Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1982 Apr;79(8):2678–2682. doi: 10.1073/pnas.79.8.2678

Reductive metabolism of carbon tetrachloride by human cytochromes P-450 reconstituted in phospholipid vesicles: mass spectral identification of trichloromethyl radical bound to dioleoyl phosphatidylcholine.

J R Trudell, B Bösterling, A J Trevor
PMCID: PMC346264  PMID: 6953422

Abstract

It has been proposed that covalent binding of reactive metabolites to liver membrane constituents may be responsible for the hepatoxicity of carbon tetrachloride. This study demonstrates that trichloromethyl free radical is the major reductive metabolite of carbon tetrachloride by cytochrome P-450 and that this free radical is capable of binding to double bonds of fatty acyl chains of the phospholipids in the membrane surrounding cytochrome P-450. The structural identification of the reactive free radical metabolite and the product of its addition to phospholipids was accomplished by use of a reconstituted system of human cytochromes P-450, NADPH-cytochrome P-450 reductase, and cytochrome b5 in phospholipid vesicles. The reconstituted vesicles contained a mixture of dioleoyl phosphatidylcholine and egg phosphatidylethanolamine that served as both structural components and targets for trichloromethyl free radical binding. After incubation of these vesicles under a N2 atmosphere in the presence of NADPH with 14CCl4, the phospholipids were extracted and then separated by high-pressure liquid chromatography. The dioleoyl phosphatidylcholine fraction was transesterified and the resulting single 14C-labeled fatty acid methyl ester was purified by reverse-phase chromatography. Desorption chemical ionization mass spectrometry with ammonia as reagent gas as well as desorption electron-impact mass spectrometry permitted identification of the molecular structure as a mixture of 9- and 10-(trichloromethyl)stearate methyl esters.

