Laboratory Animals >
Volume 42, Number 4 >
Pp. 495-504
Papers |
* Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan, ROC;
Division of Gastroenterology, Department of Internal Medicine, Cardinal Tien Hospital, Fu Jen Catholic University, Taipei, Taiwan, ROC;
Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, ROC;
Department of Pathology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, ROC;
** School of Pharmacy, National Defense Medical Center, Taipei, Taiwan, ROC;
Department of Research and Development, National Defense Medical Center, 161 Minchuan East Road, Sector 6, Taipei, Taiwan 114, ROC
Correspondence: O Y P Hu. Email: hyp{at}ndmctsgh.edu.tw
 | Summary |
|---|
 Top Summary Materials and methodsResultsDiscussionReferences |
|---|
Key Words: Galactose single point method • quantitative liver function • rat • isoniazid • hepatotoxicity
Several tests, both invasive and non-invasive, have been used to assess hepatic function in rats, monitor the progression of hepatic injury and screen for liver disease. The usual parameters of hepatotoxicity include: (a) indicators of hepatocellular injury: alanine aminotransferase (ALT) and aspartate aminotransferase (AST); (b) indicators of hepatobiliary injury: alkaline phosphatase (ALP) and total bilirubin; (c) supplemental indicators of hepatic synthetic function: total protein, albumin, triglycerides, cholesterol, glucose, urea, activated partial thromboplastin time and prothrombin time (Weingand et al. 1996, O'Brien et al. 2002, Provost et al. 2003, Boone et al. 2005). Currently, there is no routinely used method to quantitatively measure the residual liver function in rats.
Quantitative measurement of the entire liver function involving all processes that take place in the liver is fundamentally not possible (Bircher 1986). A number of methods or drug clearance evaluations have been reported to estimate human liver function (Roberts et al. 1978, Homeida et al. 1979, Baker et al. 1983, Renner et al. 1984). The use of drugs as liver function indicators may increase the burden on liver cells, as well as a certain degree of damage. Measurements of the metabolic capacity of a certain compound, such as galactose, can assume a metabolic rate-limiting step in the liver, making it possible to obtain a representative value of residual liver function (Keiding et al. 1977, 1982).
In patients with chronic hepatitis, cirrhosis and hepatocellular carcinoma, our laboratory has previously shown that the galactose single point (GSP) method is well correlated with the severity of liver disease (Tang & Hu 1992). This GSP method has been successfully applied to drugs which are extensively metabolized or excreted from the liver, such as promazine and cefoperazone clearance in patients with various liver diseases (Hu et al. 1994, 1995b,c). This method has also been recommended by the US Food and Drug Administration (FDA) in the guidance for industry pharmacokinetics in patients with impaired hepatic function (FDA 2003).
Galactose is a naturally forming sugar with a high extraction ratio that is 90% metabolized in the liver. The metabolism of galactose occurs through a cytosolic pathway independent of the cytochrome P450 (CYP) system. Thus, there is less variation in its metabolism because of drug induction or inhibition, or genetic polymorphism (Becker 1998). In the liver, galactose is catalysed by galactokinase through epimerization, converting to glucose-1-phosphate. The reaction with galactokinase is the rate-limiting step of the galactose metabolism in the hepatocyte (Ballard 1966, Keiding et al. 1977, Craik & Elliott 1980). The fact that galactose has a high extraction ratio, however, makes the metabolism of galactose dependent on liver blood flow and hepatic functional mass. To reflect the hepatic enzymatic activity, the metabolic pathway must be saturated by giving relatively large doses of galactose (Tygstrup 1964, Henderson et al. 1982). At high concentrations in rats and humans, galactose follows Michaelis-Menten kinetics (Keiding 1973, Hu et al. 1995a). The galactose elimination capacity (GEC) test was proposed several years ago as a quantitative test to measure human liver function (Lindskov 1982). However, the multiple blood samples required to establish the decrease of galactose concentration made the test difficult in clinical practice, and therefore led to the more practical investigation of the use of the GSP method in the assessment of human liver function (Tang & Hu 1992).
