Int. I. Exp. Path. (1992), 73, 449-453
The effects of supra-therapeutic doses of rifampicin on liver function in the perfused rat liver S.M. Khedun, B. Maharaj and W.P. Leary Department of Experimental and Clinical Pharmacology, University of Natal Medical School, Durban,
South Africa Received for publication 6 September 1991 Accepted for publication 16 March 1992
Summary. The isolated liver perfusion model was used to study the effects of supra-therapeutic doses of rifampicin on hepatic gluconeogenesis and bromosulphthalein (BSP) clearance from the perfusate and biliary excretion of the dye in the rat. Three groups of rats randomly assigned to a control and two experimental groups were studied; those in the experimental groups were given 4 mg rifampicin per os daily for 90 days. The control group was untreated. The livers of the control and one experimental group were perfused with a medium containing pyruvate and subsequently these livers were perfused with a medium containing bromosulphthalein. The livers of the second experimental group were subjected to histological examination. The rate and the concentration ofglucose was decreased, lactate levels and lactate: pyruvate ratios were increased in the experimental animals. The mean perfusate BSP and biliary excretion of the dye was decreased in the experimental group. Fatty change was present in the livers of rifampicin treated rats. This study demonstrates that the isolated liver model has proved to be both suitable and useful for the study of the effects of drugs and that chronic administration of supra-therapeutic doses of rifampicin to rats adversely affected liver function. It also produces histological evidence of hepatic damage in rats.
Keywords: rifampicin, BSP, gluconeogenesis, fatty change, isolated liver perfusion, rat Numerous reports have been published showing rifampicin to be a potent antituberculous drug, comparable with isoniazid in its activity in both animals and humans (Canneti et al. 1968; Pine 1971). Rifampicin is an antibiotic derived from the family of rifamycins; it is produced by Streptomyces mediterranei and mainly used for its antituberculous action (Vall-Spinosa et al.
1970). It is also a drug of choice for chemoprophylaxis of meningococcal disease and shows promise in the treatment of certain non-mycobacterial disease and as an adjunct in the treatment of leprosy. Rifampicin is also used in the treatment of legionnaire's disease. Rifampicin has achieved first line status in the treatment of tuberculosis. The adminisCorrespondence: S.M. Khedun, Department of Experimental and Clinical Pharmacology, University of Natal Medical School, 719 Umbilo Road, Congella, Durban, South Africa. 449
450
S.M. Khedun et al.
tration of therapeutic doses of this compound has been complicated by the development of hepatic damage in the form of hepatitis (Canneti et al. 1968; Scheuer et al. 1974). The recommended daily dosage of this drug in adults is 10 mg/kg, with a maximum of 600 mg. The isolated perfused liver appears to be a useful experimental model for the study of the effects of drugs on the liver. The present study was undertaken to investigate the suitability of the perfused rat liver ex vivo for the study of the effects of rifampicin on hepatic function when the substance was administered to a group of rats for a 3-month period in doses exceeding those that are recommended.
Materials and methods Thirty male Wistar rats (190-210 g) of the University of Natal inbred strain were kept in stainless steel cages with plastic bottoms. Each animal was individually earmarked for identification and randomly assigned to a control and two experimental groups, each consisting of 10 rats. All the animals were fed on the same diet in the form of commercial rat cubes. Water was given ad libitum. The animals in the experimental group were given 4 mg rifampicin per os daily for 90 days. The control group was untreated. The livers of animals in the experimental and control group were perfused for 75 minutes with a medium containing pyruvate (500 gM), a gluconeogenic precursor, and subsequently these livers were perfused with a medium containing bromosulphthalein (10 mg/100 ml) for an additional 30 minutes. The livers of the rats in the second experimental group were subjected to histological examination.
Liver perfusion Livers were perfused in situ with a synthetic recycling medium (Hems et al. 1966; Krebs & Henseleit, 1932) as one giving optimum rates of gluconeogenesis.
Results The integrity of the liver throughout the perfusion was tested by LDH activity in the perfusate and by histological observation. Except for a rapid increase immediately after surgery, most likely due to damage of some cells in the course of operation, the LDH activity value remained constant for up to 1.5 hours from the beginning of the perfusion (Table 1). The histological observation of tissue performed at 30, 60 and 90 minutes after surgery showed preserved hepatic structure without hepatocellular damage. The same results were obtained in control and experimental groups. Only the clear, yellowish coloured livers were considered as being well perfused (n =16); those of red colour were discarded (n =4).
