The effect of partial hepatectomy on the metabolism, distribution, and nephrotoxicity of para-methylthiobenzamide in the rat.

Para-Methylthiobenzamide (PMTB) produces injury to the liver and kidney. Toxicity is mediated via its biotransformation to a reactive S,S-dioxide metabolite. The objective of this study was to examine the role of hepatic metabolism in the production of PMTB-induced renal toxicity. Renal injury was assessed in partially hepatectomized and sham-operated rats and the effect of this procedure on the distribution and metabolism of PMTB was examined. The in vitro oxidation of PMTB and [14C]thiobenzamide by rat kidney microsomes was also examined. Plasma urea levels and renal cortical slice uptake of organic ions were used to monitor renal function. Partial hepatectomy alone did not alter renal function nor raise blood urea nitrogen levels. Nephrotoxicity resulted when a nonnephrotoxic dose of PMTB (1.2 mmol/kg) was given to partially hepatectomized rats. An HPLC method was used for measurement of PMTB and its metabolites para-methylthiobenzamide S-oxide (PMTBSO) and para-methylbenzamide (PMBA) in plasma and kidney. Hepatectomy delayed the removal of this dose of PMTB from plasma and allowed greater concentrations of PMTB and PMTBSO to accumulate in plasma and kidney at 6 and 15 hr. The level of PMBA was similar in both groups at 6 hr, but was increased in plasma and kidney of the hepatectomized group at 15 hr. Kidney microsomes rapidly converted PMTB to PMTBSO and small amounts of PMBA. [14C]TB was oxidized by microsomes to thiobenzamide S-oxide, benzamide, and covalently bound metabolites. The results indicate that partial hepatectomy lowered the threshold for the expression of nephrotoxicity by PMTB. This procedure is associated with an increased renal accumulation of PMTB and PMTBSO, which are both sequentially transformed to the toxic metabolite.

Determination of p-methylthiobenzamide and p-methylthiobenzamide-S-oxide from rat plasma using solid-phase extraction and high-performance liquid chromatography.

p-Methylthiobenzamide (PMTB) is a thiocarbonyl compound exhibiting marked hepatotoxicity and nephrotoxicity. We describe a high-performance liquid chromatographic method for analyzing PMTB and a metabolite, p-methylthiobenzamide-S-oxide (PMTBSO), from rat plasma using a solid-phase extraction technique. In this way, PMTB and PMTBSO can be extracted from 0.5 ml of plasma and separation achieved by an ODS analytical column in as little as 9 min. The mobile phase used was methanol-water (55:45, v/v) and the wavelength for detection was 290 nm. The limits of detection in plasma were 15 ng/ml for PMTB and 33 ng/ml for PMTBSO; the absolute recovery from spiked plasma samples was greater than 84.4% for both compounds and the internal standard. The method was linear throughout the range used with correlation coefficients greater than 0.969. The intra-day accuracy ranged from 1.52 to 15.23% relative error for the PMTB concentration range 151-3025 ng/ml; accuracy of 4.97% or less was obtained for PMTBSO concentrations of 1672-20,068 ng/ml. The intra-day precision (coefficient of variation) of the procedure was found to be no greater than 5.28% for PMTB and 7.9% for PMTBSO. Inter-day accuracy and precision measurements were similar.

Nephrotoxicity of para-substituted thiobenzamide derivatives in the rat.

