Cigarette smoke-induced protein oxidation and proteolysis isexclusively caused by its tar phase: prevention by vitamin C
Koustubh Panda a,*, Ranajoy Chattopadhyay a, Dhrubajyoti Chattopadhyay a,Indu B. Chatterjee a
a Dr B.C. Guha Centre for Genetic Engineering and Biotechnology and the Department of Biochemistry,Calcutta Uni�ersity College of Science, Calcutta 700019, India
Received 3 January 2001; received in revised form 22 May 2001; accepted 22 May 2001
Keywords: Cigarette smoke; Tar phase cigarette smoke; Oxidative damage of proteins; Vitamin C; Antioxidants; Free radicals
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1. Introduction
Cigarette smoke (CS) has been implicated withvarious degenerative pulmonary and cardiovascu-lar diseases like bronchitis, emphysema, myocar-dial infarction as well as lung cancer and othermalignancies (U.S. Surgeon General’s Report,1985; Shah and Helfant, 1988; Sherman, 1991;
* Corresponding author. Present Address: Department ofImmunology, NB30, Lerner Research Institute, The ClevelandClinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195,USA. Tel.: +1-216-445-9761; fax: +1-216-444-9329.
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Frank, 1993; Bartecchi et al., 1994). Many of thehealth debilitating effects of CS have been associ-ated with its characteristic oxidative prowess(Janoff et al., 1983; Loeb et al., 1984; Shalini etal., 1994; Hulea et al., 1995; Veyssier, 1997;Kleages et al., 1998). The oxidative potency of CSis revealed by its capacity to oxidize proteins (Coxand Billingley, 1984; Reznick et al., 1992; Cross etal., 1993; Panda et al., 1999), lipids (Frei et al.,1991; Mahfouz et al., 1995; Santanam et al., 1997)and DNA (Kiyosawa et al., 1990; Asami et al.,1997), a property, which can cumulatively causeextensive damage to our tissues and organs andmay in turn explain why cigarette smoking isassociated with a large number of degenerativediseases. In fact, the numerous free radical speciespresent in CS have been held responsible for theobserved oxidative damage and the consequenthealth damage (Church and Pryor, 1985; Cross,1987; Kasai, 1989; Pryor, 1997). Whole phase CScontains about 4000 components, out of whicharound 3000 components are present in its gasphase and 1000 components in the particulate, tarphase (Schumacher et al., 1997). Ever since it wasrealized that the oxidants present in CS have apredominant role in the pathogenesis of variousdegenerative diseases, there have been sustainedattempts to elucidate their role in disease cau-sation as well as isolate and characterize them.We approached this, in part, by separately study-ing the contribution of the two physically separa-ble phases of CS, the gas phase and the tar phasein the CS-induced oxidative damage of proteins.Among the free radicals present in CS, the gasphase contains small oxygen and carbon-centredradicals which are extremely unstable (Pryor etal., 1983; Church and Pryor, 1985). We havereported before that when the gas phase is passedinto a physiological buffer similar to that of thehuman respiratory tract lining fluid (RTLF), theresulting solution is unable to produce significantoxidation of proteins indicating the transient na-ture of the gas phase oxidants (Panda et al.,1999). We have also reported that aqueous extractof whole phase CS contains some stable oxidantswhich cause marked oxidative damage of proteins(Panda et al., 1999). Here, we demonstrate thatthe stable oxidants of the whole phase CS are
exclusively present in the tar phase, or more pre-cisely the aqueous extract of tar. The tar phasecan almost wholly mimic the oxidative behaviorof the whole phase CS in oxidation of humanplasma proteins, bovine serum albumin and theextensive oxidative degradation of guinea pig lungand heart microsomal proteins. As regards themechanism of the tar phase-CS induced oxidativedegradation of microsomal proteins, we observedthat it is comprised of two-steps: (i) initial oxida-tion of the proteins by the tar phase CS oxidants,followed by (ii) rapid proteolytic degradation ofthe oxidized proteins by proteases present in themicrosomes. Very similar to what is observed forthe whole phase CS, the protein oxidation causedby the tar phase CS is also almost completelyprevented by ascorbic acid and only partially byglutathione. Other antioxidants including super-oxide dismutase (SOD), catalase, vitamin E, �-carotene and mannitol are also found to beineffective in preventing the tar phase CS-inducedprotein oxidation and proteolysis of microsomalproteins. This indicates that the tar phase CSapparently carries the same oxidants responsiblefor the extensive oxidative damage of proteins bywhole phase cigarette smoke.
