Korean J Hepatobiliary Pancreat Surg.  1997 Aug;1(2):83-118.

Assessment for Hepatic Injuries Induced by CCl4 and 2,2'-azobis(2-amidinopropane) dihydrochloride(AAPH) in Sprague-Dawley Rats

• Lee YJ
• Ha WS
• Park ST

Abstract

INTRODUCTION: It is now generally accepted that free radicals play an important role in the development of various forms of tissue damage and pathological events, such as heart disease, cancer, and aging. On the other hand, it is difficult to demonstrate the injurious actions of free radicals in vivo, and it has not been clearly proven experimentally whether the damage is really caused by free radicals, nor how the free radicals damage biological molecules, primarily because of the difficulty in generating free radicals in biological systems under controlled conditions. PURPOSES OF STUDY: In this study, we investigated whether the histologically different part of lesion is created in liver injuries by free radicals formed from each mechanism. we investigated the usefulness of the ICG excretion test as an early indicator of liver injury, and the significance of an increase of urinary biopterin in free radical injury. MATERIALS & METHODS: In this study, the acute hepatic injuries were induced in the Sprague-Dawley rat by intraperitoneal injection with CCl4(0.125 ml/100 gm body wt.) and AAPH(1 mg/100 gm body wt.). Control rats were treated with 0.5 ml of corn oil per 100 gm via intraperitoneal route. And then, liver function tests(ALT, AST, ALP, gamma-GTP, serum bilirubin and ICG excretion test), alpha-fetoprotein, urinary biopterin and histologic changes were serially examined. Total 130 rats were prepared, and each group of 5 rats of them were sampled at intervals of 2, 4, 8, 12, 24, 48 hours following administration of CCl4, AAPH solution. All values were expressed as mean +/- standard error. The results of all treatment groups and control group were analyzed by ANOVA with Duncan's multiple range test for variable. And then, the correlations between the increasing rates of urinary biopterins, ICG T1/2 and those of classic liver enzyme activities, serum bilirubin levels were analyzed for statistical significance by Pearson correlation analysis. In this analysis, a p-value below 0.05 was considered to be statistically significant.
RESULTS
1. Activities of liver enzymes(serum AST, ALT, ALP and gamma-GTP) and levels of serum bilirubin significantly increased in all the treatment groups compared to control. As time passed, these levels continued to increase(p<0.05). 2. In comparison with AAPH treatment group, serum AST and ALT of CCl4 treatment group increased more significantly(p<0.05). 3. In ICG excretion test, ICG T1/2 of all the treatment groups were greatly prolonged from about 4 - 12 hours. Compared to AAPH treatment group, ICG T1/2 of CCl4 treatment group was more significantly prolonged(p<0.05). 4. As time passed, urinary biopterin levels of all the treatment groups increased from about 24 hours. Urinary biopterin levels of AAPH treatment group increased more significantly than in CCl4 treatment group from about 48 hours(p<0.05). 5. In histopathologic view, CCl4 treated hepatic acini showed that hepatocytes in centrilobular zone were degenerated. But, AAPH treated livers showed that all hepatocytes were degenerated. 6. Passive hemagglutination tests of alpha-fetoprotein showed negative reaction in all the treatment groups and control group. 7. Correlations were statistically significant(p<0.05) between the increasing rates of urinary biopterin levels and those of classic liver enzyme activities(except, serum AST), and serum bilirubin levels in CCl4 induced hepatotoxicity. 8. In AAPH induced hepatotoxicity, the increasing rates of urinary biopterin levels correlated with those of classic liver enzyme activities(serum ALT, AST, ALP, -GTP) and serum bilirubin levels(p < 0.05). 9. The increasing rates of ICG T1/2 correlated significantly with those of liver enzyme activities and serum bilirubin in CCl4 and AAPH induced hepatotoxicity(p<0.05), but there were no statistically significant correlations between the increasing rates of ICG T1/2 and those of serum ALT in CCl4 induced hepatotoxicity. 10. And also, the increasing rates of ICG T1/2 correlated significantly with those of urinary biopterin in CCl4 and AAPH induced hepatotoxicity(p<0.05).
CONCLUSION
We observed in this study that the difference in hepatic lesions induced by free radicals might be closely related to the site where free radicals had been generated. We indirectly found that the tissue damage is caused by free radicals. There were excellent correlations between urinary biopterin levels, ICG excretion test and classic liver enzyme activities, and serum bilirubin levels in acute hepatic injuries induced by free radicals from CCl4 and AAPH. Also, we determined that ICG excretion test is a saturable process for evaluation of acute hepatic injury. The urinary biopterin levels in CCl4 treated groups were significantly different from those of AAPH treated groups suggesting that an unknown mechanism is concerned with free radical induced biopterin elevation. However, the level of urinary biopterin in free radical injury may be a useful complementary index.

MeSH Terms

Aging
alpha-Fetoproteins
Animals
Bilirubin
Biopterin
Corn Oil
Free Radicals
Hand
Heart Diseases
Hemagglutination Tests
Hepatocytes
Injections, Intraperitoneal
Liver
Rats
Rats, Sprague-Dawley*
Bilirubin
Biopterin
Corn Oil
Free Radicals
alpha-Fetoproteins