Histological examination, plasma urea nitrogen levels (BUN), and renal cortical slice uptake of paminohippurate (PAH) or tetraethylammonium (TEA) were used to assess the nephrotoxicity of thiobenzamide and its para-substituted derivatives in Sprague-Dawley rats. Intraperitoneal injection of p-methylthiobenzamide (PMTB) to rats resulted in dose-dependent nephrotoxicity as judged by increased BUN levels, decreased TEA uptake and histologic examination of the kidney. Para-methoxythiobenzamide and PMTB were more potent nephrotoxins than thiobenzamide, which was itself minimally nephrotoxic. Para-methylthiobenzamide-S-oxide (PMTBSO) was more nephrotoxic than PMTB. Rats were pretreated with 1-methyl-1-phenylbenzoylthiourea (MPBTU), a non-toxic arylthiourea which inhibits the metabolism and toxicity of thiocarbonyl compounds. The nephrotoxicity and hepatotoxicity of PMTB was reduced by treatment with MPBTU 30 min prior to PMTB. Pretreatment with MPBTU protected against the renal toxicity of PMTBSO. The results indicate that electron donating para-substituted thiobenzamides produce dose-dependent renal injury, dependent upon oxidative biotransformation.

Effect of reserpine on N-methylthiobenzamide-induced pulmonary edema: role of lung norepinephrine and hypothermia.

N-Methylthiobenzamide (NMTB) is a pneumotoxin which causes pulmonary edema and hydrothorax in rodents. Reserpine has been shown to attenuate the pneumotoxicity induced by NMTB. Some of that evidence suggests that the protection afforded by reserpine occurs independently of its capacity to reduce peripheral 5-hydroxytryptamine (5-HT). We therefore investigated 2 other pharmacologic properties of reserpine, namely: (1) its capacity to reduce lung norepinephrine (NE); and (2) its capacity to induce hypothermia, in order to more fully understand its mechanism of protection. Pretreatment of mice or rats with 6-hydroxydopamine at a dose which reduced lung NE by approximately 80% did not affect the pneumotoxic response to NMTB. Thus a decrease in lung NE probably does not account for reserpine's protective effect. An investigation of reserpine's effects on core temperature revealed that mice dosed with a combination of reserpine + NMTB presented with core temperatures lower than mice treated with either compound alone. Mice placed in a cold environment (2 degrees C) and dosed with NMTB presented with hypothermia and an attenuated toxic response to NMTB. Thus a reserpine-induced hypothermia could be allowing for a reduction of NMTB metabolism and consequent diminution of toxicity. These observations suggest that reserpine's capacity to protect animals against NMTB-induced pulmonary edema may in part be due to its capacity to induce hypothermia.

Effect of 2-butanol and 2-butanone on rat hepatic ultrastructure and drug metabolizing enzyme activity.

The effect of a single oral dose of 2-butanol (2.2 ml/kg) or 2-butanone (1.87 ml/kg) on hepatic ultrastructure and drug-metabolizing enzyme activity was studied in the rat. A 135-197% increase in acetanilide hydroxylase activity was found in rats sacrificed 12-40 h after dosing with 2-butanol or 2-butanone. A 40-h pretreatment with 2-butanone produced a 155% increase in aminopyrine N-demethylase activity. NADPH-cytochrome c reductase activity and the concentrations of cytochromes P-450 and b5 were largely unaltered 2-40 h after dosing with either agent. Electron microscopic examination of hepatocytes from rats sacrificed 16 h after 2-butanol or 2-butanone revealed a marginal increase in the prevalence of smooth endoplasmic reticulum. However, by 40 h, there was a marked proliferation of the smooth endoplasmic reticulum and reduction in rough endoplasmic reticulum in response to both agents. The most marked potentiation of CCl4 hepatotoxicity occurred when rats were pretreated with 2-butanol or 2-butanone 16 h before CCl4 administration. The coincidental finding of maximal CCl4-induced hepatic injury and elevation of microsomal xenobiotic activity within the same time frame following 2-butanol or 2-butanone supports the hypothesis that aliphatic alcohols and ketones potentiate CCl4 hepatotoxicity by enhancing biotransformation of the halocarbon to cytotoxic metabolites.

The involvement of serotonin in the pneumotoxicity induced by N-methylthiobenzamide.