Gluconeogenesis Pyruvate (500 gM) was added 35 minutes after the perfusion was started; the average lactate concentration was 2.63 mmol/l at the end ofthe perfusion and formation was at an average rate of 0.04 mmol/min/g wet weight of liver in the control group whereas the concentration was 3.83 mmol/l and formation was at an average rate of 0.1 mmol/min/g wet weight of liver in the experimental group. Glucose formation was
Table 1. LDH activity in the perfusate at different times after surgery Time after surgery
LDH activity
(min)
(nmol/h/ml)
10 20 30 40 50 60 70 80 90
2.64 1.05 1.03 1.05 1.03 1.04 1.03 1.03 1.04
Effects of supra-therapeutic rifampicin on rat liver function 45 I linear in both groups at a mean rate of demonstrated by comparison of the observed formation of 0.09 mmol/min/g wet weight of rates of disappearance of BSP in the rifampiliver in the control group compared to 0.02 cin treated animals compared to those of the mmol/min/g wet weight in the rifampicin- controls. The concentration of BSP was 8.2 treated rats. The mean glucose concentra- jug/ml at 10 min and 4.1 jug/ml at 30 minutes in the experimental group comtions at the end of perfusion were 5.42 mmol/l in the control group compared to pared to 7.8 jg/ml at 10 minutes and 1.3 ,ug/ 2.07 mmol/l in the experimental group. The ml at 30 minutes in the controls. The excretion rate of BSP into the bile was lactate: pyruvate ratio was 24 in the experimental group and 12 in the control decreased in the rifampicin treated group. group. The rate of glucose formation and The excretion of BSP into the bile of the glucose concentration was decreased and control group increased during the 30lactate: pyruvate ratios were increased in the minute collection from 0.6 at 10 minutes to 8.2 mg/ml at 30 minutes, whereas it experimental group compared to controls increased from 2.6 at 10 minutes to 3.9 mg/ (P
The effects of supra-therapeutic doses of rifampicin on liver function in the perfused rat liver S.M. Khedun, B. Maharaj and W.P. Leary Department of Experimental and Clinical Pharmacology, University of Natal Medical School, Durban,
South Africa Received for publication 6 September 1991 Accepted for publication 16 March 1992
Summary. The isolated liver perfusion model was used to study the effects of supra-therapeutic doses of rifampicin on hepatic gluconeogenesis and bromosulphthalein (BSP) clearance from the perfusate and biliary excretion of the dye in the rat. Three groups of rats randomly assigned to a control and two experimental groups were studied; those in the experimental groups were given 4 mg rifampicin per os daily for 90 days. The control group was untreated. The livers of the control and one experimental group were perfused with a medium containing pyruvate and subsequently these livers were perfused with a medium containing bromosulphthalein. The livers of the second experimental group were subjected to histological examination. The rate and the concentration ofglucose was decreased, lactate levels and lactate: pyruvate ratios were increased in the experimental animals. The mean perfusate BSP and biliary excretion of the dye was decreased in the experimental group. Fatty change was present in the livers of rifampicin treated rats. This study demonstrates that the isolated liver model has proved to be both suitable and useful for the study of the effects of drugs and that chronic administration of supra-therapeutic doses of rifampicin to rats adversely affected liver function. It also produces histological evidence of hepatic damage in rats.
Keywords: rifampicin, BSP, gluconeogenesis, fatty change, isolated liver perfusion, rat Numerous reports have been published showing rifampicin to be a potent antituberculous drug, comparable with isoniazid in its activity in both animals and humans (Canneti et al. 1968; Pine 1971). Rifampicin is an antibiotic derived from the family of rifamycins; it is produced by Streptomyces mediterranei and mainly used for its antituberculous action (Vall-Spinosa et al.
