Indian Journal of Experimental Biology Vol. 39, May 2001, pp. 436-440

Protective effect of N-acetylcysteine in isoniazid induced hepatic injury in growing rats

S Attri

Department of Biochemistry, Govt. Medical College, Chandigarh 160 032, India

and

S VRana*, K Vaiphie+, R Katyal , CP Sodhi, S Kanwar & K Singh

*Department of Gastroenterology and •rathology, Postgraduate Insti tute of Medical Education and Research, Chandigarh 160 012, India

Received 10 April 2000; revised 22 January 2001

Status of oxidative/antioxidativc profile was the mechanistic approach to inumerate the nature of protection by N­acetylcysteine (NAC) in isoniazid (INH) exposed experimental animals. Analysis of lipid peroxidation, th iol levels, cytochrome P450, superoxide dismutase (SOD), catalase, glutathione peroxidase, reductase and transferase were estimated in liver along with the body and liver weight of animals and histological observations. Isoniazid exposure to animals resulted in no change in body and liver weights. Thiols, lipid peroxidation, catalase, SOD glutathione peroxidase, reductase, transferase and cytochrome P450 level s were altered with INH exposure. Supplementation of NAC with INH protected the ani mals against hepatotoxic reactions by minimizing the free radical induced tissue injury and overall maintenance of the endogenous scavengers of free radicals.

The aminothiol, N-acetyl-L-Cysteine (NAC), an analogue and one of the precursors of glutathione (GSH) is capable of rapidly replenishing the depleted intracellular GSH levels'. The intracellular effects of NAC are amenable to the fact that it enriches the thiol pool in the cellular environment and by acting as a precursor of GSH, triggers many of the enzymatic and non-enzymatic protective mechanisms of cellular defense system2

• Most of the circulating GSH originates from liver3 and hepatocytes are known to contain the highest concentrations (5-1 0 mmol/1). GSH homeostasis is disturbed in excessive conjugation reactions of GSH with xenobiotic or endogenous reactive intermediates formed during lipid peroxidation4

. Formation of reactive oxygen species (ROS) can occur during xenobiotic metabolism and could be a mechanism for initiation of hepatotoxicity. ROS appear to have broader significance in the production of tissue injury under conditions of oxidative stress5

·6

.

N-acetylcysteine at the cellular level suppresses the production of reactive oxygen metabolites from human neutrophils during their oxidative burst in vitro7

• The ability of NAC to support the GSH system and to scavenge ROS represents one of the major

*Author for correspondence: House No. 137115-A, Chandigarh 160 015, India E-mail: svrana25@hotmail.com

mechanisms explaining its multiple therapeutic properties and the antioxidant, antigenotoxic and anticarcinogenic effects2

·6

.

Isoniazid (INH), a sheet anchor of antitubercular therapy, induced hepatic injury and was oxidative in nature8

. The role of ROS, thus the oxidative stress, has been recently highlighted in INH-induced hepatotoxicity in experimental animals9

·10

.

Keeping above in view, the present study has been designed to understand the protective effect of AC on INH induced oxidative hepatic inj ry in young rats.

Materials and Methods Young male wistar rats (24) of about 40g body

weight were divided into 4 groups of 6 rats each . Purified powder of INH was administered ip to animals, at a dose of 50 mg/kg body weight daily in 4 mllkg sterile distilled water for 3 weeks. NAC (Sigma, USA) was administered ip either alone or simultaneously alongwith INH, at a dose of 100 mg/kg body weight 11 daily in 4 ml/kg sterile distilled water for 3 weeks. Similarly, sterile water (4 mllkg) was administered by the same route to control animals.

Rats were fasted for 12 hr before sacrificing by cervical dislocation under light anaesthesia. Liver tissue was excised immediately and quickly cooled in

AlTRI eta/.: PROTECTIVE EFFECT OF N-ACETYLCYSTEINE ON HEPATIC INJURY 437

a beaker resting in an ice bath followed by perfusion with ice-cold normal saline. Histopathological investigations of tissue of control and treated rats were carried out on H & E stained sections.

