Toxicology Letters, 58 (1991) 3741 0 1991 Elsevier Science Publishers B.V. 0378-4274/91/$3.50 ADONIS
37
0378427491000996
TOXLET 02603
The effect of methimazole on thioamide bioactivation and toxicity
M.J. Ruse and R.H. Waring School of Biochemistry, The University of Birmingham, Birmingham (U.K.)
(Received 29 November 1990) (Accepted 27 February 1991) Key words: Thioamide; Ethionamide; Thionicotinamide;
FAD-containing
mono-oxygenase; S-Oxidation;
Hepatotoxicity SUMMARY The hepatotoxicity induced by administration of ethionamide and thionicotinamide (TNA) was shown to be decreased by pre-administration of methimazole (MMI). Pre-administration of MM1 was also shown to decrease the levels of excretion of TNA S-oxide. This indicates that thioamide S-oxidation, mediated by the flavin-containing mono-oxygenase, may be linked to the initiation of hepatotoxicity induced by these thioamides. SK&F-525-A, the cytochrome P-450 inhibitor, did not affect either thioamide-induced toxicity or levels of excretion of TNA S-oxide; however, the role of the P-450 isoenzymes cannot be totally ruled out.
INTRODUCTION
Ethionamide (ETH) (Trescatyl; 2-ethyl-4-pyridinecarbothiamide) is prescribed in second-line therapy against tuberculosis. However, its use has been limited by the high incidence of gastric intolerance [l] and hepatotoxicity [2]. Activity may be related to the isothionicotinamide group as thionicotinamide (TNA), an analogue of ethionamide, was shown to have no effect against tuberculosis in the standard mouse assay [31The mechanisms of ETH-induced hepatotoxicity are not well understood [2]. The hepatotoxicity induced by two other thioamide compounds, namely thioacetamide and thiobenzamide, has been associated with their oxidative biotransformation to S-oxidised metabolites [48].
Address for correspondence: M.J. Ruse, School of Biochemistry, The University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K.
38
The S-oxidation of thioacetamide [4,9] and thiobenzamide [5,10] is mainly mediated by the flavin-containing mono-oxygenase (FMO) and prior administration of FM0 inhibitors, such as methimazole (MMI), has been shown to decrease the hepatotoxicity induced by these thioamides [4-6,8]. Classical inhibitors of the cytochrome P-450 mono-oxygenase, such as SK&F-525-A, were shown to have no effect on thioamide- induced hepatotoxicity [4,6]. The involvement of S-oxidation in the hepatotoxicity induced by ETH and TNA has not been previously investigated, but both thioamides are known to form S-oxide metabolites in a biotransformation mediated mainly by the FM0 [ 111.The effects of MM1 and SKF-525-A on the hepatic disturbances (cytochrome P-450 levels, fatty liver characterised by liver weight increase and histochemical staining) caused by these thioamides has been investigated. The effect of both of these mono-oxygenase inhibitors on the in vivo S-oxidation of TNA was also determined. MATERIALS
AND
METHODS
TNA and ETH were obtained from Sigma Chemicals (Poole, Dorset, U.K.). Male Wistar rats (20&250 g), used throughout this study, were housed in metabolism cages at 22°C. Rats had free access to standard diet and water; they were starved overnight prior to killing. TNA and ETH were administered orally in 1 ml 3% methylcellulose (Colagel, Eli Lilly and Co. Ltd., Basingstoke, U.K.). MM1 and SK&F-525-A were given intraperitoneally at a dose of 10 and 7.5 mg/lOO g body wt., respectively, 30 min before thioamide administration according to Chieli and Malvaldi [6]. Animals were killed by cervical dislocation 24 h after thioamide administration. The liver was immediately removed, blotted dry and weighed. Microsomes were prepared according to Hanzlik and Cashman [lo] and the cytochrome P-450 levels measured according to Omura and Sato [12]. Liver sections from 2 lobes were also immediately fixed in 10% formal saline, frozen sections cut and stained with Oil Red 0 to check fat deposition. The urinary excretion of TNA and TNA S-oxide was determined as previously described [ 131. Statistical analyses were performed using Student’s t-test, assuming PcO.05 as the significance level. RESULTS
AND DISCUSSION
