Effect of different doses of carotenoids in isoniazid-rifampicin induced hepatotoxicity in rats

Isoniazid and rifampicin are the safest known drugs used in the treatment of tuberculosis (TB). These drugs however are potentially hepatotoxic. This hepatotoxicity is manifested by elevation in liver transaminases and rise in bilirubin. A greater number of TB patients develop hepatotoxicity when these two drugs are used in combination.[1] Liver toxicity has been successfully demonstrated in experimental models,[2,3] associated with increase in lipid peroxidation and suppression of the antioxidant system. Due to the large number of patients developing hepatotoxicity, various hepatoprotective agents have been tried for many years against INH–RIF toxicity in rats.[4]

Carotenoids are a group of fat-soluble compounds, which are consumed frequently as part of the diet. The beneficial effects contributed by carotenoids are numerous.[5] It has been reported in several studies that carotenoids protect the liver against potential toxins both in vitro6 and in vivo.[7] This hepatoprotective effect of carotenoids is attributed to the inhibition of reactive metabolites and their radical scavenging activity.[8] Many studies have shown that these dietary carotenoids act as effective antioxidants and prevent liver cell damage. Carotenoids (beta-carotene) have shown hepatoprotective effect in rats in paracetamol-induced hepatotoxicity.[9] Beta-carotene and other carotenoids are efficient singlet oxygen quenchers and thus may be important in protecting against INH+RIF induced hepatotoxicity.

Therefore, the purpose of this study was to evaluate the hepatoprotective effect of different doses of carotenoids against INH+RIF induced hepatotoxicity in rats.

Methods

Young wistar rats (100–150 g) were used in these experiments. They were individually housed in cages, and fed standard diets and water ad libitum. The animals were acclimatized before beginning the experiments. Body weights of these rats were monitored sequentially in control as well as treated groups.

INH and RIF were gifts from Lupin Laboratories, India. Carotenoids were obtained from Parry’s Nutraceuticals; Chennai in soft gel form and each soft gel contained 10 mg of carotenoids with the following composition:

Total Carotenoids = 10 mg (All–trans beta–carotene = 4.20 mg, 9 Cis beta–carotene = 3.74 mg, Dicis beta–carotene = 1.12 mg, Alpha carotene = 0.64 mg. Xanthophylls = 0.30 mg). Kits of serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), bilirubin (Bil) and alkaline phosphatase (ALP) were obtained from Span Diagnostic, India Ltd. All animal procedures were reviewed and approved by the Animal Ethics Committee of Post Graduate Institute of Medical Education and Research, Chandigarh, India.

Dose administration protocol

The animals in the present study were divided into the following groups and were treated for a period of 28 days. All doses were given intragastrically to the animals.

1. Control group: Normal saline treated animals (n = 8).

2. INH+RIF group: 50 mg/kg body weightday of INH + RIF each treated animals (hepatotoxic dose, according to Pal et al., 2006) (n = 8).

3. 2.5 mg Carotenoid group: 2.5 mg/kg body weight/day of carotenoids (n = 8).

4. 2.5 mg Carotenoid+INH+RIF group: 2.5 mg/kg body weight/ day of carotenoids + 50 mg/kg body weight/day of INH + RIF each (n = 12).

5. 5 mg Carotenoid group: 5 mg/kg body weight/day of carotenoids (n=8).

6. 5 mg Carotenoid+INH+RIF group: 5 mg/kg body weight/ day of carotenoids + 50 mg/kg body weight/day of INH + RIF each (n = 12).

7. 10 mg Carotenoid group: 10 mg/kg body weight/day of carotenoids (n=8).

8. 10 mg Carotenoid+INH+RIF group: 10 mg/kg body weight/ day of carotenoids + 50 mg/kg body weight/day of INH + RIF each (n = 12).

9. 20 mg Carotenoid group: 20 mg/kg body weight/day of carotenoids (n=8).

10. 20 mg Carotenoid+INH+RIF group: 20 mg/kg body weightday of carotenoids + 50 mg/kg body weight/day of INH + RIF each (n = 12).