As we wanted to know how GSP would reflect the degree of hepatic injury in rats, we chose to use the hepatotoxin, carbon tetrachloride (CCl4), for its known severe hepatotoxicity and isoniazid (INH) for its mild hepatotoxicity. In the liver, INH is first metabolized into acetylisoniazid via N-acetyltransferase, followed by hydrolysis to acetylhydrazine (Mitchell et al. 1976). Hydrazine, a toxic intermediate product of INH metabolism can be produced both directly (from INH) and indirectly (from acetylhydrazine). The severity of INH-induced hepatocellular damage has a positive correlation with the plasma hydrazine concentrations (Sarich et al. 1996). Both hydrolysis reactions involve an amidase enzyme. Sarich et al. (1999) reported that bis-p-nitrophenyl phosphate (BNPP), an amidase inhibitor, prevented INH-induced hepatocellular damage. The protective mechanism is most likely through the inhibition of hydrazine production from INH.
On these grounds, the present study was designed with the aim of investigating the suitability of the GSP method as an indicator of residual liver function in rats. This GSP value was determined in dried blood samples from the tail vein and compared with plasma AST and ALT activities, and with a score obtained from the histological examination of the respective liver samples.
| Materials and methods |
|---|
|
TopSummaryMaterials and methodsResultsDiscussionReferences |
|---|
Animals
Male Sprague-Dawley rats weighing 300–325 g were obtained from the National Applied Research Laboratories and National Laboratory Animal Center (Taiwan). All the experiments were approved by the local Institutional Review Board according to the Helsinki recommendations and were performed in adherence to the National Institutes of Health guidelines for the treatment of animals. All the rats were housed in a room with air/humidity control, with a 12 h light/dark cycle, and allowed access to food and water ad libitum throughout the experiment. All the rats were intraperitoneally (i.p.) anaesthetized with sodium pentobarbital (50 mg/kg). Polyethylene catheters were placed in the right or left internal jugular vein for the administration of galactose. Catheter insertion was performed by a cut-down technique and the distal end of the catheter was tunnelled under the skin and externalized through an incision in the back of the neck. Postoperative discomfort was minimized by giving all animals 5 mg/kg carprofren (Sigma) by subcutaneous injection. After the surgery was completed, the rats were fasted overnight (approximately 16 h) during recovery, but were allowed water ad libitum.
Experimental design
GSP in rats
Rats (n = 8/group) were given i.p. CCl4 injections (10% solution in corn oil) of 1 mL/kg body weight. Control groups were injected with corn oil vehicle only. The galactose tests were assayed 24 h after CCl4 treatment in order to study the functional liver cell mass. Galactose was injected intravenously (i.v.) as a bolus (40% galactose, 0.5 g/kg body weight) saturating the catabolic enzyme system. The rate of elimination was determined by the phosphorylation of galactose by galactokinase, which is located in the cytoplasm of hepatocytes.
Dried blood samples were taken from the tail vein at 5, 10, 15, 30, 45, 60 and 90 min after injection. A colorimetric galactose dehydrogenase (GADH) method was used to measure galactose levels using a modification of the neonatal screening test (Interscientific GAL570 nm, USA). The concentration range of the calibration curve was 50–1000 µg/mL. Within-day variations were evaluated by standard deviation and percent coefficient of variation (CV) for each concentration. A maximum of 10% CV was permitted. Day-to-day variations were also checked by comparing the slope and intercept of the calibration curves.
Isoniazid injection procedures Three study groups were investigated. The first treatment group (n = 7) involved INH i.p. injections of 150 mg/kg per day (dissolved in saline) alone for 21 days. The second treatment group (n = 6) involved INH and BNPP i.p. injections for 21 days. BNPP (25 mg/kg) was dissolved in saline after heating to approximately 60°C, cooled and administered 30 min before the INH injections. The third control group (n = 20) received i.p. injections of saline solution. The GSP method was applied before and 21 days after treatment.
