Glucuronidation, oxidative metabolism, and bioactivation of enterolactone in rhesus monkeys

Enterolactone (ENL) and enterodiol (END) are mammalian lignans derived from the plant lignans matairesionol (MAT), secoisolariciresinol (SECO), and other dietary precursors. ENL was found to undergo extensive glucuronidation with rhesus liver microsomes to form O-glucuronides at both phenolic hydroxy groups. In addition to glucuronidation, ENL was found to be a good substrate for oxidative metabolism. The major products had a m/z of 313 or 295 by LC-MS analysis in negative ion mode and were determined to be products of monohydroxylation of ENL. The m/z 295 products were the result of a dehydration of the m/z 313 in the MS source. Addition of N-acetylcysteine (NAC) to the NADPH incubations resulted in a decrease of at least 2 major monohydroxylated products and the formation of a major and several minor new products with a m/z of 474. The major adduct was isolated, purified for NMR, and confirmed to be the NAC adduct of the ENL catechol. Incubations of ENL with liver microsomes containing UDPGA, NADPH, and NAC resulted in the formation of ENL-glucuronides; no NAC adducts were detected by LC-MS. Incubations of ENL with human and rhesus hepatocytes resulted in several metabolites. The major metabolites in hepatocytes were the glucuronic acid conjugates; minor amounts of the sulfate conjugate(s) and monohydroxylated products were also detected by LC-MS. Glutathione or other thiol adducts were not detected in hepatocytes. Conclusion. The high efficiency and specificity for the glucuronidation of ENL decrease its potential toxicity via CYP450 bioactivation.     

Glucuronidation of haloperidol by rat liver microsomes: involvement of family 2 UDP-glucuronosyltransferases

The purposes of this study were to develop a HPLC method to assay for haloperidol glucuronide (HALG); to apply this assay method to the in vitro determination of haloperidol (HAL) UDP-glucuronosyltransferase (UGT) enzyme kinetics in rat liver microsomes (RLM); and to identify the UGT isoforms catalyzing glucuronidation of HAL in rats. Incubation of Brij-activated RLM with HAL and UDP-glucuronic acid (UDPGA) in TRIS pH 7.4 buffer resulted in the formation of a single peak in the HPLC chromatogram at 270 nm. The identity of this peak was confirmed to be that of HALG by 1) β-glucuronidase hydrolysis; 2) incubation without UDPGA; 3) UV spectral analysis; and 4) LC/MS/MS to yield the expected mass of 552.1. Enzyme kinetic studies using single enzyme Michaelis-Menton model showed an apparent V_max = 271.9 ± 10.1 pmoles min⁻¹ mg protein⁻¹ and K_m = 61 ± 7.2 μM. Glucuronidation activity in homozygous Gunn (j/j) rats was approximately 80% as compared to Sprague-Dawley RLM. HALG formation was approximately doubled in PB-induced RLM. There was no increase in glucuronidation activities in 3MC-induced RLM. The Gunn rat and the PB-induced RLM data suggest predominant but not exclusive involvement of the UGT2B family in the formation of HALG. Because the UGTs exhibit overlapping substrate specificities and most substrates are glucuronidated by more than one isoform, inhibition studies with UGT2B1 substrate probe testosterone and the UGT2B12 substrate probe borneol were conducted. UGT2B1 and UGT2B12 exhibited 40% and 90% inhibition of HAL glucuronidation, respectively. Thus, UGT2B12 and UGT 2B1 isoforms are responsible for catalyzing HAL glucuronidation in rats. Our HPLC assay provides a specific and sensitive technique for the measurement of in vitro HAL-UGT activity.     