Where n was the number of animals comprising the study.

INH–RIF and carotenoids feeding studies

INH and RIF were prepared in sterile distilled water and pH of RIF solution was adjusted to 3 with 0.1 N HCl.[10] SGOT and SGPT were assayed by the method of Reitman and Frankel, 1957.[11] Bilirubin was measured by the method of Powell, 1944.[12] ALP was assayed by the method of Bergmeyer, 1963[13] Four doses of carotenoids were given to animals for four weeks, according to the method used by Krutovikikch et al, 1980.[14] The minimum dose of carotenoids required to achieve maximum hepatoprotection was tested for. Hepatoprotection after carotenoid administration was monitored serially in all the groups.

Criteria for hepatotoxicity and hepatoprotection

The criteria analysed for measuring hepatotoxicity were the presence of histological lesions such as hepatotoxic necrosis, portal triaditis and intralobular inflammation along with elevated transaminases (more than three times the upper normal limit along with high bilirubin). Criteria for hepatoprotection included absence of histological lesions as

well as normalisation of liver transaminases by co– administration of carotenoids.

Histopathological protocol

After excision, trimmed and fixed in 10% formal saline in all the groups. Light microscopic examination of the liver was undertaken after preparing routine H & E slides. Attention was particularly focused on hepatocyte injury and cellular reaction in portal tracts. Special staining i.e. reticulin, AFB and PAS were carried out whenever it was necessary.

Results

There was no effect in any of the treatment groups on body weight and relative liver weight (Table 1). Rise in the levels of SGOT, SGPT, Bil and ALP are the biochemical indicators for hepatocellular injury. In our experiment, these biochemical parameters were serially monitored before starting the experiments at 14, 21 and 28 days of treatment. INH+RIF treated group experienced a 3 fold rise in SGOT/SGPT values from their baseline at 28 days of treatment (Figures 1 and 2). Bilirubin and ALP were also significantly (p<0.001) raised in INH+RIF treated group at 28 days of treatment when compared to baseline values (Figures 3 and 4). Data of 2.5 and 5 mg/kg b.wt (body weight)/day of carotenoids on serum SGOT, SGPT, Bil and ALP did not show any significant difference at 14, 21 and 28 days as compared to their base line values (Figures1, 2, 3 and 4). Findings from 2.5 mg/kg b.wt / day of carotenoid supplementation along with hepatotoxic dose of INH+RIF showed that this lower dose of carotenoids did not show any significant decrease as compared to hepatotoxic group after 28 days of treatment.

Another dose supplement of carotenoids (5 mg/kg b.wt/ day) was also used along with hepatotoxic dose of INH+RIF for 28 days. The levels of SGOT, SGPT, Bil and ALP showed no significant decrease in 5 Carotenoids+INH+RIF group, as compared to the hepatotoxic group (Figures 1,2,3 and 4) up to 28 days and the values were significantly higher when compared to the control group. Animals treated with 10 and 20 mg/kg b.wt/day of carotenoids also did not show any significant difference in serum SGOT, SGPT, Bil and ALP at 14, 21 and 28 days when compared to their baseline values (Figures 5, 6, 7 and 8) of these parameters. Animals supplemented with 10 mg/kg b.wt/day of carotenoids along with the hepatotoxic dose of INH + RIF for 28 days showed better effect on SGOT, SGPT, Bil and ALP activity. There was significant decrease in these parameters as compared to hepatotoxic dose treated group (Figures 5,6,7 and 8). Higher dose of carotenoids (i.e. 20 mg/ kg b.wt/day) also indicated significant decrease in the activity of serum SGOT, SGPT, Bil and ALP in 20 Carotenoids+INH+RIF treated group as compared to INH+RIF treated group (Figures 5,6,7 and 8), but the values were not comparable to control group.