Enzyme measurements
For plasma enzyme measurements, blood samples were collected from conscious animals (blood drops from tail) into heparinized tubes before galactose injection, then centrifuged at 5000 rpm for 10 min at room temperature and the separated plasma assayed within 2 h. Plasma enzyme activities were determined at 37°C (AST and ALT) using a Beckman Auto Analyzer (type Synchron LXi 725; Beckman Instruments, USA) with reagent kits provided by the manufacturer (AST: 
-ketoglutarate, malate dehydrogenase, L-aspartate, NADH; ALT: -ketoglutarate, lactate dehydrogenase, L-alanine, NADH) (Henry et al. 1960).
Light microscopy and electron microscopy At the end of the treatment, the animals were sacrificed under diethyl ether anaesthesia. The liver was immediately removed for histological and electron microscopy analysis. For light microscopy, liver specimens (n = 6, each group) were fixed in 10% phosphate-buffered formalin, dehydrated and embedded in paraffin. Each paraffin-embedded block was cut into 5 µm sections and then stained with haematoxylin & eosin, and periodic acid Schiff for histological observation. For electron microscopy, liver sections (n = 3, each group) were washed in cacodylate buffer (0.1 mmol/L, pH 7.4), post-fixed for one hour in 20% aqueous osmium tetroxide, dehydrated in graded alcohol and embedded in Spurr resin. Ultra-thin sections were cut with diamond knives, stained with uranyl acetate and lead citrate, and examined with a transmission electron microscope (Hitachi 600, Hitachi Co, Japan).
Liver histology was assessed by a pathologist (H S Lee) ‘blinded’ to the results of the liver biochemistry and GSP values. Histology was graded by the histologic activity index (HAI) according to the criteria of Knodell et al. (1981). The total HAI score is divided into two components: HAI-necroinflammation (HAI-NI) and fibrosis. The HAI-NI score includes three components (0–10, piecemeal necrosis; 0–4, lobular necrosis and inflammation; 0–4, portal inflammation).
Statistical analysis
The results were presented as mean ± standard deviation (SD). Means between the two samples were compared with the Student's t-test or the paired t-test. Analysis of variance (ANOVA) with post hoc Bonferroni corrections of t-tests (SigmaStat version 2.03, SPSS Inc) was used to compare the control and treatment groups' data from the biochemical parameters and GSP. Comparison of continuous variables with skewed distribution between the two groups was tested by a non-parametric test, such as the Mann-Whitney U-test. Correlation between different continuous variables was tested by Spearman's rank correlation. A two-tailed P value <0.05 is considered to be statistically significant.
| Results |
|---|
|
TopSummaryMaterials and methodsResultsDiscussionReferences |
|---|
|
|
|
|
|
|
| Discussion |
|---|
|
TopSummaryMaterials and methodsResultsDiscussionReferences |
|---|
INH has been widely used as a medicine in the prophylaxis and treatment of tuberculosis. The incidence of anti-tuberculosis drug-induced hepatitis ranges from 1% to 36%, depending on different regimens and definitions of hepatic injury (Yamamoto et al. 1986, Wu et al. 1990, Huang et al. 2003). Of the various anti-tuberculosis regimens, INH is the main drug that causes hepatotoxicity (Mitchell et al. 1976, Yamamoto et al. 1986, Wu et al. 1990, Lee 1995). We showed here, through the histopathological evaluation of INH-induced hepatotoxicity in rats, the hepatocellular disintegration and vacuolation in the centrilobular region, even though there was no evidence of liver cell necrosis throughout the whole course. Such types of changes have also been observed in experimental rats treated with 100 mg/kg of INH for 21 days intraperitoneally, consistent with our data (Yue et al. 2004). Electron microscopic examination also revealed marked proliferation of rER. A similar type of hepatic injury in rats was produced by giving 50 mg/kg of INH intraperitoneally for two weeks. Hence, the higher production of the reactive oxygen metabolites in INH treatment can be speculated (Sodhi et al. 1996).