Association of SULT1A1 and UGT1A1 polymorphisms with breast cancer risk and phenotypes in Russian women

Estrogens are critical for breast cancer initiation and development. Sulfotransferase 1A1 (SULT1A1) and UDP-glucuronosyltransferase 1A1 (UGT1A1) conjugate and inactivate both estrogens and their metabolites, thus preventing estrogen-mediated mitosis and mutagenesis. SULT1A1 and UGT1A1 are both polymorphic, and different alleles encode functionally different allozymes. We hypothesize that low-activity alleles SULT1A1*2 and UGT1A1*28 are associated with higher risk for breast cancer and more severe breast tumor phenotypes. We performed a case-control study, which included 119 women of Russian ancestry with breast cancer and 121 age-matched Russian female controls. We used PCR followed by pyrosequencing to determine the SULT1A1 and UGT1A1 genotypes. Allele UGT1A1*28 was present at a higher frequency than the wild-type UGT1A1*1 allele in breast cancer patients as compared to controls (P = 0.002, OR = 1.79, CI 1.23–2.63). Consistently, the frequency of genotypes that contain allele UGT1A1*28 in the homozygous or the heterozygous state was greater in breast cancer patients as compared with the frequency of the wild-type UGT1A1*1/*1 genotype (P = 0.003, OR = 4.00, CI 1.49–11.11 and P = 0.014, OR = 2.04, CI 1.14–3.57, respectively). Individuals carrying allele UGT1A1*28 in the homo-or heterozygous state had larger breast tumors (>2 cm) as compared to the group with high-activity genotypes (P = 0.011, IR = 3.44, CI 1.42–8.36). No association was observed between any of the SULT1A1 genotypes and breast cancer risk or phenotypes. Our data suggest that UGT1A1, but not SULT1A1, genotypes are important for breast cancer risk and phenotype in Russian women. Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 2, pp. 263–270. The article was translated by the authors.     

Endoplasmic reticulum: a metabolic compartment

Several biochemical reactions and processes of cell biology are compartmentalized in the endoplasmic reticulum (ER). The view that the ER membrane is basically a scaffold for ER proteins, which is permeable to small molecules, is inconsistent with recent findings. The luminal micro-environment is characteristically different from the cytosol; its protein and glutathione thiols are remarkably more oxidized, and it contains a separate pyridine nucleotide pool. The substrate specificity and activity of certain luminal enzymes are dependent on selective transport of possible substrates and co-factors from the cytosol. Abundant biochemical, pharmacological, clinical and genetic data indicate that the barrier function of the lipid bilayer and specific transport activities in the membrane make the ER a separate metabolic compartment.     

Induction of human UGT1A1 by a complex of dexamethasone-GR dependent on proximal site and independent of PBREM

UDP-glucuronosyltransferase 1A1 (UGT1A1) plays a key role to conjugate bilirubin and prevent jaundice. There are two major elements for the induction of UGT1A1, such as PBREM (-3483/-3194), far from the promoter site, and HNF1 (-75/-63), near to the promoter site. In a previous report, we showed that the proximal HNF1 site is essential for the induction of UGT1A1 by glucocorticoid receptor (GR). In this report, we investigated the influence of PBREM on the induction of the UGT1A1 reporter gene by GR and PXR with dexamethasone (DEX). We confirmed that GR was transferred from cytosol into the nucleus in 15-30 min by DEX stimulation, but HNF1 was not. We constructed a reporter gene containing PBREM to compare the induction of the reporter gene without PBREM by DEX-GR. The results show that induction of the reporter gene with PBREM by DEX at 100 muM is the same level as the induction of the reporter gene without PBREM, although PBREM contains GRE. Co-transfection of hGR with the reporter gene did not show any influence of the induction of the reporter gene between the vector with and without PBREM. Meanwhile, by co-transfection of hPXR, the induction of the reporter gene with PBREM was significantly more than the induction of the reporter gene without PBREM at 100 muM DEX. This supports that hPXR induced UGT1A1 through PBREM by DEX. These results showed that PBREM has no relation with the induction by DEX-GR but the proximal site of UGT1A1 may function in stimulation by DEX-GR.     