2. Material and methods
Ascorbic acid, reduced glutathione, SOD, �-to-copherol, �-carotene, sodium dodecyl sulfate(SDS), acrylamide, bisacrylamide, ammoniumpersulfate, Coomassie Brilliant Blue, N-�-p-tosylL-lysine chloromethyl ketone (TLCK) and N-to-syl-L-phenylalanine chloromethyl ketone (TPCK)were obtained from Sigma, USA. �-Tocopherolwas a gift from Merck (India). Catalase (SODfree) was obtained from the CSIR Centre forBiochemicals (New Delhi, India). 2,4-Dinitro-phenyl hydrazine was procured from Merck(Darmstadt, Germany) and bovine serum albuminfrom Boehringer-Mannheim (Germany). All otherchemicals used were of analytical grade.
Male shorthaired guinea pigs (400–500 g) agedbetween 5 and 7 months were procured from theAnimal House of the Indian Institute of ChemicalBiology, Calcutta and were used for all experi-
K. Panda et al. / Toxicology Letters 123 (2001) 21–32 23
mental purposes. Guinea pigs, like humans, areincapable of synthesizing ascorbic acid (Chatterjee,1973) and also several metabolic characteristics inguinea pigs are similar to that of humans (Stith andDas, 1982). Thus, the results obtained with guineapigs might be applicable to humans.
2.1. Preparation of whole phase cigarette smoke(CS) solution
One milliliter of 50 mM potassium phosphatebuffer, pH 7.4, was placed in a 250-ml glassErlenmeyer flask with a side arm. A commercialfilter-tipped cigarette (74 mm) with tar content of25 mg was mounted in a 500-�l pipette tip thatpenetrated the hole in the stopper of the flask andextended down past the side arm connected to awater pump and ended about half way from thebottom of the flask containing the buffer solution.The cigarette was lit and puffs of CS were intro-duced into the flask for 5 s by applying mildsuction of 4 cm water. The smoke was released onto the surface of the buffer without bubbling andallowed to dissolve in it for 25 s with gentleshaking. The whole process was so devised as tosimulate the manner in which the respiratory tractlining fluid (RTLF) is exposed to CS during theprocess of smoking by humans. The resultant darkyellow colored solution was filtered through a0.22-�m filter and is termed whole phase CSsolution.
2.2. Preparation of tar phase cigarette smoke(CS) solution
The set-up for the preparation of the tar phaseCS solution was the same as that for the wholephase CS solution except that the Erlenmeyer flaskwas in this case dipped in a mixture of ice and saltto allow condensation of the tar. The cigarette waslit and puffs of CS were introduced into the flaskby a mild suction of 4 cm water. The tar wasallowed to condense and settle to the bottom of theflask. Altogether, tar from five cigarettes was col-lected, extracted with 5 ml of 50 mM potassiumphosphate buffer, pH 7.4 and filtered through a0.22-�m Millipore filter. The filtrate thus obtainedis termed tar phase CS solution or tar extract.
2.3. Preparation of gas phase cigarette smoke(CS) solution
The gas phase cigarette solution was prepared bythe same procedure as for the whole phase CSsolution except that the Erlenmeyer flask wasreplaced by a boiling tube (50 ml) with a side armand a 0.22-�m Millipore filter unit was placedbetween the lit cigarette and the tube passing downthe stopper of the boiling tube to trap the particu-late tar phase. The Millipore filter was changedafter every 2 min to avoid clogging of the filter andallow unhindered passage of the gas phase CS. Foreach cigarette, four filters were used. The waterpump was replaced by a LKB Bromma 2016vacuum pump and a suction of 30 cm water wasused to draw and bubble the colorless gas phase CSinto 1 ml of physiological buffer (50 mM potas-sium phosphate buffer, pH 7.4) contained in theboiling tube.
2.4. Preparation of microsomes
Microsomal suspension from guinea pig lungand heart, washed free of ascorbate, containing 10mg protein/ml was prepared as described before(Mukhopadhyay and Chatterjee, 1994). Proteinwas estimated by the method of Lowry et al.(1951).
2.5. Human plasma
Human plasma was collected from blood of fourdifferent non-smokers and was used after extensivedialysis to free it from ascorbic acid.