Our laboratory, using a similar model of INH-induced hepatotoxicity in rats and the pretreatment of rats with BNPP (an amidase inhibitor) 30 min before injection of INH for 21 days, was carried out and produced consistent clinical manifestations. Compared with INH treatment alone, however, there were significant differences in average GSP values for the control and INH-BNPP-treated groups, whereas AST and ALT activities were almost indistinguishable. Plasma AST and ALT activities are very sensitive markers but mainly for hepatocellular necrosis. Hepatic AST and ALT activities failed to indicate mild hepatotoxicity in our experimental models. This failure may be adjusted using GSP as a quantitative liver function biomarker. This may be one of the reasons why GSP is more sensitive than AST and ALT in distinguishing the residual liver function of rats among the control, INH- and INH-BNPP-treated groups (P < 0.001). Among these controls (CCl4-control and INH-control) and the experimental groups (CCl4, INH and INH-BNPP) in the present study, excellent correlation was observed between GSP and HAI-NI scores (r = 0.748, P < 0.001), which suggested that GSP is a potential biomarker of drug-induced hepatocellular toxicity.
Of the residual liver function measured, the GSP value consistently increased by INH-treatment (mild) and CCl4-induced (severe) hepatotoxicity in various degrees of liver injury. Previously, aminotransferases were known to carry little prognostic importance and to be poor indicators of severity (O'Brien et al. 2000). Surprisingly, AST and ALT had a good correlation with GSP values, because of markedly elevated AST and ALT values in the CCl4-treated rats, reflecting severe hepatotoxicity.
Finally, GSP takes the 60 min galactose blood concentration as a measure of residual liver function. Therefore, GSP concentration possibly reflects both the change in enzyme activity and hepatic blood flow (Tang & Hu 1992). To accurately estimate the value of GSP, the calibration curve is of vital importance. Duplicate measurements should be done in six different concentrations for each subject's calibration curve, which covers high and low galactose concentrations after administration. Traditionally, galactose concentration was measured by applying GADH in dried blood spots using fluorometric assays (Fujimura et al. 1981, Orfanos et al. 1986, Yamaguchi et al. 1989). Here, a modified colorimetric GADH method (Diepenbrock et al. 1992) was used to measure values of galactose in dried blood specimen from the rat tail vein.
In conclusion, the GSP method has been proven to be a simple and useful tool for the quantitative determination of rat liver function, based primarily on marked GSP elevation following CCl4- or INH-induced extensive and severe hepatic necrosis, with a high sensitivity for detection even at the early stages of liver disease.
