Birgit L. Coffman

Genetic predisposition to the metabolism of irinotecan (CPT-11): Role of uridine diphosphate glucuronosyltransferase isoform 1A1 in the glucuronidation of its active metabolite (SN-38) in human liver microsomes

Irinotecan (CPT-11) is a promising antitumor agent, recently approved for use in patients with metastatic colorectal cancer. Its active metabolite, SN-38, is glucuronidated by hepatic uridine diphosphate glucuronosyltransferases (UGTs). The major dose-limiting toxicity of irinotecan therapy is diarrhea, which is believed to be secondary to the biliary excretion of SN-38, the extent of which is determined by SN-38 glucuronidation. The purpose of this study was to identify the specific isoform of UGT involved in SN-38 glucuronidation. In vitro glucuronidation of SN-38 was screened in hepatic microsomes from normal rats (n = 4), normal humans (n = 25), Gunn rats (n = 3), and patients (n = 4) with Crigler-Najjar type I (CN-I) syndrome. A wide intersubject variability in in vitro SN-38 glucuronide formation rates was found in humans. Gunn rats and CN-I patients lacked SN-38 glucuronidating activity, indicating the role of UGT1 isoform in SN-38 glucuronidation. A significant correlation was observed between SN-38 and bilirubin glucuronidation (r = 0.89; P = 0.001), whereas there was a poor relationship between para-nitrophenol and SN-38 glucuronidation (r = 0.08; P = 0.703). Intact SN-38 glucuronidation was observed only in HK293 cells transfected with the UGT1A1 isozyme. These results demonstrate that UGT1A1 is the isoform responsible for SN-38 glucuronidation. These findings indicate a genetic predisposition to the metabolism of irinotecan, suggesting that patients with low UGT1A1 activity, such as those with Gilberts syndrome, may be at an increased risk for irinotecan toxicity.

Differential glucuronidation of bile acids, androgens and estrogens by human UGT1A3 and 2B7.

In this work, UDP-glucuronosyltransferases (UGTs), UGT1A3, 2B7(H268) and 2B7(Y268), stably expressed in human embryonic kidney cells (HK293) were used to assess glucuronidation activities with a variety of steroid hormone and bile acid substrates. The rate of synthesis of carboxyl- and hydroxyl-linked glucuronides was determined under optimal reaction conditions. Expressed UGT1A3 catalyzed bile acid glucuronidation at high rates exclusively at the carboxyl moiety for all compounds tested. In contrast, UGT1A4 catalyzed bile acid glucuronidation at very low rates exclusively at the 3alpha-hydroxyl function. Both UGT2B7 allelic variants glucuronidated the bile acid substrates at both carboxyl and hydroxyl moieties, however, the 3alpha-hydroxyl position was preferentially conjugated compared to the carboxyl function. Similarly, androsterone, a 3alpha-hydroxylated androgenic steroid, was glucuronidated at very high rates by expressed UGT2B7. Of the estrogenic compounds tested, UGT2B7 catalyzed the glucuronidation of estriol at rates comparable to those determined for androsterone. Other structural discrimination was found with UGT2B7 which had activity toward estriol and estradiol exclusively at the 17beta-OH position, yielding the cholestatic steroid D-ring glucuronides.

Identification of N-methylprotoporphyrin IX in livers of untreated mice and mice treated with 3,5-diethoxycarbonyl-1,4-dihydrocollidine: Source of the methyl group

Abstract Administration of the porphyrogenic agent, 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) to mice, leads to the accumulation of N-methylprotoporphyrin IX in liver. This porphyrin is a potent inhibitor of ferrochelatase activity and accounts for the porphyria produced after DDC administration. The N-methylprotoporphyrin IX extracted from DDC-treated mice is primarily of one isomeric form, as shown by nuclear magnetic resonance spectroscopy. The methyl group of N-methylprotoporphyrin IX isolated from DDC-treated mice is derived mostly from the 4-methyl group of DDC. The transfer of this methyl group and its subsequent covalent attachment to protoporphyrin IX may be mediated by a form of hepatic microsomal cytochrome P-450. N-Methylprotoporphyrin IX is also found in livers of untreated mice at levels that are low but significant.