N-Methylthiobenzamide (NMTB) and alpha-naphthylthiourea (ANTU) are pneumotoxicants which cause pulmonary edema and hydrothorax. Recently a role was assigned to serotonin (5-hydroxytryptamine, 5-HT) in the pneumotoxic response to ANTU (D.E. Mais and T.R. Bosin, 1984, Toxicol. Appl. Pharmacol. 74, 185-194). We therefore investigated the participation of 5-HT in NMTB-induced pneumotoxicity. Pulmonary clearance of 5-HT was studied after NMTB or ANTU using the rat isolated perfused lung. Lung 5-HT uptake was not depressed 5 hr after ANTU or NMTB, but was depressed 12 hr after compound administration. At both time points lungs were edematous as judged by lung wet weight to body weight ratios. Pretreatment with reserpine, a drug known to deplete 5-HT, did not affect the NMTB-induced decrease in lung 5-HT uptake, but did diminish the increased lung wet weight to dry weight ratios seen after NMTB administration in rats and mice and the increased lung wet weight to body weight ratios in mice. NMTB induces a dose-dependant increase in the incorporation of [14C]thymidine into mouse pulmonary DNA. This increase was attenuated, but not abolished, by pretreatment with reserpine. Reserpine did not alter survival time after NMTB or ANTU and did not shift the 14-day LD50 of NMTB. These data suggest that 5-HT is not a primary mediator in the pneumotoxic response to these thiono-containing compounds.

Comparison of the toxicity of methylcyclopentadienyl manganese tricarbonyl with that of its two major metabolites.

Pneumotoxicity and lethality resulting from administration of methylcyclopentadienyl manganese tricarbonyl (MMT) and its 2 major metabolites in rats were compared. Following i.p. injection, MMT was found to have an LD50 value of 12.1 mg/kg. Neither of the metabolites appeared to have significant acute toxicity even when doses as high as 250 mg/kg were given. This impressive difference in toxicity may be due in part to changes in solubility of the metabolites, allowing for (1) decreased distribution into the central nervous system, coupled with (2) a more rapid excretion rate. This suggests that the oxidative metabolism of MMT that results in the formation of these metabolites is an important detoxifying pathway.

The role of metabolism in N-methylthiobenzamide-induced pneumotoxicity.

N-Methylthiobenzamide (NMTB) produces pulmonary edema, hydrothorax, and death in rodents. The objectives of the present studies were to establish a relationship between the lethality of NMTB and its pneumotoxicity and to explore the role of S-oxidation in these events. Pulmonary injury was assessed by measuring [14C]thymidine incorporation into pulmonary DNA. Administration of NMTB resulted in increased pulmonary [14C]thymidine incorporation in both rats and mice. These increases were blocked in both species by pretreatment of animals with sublethal doses of NMTB. However, the lethality of NMTB was not blocked in mice by prior administration of NMTB even though this procedure has been shown to protect rats. 1-Methyl-1-phenyl-3-benzoylthiourea (MPBTU) protected both rats and mice from lethal doses of NMTB and blocked NMTB-induced increases in pulmonary [14C]thymidine incorporation. N-Methylthiobenzamide S-oxide (NMTBSO), a metabolite of NMTB, produced lung injury which was similar to that produced by NMTB. NMTBSO was more potent than NMTB when administered iv, but not when given ip. The role of hepatic metabolism in NMTB pneumotoxicity was examined by administering NMTB to rats which had either undergone partial hepatectomy or been pretreated with N-octylimidazole. Neither of these procedures diminished the lethality of NMTB. These data suggest that NMTB lethality is mediated by pulmonary injury resulting from NMTB S-oxidation in the lung.

Oxidation of N-methylthiobenzamide and N-methylthiobenzamide S-oxide by liver and lung microsomes.