2.6. Incubation system
Incubation mixtures using human plasmaproteins, bovine serum albumin or microsomalsuspension contained 1 mg of protein and 50 �l(equivalent to one-twentieth of a cigarette) ofwhole phase CS solution, tar phase CS solution orgas phase CS solution in a final volume of 200 �lof 50 mM potassium phosphate buffer, pH 7.4.Incubation was carried out in a 1.5-ml Eppendorftube at 37 °C with occasional shaking for 1 h.
SDS–PAGE of microsomal proteins was car-ried out as described before (Panda et al., 1999).
2.8. Assay of carbonyl content
Protein carbonyl was measured by reactionwith 2,4-dinitrophenyl hydrazine (DNPH) follow-ing the method of Levine et al. (1990) with mod-ifications. After incubation of albumin/humanplasma proteins or microsomes with CS (in thepresence of PMSF (10 �g/100 ml) and EDTA (2mM) to minimize proteolytic degradation due toprotein oxidation), the proteins were precipitatedwith 200 �l of 20% trichloroacetic acid solutionand the protein carbonyl formation was estimatedas described before (Panda et al., 1999). Wholephase CS solution as well as the tar phase CSsolution contained substantial colored materials,which were absorbed by the proteins and couldnot be completely removed by the wash proce-dures prior to the assay. The absorption of suchcolor was observed to reach its maximum level ataround 4 min of incubation. Thus, to eliminateerrors in phenylhydrazone scoring due to suchcolor absorption by the proteins, control incuba-tion tubes were set up where the incubation ofalbumin/human plasma proteins/microsomes withwhole phase CS/tar phase CS solution was carriedout up to 4 min and then stopped by the additionof 20% trichloroacetic acid. The phenylhydrazonevalues so obtained from these control tubes werededucted from the gross phenylhydrazone valuesof the experimental tubes to arrive at the proteincarbonyl values accumulating due to oxidation ofproteins only. For assays involving the colorlessgas phase CS solution, the 2,4 DNPH was directlyadded to the TCA precipitated incubated proteinand the phenylhydrazone formation directly de-termined therefrom (Panda et al., 1999).
2.9. Measurements of bityrosine formation,tryptophan loss and loss of protein thiols
Assay of the oxidative parameters of bityrosineformation, tryptophan loss and loss of protein
thiols was done as described before (Mukhopad-hyay and Chatterjee, 1994; Panda et al., 1999).
3. Results
3.1. Tar phase CS solution-induced degradationof microsomal proteins
Fig. 1 shows that tar phase CS solution inducesextensive degradation of guinea pig lung (Fig. 1,lane 2) and heart (Fig. 1, lane 5) microsomalproteins indicating that the damage is not tissue-specific. The extent of degradation is comparable
Fig. 1. SDS–PAGE showing degradation of guinea pig lungand heart microsomal proteins by tar extract of CS andprotection by ascorbate. Microsomal suspension (1 mgprotein) was incubated with 50 �l of tar extract (equivalent toone-twentieth of a cigarette) at 37 °C for 1 h as described inSection 2. After incubation, 40 �l of the incubated mixturewere subjected to SDS–PAGE using 10% gel. The gel wasstained with Coomassie Brilliant Blue R-250. Degradation ofproteins as evidenced by loss of protein bands was quantitatedusing a LKB 2202 ultrascan laser densitometer. Lane 1, lungmicrosomes incubated without tar extract; lane 2, incubatedwith 50 �l of tar extract; lane 3, incubated with 50 �l tarextract in the presence of ascorbate (100 �M); lanes 4–6, aresame as lanes 1–3 except that lung microsomes were replacedby heart microsomes.
K. Panda et al. / Toxicology Letters 123 (2001) 21–32 25
Fig. 2. SDS–PAGE showing the effect of gas phase CS onlung microsomal proteins in the absence and presence ofascorbic acid (100 �M). Gas phase CS was directly passed intothe microsomal suspensions (1 mg protein) separately in theabsence and presence of 100 �M ascorbate. The samples werethen incubated at 37 °C in a final volume of 200 �l, asdescribed in Section 2. After 1 h of incubation, 40 �l of theincubation mixtures were taken and subjected to polyacry-lamide gel electrophoresis under denaturing conditions using a7.5% gel. Lane 1, lung microsomes incubated without exposureto gas phase CS; lane 2, 1 mg protein equivalent of lungmicrosomes after incubation with gas phase CS from one-twentieth equivalent of a cigarette in the absence of ascorbate;lane 3, 1 mg protein equivalent of lung microsomes incubatedwith gas phase CS from one-twentieth equivalent of a cigarettein the presence of 100 �M ascorbate.
