. The resultsare presented in Fig. 3. Exposure to a single puff of CS increasedfluorescent intensity by 3.2 times (p < 0.02) compared to the con-trol. Incubation with 1 lmol of pure acrolein increased the fluores-cence by 3.1 times (p < 0.06), while exposure to 200 lmol ofacetaldehyde showed an insignificant increase in fluorescence.Pre-incubation with NAC partially neutralized ROS generation(Fig. 4II).

3.6. Detection of GSH and GSSG rate

A single puff of CS caused an average reduction of GSH levels to34% of the initial content (Fig. 5I) (p < 0.01), two and three puffs al-most obliterated GSH activity, as did acrolein (1 and 10 lmol)(Fig. 5II). Exposure to acetaldehyde left GSH levels similar to thecontrol. No parallel elevation of GSSG was observed (Fig. 5I and II).

4. Discussion

The first objective of the current study was to examine the ef-fect of CS and its aldehydic constituents on intracellular proteins

to reactive oxygen species and free radicals within the cytosol. 4I: (A) Air subjectedure to 200 lmol acetaldehyde, (D) after exposure to 1 lmol acrolein. 4II: Average

keratinocytes exposed to CS with and w/o pre-incubation with NAC (⁄statistically

Fig. 5. GSH and GSSG concentration ratio following exposure to CS and toaldehydes. 5I: GSH and GSSG activity ratio following an increasing number of CSpuffs. 5II: GSH and GSSG levels after saturated (acetaldehyde) and exposure tounsaturated (acrolein) aldehydes (⁄statistically significant).

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of keratinocytes. The use of the smoking apparatus applied in theabove experiments precisely simulates the exposure of keratino-cytes to CS in the oral cavity. The cultures in media were exposedto the smoke in the same way oral mucosal cells, bathed in saliva,are exposed to the fumes in smokers’ mouths. Additionally, the cul-tures were exposed to the same CS as smokers in real life, in con-trast to related studies (Park et al., 2012; Kreindler et al., 2005),where CS extract was used, thereby potentially altering the con-tents. Thus, direct local exposure of the cells to dissolved aldehydesoriginating in CS was achieved and examined.

Among CS aldehydes, acrolein is probably the most chemicallyactive, and has numerous biological effects including regulationof signaling molecules and activation of transcription factors(Stevens and Maier, 2008). It readily reacts with cysteine, histidine,and lysine residues of proteins, generating protein-carbonyl deriv-atives (Esterbauer et al., 1991; Uchida et al., 1998). In cardiomyo-cytes acrolein caused contractile dysfunction and protein adductformation (Luo et al., 2007). In skeletal myotubes acrolein, butnot acetaldehyde, was shown to activate a signaling cascade result-ing in muscle catabolism (Rom et al., 2013). Formation of acrolein-protein adducts and protein carbonylation have been identified asfactors causing various pathologic disorders (O’Brien et al., 2005)(Uchida et al., 1998). For example, carbonylation caused by CSaldehydes was revealed in lung fluid (Rahman and MacNee,1999; Yao and Rahman, 2011) and cardiomyocytes (Luo et al.,2007). In the oral cavity, the first organ to encounter CS, carbonyl-ation was observed in gingival fibroblasts (Colombo et al., 2012)and enzymatic dysfunction was induced in salivary proteins(Nagler et al., 2000; Weiner et al., 2008; Avezov et al., 2014). Acro-lein high chemical reactivity is due to its double bond which reactswith -SH (thiol) groups of proteins (Kehrer and Biswal, 2000). In-deed, in the current study, a dose dependent intracellular proteincarbonylation was observed in cells treated with CS resemblingthe carbonylation in cultures incubated with acrolein. Exposure

to acetaldehyde showed no carbonyl modification (Figs. 2II andIII and 3II and III). This may indicate that dissolved unsaturatedaldehydes from CS traverse cell membranes and are responsiblefor carbonylation occurring within the cells. In the experimentswhere cell cultures were pre-incubated with NAC, which is capableof penetrating the cell membranes (as opposed to GSH) and boostthe -SH containing entities, carbonylation was partially avoided.Replacing the culture media before the exposure experiments en-sured that the reaction with aldehydes was entirely intracellular.It is noteworthy that the addition of NAC prevented carbonylationwith low doses of CS (one and two puffs) and acrolein (1 lmol)exposure, but not with the high doses of exposure (three puffs ofCS and 10 lmol, respectively). This is probably due to the overloadof aldehydes, making the addition of 1 mM of NAC insufficient.

The second objective was to observe the antioxidant response inkeratinocytic cells. The general cellular oxidant level was elevatedupon exposure to CS and acrolein, and only a minor elevation wasobserved upon exposure to acetaldehyde (Fig. 4). Cigarette smokecontains high levels of ROS and RNS which explains the increasein the cellular oxidative state in cells exposed to the smoke. Butpure acrolein, which is not a free radical, was not expected to raisethe cellular oxidative status. The assumption is that acrolein in-creases the cellular oxidants via a ‘‘secondary’’ mechanism. Acro-lein double bond can react with the thiol group of GSH, anddiminish its availability for the antioxidant function. Consequently,intracellular ROS are elevated as shown by the DCF assay. Acetalde-hyde that has no reactive double bond does not react with GSH. In-deed, Fig. 5 demonstrates lower GSH levels in acrolein but not inacetaldehyde treated cells. Moreover, under OS conditions, parallelGSSG elevation is expected as a result of GSH oxidation. But in theabove experiments, no GSSG elevation was observed. It was previ-ously proposed (Luo et al., 2007; Colombo et al., 2012) that underCS exposure, GSH-a,b-unsaturated aldehyde adducts are, GSH-a,b-unsaturated aldehyde adducts are formed and prevent itsoxidation to GSSG and subsequent regeneration by glutathionereductase. The above results support these findings and show anacute reduction in cellular GSH levels upon acrolein exposure. Itis also known that acrolein is eliminated systemically by conjuga-tion with GSH in the liver (Stevens and Maier, 2008). Nonetheless,in the above experiments a local effect under direct and notsystemic exposure was observed.

Finally, under CS and aldehyde exposure, intracellular activityof the LDH enzyme was conserved (Figs. 2IV and 3IV). This is op-posed to its activity reduction in saliva under similar conditions(Avezov et al., 2014). In another work (Sticozzi et al., 2012), acro-lein also increased intracellular carbonyl levels in HaCaT cells,but did not affect Scavenger Receptor B1 (SR-B1) expression. Seem-ingly we can assume that the intracellular presence of an antioxi-dant system such as GSH is capable of neutralizing part of thedamage caused by aldehydes. In addition, total concentration ofproteins within cells is higher than in saliva, especially in keratinproducing cells such as HaCaT. Higher aldehyde concentrationsmight be required for chemical alterations such as carbonylationto cause activity reduction in enzymes in these cells. Either way,despite the fact that OS and total protein carbonylation wasobserved within the cells, intracellular enzymatic activity,(represented by LDH activity), was preserved.

In conclusion, a,b-unsaturated aldehydes from CS have a role inintracellular protein carbonylation and oxidative stress elevationin keratinocytes, probably due to the reduction in GSH levels.

Conflict of Interest

The authors declare that they have no competing financial ornon-financial interests.

K. Avezov et al. / Toxicology in Vitro 28 (2014) 485–491 491

Acknowledgments

This work was supported by Rappaport Institute Grant and aGrant from The Krol Foundation of Barnegat, NJ to AZR.

Appendix A. Supplementary material

Supplementary material associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.tiv.2014.01.004.

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