Full text

PDF
2678

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ahr H. J., King L. J., Nastainczyk W., Ullrich V. The mechanism of chloroform and carbon monoxide formation from carbon tetrachloride by microsomal cytochrome P-450. Biochem Pharmacol. 1980 Oct 15;29(20):2855–2861. doi: 10.1016/0006-2952(80)90022-2. [DOI] [PubMed] [Google Scholar]
  2. BUTLER T. C. Reduction of carbon tetrachloride in vivo and reduction of carbon tetrachloride and chloroform in vitro by tissues and tissue constituents. J Pharmacol Exp Ther. 1961 Dec;134:311–319. [PubMed] [Google Scholar]
  3. Benedetti A., Casini A. F., Ferrali M., Comporti M. Early alterations induced by carbon tetrachloride in the lipids of the membranes of the endoplasmic reticulum of the liver cell. I. Separation and partial characterization of altered lipids. Chem Biol Interact. 1977 May;17(2):151–156. doi: 10.1016/0009-2797(77)90081-3. [DOI] [PubMed] [Google Scholar]
  4. Bonfils C., Balny C., Maurel P. Direct evidence for electron transfer from ferrous cytochrome b5 to the oxyferrous intermediate of liver microsomal cytochrome P-450 LM2. J Biol Chem. 1981 Sep 25;256(18):9457–9465. [PubMed] [Google Scholar]
  5. Bösterling B., Stier A., Hildebrandt A. G., Dawson J. H., Trudell J. R. Reconstitution of cytochrome P-450 and cytochrome P-450 reductase into phosphatidylcholine-phosphatidylethanolamine bilayers: characterization of structure and metabolic activity. Mol Pharmacol. 1979 Jul;16(1):332–342. [PubMed] [Google Scholar]
  6. Cohen E. N., Trudell J. R., Edmunds H. N., Watson E. Urinary metabolites of halothane in man. Anesthesiology. 1975 Oct;43(4):392–401. doi: 10.1097/00000542-197510000-00004. [DOI] [PubMed] [Google Scholar]
  7. Erickson S. K., Bösterling B. Cholesterol 7 alpha-hydroxylase from human liver: partial purification and reconstruction into defined phospholipid-cholesterol vesicles. J Lipid Res. 1981 Jul;22(5):872–876. [PubMed] [Google Scholar]
  8. Hjelmeland L. M. A nondenaturing zwitterionic detergent for membrane biochemistry: design and synthesis. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6368–6370. doi: 10.1073/pnas.77.11.6368. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kalyanaraman B., Mason R. P., Perez-Reyes E., Chignell C. F., Wolf C. R., Philpot R. M. Characterization of the free radical formed in aerobic microsomal incubations containing carbon tetrachloride and NADPH. Biochem Biophys Res Commun. 1979 Aug 28;89(4):1065–1072. doi: 10.1016/0006-291x(79)92116-8. [DOI] [PubMed] [Google Scholar]
  10. Lai E. K., McCay P. B., Noguchi T., Fong K. L. In vivo spin-trapping of trichloromethyl radicals formed from CCl4. Biochem Pharmacol. 1979 Jul 15;28(14):2231–2235. doi: 10.1016/0006-2952(79)90212-0. [DOI] [PubMed] [Google Scholar]
  11. Poyer J. L., McCay P. B., Lai E. K., Janzen E. G., Davis E. R. Confirmation of assignment of the trichloromethyl radical spin adduct detected by spin trapping during 13C-carbon tetrachloride metabolism in vitro and in vivo. Biochem Biophys Res Commun. 1980 Jun 30;94(4):1154–1160. doi: 10.1016/0006-291x(80)90540-9. [DOI] [PubMed] [Google Scholar]
  12. Reynolds E. S. Liver parenchymal cell injury. IV. Pattern of incorporation of carbon and chlorine from carbon tetrachloride into chemical constituents of liver in vivo. J Pharmacol Exp Ther. 1967 Jan;155(1):117–126. [PubMed] [Google Scholar]
  13. SINGLETON W. S., GRAY M. S., BROWN M. L., WHITE J. L. CHROMATOGRAPHICALLY HOMOGENEOUS LECITHIN FROM EGG PHOSPHOLIPIDS. J Am Oil Chem Soc. 1965 Jan;42:53–56. doi: 10.1007/BF02558256. [DOI] [PubMed] [Google Scholar]
  14. Sipes I. G., Krishna G., Gillette J. R. Bioactivation of carbon tetrachloride, chloroform and bromotrichloromethane: role of cytochrome P-450. Life Sci. 1977 May 1;20(9):1541–1548. doi: 10.1016/0024-3205(77)90446-5. [DOI] [PubMed] [Google Scholar]
  15. Spatz L., Strittmatter P. A form of cytochrome b5 that contains an additional hydrophobic sequence of 40 amino acid residues. Proc Natl Acad Sci U S A. 1971 May;68(5):1042–1046. doi: 10.1073/pnas.68.5.1042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Trudell J. R., Bösterling, Trevor A. 1-Chloro-2,2,2-trifluoroethyl radical: Formation from halothane by human cytochrome P-450 in reconstituted vesicles and binding to phospholipids. Biochem Biophys Res Commun. 1981 Sep 16;102(1):372–377. doi: 10.1016/0006-291x(81)91531-x. [DOI] [PubMed] [Google Scholar]
  17. Uehleke H., Hellmer K. H., Tabarelli S. Binding of 14 C-carbon tetrachloride to microsomal proteins in vitro and formation of CHC1 3 by reduced liver microsomes. Xenobiotica. 1973 Jan;3(1):1–11. doi: 10.3109/00498257309151495. [DOI] [PubMed] [Google Scholar]
  18. Wolf C. R., Harrelson W. G., Jr, Nastainczyk W. M., Philpot R. M., Kalyanaraman B., Mason R. P. Metabolism of carbon tetrachloride in hepatic microsomes and reconstituted monooxygenase systems and its relationship to lipid peroxidation. Mol Pharmacol. 1980 Nov;18(3):553–558. [PubMed] [Google Scholar]
  19. Wolf C. R., Mansuy D., Nastainczyk W., Deutschmann G., Ullrich V. The reduction of polyhalogenated methanes by liver microsomal cytochrome P450. Mol Pharmacol. 1977 Jul;13(4):698–705. [PubMed] [Google Scholar]
  20. Yasukochi Y., Masters B. S. Some properties of a detergent-solubilized NADPH-cytochrome c(cytochrome P-450) reductase purified by biospecific affinity chromatography. J Biol Chem. 1976 Sep 10;251(17):5337–5344. [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES