Ind ian Journal of Exp,'rilll-:ntal B ioiogy Vol. 37, De.:cmber 19"'1 . Pl'. ! 1%- :20(j

Effect of ciga re tte smoke on lipid peroxidJtion and antioxidant enzymes in albino rat

S. Baskaran*, S. Lakshmi" & P. R. Pra~ad b

DepJrlment or Biochcmistry* & hIRT_ Perundurai Medi cal Coll ege & Research Cenle r. Perundllrai 61X()'i .I. I ntiI:!.

"Dcpartment of Zooil':;y, Al:lmclu Ang:lppan College for W0I11cn. Komara pa layalll () IS I i:U , India .

Received' Sr' p/clIlba 1998; rel'is('d 3 illlw 19519

Effect llf cigarette smoke (11\ li pid peru xici:1 tion (LPX) and antioxidant enzymc, like cata lase tCAT). slIpcroxiLie di,mutasc ( OD), glulath ione pcrox i d~se (GSH-Px) amI glutat hione-S- tlansfera;c (GST) ;11 varioll s org,\I,s like hnlin . heart, lu ng, liver and kidney of the albino rJt ~ exposed !() cigarelle smoke fi)r ~O minlcby for a period of :W days were :l~s aycd. It was observed that the li pid p roxide levels in liver, lung and kidney were enhanced in case of animals exposed [() cigare tte smlJke, wherea ' brain and heart did not show any change as compared to control <lnimals. The act ivit y or the ant iox id :!11l enzymcs WJS a!s!) ele '/3teu in iiv,:r. iu ng and kid ney of the test animals whereas, br"in ,md heart did not ,how <lny ch:lI1ge in (he acti viti e~ Dr all o r these antioxidant enzymes except glut:Hh ivne-s-l r::msfer:Jsc ',vi1i ch was in cl ea,cd in brain al ~ ,). The i 'vel of reduced glll t3thionc «(;S H) was lowered in !;vcr. lung 3ncl kid ney of the tested ::mim3is when compared with the control illlimals but there was no significant ch<lnge in brain and heart . The results of ollr study suggest that cigarcll e smoke induces lipid peroxidatio n in liver, lUll,!! and kidney, and the antioxidant enzymes leve ls wae cnh ~lIlceJ in order 10 pnllcc i these tissues against the deleterious erreu of the oxygen derived rree radical:, . The dql let ioll or red uced glut athione in these organ~ cou ld be clue to it 's ut il ilati on hy the tissues to mop of( th.: free rad ica ls.

Lip id perox idation (LPX) has gained more impor­talll'e nowadays because of it ';.: in vo lvement in pathog nes is of many disea<;es like atherosclerosist, cancer2 , di abetes mell itu s' and myocardia l infarction4

and also in ageing5, Free rad i .:b or rClc tive oxygen

species (ROS) are produced ill \'iv(} from va rious

bi ochemica l reacti on and also frc'm the respiratory chain as a result of occasional leak age6

. These free radicals are the main cul prits in lipid perox idation . A free rad ical may be <.m atom or a mo lecule with one or more unpaired e lectrons like sllperoxide anion rad ical , hydroxy l radical, pe roxyl rad ica l and alkoxyl radical. T he free radical s are capable of independent existence and can cause ox idative ti ssue damage7

,

The non-radical oxidan ts like hydrogen perox ide and hypochl orous acid which do not posses unpaired ekctroll s are a lso capab le of inciting ox idative ti ssue damageS, Oxidative stress can increase the rate of product ion of free radicals and hence induces lipid peroxidati on. Antioxidants are natural defen se mechan isms existing in our system and these are capable of scavenging the de leterious free radicab. T hese antioxidants are of three types : (a) antiox idan t enzymes like sllperoxide di smutase (EC I. J 5.1.1 ),

*Correspondent author.

cata lase (EC 1,11 . 1.6), g lutathipne perox iclast' (EC 1.1 1.! .9), g lu iathione-S-t ran sferase (EC 2.5.1.1 8) etc., (b) Ant ioxidant vitami ns li ke be ta carotene (pr cursor of vitamin A), a lpha tocopherol (v itam in E) :md ascorb ic acid (\' itamin C) and (c) . on prote in thi ol/ reduced g lutath ione. The de leterious e ffec t of the free radica ls are kept under c hec k by a de lica te ba lance between the ratE' of the ir producti c1n and the rate of thei r e li mipation by the di ffe ren t defense mechanisms, and any shift in thi s de licate ba lance will lead to ce llul ar damage'). C igarett e smoke is

produced by incomple te combusti on of tobacco . It is a heterogenous ae rosol conta in ing marc than 4000 substances 10. Cigare tte smoke contai ns varioll s

cytotoxic, mutagenic and carc inogenic agents li ke biphenyl and po lycyc lic a romatic hydroca rbons. Besides a brge va ri ety of to' ic compoll nds, it al so conta ins many ox idants like oxygen, n itrOll s ox ide etc and free radica ls. Hence, it is capabl e o f initiating or