Liver tissues were homogenised in 100 mM potassium phosphate buffer as per Huang and Iwu 12

•

Total , protein and non-protein thiols were estimated in crude homogenate 13

. Lipid peroxidation was measured in crude homogenate as per Ohkawa et al 14

•

Enzyme assays-All enzyme assays were carried out in post- mitochondrial supernatant (PMS). Cytochrome P450 (Ref. 15) superoxide dismutase (SOD) 16

, catalase 17, glutathione peroxidase 18 and

gl utathione reductase 19 were assayed. Glutathione-S­transferases were assayed using 1-chloro-2, 4-dinitrobenzene (CDNB), 2,3-dinitro -4-chlorobenzene (DCNB) and ethacrynic ac id (EA) as substrates following Habig et aP0

• Lowry's method21 was employed for estimation of tissue proteins.

Statistical analysis-Statistical analysis was applied as reported earlier10

.

Results and Discussion Effect of INH and NAC on various biochemical

parameters are given in Tables 1-4. INH and NAC treatments, either alone or in

combination had no effect on body weights of animals. Similarly, relative liver weights were also not affected by any of the drug treatments.

Hepatic injury--Hi sotological observations are shown in Figs 1-4. Portal tri aditis of mild to moderate degree was the main finding on histopathology (Fig. 2). The infiltration was observed in hepatic lobules in INH treated animals (Fig. 3). Co­administration of NAC with INH was found to have completely ameliorated the induction of hepatic lesions by INH as evidenced by the normal morphology of rat liver (Fig. 4).

By augmenting cellular antioxidative system, cells can be protected against the oxidative injuries

Table 1- Effect of INH and NAC on Total, protein bound and non protein thiols.

Groups

Control (C)

INH

lNH+NAC

NAC

[Values are mean± SO for 6 rats]

Hepatic thiol s ().! mole/g tissue) Totalthiols Protein Non protein

13.32 ± 0.54

12.0 1±0.41 *

13.20 ± 0.49

13.44 ± 0.46

bound bound

10.09 ± 0.05

9.41 ± 0.43*

9.87 ± 0.24

9.97 ± 0.22

3.23 ± 0.39

2.60 ± 0.41 *

3.35 ± 0.46

3.47 ± 0.38

P value *<0.01 between control & 1NH

produced by various drugs and chemicals. NAC happens to be least toxic with best ability to be a precursor of GSH22

•

In the present study INH and NAC, either individually or in combination, did not have any effect on body weights and relative liver weights. These results are in accordance with earlier studies9

·23

·24 reporting no change in relative li ver

weights of animals exposed to INH.

Hepatotoxicity produced by INH was revealed by histological changes wherein mild to moderate degree of portal triaditis with occasional collection of inflammatory cells in the hepatic lobules was

Table 2- Effect of INH and NAC on lipid peroxidation, catalase and superoxide dismutase.

Groups

Control (C)

INH

INH+NAC

NAC

[Values are mean± SO for 6 rats]

Lipid Catalase peroxidation ().! mol/min /mg

(nmol MOA/g protein) tissue /IOmin)

117 ±7 312 ± 33

133 ± 9* 181±22**

113 ± 17 289 ± 28

101 ± 13* 303 ± 31

Superoxide-dismutase (IU)

14.7 ±2.1

17.7 ± 1.86*

18.2 ± 1.76**

14.2 ± 2.64

P values; *<0.05 between control vs INH and Control vs NAC; **<0.0001 control vs INH Controls vs INH+NAC.

Table 3-Effect of INH and NAC on Glutathione peroxidase, glutathione reductase and cytochrome P45o

Groups

Control (C)

INH

INH+NAC

NAC

[Values are mean± SO for 6 rats]

Glutathione Glutathione peroxidase reductase

Cytochrome p450

(nmollminl mg protein)

460 ± 67 25 .3±2. 1 0.42 ± 0.05

294 ± 39** 29.0 ± 1.5** 0.485±0.052""

438 ± 86 32.3 ± 2.13*** 0.468±0.052*

471 ±58 28.0 ± 1.72* 0.408± 0.053

P values; *<0.05 between control vs INH and control vs NAC and Control vs INH+NAC; **<0.01 control vs INH ; and***<O.OOl control vs INH+NAC.