Histological changes

The liver tissues of all the animals were examined histologically. The control group showed normal morphology in all animals (Figure 9). In INH+RIF treated group, 6/8 (75%) of animals showed moderate to heavy lobular inflammation and moderate portal triaditis with piecemeal necrosis (Figure 10 and Table 2). Rest 2/8 (25%) of animals showed focal lobular inflammation with moderate to heavy portal triaditis (Table 2). Liver histology of the groups treated with 2.5, 5, 10 and 20 mg/kg b.wt/day of carotenoids showed normal morphology in all animals. Histological examination revealed correlation with biochemical parameters in the present study, as liver function tests were found to be increased with hepatocellular necrosis in 2.5 Carotenoids+INH+RIF treated group. Animals in this group, 3/12 (25%) showed moderate to heavy lobular inflammation with moderate portal triaditis and 7/12 (75%) showed focal lobular inflammation, moderate portal triaditis with piecemeal necrosis (Table 3). Thus, this group of animals showed no evidence of hepatoprotection. Both the lobes of liver in 5 Carotenoids+INH+RIF treated group showed focal lobular inflammation and moderate portal triaditis with piecemeal necrosis in 7/12 (58.3%) rats (Table 2). Rest 5/12 (41.7%) animals showed inflammation with moderate portal triaditis (Table 3). Thus, this dose of carotenoids also could not provide clear evidence of hepatoprotection in hepatotoxic dose treated animals. Histology of 10 Carotenoids+INH+RIF treated group have better hepatoprotection as indicated by normalization of the liver morphology in histology four of the twelve animals studied. 1/12 (8.3%) animal showed focal lobular inflammation with moderate portal triaditis (Fig. 11) and 7/12 (58.3%) showed lobular inflammation, moderate portal triaditis with piecemeal necrosis (Table 3). In the group treated with 20-mg/kg b.wt/day of carotenoids along with hepatotoxic dose of INH+RIF showed partial protection in 4 animals, as there was normalization of the liver histology. In 3/ 12 (25%) animals, there was focal lobular inflammation with mild portal triaditis (Table 3). One animal out of 12 (8.3%) had moderate portal inflammation with intralobular inflammation. In the rest 4 animals, there was focal lobular inflammation, heavy portal inflammation with piecemeal necrosis (Figure 12).

Discussion

The main finding of this study was that carotenoid administration in rats was associated with a partial decrease in INH+RIF induced hepatotoxicity. INH + RIF along with 1 mg carotenoids treated animals could result in partial protection against the toxic dose of INH–RIF, which was evident in the unprotected animals. The primary reason for observed protective effects of the carotenoids against INH+RIF toxicity may be attributed to the antioxidant effect of these compounds. Many studies have shown that carotenoids behave as antioxidants by quenching free radicals and reactive oxygen species in animal models.[15] INH+RIF toxicity has reportedly produced oxidative stress in animals[16,17] and further free radicals. These free radicals in turn result in the generation of lipid hydroperoxide and cause cellular injury. This cellular injury further leads to leakage of transaminases, Bil and ALP from hepatocytes whose levels were increased. We observed similar type of elevated transaminases in the present study after 28 days of treatment of INH+RIF. Therefore, in the present study it is possible that beta–carotene and other carotenoids through their antioxidant properties prevented membrane lipid peroxidation produced by INH+RIF treatment. These carotenoids would have terminated the peroxyl radical mediated reactions and further decreased the cellular injury. Similar types of results were seen in carbon tetrachloride intoxication in rats by Chidambara Murthy et al, (2005)[18] in which carotenoids (100 µg/kg b.wt/day) demonstrated hepatoprotective effect through their antioxidant behavior. Therefore, this study presents an attractive and novel idea that carotenoids have a hepatoprotective effect against INH–RIFinduced toxicity in rats. INH+RIF treated animals did not show any significant reduction either in body or in relative liver weight. Similar results were observed in earlier studies from the same laboratory.[19,20 ]Supplementation of carotenoids in the present study also did not indicate any effect neither in body or in relative liver weight as compared to controls. This is in accordance with previous studies in which supplementation of various antioxidants i.e. garlic,[21] carotenoids,[22] N–acetylcysteine[23] had reported no effect on the body or liver weight of animals.

