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.

Half-life and vitreous clearance of trifluorothymidine after intravitreal injection in the rabbit eye.

To determine the half-life and vitreous clearance of trifluorothymidine, a drug that demonstrates good toxicity against cytomegalovirus (CMV), we injected 200 micrograms of the drug intravitreally in the eyes of nine New Zealand white rabbits. None of the rabbits showed any adverse reactions to the injection or the drug. The rabbits were killed at 3, 6, 12, 24 or 72 hours. High-performance liquid chromatography was used to measure the vitreous trifluorothymidine concentration. The half-life of the drug was found to be 3.15 hours, and the vitreous concentration remained above the ID50 (concentration required to inhibit cytopathic effects by 50% compared with control cultures) for CMV for about 30 hours. We conclude that trifluorothymidine may be of future benefit in the management of CMV retinitis.

Mechanism of defluorination of enflurane. Identification of an organic metabolite in rat and man.

Difluoromethoxydifluoroacetic acid (CHF2OCF2CO2H) has been identified as a metabolite of enflurane (CHF2OCF2CHCIF) in rat liver microsomes in vitro and in human urine by gas chromatography mass spectrometry. The formation of the metabolite in rat liver microsomes was dependent upon the presence of NADPH and O2, and was inhibited when SKF 525-A or CO/O2 (8:2, v/v) were present in the reaction mixture. When the C-H bonds of the CHCIF group of enflurane or of the CHCI group of isoflurane (CHF2OCHCICF3) were replaced with a C-CI bond, virtually no fluoride ion was produced from either derivative in rat liver microsomes. These results indicate that cytochrome P-450 catalyzes the oxidative dehalogenation of CHF2OCF2CHCIF at its CHCIF group to form CHF2OCF2CO2H and chloride and fluoride ions. In contrast, the CHF2 group does not appear to be appreciably susceptible to metabolic oxidative dehalogenation. These results can be used for the more rational design of new inhalation anesthetics that would not be appreciably metabolized to the potential kidney toxin, F-.

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.

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.

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.

The formation of diglutathionyl dithiocarbonate as a metabolite of chloroform, bromotrichloromethane, and carbon tetrachloride.

One hour after the intraperitoneal administration of CHCl3, CBrCl3, or CCl4 to phenobarbital (PB)-treated rats, hepatic GSH levels decreased to 30, 59, and 88% of control levels, respectively; after 4 hr, the GSH levels had returned to 46, 65, 99%, respectively, of control levels. When incubated for 15 min in air with rat liver microsomes from PB-treated rats, a NADPH-generating system, and GSH (5 mM), all of the compounds were converted to diglutathionyl dithiocarbonate (GSCOSG). The rate of conversion of CHCl3, CBrCl3, and CCl4 to GSCOSG was 180, 58, and 8 nmol per mg of protein per 15 min, respectively. The GSCOSG was also identified in bile by 13C-NMR spectroscopy and HPLC as an in vivo metabolite of CHCl(3), CBrCl3, and CCl4. After the administration of CHCl3, CBrCl3, and CCl4, 2.89, 0.64, or 0.11 mumol of GSCOSG, respectively, was excreted in 6 hr. These results suggest that CHCl3, CBrCl3, and CCl4 are metabolized in vitro and in vivo to phosgene (COCl2), which reacts with GSH to produce GSCOSG. The reaction of GSH with COCl2 may be responsible at least in part for the GSH-depleting properties of CHCl3, CBrCl3, and CCl4, inasmuch as the relative amounts of formation of GSCOSG in vitro and in vivo paralleled their relative GSH-depleting activities.