Table 4-Effect of INH and NAC on Glutathione-S-transferases using CONB, OCNB and EA as substrates

[Values are mean± SO for 6 rats]

Groups Glutathione -S-transferases (nmol/min/mg protein) CONB OCNB EA

Control (C) 626± 62 20.2± 2.9 14.2 ± 2.32

INH 551 ± 65* 18.0 ± 3.2 16.8 ± 1.47*

INH+NAC 609 ± 46 19.8 ± 1.7 17.5 ± 2.6*

NAC 678 ±58* 22.17 14.8 ± 2.32

P values; *<0.05 between control vs INH and control vs NAC and Control vs lNH+NAC

438 INDIAN 1 EXP BIOL, MAY 200 I

observed. The histopathological pattern of liver injury in the present study was similar as reported by Mitchell et aP5

•

Co-administration of NAC alongwith INH protected experimental animals against hepatotoxic reactions. These results are supported by earlier studies 16

•27

. The hepatoprotection by NAC observed in the present study may provide an additional support to the oxidative stress mechanism of INH induced hepatic injury.

Exposure of animals to INH resulted in depletion of hepatic thiols (protein as well as non-protein bound) which are critical components of antioxidant defense system and made the animals susceptible to oxidative

stress (Table 1). This was evident from increased lipid peroxidation observed in these animals (Table 2). N­acetylcysteine was found to significantly prevent the alteration in antioxidative profi les and protected the animals against drug-induced lipid peroxidation. As NAC has been shown to inhibit lipid peroxidation in the present study, it is likely that protein thiol groups were spared from the attack of lipid radicals or reactive lipid soluble aldehydes.

Total cytochrome P4so levels were induced by INH treatment indicating more production of reactive metabolites and superoxide radicals. NAC did not reduce levels of cytochrome P450 in INH exposed animals in the present study. This Is m accordance

Figs 1-4-Photomicrograph of rat liver showing (I) normal morphology of control animal including one portal tract and one central vein; (2) portal tract inflammation (arrows) in INH treated animal; (3) infilteration in hepatic lobules (arrow heads) in INH treated animal; and (4) normal morphology in INH and NAC treated animals (all Figs H & E, x520).

A TIRI et a . . : PROTECTI VE EFFECT OF N-ACETYLCYSTEINE ON HEPATIC INJURY 439

with experimental study by De Flora et at28.

In the present study, INH exposed animals exhibited significantly enhanced SOD activity. This may be, as the liver is trying to remove more superoxide radicals formed due to INH. Supplementation of NAC did not alter the INH induced SOD activity because NAC may be unable to remove superoxide radicals formed due to INH.

The activities of both H20 2 scavengers i.e. catalase and glutathione peroxidase showed a severe decline with INH treatment which is in accordance with the previous investigations8

-10

. The decreased activities of these enzymes can be explained on the basis that superoxide radicals when produced in excess may inactivate these H20 2 scavengers29

• Simultaneous administration of NAC with INH prevented the decrease of catalase and glutathione peroxidase activities either directly or via GSH.

Supplementation of NAC significantly prevented the depletion of non-protein thiols (GSH) in INH exposed animals (Table l). Similar kind of mechanism has been proposed for NAC hepatoprotection in case of acetaminophen30

•

A significant decrease in protein thiols has been observed in INH treated animals in the present study (Table I). The depletion of protein thiols has been proposed as a critical event in the lethal injury of hepatocytes exposed to menadione or adriamycin induced acute oxidative stress31

• Supplementation of NAC prevented the loss of protein thiols in INH treated animals (Table l). INH exposure significantly enhanced glutathione reductase activity, an enzyme that regenerates reduced glutathione from oxidised glutathione (Table 3). Increase in this enzyme activity may be associated with GSH depletion which is in accordance to Joshi et al8

-10

• Supplementation of NAC to INH treated animals and to controls also stimulated significantly the glutathione reductase enzyme activity. Similar effect of NAC has also been reported2

•

The present results of alteration in glutathione transferase enzyme (CDNB) activity shown in Table 4 are in accordance with Aniya and Naito32 who observed that the microsomal GST (CDNB) activity was decreased after 80 min perfusion of liver with H20 2. The enyzme activity against EA as substrate was found to be stimulated by treatment with INH as reported earlier also8

-10

• Supplementation of NAC prevented the decrease of GST (CDNB) activity in INH treated rats probably by providing adequate GSH for functioning of GST.

The present study indicates that simultaneous administration of NAC alongwith INH protected the experimental animals against hepatotoxic reactions. It can be concluded that NAC is a valuable tool for protection against hepatotoxicity induced by INH in an animal model by increasing the antioxidant defense systems, minimizing the free radical induced tissue injury and overall maintenance of the endogenous scavengers of free radicals.

Acknowledgement The study was financially supported by Department

of Science and Technology, Chandigarh. Lupin Laboratories India is acknowledged for supplying the purified JNH powder as a gift item.

References I Ellenhorn M J & Barceloux D G, in Medical toxicology;

Diagnosis and treatment of human poisoning: (Elsevier, New York) 1988, 162.

2 De Flora S, Balansky R, Bennicelli C, Camoirano A, D'Agostini F, Izzotti A & Cesarone, C F, Mechanisms of anticarcinogenesis the example of N-acetylcysteine, in Drugs, diet and disease, Mechanistic approaches to cancer. Vol. I ; edited by C Loannides & D F V, Lewis. (Ell in Harwood, Heme!, Hempstead, UK) 1995, 151.

3 Lauterberg B H, Smith C V, Hughes H & Mitchell I R, Biliary excretion of glutathione and glutathione disulphide in the rat, J Clin In vest, 73 ( 1984) 124.

4 Ishikawa T & Sics H, Glutathione as an antioxidant, Toxicological aspects, in Glwathione Chemical, biochemical and medical aspects, edited by D Dolplain, R Poulron & D Avramovic, Wiley, New York, 1989, 85.

5 Farber J L, Kyle M E & Coleman 1 B, Mechanism of cell injury by artivated oxygen species, Lab Invest, 62 ( 1990) 670.

6 Sics H, Oxidative stress from basic research to clinical application, Am J Med, 91 ( 1991 ) 31.

7 Gressier B, Cabanis A, Lebegue S, Burner C, Dine T, Luyckx M, Cazin M & Ca"-in 1 C, Comparison of the in virro effects of two thion-containing drugs on human neutrophil hydrogen peroxide production, Meth Find Exp Clin Pharmacal, 15 ( 1993) I 0 I.

8 Sodhi C P, Rana S V, Mehta S K, Vaiphei K, Attri S, Thakur S & Mehta S, Study of oxidative stress in isoniazid induced hepatic injury in young rats with and without protein energy malnutrition, J Biochem Toxicol, II ( 1996) 139.

9 Sodhi C P, Rana S V, Mehta S K, Vaiphei K, Attri & Mehta S, Study of oxidative stress in INH-RMP induced hepatic injury in young rats, Drug Chem Toxico/, 20 ( 1997) 255.

10 Sodhi C P, Rana S V, Mehta S, Vaiphei K, Gael R C & Mehta S K, Study of oxidative stress in rifapicin induced hepatic injury in growing rats with and without protein energy malnutrition , Hum Exp Toxicol, 16 (1997) 315.

11 Attri S, Vaiphei K, Rana S V, Sodhi C P & Singh K, N­acetylcysteine confers hepatoprotection 111 isoniazid rifampicin induced hepatic injury - a histological study Indian J Gastroenterol, 15 ( 1996) A52.

440 INDIAN J EXP BIOL, MAY 2001

12 Huang C J & Fwu M L, Degree of protein deficiency affects the extent of the depression of antioxidative enzyme activity and the enhancement of ti ssue lipid peroxidation in rats, J Ntar, 123 (1993) 803.

13 Sedlak J & Lindsay R H, Estimation of total, protein- bound and non-protein sulphydyrl groups in ti ssue with Ellman's reagent , Anal Biochem, 25 ( 1968) 192.