1970). It is also a drug of choice for chemoprophylaxis of meningococcal disease and shows promise in the treatment of certain non-mycobacterial disease and as an adjunct in the treatment of leprosy. Rifampicin is also used in the treatment of legionnaire's disease. Rifampicin has achieved first line status in the treatment of tuberculosis. The adminisCorrespondence: S.M. Khedun, Department of Experimental and Clinical Pharmacology, University of Natal Medical School, 719 Umbilo Road, Congella, Durban, South Africa. 449
450
S.M. Khedun et al.
tration of therapeutic doses of this compound has been complicated by the development of hepatic damage in the form of hepatitis (Canneti et al. 1968; Scheuer et al. 1974). The recommended daily dosage of this drug in adults is 10 mg/kg, with a maximum of 600 mg. The isolated perfused liver appears to be a useful experimental model for the study of the effects of drugs on the liver. The present study was undertaken to investigate the suitability of the perfused rat liver ex vivo for the study of the effects of rifampicin on hepatic function when the substance was administered to a group of rats for a 3-month period in doses exceeding those that are recommended.
Materials and methods Thirty male Wistar rats (190-210 g) of the University of Natal inbred strain were kept in stainless steel cages with plastic bottoms. Each animal was individually earmarked for identification and randomly assigned to a control and two experimental groups, each consisting of 10 rats. All the animals were fed on the same diet in the form of commercial rat cubes. Water was given ad libitum. The animals in the experimental group were given 4 mg rifampicin per os daily for 90 days. The control group was untreated. The livers of animals in the experimental and control group were perfused for 75 minutes with a medium containing pyruvate (500 gM), a gluconeogenic precursor, and subsequently these livers were perfused with a medium containing bromosulphthalein (10 mg/100 ml) for an additional 30 minutes. The livers of the rats in the second experimental group were subjected to histological examination.
Liver perfusion Livers were perfused in situ with a synthetic recycling medium (Hems et al. 1966; Krebs & Henseleit, 1932) as one giving optimum rates of gluconeogenesis.
Results The integrity of the liver throughout the perfusion was tested by LDH activity in the perfusate and by histological observation. Except for a rapid increase immediately after surgery, most likely due to damage of some cells in the course of operation, the LDH activity value remained constant for up to 1.5 hours from the beginning of the perfusion (Table 1). The histological observation of tissue performed at 30, 60 and 90 minutes after surgery showed preserved hepatic structure without hepatocellular damage. The same results were obtained in control and experimental groups. Only the clear, yellowish coloured livers were considered as being well perfused (n =16); those of red colour were discarded (n =4).
Gluconeogenesis Pyruvate (500 gM) was added 35 minutes after the perfusion was started; the average lactate concentration was 2.63 mmol/l at the end ofthe perfusion and formation was at an average rate of 0.04 mmol/min/g wet weight of liver in the control group whereas the concentration was 3.83 mmol/l and formation was at an average rate of 0.1 mmol/min/g wet weight of liver in the experimental group. Glucose formation was
Table 1. LDH activity in the perfusate at different times after surgery Time after surgery
LDH activity
(min)
(nmol/h/ml)
10 20 30 40 50 60 70 80 90
2.64 1.05 1.03 1.05 1.03 1.04 1.03 1.03 1.04
Effects of supra-therapeutic rifampicin on rat liver function 45 I linear in both groups at a mean rate of demonstrated by comparison of the observed formation of 0.09 mmol/min/g wet weight of rates of disappearance of BSP in the rifampiliver in the control group compared to 0.02 cin treated animals compared to those of the mmol/min/g wet weight in the rifampicin- controls. The concentration of BSP was 8.2 treated rats. The mean glucose concentra- jug/ml at 10 min and 4.1 jug/ml at 30 minutes in the experimental group comtions at the end of perfusion were 5.42 mmol/l in the control group compared to pared to 7.8 jg/ml at 10 minutes and 1.3 ,ug/ 2.07 mmol/l in the experimental group. The ml at 30 minutes in the controls. The excretion rate of BSP into the bile was lactate: pyruvate ratio was 24 in the experimental group and 12 in the control decreased in the rifampicin treated group. group. The rate of glucose formation and The excretion of BSP into the bile of the glucose concentration was decreased and control group increased during the 30lactate: pyruvate ratios were increased in the minute collection from 0.6 at 10 minutes to 8.2 mg/ml at 30 minutes, whereas it experimental group compared to controls increased from 2.6 at 10 minutes to 3.9 mg/ (P