| References |
|---|
|
TopSummaryMaterials and methodsResultsDiscussionReferences |
|---|
Ballard FJ (1966) Purification and properties of galactokinase from pig liver. Biochemical Journal 98, 347–52[Medline]
Becker M (1998) 13C breath test for measurement of liver function. Gut 43 (suppl. 3), S25–7
Bircher J (1986) Assessment of prognosis in advanced liver disease: to score or to measure, that's the question. Hepatology 6, 1036–7[Medline]
Boone L, Mever D, Cusick P, et al. (2005) Selection and interpretation of clinical pathology indicators of hepatic injury in preclinical studies. Veterinary Clinical Pathology 34, 182–8[Medline]
Bruckner JV, MacKenzie WF, Muralidhara S, Luthra R, Kyle GM, Acosta D (1986) Oral toxicity of carbon tetrachloride: acute, subacute, and subchronic studies in rats. Fundamental and Applied Toxicology 6, 16–34[Medline]
Chopra P, Roy S, Ramalingaswami V, Nayak NC (1972) Mechanism of carbon tetrachloride hepatotoxicity. An in vivo study of its molecular basis in rats and monkeys. Laboratory Investigation 26, 716–27[Medline]
Craik JD, Elliott KR (1980) Transport of D-fructose and D-galactose into isolated rat hepatocytes. Biochemical Journal 192, 373–5[Medline]
Diepenbrock F, Heckler R, Schickling H, Engelhard T, Bock D, Sander J (1992) Colorimetric determination of galactose and galactose-1-phosphate from dried blood. Clinical Biochemistry 25, 37–9[Medline]
Food and Drug Administration (2003) Guidance for Industry: Pharmacokinetics in Patients with Impaired Hepatic Function: Study Design, Data Analysis, and Impact on Dosing and Labeling. US Department of Health and Human Services, FDA, Center for Drug Evaluation and Research, Center for Biologics Evaluation and Research
Fujii T (1997) Toxicological correlation between changes in blood biochemical parameters and liver histopathological findings. Journal of Toxicological Sciences 22, 161–83[Medline]
Fujimura Y, Ishii S, Kawamura M, Naruse H (1981) Microdetermination of galactose and galactose 1-phosphate in dried blood spots. Analytical Biochemistry 117, 187–95[Medline]
Henderson JM, Kutner MH, Bain RP (1982) First-order clearance of plasma galactose: the effect of liver disease. Gastroenterology 83, 1090–6[Medline]
Henry RJ, Chiamori N, Golub OJ, Berkman S (1960) Revised spectrophotometric methods for the determination of glutamic-oxalacetic transaminase, glutamic-pyruvic transaminase, and lactic acid dehydrogenase. American Journal of Clinical Pathology 34, 381–98[Medline]
Homeida M, Roberts CJ, Halliwell M, Read AE, Branch RA (1979) Antipyrine clearance per unit volume liver: an assessment of hepatic function in chronic liver disease. Gut 20, 596–601
Hu OY, Hu TM, Tang HS (1995a) Determination of galactose in human blood by high-performance liquid chromatography: comparison with an enzymatic method and application to the pharmacokinetic study of galactose in patients with liver dysfunction. Journal of Pharmaceutical Sciences 84, 231–5[Medline]
Hu OY, Tang HS, Chang CL (1995b) Novel galactose single point method as a measure of residual liver function: example of cefoperazone kinetics in patients with liver cirrhosis. Journal of Clinical Pharmacology 35, 250–8[Abstract]
Hu OY, Tang HS, Sheeng TY, Chen TC, Curry SH (1995c) Pharmacokinetics of promazine in patients with hepatic cirrhosis – correlation with a novel galactose single point method. Journal of Pharmaceutical Sciences 84, 111–14[Medline]
Hu OY, Tang HS, Chang CL (1994) The influence of chronic lobular hepatitis on pharmacokinetics of cefoperazone – a novel galactose single-point method as a measure of residual liver function. Biopharmaceutics and Drug Disposition 15, 563–76[Medline]
Huang YS, Chern HD, Su WJ, et al. (2003) Cytochrome P450 2E1 genotype and the susceptibility to antituberculosis drug-induced hepatitis. Hepatology 37, 924–30[Medline]
Keiding S (1973) Galactose elimination capacity in the rat. Scandinavian Journal of Clinical and Laboratory Investigation 31, 319–25[Medline]
