Sulfation and glucuronidation of acetaminophen by cultured hepatocytes reproducing in vivo sex-differences in conjugation on matrigel and type 1 collagen

  • Robert E. Kane
  • Joseph Tector
  • John J. Brems
  • Albert Li
  • Donald Kaminski
Regular Papers


The sulfate and glucuronide conjugation of acetaminophen (APAP) by hepatocytes cultured on Matrigel or type 1 collagen was compared to APAP metabolism in vivo. The metabolic fate of low (15 mg/kg), medium (125 mg/kg), and high (300 mg/kg) doses of APAP injected intraperitoneally were determined in male and female rats. Males excreted more APAP as the sulfate conjugate than females, which correlated with the twofold greater APAP sulfotransferase activity in the male vs. females (301±24 vs. 156±18 pmol · mg−1 protein · min−1). Also, as sulfate conjugation became saturated, there was a dose-related shift in APAP metabolism from sulfate to glucuronide conjugation in both sexes. After death, the livers of the same animals were perfused with collagenase and the hepatocytes cultured in modified Waymouth’s medium on either Matrigel or rat-tail collagen, with various doses of APAP (0, 0.125, 0.25, 0.5, and 1.0 mM). Sex differences in APAP sulfation and glucuronidation persisted in culture for up to 4 days, with sulfation predominating in the male similar to in vivo. With increasing APAP concentration (dose), there was a saturation of sulfate conjugation and a shift to glucuronidation as observed in vivo. Sex differences in APAP sulfation and glucuronidation were no longer significant by Day 4 in culture. Sulfation, and to a lesser extent, glucuronidation, were more stable on Matrigel than collagen. We concluded that APAP metabolism of freshly isolated hepatocytes could replicate in vivo sex differences in conjugation, and that Matrigel was superior to collagen as substrate.

Key words

UDP-glucuronyltransferase cells cultured liver acetaminophen sex characteristics sulfotransferase 


