Toxic. in Vitro Vol. 5, No. 3, pp. 219-224, 1991 0887-2333/91 $3.00 + 0.00 Printed in Great Britain.All rights reserved Copyright © 1991 Pergamon Pressplc THE EFFECTS OF BUPRENORPHINE ON THE METABOLISM OF HUMAN HEPATOCYTES X. PONSODA, R. JOVER, M. J. G6MEz-LEcn6N, R. FABRA*, R. TRULLENQUE* and J. V. CAS'I'ELL Unidad de Hepatologia Experimental, Centro de lnvestigaci6n, Hospital Universitario La Fe, SVS. Avenida de Campanar 21, E-46009 Valencia and *Serviciode Cirugia General A. Hospital General de Valencia, SVS. Avenida del Cid, Valencia, Spain (Received 13 June 1990; revisions received 24 September 1990) Abstract--Adult human hepatocytes cultured in chemically defined conditions were used as a biological model to examine the metabolic effects of buprenorphine on the human liver. Cell extension and mono- layer formation of human hepatocytes were affected in a dose-dependent manner after 24 hr of exposure to the drug. According to the several endpoints evaluated (cellular protein, intracellular lactate dehydro- genase activity and the MTT test), the half-maximal cytotoxic effect (ICso) of buprenorphine was close to 100/~M. Longer exposure of hepatocytes to buprenorphine (72 hr) increased its cytotoxicity, and the ICso of the drug was reduced to 50/~M. Lower concentrations of the drug (in the 5-50-#M range) significantly impaired the metabolic functions of the hepatocytes. Incubation of the cells with 40/~M-buprenorphin¢ for 24 hr reduced the glycogen content to 60% of the initial content and 50/~M-buprenorphine inhibited giycogen synthesis in giucagon-depleted human hepatocytes by about 40%. Albumin synthesis was the most sensitive metabolic parameter, and 24-hr exposure of hepatocytes to I0/~M-buprenorphine, a concentration with no apparent cytotoxic effects, reduced albumin synthesis to 50%. Urea synthesis was moderately affected by buprenorphine. Glutathione content was reduced in a dose-dependent manner by the drug, reaching a minimum value (60% of control values) after 6 hr of exposure to 50/~ M of the opiate. Analysis of the data on the therapeutic dosage of buprenorphine and other opiates showed that the toxicity risk index of buprenorphine, like that of meperidine, lies somewhere between that of morphine and methadone. INTRODUCTION Liver pathology among patients on opiate therapy and narcotic addicts is very frequent. The pathogenesis of liver dysfunction in such individuals has been de- bated for many years (Gelb et al., 1977; Gorodetzky et al., 1968; Kreek et al., 1972; Marks and Chapple, 1967). While several studies concluded that liver alterations in drug addicts were the result of previous viral infection, impurities of the self-administered drug or alcohol abuse (Gorodetzky et al., 1968), other clinical studies attributed the liver dysfunction directly to morphine, heroin (Marks and Chapple, 1967) or methadone (Walter, 1969). Recent experiments with animals have shown that opiates by themselves produced morphological and metabolic alterations in the liver (Borgia et al., 1982; Chang and Ho, 1979; Ekl6w-LAstbom et al., 1986; James et al., 1982; Nagamatsu et al., 1986). Moreover, previous results from our laboratory showed that opiates alter the biochemical functionality of cultured human hepatocytes (G6mez-Lech6n et al., 1987). Taken together, this evidence indicates that opiates can act as intrinsic hepatotoxins and could represent per sea risk to man after chronic use or inappropriate self-administration. Buprenorphine is a semi-synthetic opiate derived from tebaine, and closely related in structure to Abbreviations: GSH = glutathion¢; LDH = lactate dehydro- genase; TRI = toxicity risk index: the quotient between the plasma concentration after drug administration to humans and the toxic concentration to cells (IC~o). morphine, with mixed agonist-antagonist properties (Heel et al., 1979). Buprenorphine is claimed to have a more prolonged action (Dowing et al., 1977), with less risk of physical dependence (Jasinski et al., 1978) and 25-50 times more potency as an analgesic than morphine (Houde, 1979). It has also been shown to be three times more potent than naloxone as an antagonist of morphine (De Castro and Paramentier, 1976), and to induce tolerance development in the host to a limited extent. Over the past few years, increasing misuse of buprenorphine by opiate addicts has been observed (Strang, 1985), and as buprenor- phine is structurally related to opioids, its use could represent a potential hepatotoxic risk to man. In the present paper we examined the cytotoxic and metabolic effects of buprenorphine on human hepato- cytes after exposure of cells to concentrations in the range found in human serum after therapeutic or abuse administration. MATERIALS AND METHODS Drugs. Buprenorphine, (6R,7R, 14S)- 17-cyclopropyl- methyl-7,8-dihydro-7-[(IS)-l-hydroxy-l,2,2-trimethyl- propyl]-60-methyl-6,14-ethano- 17-normorphine (RX 6029-M), was obtained from Laboratorios Esteve S.A. (Spain), then dissolved in l mM-HCI and filtered through a 0.22-/~M membrane to prepare a 3-ram stock solution. Ready-to-use dilutions were made in culture medium and prepared fresh every day. Final concentrations of buprenorphine added to the cells were between 5 x 10-(M and 10 -7 M. 219