14 Ohkawa H, Ohishi N & Yagi K, Adenosine acts as an endogenous acti vator of the cellular antioxidant defense system, Biochem Biophys Res Com1111111, 20 I ( 1994) 508.

15 Omura T & Sato R, The ca rbon monoox ide binding pigment of liver mi crosomes II Solubili za tion, purification and properties, J Bioi Chem, 239 ( 1964) 2370.

16 Kono Y, Generation of superoxide radical during autoox idation of hydroxy lamine and an assay for superox ide dismutase, Arch Biochem Biophys 186 ( 1978) 189.

17 Luck N, Catalase estimati on, in Methods in en:ymatic analysis, (Academic Press, New York) 1974, 886.

18 Carlberg I & Mannervik B, Purification by affin ity chromatography of yeast glutathione reductase, the enzy me responsible for the NADPH dependent reduction of the mixed disulfide of co-enzy me A and glutathione, Biochem Biophys Acta 484 ( 1977) 268.

19 Flohe L & Gungler W A, Assay of glutathione peroxidase, Methods Enzymol, 105 (1984) 114.

20 Habig W H, Pabst M J & Jakoby W B, Glutathione-s­transferasese, the first step in mercapoturic acid formation, J Bioi Chem, 249 ( 1974) 7130.

21 Lowry 0 H, Rosebrough N J, FarrA L, Randall R J, Protein measurement with the Foli n phenol reagent, J Bioi Chem 193 (195 1) 265.

22 Bananomi L & Gazzaniga A, Toxicological pharmakinetic and metablic studies of acetylcysteine, Eur J Resp Dis, 61 (1980) 45.

23 Powell -Jackson P R, Tredger J M, Smith H M, Davis M & Williams R, Effect of isoni azid administration on selected rat and mouse hepatic microsomal mixed function oxidase and

in vitro [14C]acetylhydrazine-deri ved covalent bindi ng, Biochem Pharmacal 3 1 ( 1982) 4031 .

24 Tredger J M, Smith H M, Powell-Jackson P R, Davis M & Williams R, Effect of rifampicin on the mouse hepatic mi xed functi on oxidase system, Biochem Pharmacal, 30 ( 198 1) 1043.

25 Mitchell J R, Zimmermer H J, Ishak K G, Thorgeirson 0 P, Timbrel! J A, Snodgrass W R & Nelson S D, Isoniazid li ver InJury: Clinical spectrum, pathology and probable pathogenesis, Ann Intern Med 84 ( 1976) 181 .

26 Bruno M K, Cohen S D & Khairallah E A, Antidotal effecti veness of N-acetylcysteine in reversing acetaminophen induced hepatotoxicity. Enhancement of the proteo lysis of arylated proteins, Biochem Pharmacal 37 ( 1988) 43 19.

27 Harrison PM, Wendon J A, Gimson A E S, Alexander G J M & Willi ams R, Improvement by acetylcysteine of hemodynamics and oxygen transport in fu lmi nant hepatic failure, N Eng/ J Med, 324 ( 199 1) 852.

28 De FloraS, Bennicelli C, Camoirano A. Serra D, Romano M, Rossi G A, Morelli A & De Flora A, In 1•ivo effects of N­acety lcysteine on gl utathione metabolism and on the biotransformation of carcinogenic and/or mutagenic compounds, Carcinogenesis 6 ( 1985) 1735.

29 Blum J & Fridovich I, Inact ivati on of glutath ione peroxidase by superox ide radical, Arch Biochem Biophys 240 ( 1985) 500.

30 Al-mustafa Z H, AI-Aii A K, Qaw F S & Abdul-eader Z, Cimetidine enhances the hepatoprotcctive action of N­acetylcysteine in mice treated with toxic doses of paracetamol, Toxicology 121 ( 1997) 223.

31 Brent J A & Rumack B H, Role of free radicals in tox ic hepatic inj ury, I Free radical biochemistry, Clin Toxicol, 3 1 (1993) 139.

32 Aniya Y & Naito R, Ox idative stress induced act ivation of microsomal glutath ione-s-transferase in isolated rat li ver, Biochem Plwrmaco/, 45 (1993) 37.