microsomal suspension in 50 mM potassiumphosphate buffer pH 7.4, did not produce anydegradation of the lung microsomal proteins (Fig.2, lane 2). In fact, it had an almost identicalSDS–PAGE profile as the gas phase CS-un-treated fraction (Fig. 2, lane 1) as well as the onetreated with gas phase CS in the presence of 100�M ascorbate (Fig. 2, lane 3). This clearly indi-cated that the CS-induced degradation of micro-somal proteins is caused exclusively by the tarphase of whole CS and not its gas phase.
3.2. Oxidation of microsomal proteins and effectsof antioxidants
That tar phase CS solution caused oxidation ofmicrosomal proteins was evidenced by the time-dependent formation of protein carbonyl (Fig. 3).Ascorbate (100 �M) almost completely preventedthis protein carbonyl formation (Fig. 3). Glu-tathione (0.5 mM) was partially effective (20%),
Fig. 3. Time-dependent formation of protein carbonyl inguinea pig lung microsomes induced by tar phase CS solutionand protection by ascorbate. Conditions of incubation are thesame as in Fig. 1 except that the incubation was carried out inthe presence of PMSF (10 �g/100 ml) and EDTA (2 mM) tominimize protein loss due to oxidative degradation prior toscoring the carbonyl content. Measurement of carbonyl isgiven in Section 2. Carbonyl values obtained with microsomes(0.25 nmol) in the absence of tar were subtracted from respec-tive experimental data. Data represent means of four indepen-dent measurements; S.D.�10%.
to that observed with whole phase CS solution,reported before (Panda et al., 1999). The degrada-tion was almost completely prevented by 100 �Mascorbic acid (Fig. 1, lanes 3 and 6). Glutathione(0.5 mM) was only partially effective and otherantioxidants, e.g. SOD (50 �g), catalase (40 �g),vitamin E (20 �M), �-carotene (20 �M), andmannitol (10 mM), were ineffective (figure notshown). No degradation of proteins was observedwhen the microsomes were incubated in the ab-sence of tar phase CS solution (Fig. 1, lanes 1 and4). In contrast, equivalent amounts of the gasphase CS, when bubbled into a guinea pig lung
K. Panda et al. / Toxicology Letters 123 (2001) 21–3226
Table 1Effect of ascorbate and other scavengers of reactive oxygen species on tar phase CS solution-induced carbonyl formation in guineapig lung microsomal proteins in the presence and absence of DFO
Absence of DFO Presence of DFOAntioxidants Concentrations used
Carbonyl Percentage inhibition Carbonyl Percentage inhibitionformed formed
500 �MGSH 1.08�0.08 20.0 1.08�0.04 20.01.35�0.04Mannitol Nil10 mM 1.35�0.07 Nil1.34�0.09 Nil 1.35�0.06 Nil20 �MVitamin E
Preparation of tar phase CS extract, incubation systems and estimation of protein carbonyl formation (nmol/mg protein) aredescribed in Section 2. The conditions of incubations are as in Fig. 3. The concentration of DFO used was 20 �M. Vitamin E wasused as a dispersion in sodium deoxycholate solution (final concentration 0.02%). Values represent means of four independentmeasurements; S.D.�10%.
but other antioxidants including SOD (50 �g),catalase (40 �g), vitamin E (20 �M), �-carotene(20 �M), and mannitol (10 mM), were ineffective(Table 1). This would indicate that O2
−, H2O2 andOH� were not involved in protein oxidationcaused by tar phase CS solution. Also, free ironwas not involved in the protein oxidation inducedby tar phase CS because desferrioxamine (20 �M),a strong chelator of Fe(III), had virtually noeffect (Table 1). The oxidative potency of the tarphase CS solution was almost stable up to 4 h at25 °C in the pH range of 7.4–8.0. The tar phaseCS solution oxidized microsomal proteins in theabsence of oxygen (nitrogen atmosphere). Thisshowed that the stable oxidants present in the tarsolution oxidized the proteins directly withoutbeing generated by interaction with atmosphericoxygen. Again, gas phase CS solution, preparedfrom equivalent amounts of whole phase CS vir-tually failed to produce any protein carbonyl for-mation or oxidation of the microsomal proteins(Table 2).
Tar phase CS solution-induced oxidative dam-age of microsomal proteins was also evidenced bythe formation of bityrosine (Fig. 4, Panel A) aswell as loss of tryptophan residues (Fig. 4, PanelB) and protein thiols (Fig. 4, Panel C). Interest-ingly, in all these cases, ascorbate (100 �M) al-
most completely prevented the damage (Fig. 4,Panels A–C). The protection provided by thepresence of 100 �M ascorbate to the tar phase CSsolution-induced oxidation of the lung microso-mal proteins was almost 97% for protein carbonylformation (Table 1), 92% for bityrosine formation(Fig. 4, Panel A), 90% for loss of tryptophanresidues (Fig. 4, Panel B) and 93% for loss ofprotein thiols (Fig. 4, Panel C). Notably, thesevalues are very close to that observed with wholephase CS solution (Panda et al., 1999). Assay ofthe above parameters of oxidative damageshowed virtually no change when the tar phaseCS solution was replaced by gas phase CS solu-tion, indicating that the gas phase CS had nodirect role in the observed whole phase CS solu-tion-induced oxidation of the microsomal proteins(Table 2). The comparative contributions ofequivalent amounts of the tar phase CS solutionand the gas phase CS solution in the whole phaseCS solution-induced guinea pig lung microsomalprotein oxidation is shown in Table 2. It is appar-ent that of the total oxidative damage induced bythe whole phase CS solution, the tar phase CSsolution accounts for almost 90% of the proteincarbonyl formation, 77% of the bityrosine forma-tion, 82% of the loss of tryptophan residues and82% of the loss of protein thiols (Table 2). In
K. Panda et al. / Toxicology Letters 123 (2001) 21–32 27
contrast, the comparative contributions of the gasphase CS solutions are almost insignificant withvalues of 0.67, 0.43, 0.37 and 0.3%, respectively(Table 2).
3.3. Oxidation of human plasma proteins andbo�ine serum albumin
Besides the microsomal proteins, the oxidantsin the tar phase CS solution or tar extract oxi-dized ascorbate-free human plasma proteins aswell as bovine serum albumin (Fig. 5). The oxida-tion in both the cases was again almost com-pletely prevented by ascorbate (100 �M). Hereagain the gas phase CS solution failed to producesignificant oxidation of the proteins (figure notshown). However, when gas phase CS is directlybubbled into a solution of albumin it producedsignificant oxidation (9�1 nmol/mg protein) ofthe pure protein unlike that observed for microso-mal proteins indicating that the oxidants in thegas phase CS are transient or unstable in nature.Unlike the tar phase CS-induced protein oxida-tion such gas phase CS-induced oxidation of albu-min was prevented by both SOD (50 �g) andcatalase (40 �g).
3.4. Mechanism of protein degradation by tarphase CS solution
As demonstrated before with whole phase CSor aqueous extract of CS (Panda et al., 1999), tarphase CS solution-induced microsomal proteindegradation was also found to be a two-stepprocess. Oxidation of the proteins by the tarphase rendered the oxidized proteins more suscep-tible to proteolysis as observed in lanes 2 and 5 in
Fig. 1. However, no such phenomenon was ob-served with the gas phase CS solution (Fig. 2, lane2). When microsomal proteins were incubatedwith tar phase CS solution in the presence ofPMSF (10 �g/100 ml) and EDTA (2 mM)[protease inhibitor], oxidation of the proteins wasnot inhibited as evidenced by protein carbonylformation (Fig. 3 and Table 1) but degradation ofthe proteins was completely arrested (Fig. 6, lane3), indicating the failure of the microsomalproteases to degrade the oxidized proteins in thepresence of PMSF and EDTA. However, whenthe oxidized microsomes were washed free of thePMSF and EDTA and incubated with 1 �g eachof trypsin and chymotrypsin for 10 min at roomtemperature, there was rapid proteolytic degrada-tion of the oxidized microsomal proteins (Fig. 6,lane 4). When tar phase CS solution-untreatedmicrosomes were similarly treated, there was vir-tually very little degradation of the microsomalproteins (Fig. 6, lane 6), indicating that prioroxidation of the target proteins was an essentialprerequisite for triggering rapid proteolysis by theproteases. When 100 �M ascorbate was presentduring the incubation of the microsomes with tarphase CS solution, there was very little degrada-tion of the microsomal proteins when subse-quently incubated with trypsin (1 �g) andchymotrypsin (1 �g) (Fig. 6, lane 7), indicatingthat prevention of tar phase CS solution-inducedoxidation of the microsomal proteins, as evi-denced by carbonyl formation (Fig. 3 and Table1), prevented subsequent rapid proteolysis by theproteases. In fact, when the native microsomeswere incubated with 100 �M ascorbate undersimilar conditions no change was observed (Fig.6, lane 9). Such rapid degradation of the tar phase
Table 2Comparative oxidation of guinea pig lung microsomal proteins (1 mg) by equivalent amounts of whole phase CS solution, tar phaseCS solution and gas phase CS solution under identical conditions of incubation and assay
Whole phase CS solution Tar phase CS solution Gas phase CS solutionParameter of oxidative damage
51.2�2.8Tryptophan loss (Fluorescence units) 0.23�0.0162.6�3.237.6�2.2 0.14�0.00646.1�2.8Loss of protein thiols (nmol)
Conditions of incubation and assay are described in Section 2. Values are means of four independent experiments; S.D.�10%.
