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Anthracycline resistance of P388 daunorubicin-resistant cells cannot be accounted for merely by differences in drug uptake and retention; protection against intracellular drug was also indicated. Cytotoxicity of daunorubicin may be partially due to the formation of free radicals and reactive oxygen species (hydrogen peroxide, hydroxyl radical, singlet oxygen, and superoxide anion radical). Protection against free radicals and peroxides is largely dependent upon the availability of reduced glutathione, which in turn requires NADPH for its continual regeneration. Pentose phosphate cycle (also called hexose monophosphate shunt) is known to provide NADPH for maintenance of glutathione. Activities of the two NADPH-producing dehydrogenases of the cycle, glucose-6-phosphate and 6-phosphogluconate dehydrogenase, were 40% higher (P less than 0.05) and activity of the cycle in intact cells was 2-fold higher in the resistant than the sensitive cells. The cycle was as active in these cells as it is known to be in macrophages, indicating a very effective protection against oxidative stress, free radicals, and alkylating electrophiles. Elevated activity of the pentose phosphate pathway in drug-resistant cells can represent a mechanism of resistance against multiple structurally unrelated drugs. Efflux of daunorubicin may be aided by further metabolism to glucuronides. Daunorubicinol, a known active metabolite of daunorubicin, can be metabolized to a glucuronide by the cells and eliminated into the surrounding medium. Glucuronidation of daunorubicinol was evidenced by (a) release of daunorubicinol following glucuronidase hydrolysis of media from cell incubations with 1.8 microM daunorubicin and (b) production of radioactive glucuronide when cell homogenates were incubated with UDP-[14C]glucuronic acid plus daunorubicinol. Glucuronyltransferase activity with a broad substrate specificity was found in the cells. Using model substrates, 1-naphthol and o-aminophenol, it was determined that glucuronyltransferase activity was 4 times higher in daunorubicin-resistant than -sensitive P388 cells. Elevated glucuronyltransferase could contribute to daunorubicin and multidrug resistance.

Properties of lymphocyte glucuronyltransferase were studied in homogenates of SN1006 cells. A sensitive assay procedure for lymphocyte glucuronyltransferase was developed utilizing radioactive testosterone as the acceptor substrate and TLC for separation of the metabolite. The method is capable of detecting picomolar quantities of the product. The enzyme activity exhibited a broad pH optimum, and was subject to activation by the detergent Lubrol WX and Mn++ ions. The activity conformed to the Michaelis-Menten kinetics giving apparent Km values of 0.8 mM and 11 microM, for UDPGA and testosterone, respectively. 4-Methylumbelliferone, a-naphthol and p-nitrophenol behaved as competitive inhibitors of testosterone glucuronidation. The results indicate that the method could be used for genetic studies of human lymphocyte glucuronyltransferase, and that the enzyme is of consequence in detoxication of exogenous as well as endogenous substrates.

Specific activities of UDP-glucuronosyltransferase and glucose-6-phosphatase, a marker enzyme of endoplasmic reticulum, were measured in mitochondria and microsomes. In mitochondria the specific activity of UDP-glucuronosyltransferase represented only 7-11% of that found in microsomes, when measured in the presence of various aglycone substrates, including 4-nitrophenol, 4-methylumbelliferone, 1-naphthol, phenolphthalein, testosterone and 17 beta-estradiol. Similarly, the specific activity of glucose-6-phosphatase in mitochondrial preparations was about 80% of that found in microsomes. In conclusion, it seems that in rat liver the UDP-glucuronosyltransferase activity associated with mitochondrial fractions reflects the presence of membrane fragments derived from endoplasmic reticulum, rather than mitochondrial location of this enzyme.

Fractionation of sodium cholate solubilized microsomes from mouse liver on Sepharose CL-48 yielded three protein peaks with UDP-glucuronyltransferase activities. Of these three peaks, only peak II contained activities towards all the substrates tested: p-nitrophenol, 1-naphthol, morphine, testosterone and estrone. These glucuronyltransferase activities could not be dissociated by further chromatography on DEAE-Sepharose CL-6B and isoelectric focusing. The results show the presence of a functional form of glucuronyltransferase with a wide substrate specificity, and indicate that in addition, other forms with narrower studied specificities may also be present in mouse liver microsomes.