Metabolism of Endobiotics and Xenobiotics by UDP-Glucuronosyltransferase

Publisher Summary All organisms are exposed to a number of chemical compounds that are toxic were it not for metabolic mechanisms available to the organism to moderate their effects. Chemical substances may be xenobiotics, such as drugs, or compounds presented to the organism from environmental or dietary sources. The elimination of many such compounds and their detoxification involves different types of metabolic reactions. One of them is conjugation through glucuronic acid catalyzed by UDP-glucuronosyltransferases (UGTs). In addition to xenobiotics; endobiotics, such as steroids or bilirubin, are glucuronidated. Formation of glucuronides from xeno- and endo- biotics generally results in the formation of products that are more hydrophilic and more readily excreted by the kidney or liver. A number of UGTs have been purified to homogeneity from liver of several species, including humans. Recently identified are a number of UGTs that are important for the metabolism of xenobiotics and endobiotics, such as steroids and estrogen catechols. This chapter discusses the metabolism of important amines, opioids, and endobiotics, such as steroids and their catechol derivatives. There are details on glucuronidation of amines, glucuronidation of opioid compounds, and glucuronidation of catechol estrogens by UGTs. Many isoforms of UGT catalyze the glucuronidation of primary and secondary amines. In humans, many important therapeutic agents, such as the antihistamines, antipsychotics, and tricyclic antidepressants, are tertiary amines. Many of these tertiary amines, in humans, are converted to and excreted as quaternary ammonium-linked glucuronides. Stably expressed human UGT1.4 protein has been discovered to catalyze the N-glucuronidation of a variety of tertiary amines. To date, only human UGT1.4 has been identified to catalyze the formation of quaternary ammonium-linked glucuronides from tertiary amines. UGT2B1 and UGT1.1 have been demonstrated to catalyze the glucuronidation of opioid substances. UGT2B1 has high activity toward many opioid substrates, whereas UGT1.1 catalyzes the glucuronidation the oripavine-type opioid compounds.

Characterization of antibodies to a rabbit hepatic UDP-glucuronosyltransferase and the identification of an immunologically similar enzyme in human liver

An antibody to a UDP-glucuronosyltransferase (UDPGT) isoenzyme which catalyzes the glucuronidation of p-nitrophenol (PNP) in rabbit liver was raised in sheep and used to identify immunologically similar UDPGTs in rabbit and human livers. Immunoblotting experiments showed that the antisera specifically recognized PNP UDPGT but not estrone UDPGT purified from rabbit liver. Sheep anti-rabbit liver PNP UDPGT IgG immunoprecipitated PNP, 1-naphthol, and 4-methylumbelliferone glucuronidation activities in rabbit and human liver microsomal preparations. In rabbit liver microsomes the antibody did not immunoprecipitate estrone or estradiol glucuronidation activities. In human liver microsomes, 4-aminobiphenyl but not estriol glucuronidation activities were immunoprecipitated, suggesting that the antibody recognizes a specific UDPGT (pI 6.2) in human liver microsomes.