  1. 1.
    Bissell, D. M.; Arensen, D. M.; Maher, J. J., et al. Support of cultured hepatocytes by a laminin-rich gel. Evidence for a functionally significant subendothelial matrix in normal rat liver. J. Clin. Invest. 79:801–812; 1987.PubMedCrossRefGoogle Scholar
  2. 2.
    Bissell, D. M.; Choun, C. O. The role of extracellular matrix in normal liver. Scand. J. Gastroenterol. 23:1–7; 1988.Google Scholar
  3. 3.
    Clayton, D. F.; Darnell, J. E. Changes in liver-specific compared to common gene transcription during primary culture of mouse hepatocytes. Mol. Cell. Biol. 3:1552–1561; 1983.PubMedGoogle Scholar
  4. 4.
    Crabb, D. W.; Li, T. K. Expression of alcohol dehydrogenase in primary monolayer cultures of rat hepatocytes. Biochem. Biophys. Res. Comm. 128:12–17; 1985.PubMedCrossRefGoogle Scholar
  5. 5.
    Fraser, J. M.; Tyson, C. A.; McCarthy, C., et al. Contemporary issues in toxicology. Potential use of human tissue to toxicity research and testing. Toxicol. Appl. Pharmacol. 97:387–397; 1989.CrossRefGoogle Scholar
  6. 6.
    Galinsky, R. E.; Kane, R. E.; Franklin, M. R. Effect of aging on drug-metabolizing enzymes important in acetaminophen elimination. J. Pharmacol. Exp. Ther. 237:107–113; 1986.PubMedGoogle Scholar
  7. 7.
    Galinsky, R. E.; Levy, G. Dose- and time-dependent elimination of acetaminophen in rats: pharmacokinetic implications of cosubstrate depletion. J. Pharmacol. Exp. Ther. 219:14–20; 1981.PubMedGoogle Scholar
  8. 8.
    Galinsky, R. E.; Kane, R. E.; Franklin, M. R. Effect of aging on drug-metabolizing enzymes important in acetaminophen elimination. J. Pharmacol. Exp. Ther. 237:107–113; 1986.PubMedGoogle Scholar
  9. 9.
    Grant, M. H.; Hawksworth, G. M. The activity of UDP-glucuronyl-transferase, sulfotransferase and glutathione-S-transferase in primary cultures of rat hepatocytes. Biochem. Pharmacol. 35:2979–2982; 1986.PubMedCrossRefGoogle Scholar
  10. 10.
    Green, M. D.; Fisher, L. J. Age- and sex-related differences in acetaminophen metabolism in the rat. Life Sci. 29:2421–2428; 1981.PubMedCrossRefGoogle Scholar
  11. 11.
    Green, C. E.; Segall, H. J.; Byard, J. L. Metabolism, cytotoxicity, and genotoxicity of the pyrrolizidine alkaloid senecionine in primary cultures of rat hepatocytes. Toxicol. Appl. Pharmacol. 60:176–185; 1981.PubMedCrossRefGoogle Scholar
  12. 12.
    Guzelian, P. S.; Li, D.; Schuetz, E. G., et al. Sex change in cytochrome P-450 phenotype by growth hormone treatment of adult rat hepatocytes maintained in culture system on matrigel. Proc. Natl. Acad. Sci. USA 83:9783–9787; 1988.CrossRefGoogle Scholar
  13. 13.
    Hansen, R. J.; Jugermann, K. Sex differences in the control of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Biol. Chem. Hoppe-Seyler 368:955–962; 1987.PubMedGoogle Scholar
  14. 14.
    Hutson, S. M.; Stinson-Fisher, C.; Shiman, R., et al. Regulation of albumin synthesis by hormones and amino acids in primary cultures of rat hepatocytes. Am. J. Physiol. 252:E291–298; 1987.PubMedGoogle Scholar
  15. 15.
    Kane, R. E.; Chen, L. J. Influence of gonadal hormones upon rat hepatic acetaminophen sulfotransferases. Drug Metab. Dispos. 15:725–728; 1987.PubMedGoogle Scholar
  16. 16.
    Kleinman, H. K.; McGarvey, M. L.; Hassell, J. R., et al. Formation of a supramolecular complex involved in the reconstitution of basement membrane components. Biochemistry 25:312–318; 1983.CrossRefGoogle Scholar
  17. 17.
    Koster, H.; Halsema, I.; Scholtens, E., et al. Dose-dependent shifts in sulfation and glucuronidation of phenolic compounds in the ratin vivo and in isolated hepatocytes. The role of saturation of the phenol sulfotransferase. Biochem. Pharmacol. 18:2569–2575; 1981.CrossRefGoogle Scholar
  18. 18.
    Koster, H.; Halsema, E.; Pang, K. S., et al. Kinetics of sulfation and glucuronidation of harmol in the perfused rat liver. Disappearance of aberrancies in glucuronidation kinetics by inhibition of sulfation. Biochem. Pharmacol. 31:3023–3028; 1982.PubMedCrossRefGoogle Scholar
  19. 19.
    Lowry, O. H.; Rosbrough, N. J.; Farr, A. L., et al. Protein measurement with Folin phenol reagent. J. Biol. Chem. 193:265–275; 1951.PubMedGoogle Scholar
  20. 20.
    Maher, J. J. Primary hepatocyte culture: Is it home away from home? Hepatology 8:1162–1166; 1988.PubMedCrossRefGoogle Scholar
  21. 21.
    Mulder, G. J. Kinetics of sulfation in the rat in vivo and in the perfused rat liver. Fed. Proc. 45:2229–2234; 1986.PubMedGoogle Scholar
  22. 22.
    Raheja, K. L.; Linscheer, W. B.; Cho, C. Hepatotoxicity and metabolism of acetaminophen in male and female rats. J. Toxicol. Environ. Health. 12:143–147; 1983.PubMedCrossRefGoogle Scholar
  23. 23.
    Reese, J. A.; Byard, J. L. Isolation and culture of adult hepatocytes from liver biopsies. In Vitro 17:935–940; 1981.PubMedCrossRefGoogle Scholar
  24. 24.
    Schuetz, E. G.; Le, D.; Omiecinski, C. J., et al. Regulation of gene expression in adult rat hepatocytes cultured on a basement membrane matrix. J. Cell. Physiol. 134:309–323; 1988.PubMedCrossRefGoogle Scholar
  25. 25.
    Sekura, R. D.; Marcus, C. J.; Lyon, E. S., et al. Assay of sulfotransferase. Anal. Biochem. 95:82–86; 1979.PubMedCrossRefGoogle Scholar
  26. 26.
    Souilinna, E. M.; Pitkaranta, T. Effect of culture age on drug metabolizing enzymes and their induction in primary culture of rat hepatocytes. Biochem. Pharmacol. 35:2241–2245; 1986.CrossRefGoogle Scholar
  27. 27.
    Sweeny, D. J.; Reinke, L. A. Sulfation of acetaminophen in isolated rat hepatocytes. Relationship to sulfate ion concentration and intracellular levels of 3′-phosphoadenosine-5′phosphosulfate. Drug Metab. Dispos. 16:712–715; 1988.PubMedGoogle Scholar
  28. 28.
    Zaphiropoulos, P. G.; Mode, A.; Norstedt, G., et al. Regulation of sexual differentiation in drug and steroid metabolism. TiPS 10:149–153; 1989.PubMedGoogle Scholar

Copyright information

© Tissue Culture Association 1991

Authors and Affiliations

  • Robert E. Kane
    • 1
  • Joseph Tector
    • 2
  • John J. Brems
    • 2
  • Albert Li
    • 3
  • Donald Kaminski
    • 2
  1. 1.Department of PediatricsSt. Louis University School of MedicineSt. Louis
  2. 2.Department of SurgerySt. Louis University School of MedicineSt. Louis
  3. 3.Monsanto Co.St. Louis

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