. . d' d I I & I ' TI I I promotlt1g OXI atl ve amage -. lOug1 t1e re are lot of reports regard ing effect () f cigarette smoke on lipid perox idat ion and antiox id~!Il t e nzymes. the effec t o f c igarette smoke on lipid pe roxidati on and antiox idant enzym~s in va ri olls organs are lacking. Hence, we have unde rtaken t h i ~ study ro inves tigate the lipid peroxidation and anti ox idant status in

BASKARAN el al.: EFFECT OF CIGARETTE SMOKE ON LIPID PEROXIDATION & A TIOXIDA T ENZYMES 1197

various organs under ox idati ve . tress ue to c igarette smoke.

Materials and Met 10ds Bovill':: :,cru m albumin, 5, 5 '-Ji thio-bi '-(2-nitro­

benzoic ck :J), glut<tlhiolh .. (,ed1Iu,J I'm Ill), glUi<lthione reduc la ~e, NAOH-rccluccd COli 11 (di. aLlium sa lt), NAOPlI-rLduccd furm (tdrdsvJiLiIlI . alt) and I, I, 3, 3-tetramethoxy propane were purc hased from Sigma Chemica l, St Louis, USA. Hydrogen perox ide solution (30%) was obtained from Qualigen Fi ne Chemicals, Bombay. I-Chloro-2, 4-dinitrobenzene (CONB), EOTA, Fo lin & Ciocal teau' s ph nol reagent, thiobarbituric acid and trichloroacetic ac id were purchased from Loba-Chemie, Bombay. All other chemicals were of analytical grade and purchased I ocall y.

Inbred adult, male albino rats weighing from 200-250 g from our animal house were used for the experiment. Animals (5 rats/cage) were kept in polypropylene cages of size 43.5x29.0x 16.0 cm and fed with standard pellet diet supplied by National Institute of Nutrition , Hyderabad and water ad libitum. Animals were divided in to two groups each group comprised of 10 animal s. Group I served as control and group II served as test animals. The animals were exposed to c igarette smoke by keepi ng a bottom less rectangular metal container on top of the polypropylene cage containing rats. This metallic container contai ns two holes of about 3 cm diameter, one in the front and the other at the back of the container. A burning cigarette was introducea th rough one hole and air through another hole. The animal s were exposed to the smoke of one burning cigarette for 15 min , twice dail y, at the same time every day, morning and evening (10 .00 A.M. and 4.00 P.M.) fo r 30 days. The same brand of a locally avai lable cigarette was used throughout this experiment. Animals were sacrificed at the end of the experimental period by cervica l dislocati on, the organs like brain, heart, liver, lung and kidney were dissected out, washed in ice cold PBS and frozen immediate ly in deep freezer until further use. The organs were minced and 10% homogenate (I g, w/v) in 50 mM, phosphate buffer, pH 7.4, in cold was prepared using teflon-glass homogeni ser. The homogenate was centrifu ged at I 000 ~ for 15 min , the supernatant was stored at 4°C and used for various biochemical assays .

The lipid peroxide. lc\'e l in va ri ou. ti sslie homogenate was assayed in terms of thiobarbituri c acid react ive substances (TBARS)/ malondi aldehyde (MOA) using

I' I, , 3, 3-tetramcth oxy propane as standard '. The lipid peroxidati on \Va~ ex pressed a<; n 1110Ie.· ()f DA fo rmed per mg prot'.'in ntio '( idant en7V l11CS "uch ;1,

I 14 I I . . I I " I I . S cat a ase , g utat 110l1e pern'(l( a,e ' . p. utat ll one-. -transferase l 6 and slIrl'/"o' ide di "llllltasc l 7 anel reduced glutathione1x were a<; sayed in varioll s ti, suc homo­genates. Total protein in various tissuc homogenate was assayed by the Lowry's method I ') Mean and standard deviation values were calcu lated and statistical signi ficance of difference hetween the contro l and test groups was calcu lated uSin g Student 's t test.

Results and Discussion Table I shows the effect of cigarettc smoke on

lipid peroxide level in li ver, lung, kidney , heart and brain of rat. The lipid perox ide leve l was increased in liver, lung and kidney by 109, 67 and 5 190 respectivel y over the control va lues, .Whereas, brain and heart did not show any signifi cant change. Elevation in catalase activity in liver. lung and kid ney of the test animal s over the control animal s by 61. 128 and 39% respective ly was observed as shown in Table 2 and thi s table also shows increase ill glutathione peroxidase activity in li ver, lung and kidney of test animals by 157, 38 and 25 90 respecti vely over the control anima ls. Increase in glutath ione-s-transferase activity in liver, lung. kidncy and brai n of test animals by 160, 18. 35 ~lIld 449C respecti vely over the control ani mal s was noticed as shown in tab le 2. Superoxide disl1llltasc acti vity was also increased in liver, lu ng and kidney of tes t animals by 21, 42 and 55 % respecti vel y ow r the control values as depictcd in Table 2.

The results show th at the act ivity of the allti ox idan t enzymes were increased in all tissues stud ied except heart and bra in with the exception that the glutathione-s-transferast' was also inc reased (44%) in brain as shown in Table 2. The le vels of glut at hi one was decreased in liver, lung and kidney by 4 i, 23 and 28% respectively in test anima ls when cO l11pared with the control ani(lla ls as shown in Tab le I.

The results of our study clea rl y show th at the cigarette smoke induces lipid peroxidation in liver, lung and kidney. However, there was no alteration in lipid peroxide levels in brain and heart . There was a tremendous increase ( 109%) in lipid peroxidation in

1198 INDIAN J EXP BIOL, DECEMBER 1999

liver. It has bt:;en suggested that the metabolism of toxic compounds including free radicals mainly, though not exclusively, occurs in liver. The metabolites from liver may diffuse into various extra hepatic tissues , cause lipid peroxidation and cellular

. . 20 I I . I h Injury . n ung ti ssue a so t e re was a marked increase (67%) in lipid peroxidation leve l . Though the lung is the first target organ for c igarette smoke and it ' s massive surface area al so makes it a major target organ for the oxidative stress due to c igarette

Table I- Effect of cigarette smoke on lipid peroxidati on and level of GSH in vari ous organs of rat.

[Values are mean ± SD for 10 animals in each group]

Lipid perioxidation(a)

Organ Control Test

Liver 2.61 8±O.582 5.471± 0.782* ( 109% i )

Lung- 2.084±O.437 3.476±O.759** (67 % i )

Kidney 2.326±0.445 3.507±O.445 ** (51 % i )

Heart 2. 168±O.301 2.449±O.533 NS

Brain 2.975±O.368 2.794±O.495 NS

(a) expressed as n mole of MOA formed/ mg protein . (h) expressed in Ilg/mg protein .

Control

8.807±1.1 99

5.839±O.455

5.820±O. 182

4. 133±0.298

4 .643±1.627

Figures in parentheses show percent increase (i)/ decrease (1) as compared to control. P value : • < 0.0000 I; •• < 0.000 I ; NS: Not significant.

GSH (h)

Test

5.204±O 766* (4 1 %1)

4.534±0.626** (23 %1)

4. I 99±0. 789* (28 %1)

3.996±O. I73 NS

3.926±0.6 15 :-.IS

Table 2-Effect of cigarette smoke on the activities of the antioxidant enzymes in the various organs of rat

[Values are mean ± SO for 10 animals in each group]

Catal ase(a) Glutathione peroxidase(h) Glutathione-s-transferase«) Super ox ide di s mut;Js~~ Organ

Liver

Lung

Kidney

Heart

Brain

Control

33.519± 2.344

(61 %i)

11.723± 1.109

(128%i )

8.863± 0.924

(39% i )

7.788± 0.862

1O.947± 0.785

Test Control

53.861± 0.0413± 3.896* 0.00529

(157% i)

25A42± 0.0466± 2.329* 0.00509

(38%i )

12.326± 0.0289± 0.896* 0:00243

(25%i)

7.335± 0.02 12± 0.635 NS 0.00151

10.393± 0.0292± 1.101 NS 0.00227

Test Control Test Control

0.1063± 0.00347± 0.0091 0± IA03± 0.01246** 0.0192 0.0009* 0.202

(160% i) (2 1%i)

0.O645± 0.0192± 0.02266± 1.368± 0.00561 t 0.0008 0.0070* 0.163

(18% i) (42%i)

0.0362± 0.0107± 0.01456± 3.209± 0.00431 t 0.0008 0.0012* 0 .373

(35% i ) (55%i )

0.0202± 0.0247± 0.02446± 1.278± 0.00087 NS 0.00197 ·0.0027NS 0.3 15

0.0302± 0.0294± 0.04226± 1. 173± 0.OO330NS 0.00226 0.0023* 0 . 11 2

(44% i)

(a)One unit of enzyme acti vi ty is the amount of the enzyme required to disproporti onate hydrogen pero xide at the rate of 10.3 absorption/sec. (h)One uf,it of enzyme acti vity is defined as the amount of the enzyme requ ired to oxidize I ~ mole of NAOPH/ min. (·)One unit of enzyme acti vity is t.he amount of the enzyme requi red to conjugate 1 11 mole of CONB wi th GSHI min .

Test

1.695± 0. 232*

1.937± 0. 159.'

4.989± 0.549*

1.252± 0.274 s

1. 136± 0. 156 NS

(d)One unit o f enzyme activi ty is defin ed as the amount of the enzyme required to inhibit the opt ical density at 560 nm of chromogen production by 50%/ min. Figu res in parentheses show the percent increase (i) as compared to cont ro l. P value: *< 0.0000 I; **< 0.000 I ; '1< 0.00 I ; +< 0.0 I

BASKARAN et al. : EFFECT OF CIGARETTE SMOKE ON LIPID PE ROXIDATION & ANTIOXIDANT ENZYMES 11 99

smoke21, the increase in lipid peroxidation is less than liver ti ssue. It shows that the effect of cigarette smoke on lung is less than liver. The mucin in the fluids lining the respiratory tract presumably provide the initial as well as additional defense mechani sm against the oxidative stress so, it could minimise the impact of oxidative stress due to cigarette smoke on the lung that is why the lipid peroxidation level in lung is less than liver21 . Besides, mucin can reinforce the antioxidants present in the fluids lining the respiratory tract. Gupta et ai. 22 already reported that cigarette smoke increased lipid peroxidation in the rat lung but there was no change in the activity of the various antioxidant enzymes. They also reported that the activity of only superoxide dismutase was increased significantly by the cigarette smoke in the rat liver. Kidney, one of the important excretory organs, also showed an increase (51 %) in lipid peroxidation next to liver and lung. The study showed that cigarette smoke did not induce lipid peroxidation in heart and brain, it suggests the ex istence of a more potent antagonistic mechanism in these vital organs anyhow, further study is required to substantiate thi s view. It has also been reported that the myocardium utilizes glucose in preference to fatty acids during stress for it 's energy requirement and this decreased utilization of fatty ac ids by the myocardi al ti ssue may be one of the reasons for no change in lipid peroxide level in the heart23

.

The present study revealed that the activity of the antioxidant enzymes were increased in liver, lung and kidney of the rats exposed to cigarette smoke. The increase in activity of the glutathione peroxidase and related enzyme systems in the lung tissue of the rat exposed to cigarette smoke was also reported by york et ai. 24

, The antioxidant enzymes can detoxify the d I ·· . ?S 26 . e etenous reactive oxygen species _.. so, II1crease in the level of these enzymes could be a defense mechanism against the free radicals . The enzyme levels are elevated in the various tissues in order to cope with the tremendous increase in the production of the reactive oxygen metabolites under the oxidative stress . There was no increase in lipid peroxidation in brain ti ssue but only glutathione-s­tansferase acti vity was elevated. It appears that the lipid peroxidation induced by cigarette smoke in the brain tissue was so minimal so that it did not warrant an array of antioxidant enzymes to cope with . So, increase in activity of only one antioxidant enzyme (GST) was adequate to keep the lipid peroxidation

under control in the brain ti ssue. Red uced glutathione plays very important role in vari ous biochemica l process including lipid peroxidati on and it provides a defense mechani sm for ti ssues aga inst the reacti ve oxygen species27. GSH also forms one of the substrates for the two anti ox idant enzymes, glutathione peroxidase and glutathi one-s-transferase. The level of glutathione was found to be reduced in liver (4 1 %), lung (23%) and kidney (28 %) of the tes t animals whereas, there was no significant change in brain and heart. The decrease in the leve l of glutathione in kidney of the rats exposed to cigarette

2/01 smoke was also reported by Anand el at. . The depletion of glutathione was associated with increase in lipid peroxidation in all these organs. The decrease in glutathione leve l in these ti ssues may be a consequence of enhanced utili za ti on of thi s compound by the antioxidant enzymes, glutath ione peroxidase and glutathione-s-transferase. The maximum depletion (41 %) of glutathione was observed in liver and it could be attributed to the efflux . of glutathione from li ver to various ex tra hepatic ti ssues for their protecti on aga inst the reacti ve oxygen metabolites besides it 's consumption by the antioxidant enzymes as stated already. Thus, we have noticed a direct correlation between increase in li pid peroxidation and decrease in glutahtione level. Similar observation has been made already by va ri ous workers under different stress conditi ons l'i ·.1 1

To conclude, our findings clearly show that though there was an increase in the acti vity of antioxidant enzymes in liver, lung and kidney to protect them against the lipid peroxidation induced by the cigarette smoke, it was not adequate to cope with the copious production of free radical s, that is the reason why the lipid peroxidation could not be kept under cont ro l in these tissues. The increase in ac tivity of these antioxidant enzymes also increased the utili sat ion of glutathione hence, there was a concomitant dec rease in the level of glutathione in these ti ssues .

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