The in vitro oxidation of N-[14C]methylthiobenzamide (NMTB) and N-[14C]methylthiobenzamide S-oxide (NMTBSO) by rat lung and liver microsomes was studied. In the presence of an NADPH-generating system, NMTB was rapidly converted to NMTBSO and small amounts of N-methylbenzamide (NMBA) and covalently bound metabolites (CVB). Under similar conditions, NMTBSO was converted to NMBA and CVB. Studies with metabolic inhibitors indicate that both the S-oxidation of NMTB and its further conversion to NMBA and CVB, probably via enzymatic oxidation to the S,S-dioxide, are catalyzed by both cytochromes P-450 and the FAD-containing monooxygenase (MFMO). Based on differential effects of inhibitors with lung vs liver microsomes, it would appear that in lung microsomes the MFMO plays a significantly greater role than cytochrome P-450 in the oxidation of NMTB and NMTBSO, whereas in the liver the contribution of these two pathways is more nearly equal. 1-Methyl-1-phenyl-3-benzoylthiourea, which blocks the in vivo pneumotoxicity of both NMTB and NMTBSO, also inhibited their in vitro microsomal metabolism and CVB, suggesting that these oxidations are obligatory steps in the expression of toxicity by these compounds.

The acute toxicity of cyclopentadienyl manganese tricarbonyl in the rat.

The acute toxicity of cyclopentadienyl manganese tricarbonyl (CMT) was studied in Sprague-Dawley rats. CMT was found to produce convulsions and pulmonary edema. The ED50s for convulsion were 32 mg/kg (95% C.I. 24-42 mg/kg) p.o. and 20 mg/kg (95% C.I. 15-26 mg/kg) i.p. The LD50s for p.o. and i.p. administration were 22 mg/kg (95% C.I. 19-26 mg/kg) and 14 mg/kg (95% C.I. 10-20 mg/kg), respectively. Approximately 13-16% of the administered dose was recovered in the urine from 0 to 48 h post-dosing. The majority of this material was present as an organometallic form of manganese other than CMT. Phenobarbital pretreatment prevented the convulsions and pulmonary damage produced by a 50 mg/kg i.p. dose of CMT. Rats pretreated with CMT (5 mg/kg, i.p.) for 3 days exhibited convulsions but no deaths after treatment with a 34 mg/kg p.o. dose of CMT. These results suggest that CMT does not require metabolic activation to produce toxic effects, and that prior exposure to CMT produces tolerance.

Effect of inhibitors of alpha-naphthylisothiocyanate-induced hepatotoxicity on the in vitro metabolism of alpha-naphthylisothiocyanate.

The role of S-oxidation in the toxic bioactivation of alpha-naphthylisothiocyanate (ANIT) was investigated. The effects of several thione compounds, inhibitors and an inducer of the cytochrome P-450-dependent mixed function oxidase systems on the in vitro metabolism of ANIT and aminopyrine were determined. Ethionamide, sodium diethyldithiocarbamate (Na-DDTC) and S-methyl diethyldithiocarbamate (Me-DDTC), three agents known to undergo metabolism by an S-oxidative pathway and diminish ANIT's toxicity, inhibited the in vitro enzymatic metabolism of ANIT by rat liver microsomes. Methimazole failed to alter either the hyperbilirubinemic response of ANIT or the in vitro metabolism of ANIT. All four thione compounds (i.e., ethionamide, Me-DDTC, Na-DDTC and methimazole) inhibited the enzymatic metabolism of aminopyrine by rat liver microsomes. Me-DDTC was the most potent, whereas methimazole was the least potent inhibitor of aminopyrine metabolism. Phenobarbital, which potentiates, and SKF-525A, which inhibits the hepatotoxicity of ANIT in vivo, correspondingly stimulated or inhibited the NADPH-dependent metabolism of ANIT and aminopyrine by liver microsomes. N-Decylimidazole (NDI), another classical inhibitor of cytochrome P-450-dependent monooxygenase system, inhibited both the in vivo toxicity and in vitro metabolism of ANIT. NDI also diminished the enzymatic metabolism of aminopyrine by liver microsomes. Thus the results of this study indicate that metabolism of ANIT is intimately related to its toxicity and that ANIT probably undergoes its toxic bioactivation via a cytochrome P-450-dependent S-oxidative pathway.

Toxicity-distribution relationships among 3-alkylfurans in mouse liver and kidney.