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CS solution-oxidized microsomal proteins bytrypsin and chymotrypsin (Fig. 6, lane 4) washowever effectively prevented by inhibitors oftrypsin, N-�-p-tosyl L-lysine chloromethyl ketone(TLCK) [0.3 mM] and chymotrypsin, N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) [0.1mM] (Fig. 6, lane 5), but not by ascorbic acid(Fig. 6, lane 8). This implies that the rapid degra-dation of the tar phase CS solution-oxidized mi-crosomal proteins took place only in the presenceof proteases and once the protein was oxidized,ascorbate was incapable of preventing subsequentproteolytic degradation of the microsomalproteins. Overall, the above observations clearlyestablished the mechanism of tar phase CS solu-tion-induced oxidative degradation of microsomalproteins to be a two-step process involving: (i)initial oxidation of the microsomal proteins by theoxidants present in the tar phase CS, followed by(ii) rapid proteolytic degradation of the oxidizedproteins by proteases present in the microsomes.It also demonstrated that ascorbate by exclusivelypreventing initial oxidation of the microsomalproteins by the tar phase CS solution, prevented
Fig. 4. Effect of ascorbate on tar phase CS solution-inducedbityrosine formation (A), tryptophan loss (B) and loss ofprotein thiols (C) in lung microsomal proteins. Conditions ofincubation are the same as in Fig. 3. Concentration of ascor-bate used was 100 �M. Values are means of four independentmeasurements; S.D.�10%.
Fig. 5. Effect of ascorbate on tar extract-induced oxidation ofhuman plasma proteins and bovine serum albumin. Concen-tration of ascorbate used is 100 �M. Conditions of incubationand measurement of protein carbonyl are given in Section 2.Values are means of four independent measurements; S.D.�10%.
K. Panda et al. / Toxicology Letters 123 (2001) 21–32 29
Fig. 6. Mechanism of oxidative degradation of lung microso-mal proteins by aqueous extract of tar as evidenced by SDS–PAGE. Conditions of treatment and incubation of the proteinsare described in Section 2. Thirty microliters of the finalincubation mixture containing native or oxidized microsomeswere loaded in each lane of a 7.5% gel. Lane 1, unincubatednative microsomes; lane 2, native microsomes incubated for 1h at 37 °C without aqueous extract of tar; lane 3, aqueousextract of tar oxidized microsomes isolated in the presence ofPMSF (10 �g/100 �l)+EDTA (2 mM); lane 4, microsomestreated with aqueous extract of tar for 1 h in the absence ofascorbate and then treated with trypsin (1 �g)+chymotrypsin(1 �g) for 10 min; lane 5, aqueous extract of tar-oxidizedmicrosomes treated with trypsin (1 �g)+chymotrypsin (1 �g)for 10 min in the presence of TLCK (0.3 mM)+TPCK (0.1mM); lane 6, native microsomes treated with trypsin (1 �g)+chymotrypsin (1 �g) for 10 min; lane 7, microsomes treatedwith aqueous extract of tar for 1 h in the presence of ascorbate(100 �M) and then treated with trypsin (1 �g)+chymotrypsin(1 �g) for 10 min; lane 8, aqueous extract of tar-oxidizedmicrosomes incubated with ascorbate (100 �M) for 1 h fol-lowed by treatment with trypsin (1 �g)+chymotrypsin (1 �g)for 10 min; lane 9, microsomes incubated for 1 h at 37 °C withascorbate (100 �M) only; lane 10, trypsin (1 �g)+chy-motrypsin (1 �g) only.