ETH is known to induce fatty liver with an associated increase in liver/body weight ratio [14]. Table I shows the effects of thioamide administration, in combination with MM1 or SK&F-525-A, on the difference in liver weight, expressed as a percentage of the body weight, between control and treated animals. Administration of ETH and TNA caused an increase in liver weight compared to control values. Prior administration of SK&F-525-A did not have any effect on the difference in liver weight between control and treated groups. However, MM1 was shown to decrease significantly
39
thioamide=t animal the difference in the liver weight between control and (Table I). Administration ofTNA and ETHresulted in anexcess deposition offatin hepatocytes in the perivenous zone as characterised by staining with Oil Red 0. Prior administration of MM1 drastically reduced the incidence of fat deposition caused by thioamide administration whereas SK&F-525-A had no evident effect on fat deposition (results not shown). Both ETH and TNA significantly reduced the hepatic levels of cytochrome P-450 (Table I). Administration of SK&F-525-A in combination with either TNA or ETH did not affect the decrease in cytochrome P-450 levels induced by these thioamides. However, administration of MM1 in combination with either TNA or ETH caused a significant increase in cytochrome P-450 levels compared to levels determined after administration of thioamide alone (Table I). As with other thioamides [5,6, 81,modulation of ETH- and TNA-induced hepatotoxicity, by inhibitors of FMO, may be related to S-oxidation of these thioamides which is mediated by FM0 [l 11.S-Oxidation, primarily to the S-oxide and then to the &S-dioxide, is postulated to be a necessary step in the initiation of thioamide toxicity P51. The decrease in hepatic damage, caused by prior administration of MMI, may be TABLE I EFFECTS OF METHIMAZOLE AND SK&F-525-A ON HEPATOTOXICITY INDUCED BY ADMINISTRATION OF THIONICOTINAMIDE AND ETHIONAMIDE (n = 6 + SD) Treatment
Cytochrome P-450 levels (nmol/mg prot.)
Difference (%) in liver weight between control and treated groups
TNA TNA + MM1 TNA + SK&F-525-A
0.30+0.09 0.64f0.17a 0.20f0.12
1.33f0.28 0.86 f 0.30” 1.61 f0.24
ETH ETH + MM1 ETH + SK&F-525-A
0.35f0.15 0.53 f0.09b 0.47_+0.01
1.48+0.50 0.51 f0.05d 1.02f0.18
MM1 SK&F-525-A Control
0.73 f0.14 0.75f0.15 0.6 *0.04
0.19f0.01 0.24f0.19 _
* Difference in liver weight, expressed as a 8 body weight, between control and treated animals was calculated. as’Significant difference from TNA-administered group: “P < 0.01; “P < 0.05. b.dSignificant difference from ETH-administered group; bP
37
0378427491000996
TOXLET 02603
The effect of methimazole on thioamide bioactivation and toxicity
M.J. Ruse and R.H. Waring School of Biochemistry, The University of Birmingham, Birmingham (U.K.)
(Received 29 November 1990) (Accepted 27 February 1991) Key words: Thioamide; Ethionamide; Thionicotinamide;
FAD-containing
mono-oxygenase; S-Oxidation;
Hepatotoxicity SUMMARY The hepatotoxicity induced by administration of ethionamide and thionicotinamide (TNA) was shown to be decreased by pre-administration of methimazole (MMI). Pre-administration of MM1 was also shown to decrease the levels of excretion of TNA S-oxide. This indicates that thioamide S-oxidation, mediated by the flavin-containing mono-oxygenase, may be linked to the initiation of hepatotoxicity induced by these thioamides. SK&F-525-A, the cytochrome P-450 inhibitor, did not affect either thioamide-induced toxicity or levels of excretion of TNA S-oxide; however, the role of the P-450 isoenzymes cannot be totally ruled out.
INTRODUCTION
Ethionamide (ETH) (Trescatyl; 2-ethyl-4-pyridinecarbothiamide) is prescribed in second-line therapy against tuberculosis. However, its use has been limited by the high incidence of gastric intolerance [l] and hepatotoxicity [2]. Activity may be related to the isothionicotinamide group as thionicotinamide (TNA), an analogue of ethionamide, was shown to have no effect against tuberculosis in the standard mouse assay [31The mechanisms of ETH-induced hepatotoxicity are not well understood [2]. The hepatotoxicity induced by two other thioamide compounds, namely thioacetamide and thiobenzamide, has been associated with their oxidative biotransformation to S-oxidised metabolites [48].
Address for correspondence: M.J. Ruse, School of Biochemistry, The University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K.