The present study was designed to extend previous observations regarding toxicity of furans and related compounds to liver and kidney. It was desired to test a series of homologous 3- alkylfurans , where changes in lipophilic character might be related to changes in toxicity. Additionally, it was desired to measure distribution of toxins to the target organs to ascertain whether organ selectivity might be determined by the concentrations attained in the target organs by the toxins. A synthesis for 3- ethylfuran and 3- pentylfuran was devised, and the toxicity of these, in addition to 3- methylfuran and furan itself, to mouse liver and kidney at 2.6 mmol/kg was determined. 2- Furamide and 2- ethylfuran were used as examples of substances known to be toxic to liver and kidney, respectively. 3- Methylthiophene was also included to determine whether results with furans extend to the closely related thiophenes . Histopathological examination of both organs was done, and quantitative estimates of liver toxicity were obtained from plasma levels of glutamate-pyruvate transaminase. Renal urine concentrating ability and plasma urea nitrogen levels were useful as quantitative indices of nephrotoxicity. It was found that both 3-ethyl and 3- pentylfuran exhibited pronounced toxicity to the kidney, and that both also caused moderate liver damage. Furan caused serious damage to the liver and produced somewhat lesser effects on the kidney. Equimolar doses of 3- methylfuran did not significantly damage either organ. Among the 3-alkyl furans, there is an impression that the more volatile compounds damage lung (3- methylfuran is reported to be a potent lung toxin), with liver and kidney toxicity increasing with molecular weight, and that compounds found in higher concentration produce greater damage in liver and kidney. However, among compounds other than alkyl furans, there is no obvious correlation between toxicity and organ concentration of toxin.

Effect of thiocarbonyl compounds on alpha-naphthylisothiocyanate-induced hepatotoxicity and the urinary excretion of [35S]alpha-naphthylisothiocyanate in the rat.

The effect of disulfiram (DSF), sodium diethyldithiocarbamate (DDTC), methyl diethyldithiocarbamate (Me-DDTC), and ethionamide on the hepatotoxic response of alpha-naphthylisothiocyanate (ANIT) was studied in the rat. The hyperbilirubinemic response of ANIT was significantly inhibited by ip or po DSF pretreatment. A more marked inhibition of toxicity occurred when DSF was given via ip injection. DDTC, Me-DDTC, and ethionamide significantly inhibited ANIT-induced hyperbilirubinemia. Me-DDTC is approximately three times more potent than DDTC as an inhibitor of toxicity. Approximately 16% of a dose of [35S]ANIT was excreted in the urine as inorganic sulfate 48 hr after dosing. Me-DDTC administered simultaneously with [35S]ANIT significantly reduced urinary [35S]sulfate excretion in the first 24 hr. Ethionamide reduced urinary [35S]sulfate excretion. Pretreatment with phenobarbital which stimulates toxicity in vivo increased urinary [35S]sulfate excretion 300% in the first 12 hr. Thus, this study shows that agents which sensitize or protect rats from the toxic effects of ANIT, correspondingly stimulate or inhibit the oxidative desulfuration of [35S]ANIT in vivo.

Relative hepatotoxicity of ortho and meta mono substituted thiobenzamides in the rat.

Thiobenzamide is known to be hepatotoxic in the rat and the relative hepatotoxicity of para-substituted thiobenzamides has previously been shown to depend strictly on the electronic character of the para substituent. We have now extended this study to include ortho and meta monosubstituted thiobenzamides. Among the meta-substituted compounds, hepatotoxicity varies in strict accordance with the electronic character of the substituent, whereas the ortho-substituted compounds show no toxicity at comparable doses regardless of the nature of the substituent. Explanations for these substituent effects are provided in terms of the chemical reactivity of the compounds and their corresponding S-oxide and S,S-dioxide metabolites.

Nonciliated bronchiolar epithelial (Clara) cell necrosis induced by organometallic carbonyl compounds.