protein damage produced by the tar extract isclosely comparable to that observed with thewhole phase CS, indicating that the same stableoxidants in whole phase CS that are responsiblefor extensive oxidative damage of proteins arealso present in the tar phase CS. This is furthercorroborated by the observation that both the tarphase CS solution and whole phase CS solution-induced oxidation of proteins have almost similarprotection profiles for the antioxidants used, in-cluding vitamin C. Moreover, the virtual failureof the gas phase CS or its solution to produceoxidative modification or proteolysis of the micro-somal proteins indicates that the stable oxidativepotency exhibited by the whole phase CS is notmimicked by its gas phase. We observe the mech-anism of microsomal protein degradation by thetar phase CS to be a two-step process involving:(i) oxidation of proteins by oxidants of the tarphase CS, followed by (ii) rapid proteolytic degra-dation of the oxidized proteins by proteasespresent in the microsomes. Interestingly, a similarmechanism is observed for whole phase CS-in-duced degradation of microsomal proteins (Pandaet al., 1999).
Until now, several studies (Pryor et al., 1986;Frei et al., 1991; Takano et al., 1997) have pri-marily implicated the gas phase CS to the ob-served CS-induced oxidative damage of proteinspossibly because of the fact that it comprises threetimes as many active components compared to thetar phasecs (Schumacher et al., 1997). Conse-quently, the general mechanisms of the patho-physiological events resulting from CS-inducedoxidative damage of biological macromoleculesand tissues have been mainly centered around theunstable free radicals and reactive oxygen speciespresent in the gas phase CS including O2
−, H2O2,alkoxyl radicals, peroxyl radicals, NO and NO2
for quite a long period. We found that the gasphase oxidants were incapable of oxidizingprotein after they are dissolved in a physiologicalsolution for 30 s, which indeed disqualifies themfrom being possible candidate oxidant(s) repre-senting the stable as well as sustained oxidativepotency of the whole phase CS, which can causeextensive oxidation of proteins for up to 4 h insolution. However, the gas phase, when bubbled
the subsequent fast proteolysis by the microsomalproteases.
4. Discussion
Results presented in this paper clearly delineatethat the stable oxidants responsible for the exten-sive CS-induced oxidative damage and proteolysisof microsomal proteins of guinea pig lung as wellas oxidative damage of human plasma proteins, invitro, are exclusively present in the tar phase ofwhole phase CS. This also implies that the entireoxidative potency of the whole phase CS solutionresides in the aqueous extract of the tar phase andnot the gas phase of CS. In fact, the extent of
K. Panda et al. / Toxicology Letters 123 (2001) 21–3230
directly into a microsomal suspension, producesfeeble oxidation of the microsomal proteins(Table 2) consisting mostly of lipoproteins butsubstantial oxidation of bovine serum albumin(9�1 nmol/ mg protein), a pure protein. Furthersuch gas phase CS-induced protein oxidation isprevented by SOD and catalase quite unlike thatof the tar phase CS, denoting the presence ofstrong yet unstable oxidants like O2
−, H2O2 in thegas phase CS. It can thus be expected that the gasphase oxidants, by virtue of their instability, losetheir oxidative potency by the time they reach thelung after inhalation. In fact, this particularlyleads us to expect that any protein damage causedby the gas phase oxidants is presumably restrictedto the upper respiratory tract and unlikely reachesthe lung or other internal organs that wouldrequire penetration through the respiratory tractlining fluids much akin to the physiological bufferused for our study. On the other hand, the oxi-dants present in the tar phase CS are water solu-ble and stable at room temperature for up to 4 h.It can thus be envisaged that these may get dis-solved in the respiratory tract lining fluids(RTLF) or the blood plasma and undergo circula-tion causing extensive oxidative damage to differ-ent organs leading to the CS-associateddegenerative diseases. Indeed, Evans and Pryor(1992) demonstrated that aqueous cigarette tarextracts could damage human alpha-1-proteinaseinhibitor, a development fundamental for the ini-tiation of CS-induced emphysema. Church andPryor (1985) had also implicated the tar phaseradicals to the observed CS-induced pulmonaryand cardiovascular diseases. Zeller and Schmahl(1986) demonstrated that the tar phase of CScould cause cancer in Syrian golden hamsters,while Borish et al. (1985) showed that cigarette tarcan cause mutagenic single strand breaks inDNA. Others like Leanderson et al. and Go-palakrishna et al. have demonstrated that catecholand hydroquinone, two important compoundspresent in tar phase CS can cause extensive oxida-tive damage of lung DNA through 8-OHguanosine formation (Leanderson and Tagesson,1990) and oxidative regulation of protein kinase C(Gopalakrishna and Chen, 1994), respectively in abid to explain the cause of tar phase CS-induced
cancer and metastasis of human lung cells. Our invitro studies show that the tar solution affectslung and heart microsomes almost to the sameextent (Panda et al., 1999), but our in vivo studiesshow that the lung is more affected than the heartunder a regime of acute smoke exposure (Panda etal., 2000) presumably because lesser componentsof the inhaled CS make it to the heart than thelung, which incidentally is directly exposed to theCS during smoking.
