Richard V. Branchflower

Investigation of the mechanism of the potentiation of chloroform-induced hepatotoxicity and nephrotoxicity by methyl n-butyl ketone☆

Abstract Twenty-four hours after administration of CHCl 3 (0.5 ml/kg) to methyl n -butyl ketone (MBK)-pretreated rats, both the plasma glutamic pyruvic transaminase (GPT) and the blood urea nitrogen (BUN) levels were significantly elevated above those of sesame oil-pretreated animals. In contrast, deuterium-labeled chloroform (CDCl 3 ) (0.5 ml/kg) caused insignificant alterations in either plasma GPT or BUN levels of MBK-pretreated rats. Treatment with MBK caused a decrease in hepatic GSH levels to 61% of control values after 20 hr. When administered 18 hr after MBK pretreatment, CHCl 3 caused a further decrease in hepatic GSH levels to 20% of control values within 2 hr, whereas CDCl 3 treatment decreased them to 34% of control values. Renal GSH levels were unchanged by these treatments. Eighteen hours after administration of MBK, hepatic cytochrome P -450 levels were up by 70% but no change was detected in the amount of renal cytochrome P -450. Pretreatment of rats with MBK increased the metabolism of CHCl 3 to diglutathionyl dithiocarbonate both in vivo and in hepatic microsomal incubation mixtures containing GSH. These results suggest that MBK potentiates the hepatic toxicity produced by CHCl 3 in part by decreasing GSH levels and in part by increasing the metabolism of CHCl 3 to phosgene. Although the mechanism of potentiation of renal toxicity by MBK pretreatment remains unclear, the deuterium isotope effect on BUN levels indicates that metabolism of the CH bond of CHCl 3 may be involved in the renal toxicity.

Nephrotoxicity of chloroform: Metabolism to phosgene by the mouse kidney

In this investigation, we have attempted to determine whether chloroform (CHCl3)-induced nephrotoxicity might be due to its metabolism to phosgene (COCl2) in the kidney. We have found that kidney homogenates from DBA/2J male mice in the presence of glutathione metabolize CHCl3 to 2-oxothiazolidine-4-carboxylic acid (OTZ). This product appears to be formed by the initial trapping of COCl2 by two molecules of GSH to form diglutathionyl dithiocarbonate (GSCOSG). Kidney gamma-glutamyl transpeptidase can rapidly metabolize GSCOSG to N-(2-oxothiazolidine-4-carbonyl)-glycine which is then hydrolyzed, possibly by cysteinyl glycinase to OTZ. The finding that deuterium-labeled chloroform (CDCl3) was less nephrotoxic and depleted less renal GSH than did CHCl3 suggests that the metabolism of CHCl3 to COCl2 may also occur in the kidney in vivo and lead to nephrotoxicity.

[7] Covalent binding of electrophilic metabolites to macromolecules

Publisher Summary This chapter presents the procedures that can be employed to measure covalent binding of electrophilic metabolites to tissue macromolecules. These methods involve binding to protein in vitro and in vivo , binding to lipid in vitro and in vivo , andbinding of nonvolatile compounds. Binding to protein is relatively easy to measure and, in most cases, is higher than binding to other types of tissue macromolecules because of the relatively high intracellular protein concentration and the rapid reaction with nucleophilic groups on the proteins. Studies dealing with covalent binding to lipid have involved volatile halogenated hydrocarbons, for example, carbon tetrachloride and halothane. The determination of the binding of the metabolites of nonvolatile compounds to lipid is complicated by the difficulty in separating unbound parent or metabolites from lipid by simple extraction procedures. This problem may be overcome by thin layer chromatography or high-performance liquid chromatography, using conditions that separate the classes of lipid.

Comparison of the effects of methyl-N-butyl ketone and phenobarbital on rat liver cytochromes P-450 and the metabolism of chloroform to phosgene

It was previously shown that treatment of rats with methyl-n-butyl ketone (MBK) produced an increase in the total level of liver microsomal cytochromes P-450 and an increase in the rate of metabolism of chloroform (CHCl3) to phosgene (COCl2). In the present study it was found that MBK also produced qualitative changes in the composition of microsomal cytochromes P-450 in rat liver as determined by anion-exchange chromatography. The degree of the chromatographic changes paralleled the effect of MBK on the rate of metabolism of CHCl3 to COCl2 and CHCl3-induced hepatotoxicity, suggesting that MBK potentiated the hepatotoxicity of CHCl3, at least in part, by inducing the formation of cytochromes P-450 that metabolized CHCl3 to the hepatotoxin COCl2. In this regard, reconstitution studies with a form of cytochrome P-450 isolated from rat liver microsomes from rats treated with MBK or phenobarbital (Pb) showed unequivocally that cytochrome P-450 can metabolize CHCl3 to COCl2. Although analysis of rat liver microsomes by SDS-polyacrylamide electrophoresis and anion-exchange chromatography suggested that MBK and Pb had similar effects on the composition of cytochromes P-450, metabolism studies indicated that differences did exist.

Bone marrow toxicity in vitro of chloramphenicol and its metabolites.

The effect of chloramphenicol and several of its known and potential metabolites on DNA synthesis of rat and human bone marrow cells was investigated. The nitroso analog of chloramphenicol was the most potent inhibitor of DNA synthesis tested. Its inhibitory effect appeared to be irreversible, while that of chloramphenicol was reversible. The 14C label of the nitroso analog also bound irreversibly to viable rat bone marrow cells (9.2%) and to heat-inactivated cells (2.6%). In contrast, negligible amounts (0.02%) of the 14C label of chloramphenicol bound irreversibly to bone marrow cells. These results suggest that the bone marrow toxicity of the nitroso analog of chloramphenicol is related, at least in part, to its marked chemical reactivity.

Oxidation of carbon tetrachloride, bromotrichloromethane, and carbon tetrabromide by rat liver microsomes to electrophilic halogens

In order to determine whether CCl4, CBrCl3, CBr4 or CHCl3 undergo oxidative metabolism to electrophilic halogens by liver microsomes, they were incubated with liver microsomes from phenobarbital pretreated rats in the presence of NADPH and 2,6-dimethylphenol. The analysis of the reaction mixtures by capillary gas chromatography mass spectrometry revealed that 4-chloro-2,6-dimethylphenol was a metabolite of CCl4 and CBrCl3 whereas 4-bromo-2,6-dimethylphenol was a metabolite of CBr4. The formation of the metabolites was significantly decreased when the reactions were conducted with heat denatured microsomes, in the absence of NADPH or under an atmosphere of N2. These results indicate that the chlorines of CBrCl3 and CCl4 and the bromines of CBr4 are oxidatively metabolized by rat liver microsomes to electrophilic and potentially toxic metabolites.

A continuous-flow detector for cytochrome P-450 and cytochrome P-420.

A spectrophotometric detector for automatically monitoring levels of cytochrome P-450 and cytochrome P-420 in chromatographic column effluents is described. Levels of cytochrome P-450 and P-420 as low as 30 pmol/ml can be detected above an absorbance background noise equivalent to 10 pmol/ml of these heme proteins. Spectra and the concentrations of cytochrome P-450 and cytochrome P-420 are determined every 5 sec. The automatic flow reactor and detector offer the following advantages over existing manual methods: (i) greatly reduced analysis time, (ii) measurement of a larger number of independent samples than is practical manually, (iii) simultaneous measurement of cytochrome P-450 and its degradation product cytochrome P-420 immediately after elution from the column, thus avoiding further sample degradation, and (iv) greatly increased accuracy and threshold resolution due to highly reproducible reaction conditions and constant optics.