1. This study establishes the presence of UDP-glucuronyltransferase activity for non-steroidal as well as steroidal substrates, in cultured human B-lymphocytes. Glucuronidation of alpha-naphthol and testosterone was demonstrated in homogenates of two cell lines, SN1006 and RPMI-1788, and that of phenolphthalein, 4-methylumbelliferone, p-nitrophenol and estradiol in the cell line with the higher glucuronyltransferase activity, SN1006. 2. Kinetic studies of testosterone glucuronidation in homogenates of both cell lines revealed a similarity in the behaviour of glucuronyltransferase of these cells. Thus, comparable apparent Km values for UDPGA (0.63 mM) and for testosterone (14 microgram) were observed, although apparent maximal velocities, Vmax, differed several-fold (3.0 versus 0.55 pmol/10(6) cells per min, in SN1006 and RPMI-1788 cells, respectively). 3. Kinetic studies of glucuronidation of testosterone, estradiol, phenolphthalein, alpha-naphthol, 4-methylumbelliferone, and p-nitrophenol yielded comparable apparent Km values for UDPGA (0.56-0.67 mM), suggesting that the same, or similar, glucuronyltransferase(s) catalyse(s) glucuronidation of this wide range of substrates in lymphocytes. This was reinforced by the observation of competitive inhibition of testosterone glucuronidation by alpha-naphthol (Ki 0.25mM), 4-methylumbelliferone (Ki 0.8mM) and p-nitrophenol (Ki 0.8 mM). Thus, lymphocyte glucuronyltransferase activity with a broad substrate specificity, for steroidal and non-steroidal aglycones, is indicated.

Glucuronidation of benzo[a]pyrene (B[a]P) metabolites, generated in situ by oxidative pathways, was studied using mouse liver uninduced microsomes. No coupling was evident between UDP-glucuronyltransferase and oxygenation of B[a]P. UDPGA protected microsomal macromolecules against binding of reactive B[a]P metabolites. Superoxide, and other reactive oxygen species decreased both the overall B[a]P metabolism and glucuronidation of some B[a]P metabolic products, and caused more extensive binding to macromolecules; UDPGA was less protective in this condition. Peroxidation of microsomes differentially affected glucuronidation of various metabolites of B[a]P, and of various model substrates, indicating that multiple glucuronyltransferases are involved in the conjugation of hydroxylated metabolites of B[a]P.

Some properties of rat liver nuclear membrane-bound UDPglucuronosyltransferase were compared with those of the endoplasmic reticulum bound enzyme. The activity of nuclear membrane-bound UDPglucuronosyltransferase was stimulated only about 1.5-fold by Lubrol WX. Under the same conditions microsomal UDPglucuronosyltransferase was, as usual, highly activated (up to 10-fold), when 4-nitrophenol was the acceptor of glucuronic acid. Specific activities of the detergent-activated enzyme were similar in microsomal and nuclear membrane preparations, when the following aglycone substrates were used: 4-methylumbelliferone, 4-nitrophenol, 1-naphthol, phenolphthalein, and testosterone. Apparent Km values for UDP-glucuronic acid ranged between 0.15-0.25 mM for glucuronidation of 4-nitrophenol and 1-naphthol, by either Lubrol WX activated or non-activated, nuclear membrane-bound UDPglucuronosyltransferase. These values were comparable to those found for detergent activated microsomal enzyme. The results show a similarity in behavior of detergent-activated UDPglucuronosyltransferase regardless of subcellular membrane source and, therefore, suggest the association of the same glucuronosyltransferase with nuclear membrane and endoplasmic reticulum. A possible significance of the presence of high activity of this enzyme in nuclear membrane is discussed.

Human lymphocytes contain glucuronyltransferase activity. This enzyme requires a high concentration of uridine diphosphoglucuronic acid and can be measured with testosterone as a substrate. This finding is of interest because glucuronidation is a deactivating process for many pharmacologically active compounds that are substrates of glucuronyltransferase.