The histopathological changes observed in the present study were piecemeal necrosis, portal triaditis and focal lobular inflammation in INH+RIF treated rats. Several earlier authors have also reported similar types of histological change due to INH+RIF treatment in animals.[24,25] In the present study, the source of the carotenoids was selected from Parry’s Nutraceuticals which is 100% natural and safe. It comes from nature’s richest source of carotenoids, the micro algae Dunaliella salina. Parry’s natural beta–carotene has a composition that is superior to synthetic beta–carotene. Carotenoids from Parry’s Nutraceuticals are available in suspension form in soft gel. Lone supplementation of four dose of carotenoids (2.5, 5, 10 and 20 mg/kg b.wt/day) did not show any histological changes when compared to controls. This could be explained on the basis of the fact that high intake of carotenoids as foods or supplements is not associated with any toxic side effects.[26] Among the four doses of carotenoids, only two doses of carotenoids (i.e.10 and 20 mg/ kg b.wt/day) were able to show hepatoprotection against INH+RIF induced hepatotoxicity, as evidenced by normalization of the SGOT and SGPT levels. The partial reversal of increased serum transaminases showed trend towards returning to normal (but partially) by supplementation of carotenoids indicating partial hepatoprotective effect. Carotenoids also prevented the toxic effect of INH+RIF on bilirubin and ALP activity in serum, but partially as compared to the hepatotoxic treated group. Our study revealed that 10 and 20 mg/kg b.wt/day of carotenoids produced similar degree of hepatoprotection both biochemically and histologically at 28 days of treatment, which is difficult to explain. But, the lesser amount of carotenoids i.e. 10 mg/kg b.wt/day was selected as hepatoprotective group (dose with minimum amount and maximum protection).

Conclusion

This study indicates that carotenoids could partially prevent hepatic injury induced by INH–RIF in rats and this may be due to their antioxidant nature.

References

1. Tahaoðlu K, Ataç G, Sevim T, Tärün T, Yazicioðlu O, Horzum G, et al. The management of anti-tuberculosis drug-induced hepatotoxicity. Int J Tuberc Lung Dis. 2001;5:65–9.

2. Sodhi CP, Rana SV, Mehta SK, Vaiphei K, Attri S, Thakur S, et al. Study of oxidative stress in isoniazid–induced hepatic injury in young rats with and without protein energy malnutrition. J Biochem Toxicol. 1996;11:139–46.

3. Pal R, Vaiphei K, Sikander A, Singh K, Rana SV. Effect of garlic on isoniazid and rifampicin induced hepatic injury in rats. World J Gastroenterol. 2006;12:636–9.

4. Santosh S, Sini IK, Anandan R et al. Effect of chitosan supplementation on antitubercular drug–induced hepatotoxicity in rats. Toxicology 2006; 219: 53–9.

5. Nishino H, Murakoshi M, Mou XY, Wada S, Masuda M, Ohsaka Y, et al. Cancer prevention by phytochemicals. Oncology. 2005;691:38–40.

6. Gumpricht E, Dahl R, Devereaux MW, Sokol RJ. Beta-carotene prevents bile acid induced cytotoxicity in the rat hepatocyte: Evidence for an antioxidant and anti-apoptotic role of beta-carotene in vitro. Pediatr Res. 2004;55:814–21.

7. Murthy KN, Rajesha J, Swamy MM, Ravishankar GA. Comparative evaluation of hepatoprotective activity of carotenoids of microalgae. J Med Food. 2005;8:523–8.

8. Gerster H. Anticarcinogenic effect of common carotenoids. Int J Vitamin Nutr Res. 1993;63:93–121.

9. Kumar G, Ramu GS, Kannam V et al. Anti hepatotoxic effect of beta–carotene on paracetamol induced hepatic damage in rats. Indian Journal of Experimental Biology 2005; 43: 351–5.