Keiding S, Johansen S, Tonnesen K (1977) Kinetics of ethanol inhibition of galactose elimination in perfused pig liver. Scandinavian Journal of Clinical and Laboratory Investigation 37, 487–94[Medline]
Keiding S, Johansen S, Winkler K (1982) Hepatic galactose elimination kinetics in the intact pig. Scandinavian Journal of Clinical and Laboratory Investigation 42, 253–9[Medline]
Knodell RG, Ishak KG, Black WC, et al. (1981) Formulation and application of a numerical scoring system for assessing histological activity in asymptomatic chronic active hepatitis. Hepatology 1, 431–5[Medline]
Lee WM (1995) Drug-induced hepatotoxicity. New England Journal of Medicine 333, 1118–27
Lindskov J (1982) The quantitative liver function as measured by the galactose elimination capacity. I. Diagnostic value and relations to clinical, biochemical, and histological findings in patients with steatosis and patients with cirrhosis. Acta Medica Scandinavica 212, 295–302[Medline]
Mitchell JR, Zimmerman HJ, Ishak KG, et al. (1976) Isoniazid liver injury: clinical spectrum, pathology, and probable pathogenesis. Annals of Internal Medicine 84, 181–92
O'Brien PJ, Slaughter MR, Polley SR, Kramer K (2002) Advantages of glutamate dehydrogenase as a blood biomarker of acute hepatic injury in rats. Laboratory Animals 36, 313–21
O'Brien PJ, Slaughter MR, Swain A, et al. (2000) Repeated acetaminophen dosing in rats: adaptation of hepatic antioxidant system. Human and Experimental Toxicology 19, 277–83
Orfanos AP, Jinks DC, Guthrie R (1986) Microassay for estimation of galactose and galactose-1-phosphate in dried blood specimens. Clinical Biochemistry 19, 225–8[Medline]
Provost JP, Hanton G, Le Net JL (2003) Plasma triglycerides: an overlooked biomarker of hepatotoxicity in the rat. Comparative Clinical Pathology 12, 95–101
Renner E, Wietholtz H, Huguenin P, Arnaud MJ, Preisig R (1984) Caffeine: a model compound for measuring liver function. Hepatology 4, 38–46[Medline]
Roberts RK, Wilkinson GR, Branch RA, Schenker S (1978) Effect of age and parenchymal liver disease on the disposition and elimination of chlordiazepoxide (librium). Gastroenterology 75, 479–85[Medline]
Sarich TC, Adams SP, Petricca G, Wright JM (1999) Inhibition of isoniazid-induced hepatotoxicity in rabbits by pretreatment with an amidase inhibitor. Journal of Pharmacology and Experimental Therapeutics 289, 695–702
Sarich TC, Youssefi M, Zhou T, Adams SP, Wall RA, Wright JM (1996) Role of hydrazine in the mechanism of isoniazid hepatotoxicity in rabbits. Archives of Toxicology 70, 835–40[Medline]
Sodhi CP, Rana SV, Mehta SK, et al. (1996) Study of oxidative stress in isoniazid-induced hepatic injury in young rats with and without protein-energy malnutrition. Journal of Biochemical Toxicology 11, 139–46[Medline]
Tang HS, Hu OY (1992) Assessment of liver function using a novel galactose single point method. Digestion 52, 222–31[Medline]
Tygstrup N (1964) The galactose elimination capacity in control subjects and in patients with cirrhosis of the liver. Acta Medica Scandinavica 175, 281–9[Medline]
Weingand K, Brown G, Hall R, et al. (1996) Harmonization of animal clinical pathology testing in toxicity and safety studies. Fundamental and Applied Toxicology 29, 198–201[Medline]
Wu JC, Lee SD, Yeh PF, et al. (1990) Isoniazid-rifampin-induced hepatitis in hepatitis B carriers. Gastroenterology 98, 502–4[Medline]
Yamaguchi A, Fukushi M, Mizushima Y, et al. (1989) Microassay for screening newborns for galactosemia with use of a fluorometric microplate reader. Clinical Chemistry 35, 1962–4
Yamamoto T, Suou T, Hirayama C (1986) Elevated serum aminotransferase induced by isoniazid in relation to isoniazid acetylator phenotype. Hepatology 6, 295–8[Medline]
Yue J, Peng RX, Yang J, Kong R, Liu J (2004) CYP2E1 mediated isoniazid-induced hepatotoxicity in rats. Acta Pharmacologica Sinica 25, 699–704[Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