38
The S-oxidation of thioacetamide [4,9] and thiobenzamide [5,10] is mainly mediated by the flavin-containing mono-oxygenase (FMO) and prior administration of FM0 inhibitors, such as methimazole (MMI), has been shown to decrease the hepatotoxicity induced by these thioamides [4-6,8]. Classical inhibitors of the cytochrome P-450 mono-oxygenase, such as SK&F-525-A, were shown to have no effect on thioamide- induced hepatotoxicity [4,6]. The involvement of S-oxidation in the hepatotoxicity induced by ETH and TNA has not been previously investigated, but both thioamides are known to form S-oxide metabolites in a biotransformation mediated mainly by the FM0 [ 111.The effects of MM1 and SKF-525-A on the hepatic disturbances (cytochrome P-450 levels, fatty liver characterised by liver weight increase and histochemical staining) caused by these thioamides has been investigated. The effect of both of these mono-oxygenase inhibitors on the in vivo S-oxidation of TNA was also determined. MATERIALS
AND
METHODS
TNA and ETH were obtained from Sigma Chemicals (Poole, Dorset, U.K.). Male Wistar rats (20&250 g), used throughout this study, were housed in metabolism cages at 22°C. Rats had free access to standard diet and water; they were starved overnight prior to killing. TNA and ETH were administered orally in 1 ml 3% methylcellulose (Colagel, Eli Lilly and Co. Ltd., Basingstoke, U.K.). MM1 and SK&F-525-A were given intraperitoneally at a dose of 10 and 7.5 mg/lOO g body wt., respectively, 30 min before thioamide administration according to Chieli and Malvaldi [6]. Animals were killed by cervical dislocation 24 h after thioamide administration. The liver was immediately removed, blotted dry and weighed. Microsomes were prepared according to Hanzlik and Cashman [lo] and the cytochrome P-450 levels measured according to Omura and Sato [12]. Liver sections from 2 lobes were also immediately fixed in 10% formal saline, frozen sections cut and stained with Oil Red 0 to check fat deposition. The urinary excretion of TNA and TNA S-oxide was determined as previously described [ 131. Statistical analyses were performed using Student’s t-test, assuming PcO.05 as the significance level. RESULTS
AND DISCUSSION
ETH is known to induce fatty liver with an associated increase in liver/body weight ratio [14]. Table I shows the effects of thioamide administration, in combination with MM1 or SK&F-525-A, on the difference in liver weight, expressed as a percentage of the body weight, between control and treated animals. Administration of ETH and TNA caused an increase in liver weight compared to control values. Prior administration of SK&F-525-A did not have any effect on the difference in liver weight between control and treated groups. However, MM1 was shown to decrease significantly
39
thioamide=t animal the difference in the liver weight between control and (Table I). Administration ofTNA and ETHresulted in anexcess deposition offatin hepatocytes in the perivenous zone as characterised by staining with Oil Red 0. Prior administration of MM1 drastically reduced the incidence of fat deposition caused by thioamide administration whereas SK&F-525-A had no evident effect on fat deposition (results not shown). Both ETH and TNA significantly reduced the hepatic levels of cytochrome P-450 (Table I). Administration of SK&F-525-A in combination with either TNA or ETH did not affect the decrease in cytochrome P-450 levels induced by these thioamides. However, administration of MM1 in combination with either TNA or ETH caused a significant increase in cytochrome P-450 levels compared to levels determined after administration of thioamide alone (Table I). As with other thioamides [5,6, 81,modulation of ETH- and TNA-induced hepatotoxicity, by inhibitors of FMO, may be related to S-oxidation of these thioamides which is mediated by FM0 [l 11.S-Oxidation, primarily to the S-oxide and then to the &S-dioxide, is postulated to be a necessary step in the initiation of thioamide toxicity P51. The decrease in hepatic damage, caused by prior administration of MMI, may be TABLE I EFFECTS OF METHIMAZOLE AND SK&F-525-A ON HEPATOTOXICITY INDUCED BY ADMINISTRATION OF THIONICOTINAMIDE AND ETHIONAMIDE (n = 6 + SD) Treatment
Cytochrome P-450 levels (nmol/mg prot.)
Difference (%) in liver weight between control and treated groups
TNA TNA + MM1 TNA + SK&F-525-A
0.30+0.09 0.64f0.17a 0.20f0.12
1.33f0.28 0.86 f 0.30” 1.61 f0.24
ETH ETH + MM1 ETH + SK&F-525-A
0.35f0.15 0.53 f0.09b 0.47_+0.01
1.48+0.50 0.51 f0.05d 1.02f0.18
MM1 SK&F-525-A Control
0.73 f0.14 0.75f0.15 0.6 *0.04
0.19f0.01 0.24f0.19 _
* Difference in liver weight, expressed as a 8 body weight, between control and treated animals was calculated. as’Significant difference from TNA-administered group: “P < 0.01; “P < 0.05. b.dSignificant difference from ETH-administered group; bP