The administration of transition metal organometallic compounds such as manganese, chromium, and iron carbonyls by the i.p. route, and nickel by inhalation (mice) or intravenously (rats), resulted in selective necrosis of the nonciliated bronchiolar epithelial (Clara) cells and variable pulmonary parenchymal damage in BALB/c mice and Fischer-derived rats within 24 h of administration. The pulmonary toxicity of methylcyclopentadienyl manganese tricarbonyl (MMT), a representative of this group of compounds, was enhanced by pretreatment with piperonyl butoxide (PB), an inhibitor of the mixed-function oxidase system. This finding suggests that Clara cell necrosis can result from direct toxicity and that the specificity of toxic agents for Clara cells may not be related solely to the presence of the mixed-function oxidase system.

The effect of nephrotoxic furans on urinary N-acetylglucosaminidase levels in mice.

The effects of several potentially nephrotoxic furans on urinary levels of N-acetylglucosaminidase (NAG) were examined to determine whether this test might serve as a useful quantitative index of nephrotoxicity for this series of compounds. Whereas others have found that nephrotoxins such as sodium salicylate and biphenyl cause increases in urinary NAG, we observed that these furans, which were shown to be nephrotoxic by histological procedures, caused significant decreases in urinary levels of the enzyme. This effect was dose-related in the one case examined.

Pneumotoxic effects of thiobenzamide derivatives.

Primary thiomides such as thiobenzamide (TB) are well known hepatotoxins in the rat. Among para-substituted TB derivatives relative hepatotoxicity varies in accordance with the electronic properties of the parasubstituent. In contrast, several N-substituted TBs have been found to be potent lung toxins in rats and mice. For N-methylthiobenzamide (NMTB) the LD50 was found to be 0.315 (95% confidence interval (CI) 0.228-0.436) mmol/kg in the rat and 0.224 (95% CI 0.191--0.264) mmol/kg in the mouse. The N-mono-substituted TBs produce alveolar and perivascular pulmonary edema, together with massive pleural effusions (hydrothorax). In this regard their toxicity resembles qualitatively that of the arylthioureas. Furthermore, pretreatment of rats with sub-lethal doses of NMTB was found to protect them against subsequent challenge with supra-lethal doses. N,N-Dimethylthiobenzamide (DMTB) also causes lung injury in the rat, but only at much higher doses than with the N-mono-substituted TBs. The similarity in toxic responses elicited by the N-mono-substituted TBs and the arylthioureas is paralleled by similarities in their chemical structures and their metabolic disposition which involves (among other things) S-oxygenation by the microsomal flavin-containing monooxygenase (EC 1.14.13.8). Thus, a possible role for S-oxidized metabolites in the lung toxicity of these compounds must be considered.

Pharmacokinetics of 2-butanol and its metabolites in the rat.

A pharmacokinetic model is presented to describe the biotransformation of 2-butanol (2-OL) and its metabolites (2-butanone, 3-hydroxy-2-butanone, and 2, 3-butanediol) using in vivo experimental blood concentration. A flow limited model is developed to simulate 2-OL, 2-butanone (2-ONE), 3-hydroxy-2-butanone (3H-2B), and 2,3-butanediol (2,3-RD) blood concentrations in rats after oral administration of 2-OL. Assuming the only important site of 2-OL biotransformation is the liver, the tissues included are the liver and a volume of distribution, essentially body water in the case of 2-OL and its metabolites. A distribution coefficient is found to be necessary to describe the low concentration of 3H-2B in blood after administration of 2-OL. The need for this coefficient may be due to partitioning, binding, or altered transport rates from the liver. Inhibition of 2-ONE metabolism to 3H-2B by 2-OL has been included to explain a time delay in the appearance of 3H-2B after administration of 2-OL. Subsequent experimental verification confirms the mixed function oxidase inhibitory properties of 2-OL. The model is able to simulate blood concentrations and elimination of all four compounds after the oral administration of 2-OL. Additionally, the model also simulates the results obtained after i.v. administration of 3H-3B and 2,3-BD.