UDP-glucuronosyltransferases 1A6 and 1A10 catalyze reduced menadione glucuronidation

Menadione (2-methyl-1,4-naphthoquine), also known as vitamin K3, has been widely used as a model compound in the field of oxidative stress-related research. The metabolism of menadione has been studied, and it is known that menadione undergoes a two-electron reduction by NAD(P)H:Quinone oxidoreductase 1 (NQO1) after which the reduced form of menadione (2-methyl-1,4-naphthalenediol, menadiol) is glucuronidated and excreted in urine. To investigate which human UDP-glucuronosyltransferase (UGT) isoforms participate in the glucuronidation of menadiol reduced by NQO1 from menadione, we first constructed heterologously expressed NQO1 in Sf9 cells and tested the menadiol glucuronidating activity of 16 human recombinant UGT isoforms. Of the 16 UGT isoforms, UGTs 1A6, 1A7, 1A8, 1A9, and 1A10 catalyzed menadiol glucuronidation, and, of these, UGTs 1A6 and 1A10 catalyzed menadiol glucuronidation at much higher rates than the other UGTs. Menadiol was regioselectively glucuronidated in the manner of 4-position > 1-position by UGTs 1A7, 1A8, 1A9, and 1A10. In contrast to these UGTs, only UGT1A6 exhibited 1-menadiol-preferential glucuronidating activity. The results suggest possible detoxification pathways for quinones via NQO1 reduction followed by UGT glucuronidation.     

Natural products as modulators of the nuclear receptors and metabolic sensors LXR,FXR and RXR

Nuclear receptors (NRs) represent attractive targets for the treatment of metabolic syndrome-related diseases. In addition, natural products are an interesting pool of potential ligands since they have been refined under evolutionary pressure to interact with proteins or other biological targets.This review aims to briefly summarize current basic knowledge regarding the liver X (LXR) and farnesoid X receptors (FXR) that form permissive heterodimers with retinoid X receptors (RXR). Natural product-based ligands for these receptors are summarized and the potential of LXR, FXR and RXR as targets in precision medicine is discussed.     

Biosynthesis of the leucine derived α‐, β‐ and γ‐hydroxynitrile glucosides in barley (Hordeum vulgare L.)

Barley (Hordeum vulgare L.) produces five leucine‐derived hydroxynitrile glucosides (HNGs), of which only epiheterodendrin is a cyanogenic glucoside. The four non‐cyanogenic HNGs are the β‐HNG epidermin and the γ‐HNGs osmaronin, dihydroosmaronin and sutherlandin. By analyzing 247 spring barley lines including landraces and old and modern cultivars, we demonstrated that the HNG level varies notably between lines whereas the overall ratio between the compounds is constant. Based on sequence similarity to the sorghum (Sorghum bicolor) genes involved in dhurrin biosynthesis, we identified a gene cluster on barley chromosome 1 putatively harboring genes that encode enzymes in HNG biosynthesis. Candidate genes were functionally characterized by transient expression in Nicotiana benthamiana. Five multifunctional P450s, including two CYP79 family enzymes and three CYP71 family enzymes, and a single UDP‐glucosyltransferase were found to catalyze the reactions required for biosynthesis of all five barley HNGs. Two of the CYP71 enzymes needed to be co‐expressed for the last hydroxylation step in sutherlandin synthesis to proceed. This observation, together with the constant ratio between the different HNGs, suggested that HNG synthesis in barley is organized within a single multi‐enzyme complex.     

Polymorphisms of the uridine-diphosphoglucuronosyltransferase 1A1 gene and coronary artery disease

Bilirubin, an antioxidant in the blood, plays a role in protection from atherosclerosis. The level of bilirubin is highly correlated to the incidence of coronary artery disease (CAD). Unconjugated bilirubin is conjugated with glucuronic acid through the reaction of uridine 5′-diphosphate-glucuronosyl transferase 1A1 (UGT1A1). The interactions of CAD and the variations in the coding regions of the UGT1A1 gene have never been evaluated. The purpose of this study was to analyze the influence of the UGT1A1 variant on the incidence of CAD. There were 135 participants in this study: 61 in the experimental group, who had CAD, and 74 in the control group, who did not have CAD. The blood samples from all 135 participants were collected and assayed to clarify the relationship between bilirubin and CAD. The assay of the polymerase chain reaction and the sequence of the UGT1A1 gene were examined to find the gene’s polymorphisms. The bilirubin levels for the participants in the control group were significantly higher than for the patients in the CAD group. Although the concentration of bilirubin in the UGT1A1 variant was higher than the wild type for the patients in the CAD group, there was no significant difference in the polymorphism of UGT1A1 between the patients in the CAD group and the participants in the control group.