10. Bahri AK, Chiang GS, Timbrell JA. Acetylhydrazine hepatotoxicity. Toxicol Appl Pharmacol. 1981;60:561–9.

11. Reitman S, Frankel S. Colorimetric method for the determination of serum glutamic oxaloacetic acid and serum glutamic pyruvic transaminases. Am J Clin Pathol. 1957;28:56–63.

12. Powell WN. Serum bilirubin estimation. American Journal of Clinical Pathology. 1944;14:55.

13. Bergmeyer MVC. Method of enzymatic analysis, Academic Press, New York 1963;40:783–5.

14. Krutouskikh V, Asamato M, Tasasulea N. Differential dose– dependent effects of alpha, beta–carotene and lycopenes on gap junctional intercellular communication in rat liver in vivo. Japanese Journal of Cancer. 1980;88:1121–24.

15. Böhm V, Puspitasari-Nienaber NL, Ferruzzi MG, Schwartz SJ. Trolox equivalent antioxidant capacity of different geometrical isomer of alpha-carotene, beta-carotene, lycopenes and Zeaxanthin. J Agric Food Chem. 2002;50:221–6.

16. Chowdhury A, Santra A, Bhattacharjee K, Ghatak S, Saha DR, Dhali GK. Mitochondrial oxidative stress and permeability transition in isoniazid and rifampicin induced liver injury in mice. J Hepatol. 2006;45:117-26.

17. Srinivasan S, Pragasam V, Jenita X, Kalaiselvi P, Muthu V, Varalakshmi P. Oxidative Stress in urogenital tuberculosis patients: a predisposing factor for renal stone formation–amelioration by vitamin E supplementation. Clin Chim Acta. 2004;350:57–63.

18. Chidambara Murthy KN, Rajesha J, Vanitha A, Swamy MM, Ravishankar GA. Protective effect of Dunaliella salina- A marine microalgae against carbon tetrachloride induced hepatotoxicity in rats. Hepatol Res. 2005;33:313–9.

19. Sodhi CP, Rana SV, Mehta SK, Vaiphei K, Attari S, Mehta S. Study of oxidative stress in isoniazid–rifampicin induced hepatic injury in young rats. Drug Chem Toxicol. 1997;20:255–69.

20. Attri S, Rana SV, Vaiphei K, Sodhi CP, Katyal R, Goel RC, et al. Isoniazid and rifampicin induced oxidative hepatic injury protection by n–acetylcysteine. Hum Exp Toxicol. 2000;19:517–22.

21. Banerjee SK, Maulik M, Manchanda SC, Dinda AK, Das TK, Maulik SK. Garlic– induced alteration in rat liver and kidney morphology associated changes in endogenous antioxidant status. Food Chem Toxicol. 2001;39:793–7.

22. Seifert WF, Bosna A, Hendrius HFS etal. Beta–Carotene (provitamin A) decreases the severity of Ccl4 induced hepatic inflammation and fibrosis in rats. Liver. 1995;15:1–8.

23. Rana SV, Attri S, Vaiphei K, Pal R, Attri A, Singh K. Role of n– acetylcysteine in rifampicin–induced hepatic injury of young rats. World J Gastroenterol. 2006;12:287–91.]

24. Tasduq SA, Kaisar P, Gupta DK, Kapahi BK, Maheshwari HS, Jyotsna S, et al. Protective effect of 50% hydroalcobolic fruit extract of Emblica officinalis against anti–tuberculosis drug induced liver toxicity. Phytotherapy Research. 2005;19:193–7.

25. Tasduq SA, Singh K, Satti NK, Gupta DK, Suri KA, Johri RK. Terminalwa Chebula (fruit) prevents liver toxicity caused by sub– chronic administration of rifampicin, isoniazid and pyrazinamide in combination. Hum Exp Toxicol. 2006;25:111–8.

26. Santamaria L, Bianchi–Santamaria A, Dell’ Orti M. Carotenoids in cancer, mastalgia, and AIDS: prevention and treatment an overview. J Environ Pathol Toxicol Oncol. 1996;15:89–95.