It is, however, yet to be discovered whether theobserved tar phase CS-induced oxidative damageis caused by one major oxidant or a number ofoxidants. Identical protein oxidation is obtainedirrespective of whether the tar phase CS solution-induced oxidation is carried out in an oxygen ornitrogen atmosphere. The fact that tar phase CSalso produces significant oxidation of bovineserum albumin, a pure protein, would imply thatthe oxidants of the tar solution oxidize the tissueproteins directly without possibly being generatedsecondarily by interaction with oxygen or anysecondary cellular effect in vitro. We did attemptto study the possible contribution of several po-tential oxidants in the tar phase in line with thosereported by Pryor et al. (1983) in the observedCS-induced oxidative damage of proteins. Noneof them could singularly or in conjugation ac-count for the observed oxidative damage inducedby the tar phase CS, indicating that tar phaseoxidant(s) other than those popularly documentedare perhaps involved in the observed oxidativedamage and increased proteolysis of proteins.However, pinning those component(s) in the tarphase CS proved to be an uphill task in thepresent work, particularly because it entailed arigorous as well as random search among a com-plex mixture of about 1000 active components inthe tar phase CS (Schumacher et al., 1997). Weadmit this to be an important limitation of thepresent study and appreciate that identification ofthe responsible oxidant(s) and their precursors inthe tar phase needs to be necessarily accomplishedin future studies to have a clear idea of the exactmechanism of CS-induced oxidative damage ofproteins.
Since oxidative damage has been projected as aprominent mechanism behind the deleterious ef-
K. Panda et al. / Toxicology Letters 123 (2001) 21–32 31
fects of CS (Janoff et al., 1983; Loeb et al., 1984;Shalini et al., 1994; Hulea et al., 1995; Veyssier,1997; Kleages et al., 1998), there have been sus-tained attempts to find suitable antioxidants thatcan prevent CS-induced oxidative damage and theconsequent degenerative diseases. In a similar at-tempt, we found that vitamin C almost completelyprevented tar phase CS solution-induced oxida-tion of human plasma proteins as well as oxida-tive modification and degradation of guinea piglung and heart microsomal proteins. Other poten-tial antioxidants did not yield encouraging results.Glutathione was found to be only partially effec-tive (20%) while other antioxidants includingSOD, catalase, �-tocopherol, �-carotene and man-nitol were absolutely ineffective.
The hazardous effect of smoking is today aglobal public health problem of great concern. Alarge part of the world population continues tosmoke notwithstanding the adverse campaignsand warnings against the practice. About 15% ofsmokers are afflicted with various degenerativediseases with smoking alone being held responsi-ble for about 78% of global lung cancers (Pi-anezza et al., 1998). With the demonstration thattar-phase CS-induced oxidative damage is alsoalmost completely prevented by vitamin C likethat already observed with the whole phase CS invitro (Panda et al., 1999) as well as in vivo (Pandaet al., 2000), we re-emphasize the necessity ofadequate intake of vitamin C by smokers to avoidthe CS-induced degenerative diseases implicatedwith oxidative damage. Further our work, inhighlighting the predominant toxicity of the tarphase with respect to CS-induced oxidative dam-age, also underscores the necessity of trapping thetar phase more effectively by suitable cigarettefilters in future to reduce the health damagecaused to smokers. However, discouraging smok-ing altogether would definitely be the bestmeasure to eradicate this long-perpetuated, delete-rious practice.
Acknowledgements
This work was supported in part by a CSIRgrant.
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