Thomas R. Tephly

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.

Isolation and characterization of UGT2B15 (Y85) : a UDP-glucuronosyltransferase encoded by a polymorphic gene

Genetic polymorphisms occur in many of the drug metabolizing enzymes. However, the effect of polymorphisms in the genes encoding phase II drug metabolizing UDP-glucuronosyltransferases is still undescribed, despite the many reported cases of variations in glucuronidation activities. Characterization of the UGT2B15(Y85) cDNA, which was isolated from human prostate and LNCaP cell cDNA libraries, revealed 20 nucleotide differences between UGT2B15(Y85) and the previously characterized UGT2B15 protein UGT2B15(D85). However, only one of the two variations in the coding region leads to an amino acid change from aspartic acid to a tyrosine residue at position 85. The genomic DNA of 27 subjects were analysed by direct sequencing of polymerase chain reaction (PCR) products and demonstrated that UGT2B15(D85) and UGT2B15(Y85) are encoded by variant alleles prevalent in the Caucasian population. Expression of UGT2B15(D85) and UGT2B15(Y85) in HK293 cells demonstrated similar substrate specificities. Of the 65 potential substrates tested for activity, the proteins were active on phenolic compounds, coumarins, flavonoids, drugs and steroid hormones. Both proteins displayed similar Km values of 2.2 and 2.4 microM for androstane-3alpha,17beta-diol and dihydrotestosterone, respectively. However, results suggest that UGT2B15(Y85) has a higher Vmax than UGT2B15(D85). Specific reverse transcriptase (RT)-PCR analysis revealed expression of the UGT2B15 gene in a wide range of extrahepatic tissues including the human liver, kidney, testis, mammary gland, placenta, adipose, skin, uterus, prostate and lung. The wide expression of UGT2B15 in many tissues indicates that it is a major glucuronidation enzyme in humans.

Methanol poisoning in human subjects: Role for formic acid accumulation in the metabolic acidosis

Whereas a great deal of information is available on the etiology of methanol poisoning in the monkey, very little study has been made in human subjects. The role of formic acid in methanol toxicity in human subjects has not been established. Two patients have been studied who have presented with the characteristics of methanol poisoning--metabolic acidosis and ocular toxicity. This has made possible a confirmation of the role of formate in the toxic syndrome. Acidosis was very severe in both cases with arterial pH values of about 6.9 and plasma bicarbonate concentrations of 3 meq/liter. A sensitive and specific assay was used to measure formic acid levels in blood and other fluids. Formate accumulation was marked with initial blood levels ranging from 11.1 to 26.0 meq/liter. Decreases in blood bicarbonate concentrations of similar magnitude coincided with the increase in formate. Thus, accumulation of formic acid plays a major part in the acidosis observed in human subjects poisoned with methanol, as has been demonstrated in monkeys. Treatment involving bicarbonate administration, ethanol infusion and hemodialysis, rapidly decreased formate levels in the blood to control values. Methanol concentrations were reduced but to lesser extent than that of formate. Despite the reduction in formate and methanol concentrations in both cases, the treatment was successful in only one of the two patients.

The toxicity of methanol

Methanol toxicity in humans and monkeys is characterized by a latent period of many hours followed by a metabolic acidosis and ocular toxicity. This is not observed in most lower animals. The metabolic acidosis and blindness is apparently due to formic acid accumulation in humans and monkeys, a feature not seen in lower animals. The accumulation of formate is due to a deficiency in formate metabolism which is, in turn, related, in part, to low hepatic tetrahydrofolate (H4 folate). An excellent correlation between hepatic H4 folate and formate oxidation rates has been shown within and across species. Thus, humans and monkeys possess low hepatic H4 folate levels, low rates of formate oxidation and accumulation of formate after methanol. Formate, itself, produces blindness in monkeys in the absence of metabolic acidosis. In addition to low hepatic H4 folate concentrations, monkeys and humans also have low hepatic 10-formyl H4 folate dehydrogenase levels, the enzyme which is the ultimate catalyst for conversion of formate to carbon dioxide. This review presents the basis for the role of folic acid-dependent reactions in the regulation of methanol toxicity.

Methanol poisoning: ocular toxicity produced by formate.

Abstract After methanol administration, monkeys developed optic edema, acidosis, and formate accumulation in blood. Formaldehyde was not measurable in any fluids or tissue. Experiments were designed to study the possible role of formate in ocular toxicity. Formate was administered by iv infusion in the form of formate buffer for the purpose of maintaining pH in the normal range. The rate of formate infusion was adjusted to maintain blood formate at toxic concentration. No formaldehyde was detected. Results were similar to those described for methanol poisoning: optic disc edema with a normal vascular bed and intracellular edema with intraaxonal swelling.