The formation of N-alkylprotoporphyrin IX and destruction of cytochrome P-450 in the liver of rats after treatment with 3,5-diethoxycarbonyl-1,4-dihydrocollidine and its 4-ethyl analog

Abstract The effects of two porphyrogenic agents, 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) and 3,5-diethoxycarbonyl-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP), have been studied in rats. The administration of these compounds leads to the formation and accumulation in the liver of N-methylprotoporphyrin IX and N-ethylprotoporphyrin IX, respectively. In each case, the alkyl group of the porphyrin is derived from the 4-alkyl group of the porphyrogenic chemical. Each N-alkylporphyrin is a potent inhibitor of protoheme ferrolyase (EC 4.99.1.1) (ferrochelatase) activity. N-Methylprotoporphyrin IX is somewhat more potent than N-ethylprotoporphyrin IX as an inhibitor of ferrochelatase activity in vitro. However, more N-ethylprotoporphyrin IX accumulates in rat liver than does the N-methyl analog. Since alkylporphyrins are formed during the catabolism of heme (or hemoprotein), the effects of DDC and DDEP on hepatic microsomal cytochrome P-450 were also studied. Whereas DDC treatment led to only a slight decrease in cytochrome P-450 levels (25%), DDEP administration led to a marked decrease (75%) in the total cytochrome P-450 level. In phenobarbital- and 3-methylcholanthrene-treated rats, DDC administration did not alter the hepatic microsomal cytochrome P-450 content, while administration of DDEP to either phenobarbital-treated or 3-methylcholanthrene-treated rats led to marked reduction of levels in cytochrome P-450. Although the N-methylprotoporphyrin IX level was not increased following DDC administration to either phenobarbital- or 3-methylcholanthrene-treated rats, there was a marked increase in N-ethylprotoporphyrin IX accumulation in both phenobarbital- and 3-methylcholanthrene-treated rats after the administration of DDEP. These results suggest that DDC and DDEP react with different forms of rat hepatic microsomal cytochrome P-450.

Toxic Effects of Metals on the Synthesis and Disposition of Heme

ABSTRACT The heme biosynthetic pathway is perturbed by many chemicals and metals, and heme and hemoprotein biosynthesis may be disrupted as a result of exposure to these agents. Cobalt treatment of rats leads to marked decreases in the rate of heme biosynthesis and in the level of hemoproteins in hepatic microsomes. Cardiac heme biosynthesis in isolated perfused rat hearts is also decreased as a result of cobalt-induced decreases of δ-aminolevulinic acid synthetase activity. Lead acetate does not inhibit either heme biosynthesis or δ-aminolevulinic acid synthetase activity in isolated perfused rat hearts. Fasting of rats, which may lead to increases in hepatic δ-aminolevulinic acid synthetase activity, leads to a rapid and extensive decrease in cardiac δ-aminolevulinic acid synthetase activity and a marked decrease in the rate of cardiac heme biosynthesis. Friend erythroleukemic cells which can be induced by dimethylsulfoxide to synthesize hemoglobin show an increase in δ-aminolevulinic acid synthetase activity in response to cobalt exposure, and mutant forms of these cells which cannot be induced to synthesize hemoglobin because of a deficiency in ferrochelatase activity display an increase in δ-aminolevulinic acid synthetase activity when exposed to cobalt. Therefore, cobalt treatment may lead to different effects on the heme biosynthetic pathway in different tissues. This response may be dependent on the physiologic function of the organ and the degree of control imposed by the end product, heme, on the rate-limiting step of the pathway, δ-aminolevulinic acid synthetase.

Measurements of UDP‐ Glucuronosyltransferase (UGT) Activities

Mammalian UDP‐glucuronosyltransferases are a family of isoenzymes that catalyze the reaction of endobiotics and xenobiotics with glucuronic acid resulting in the formation of hydrophilic glucuronides. This pathway is an important step in the metabolism and subsequent excretion of many compounds that would otherwise have toxic effects. This unit describes three methods for measuring UGT activity. Thin layer chromatography is a powerful screening method and may be used to analyze multiple substrates simultaneously. The Sep‐Pak C18 cartridge extraction method has been developed to specifically separate opioid glucuronides from UDP‐glucuronic acid. Finally, the ethyl acetate extraction method is used to separate the glucuronides of bilirubin, sterols, and vile acids from UDP‐glucuronic acid. These methods may be applied to a microsomal fraction or to cultured cells transformed with cDNA for UGT.