Methanol poisoning I. The role of formic acid in the development of metabolic acidosis in the monkey and the reversal by 4-methylpyrazole

Abstract The administration of methanol (3 g/kg) to rhesus and pigtail monkeys produced signs and symptoms similar to those described for methanol poisoning in man. These were a mild central nervous system depression, a latent period of 8–12 hr when no signs were observed, followed by a severe metabolic acidosis leading to coma and death 12–33 hr after the initial administration. The gradual development of metabolic acidosis coincided with the accumulation of formic acid in the blood, and the decrease of bicarbonate in the plasma. There was an increase in the anion gap during the period of metabolic acidosis, and formic acid concentration accounted for about one-half of the increase observed. Therefore, formic acid was a major, but not the only, determinant of the metabolic acidosis. Administration of 4-methylpyrazole (50 mg/kg), a potent inhibitor of monkey hepatic alcohol dehydrogenase, produced a 75% inhibition of the rate of [ 14 C]methanol metabolism to 14 CO 2 in the monkey. During the first 36 hr following the administration of 4-methylpyrazole and methanol, no metabolic acidosis developed, no formate accumulated in the blood, and no signs of toxicity were observed. After a single dose of 4-methylpyrazole and methanol, the toxic syndrome was delayed by about 36 hr in the monkey, after which time the onset of metabolic acidosis and the accumulation of formic in blood was noted. The use of the monkey as a model for the study of methanol poisoning is presented, and the possible use of 4-methylpyrazole in the treatment of methanol poisoning is implicit.

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.

Separation, purification and characterization of three isoenzymes of UDP-glucuronyltransferase from rat liver microsomes.

Three isoenzymes of UDP-glucuronyltransferase (UDPGT) have been separated and purified from liver microsomes of untreated female rats or female rats pretreated with 3-methylcholanthrene. The UDPGT isoenzymes were purified utilizing Chromatofocusing, column isoelectric focusing, and UDP-hexanolamine Sepharose 4B affinity chromatography. UDPGT activities could also be separated during UDP-hexanolamine affinity chromatography by elution with different UDPGA (UDP-glucuronic acid) concentrations. One isoenzyme exhibits a subunit molecular weight of 56,000 and is capable of conjugating p-nitrophenol, 1-naphthol, and 4-methylumbelliferone. This isoenzyme is inducible by 3-methylcholanthrene treatment and requires high UDPGA concentrations for elution from the UDP-hexanolamine affinity column in contrast to the other UDPGT isoenzymes. A second isoenzyme was purified and displayed a subunit molecular weight of 50,000. This isoenzyme was not induced by 3-methylcholanthrene and was active towards testosterone, the 17-OH position of beta-estradiol, p-nitrophenol, and 1-naphthol. A third isoenzyme was also purified and exhibited a subunit molecular weight of 52,000. This isoenzyme conjugated androsterone and etiocholanolone and was not induced by 3-methylcholanthrene treatment. This study reports the purification of two separate and distinct rat liver UDPGT isoenzymes capable of conjugating p-nitrophenol, only one of which is inducible by 3-methylcholanthrene treatment. Also, this is the first report of the purification of a UDPGT isoenzyme active towards the 3-OH position of androgens.

Lack of a role for formaldehyde in methanol poisoning in the monkey

Abstract Methanol was administered either to untreated cynomolgus monkeys or to a folate-deficient cynomolgus monkey which exhibits exceptional sensitivity to the toxic effects of methanol. Marked formic acid accumulation in the blood and in body fluids and tissues was observed. No formaldehyde accumulation was observed in the blood and no formaldehyde was detected in the urine, cerebrospinal fluid, vitreous humor, liver, kidney, optic nerve, and brain in these monkeys at a time when marked metabolic acidosis and other characteristics of methanol poisoning were observed. Following intravenous infusion into the monkey, formaldehyde was rapidly eliminated from the blood with a half-life of about 1.5 min and formic acid levels promptly increased in the blood. Since formic acid accumulation accounted for the metabolic acidosis and since ocular toxicity essentially identical to that produced in methanol poisoning has been described after formate treatment, the predominant role of formic acid as the major metabolic agent for methanol toxicity is certified. Also, results suggest that formaldehyde is not a major factor in the toxic syndrome produced by methanol in the monkey.

Methanol poisoning. V. Role of formate metabolism in the monkey.

The accumulation of formic acid is an important factor in the production of the metabolic acidosis after methanol administration to the monkey. Formic acid accumulation following methanol administration does not occur in the rat, which oxidizes formate at rates that are markedly higher than those seen in the untreated monkey. The objectives of this study were to determine the pathway responsible for formate oxidation in the monkey and to compare the disposition of formate in the monkey with that of the rat. This information was used to increase and decrease the rate of accumulation of formate in the monkey and to increase or decrease the sensitivity of the monkey to methanol poisoning. Results show that a folate-de-pendent pathway is the major route of formate metabolism in the monkey as has been shown previously in the rat. Folate administration to the monkey increases formate oxidation. 4-Methylpyrazole treatment, after the production of metabolic acidosis and formic acidemia in the monkey, reverses the acidotic state and formic acidemia. Although the folate-deficient monkey is the most sensitive animal towards methanol poisoning, formaldehyde did not appear in the blood, body fluids, or tissues. Since formaldehyde does not accumulate in the presence of high methanol and high formate, and since reversal of formate accumulation leads to reversal of the features of the methanol poisoning syndrome, it is suggested that formate rather than formaldehyde is the major determinant of methanol poisoning in monkeys and probably in man.