Krongtong Yoovathaworn

Influence of mutations associated with Gilbert and Crigler-Najjar type II syndromes on the glucuronidation kinetics of bilirubin and other UDP-glucuronosyltransferase 1A substrates.

Objectives UGT1A1 coding region mutations, including UGT1A1*6 (G71R), UGT1A1*7 (Y486D), UGT1A1*27 (P229Q) and UGT1A1*62 (F83L), have been linked to Gilbert syndrome in Asian populations, whereas homozygosity for UGT1A1*7 is associated with the Crigler–Najjar syndrome type II. This work compared the effects of (a) the individual UGT1A1 mutations on the glucuronidation kinetics bilirubin, β-estradiol, 4-methylumbelliferone (4MU) and 1-naphthol (1NP), and (b) the Y486 mutation, which occurs in the conserved carboxyl terminal domain of UGT1A enzymes, on 4MU, 1NP and naproxen glucuronidation by UGT1A3, UGT1A6 and UGT1A10. Methods Mutant UGT1A cDNAs were generated by site-directed mutagenesis and the encoded proteins were expressed in HEK293 cells. The glucuronidation kinetics of each substrate with each enzyme were characterized using specific high-performance liquid chromatography (HPLC) methods. Results Compared with wild-type UGT1A1, in-vitro clearances for bilirubin, β-estradiol, 4MU and 1NP glucuronidation by UGT1A1*6 and UGT1A1*27 were reduced by 34–74%, most commonly as a result of a reduction in Vmax. However, the magnitude of the decrease in the in-vitro clearances varied from substrate to substrate with each mutant. The glucuronidation activities of UGT1A1*7 and UGT1A1*62 were reduced by >95%. Introduction of the Y486D mutation essentially abolished UGT1A6 and UGT1A10 activities, and resulted in 60–90% reductions in UGT1A3 in-vitro clearances. Conclusions The glucuronidation of all UGT1A1 substrates is likely to be impaired in subjects carrying the UGT1A1*6 and UGT1A1*62 alleles, although the reduction in metabolic clearance might vary with the substrate. The Y486D mutation appears to greatly reduce most, but not all, UGT1A activities.

Anti-inflammatory activity of (E)-1-(3,4-dimethoxyphenyl) butadiene from Zingiber cassumunar Roxb.

This study aimed to investigate the anti-inflammatory activity of (E)-1-(3,4-dimethoxyphenyl) butadiene (DMPBD), isolated from Zingiber cassumunar Roxb., using in vivo and in vitro models. The results show that DMPBD dose-dependently inhibited the rat ear edema induced by ethyl phenylpropiolate (EPP), arachidonic acid (AA) and 12-O-tetradecanoylphorbol 13-acetate (TPA) and it was more potent than any other standard drugs being used. In EPP-induced edema IC(50) of DMPBD and oxyphenbutazone were 21 and 136nmol per ear, respectively. The IC(50) of DMPBD and phenidone were 60 and 2520nmol per ear, respectively, in AA-induced edema whereas DMPBD was 11 times more potent than diclofenac in TPA-induced edema (IC(50)=660 and 7200pmol per ear, respectively). DMPBD and diclofenac inhibited the rat paw edema induced by carrageenan but not by platelet activating factor (PAF). In in vitro study DMPBD, aspirin and phenidone inhibited collagen-induced platelet aggregation with IC(50) of 0.35, 0.43 and 0.03mM, respectively. Whereas IC(50) of these agents in ADP, AA and PAF inductions were 4.85, 3.98 and 1.30mM; 0.94, 0.13 and 0.04mM; and 1.14, 6.96 and 2.40mM, respectively. These results indicate that DMPBD possesses a potent anti-inflammatory activity through the inhibition of CO and LO pathways and seems to have more prominent effects on the LO pathway.

Characterization of paracetamol UDP-glucuronosyltransferase activity in human liver microsomes

A specific high performance liquid chromatographic assay has been developed for the measurement of paracetamol glucuronide formation by the microsomal fraction of human liver. The procedure has been used to characterize paracetamol glucuronidation kinetics in human livers microsomes and to assess the substrate specificity of the paracetamol UDP-glucuronosyltransferase (UDPGT) activity. Paracetamol glucuronidation followed Michaelis-Menten kinetics, suggesting the involvement of a single form of UDPGT, or possibly two or more forms of UDPGT with similar affinities for paracetamol, in this reaction. Mean apparent Km and Vmax values were 7.37 +/- 0.99 mM and 4.76 +/- 1.35 nmol/min/mg, respectively. Addition of the non-ionic detergent Brij 58 to microsomal incubations resulted in approximately 50% activation of microsomal paracetamol UDPGT-activity. This contrasts to the approximately three-fold activation of 4-methylumbelliferone, morphine and 4-nitrophenol glucuronidation observed following Brij 58 treatment of human liver microsomes. The glucuronidated xenobiotics chloramphenicol, digitoxigenin monodigitoxoside, 4-hydroxybiphenyl, 4-methylumbelliferone, morphine, 1-naphthol and 4-nitrophenol were screened for inhibitory effects on paracetamol glucuronidation. Of these compounds, only digitoxigenin monodigitoxoside and 1-naphthol were found to cause significant inhibition of paracetamol UDPGT activity. Along with the results of previous studies of the kinetics and inhibitor profile of human liver glucuronidation reactions (Miners et al., Biochem Pharmacol 37: 665-671, 1988 and 37: 2839-2845, 1988), these data indicate that the model glucuronidated substrates paracetamol, morphine and 4-methyllumbelliferone may be used to differentiate at least four human liver UDPGT isozyme activities.

Optimizing bacterial expression of catalytically active human cytochromes P450: comparison of CYP2C8 and CYP2C9

Methods for the co-expression in Escherichia coli of human cytochrome P450 (CYP) 2C8 and CYP2C9 with NADPH-cytochrome P450 reductase (OxR) to produce a catalytically active system were compared. Approaches assessed were expression of a CYP:OxR fusion construct, bicistronic plasmids, simultaneous transformation with CYP and OxR plasmids, and separate expression of CYP and OxR with reconstitution of activity by mixing the bacterial membranes. Two N-terminal modifications (A3-20 and 17a-leader) of the individual P450s were additionally investigated. Each approach gave efficient expression of CYP2C8 and CYP2C9, but the bicistronic constructs under the expression conditions used gave low OxR expression and low catalytic activity. CYP expression was higher with the A3-20 construct for CYP2C9 and with the 17a-presequence construct for CYP2C8. Using torsemide as substrate, all methods gave catalytically active systems with Km values similar to human liver microsomes. Mixing bacterial membranes containing separately expressed CYP and OxR reconstituted a catalytically active system with the A3-20 construct for CYP2C9 but not for CYP2C8, and with neither of the 17a- presequence constructs. OxR co-expressed with CYP in the same membrane interacted with CYP to reconstitute activity more effectively than addition of exogenous OxR membranes. Expression construct and OxR co-expression strategy should be individualized for CYP isoforms.

The influence of metabolic gene polymorphisms on urinary 1-hydroxypyrene concentration in Thai bus drivers.

Polycyclic aromatic hydrocarbons (PAHs) are associated with an increased cancer risk. CYP1A1 and GSTs enzymes are important in metabolism of PAHs. Genetic polymorphisms of these enzymes are responsible for enzyme activity and concentration variation. The objectives of this study were to evaluate association of 1-OHP concentration with genetic polymorphisms of CYP1A1 and GSTs in Thai bus drivers. The results showed that 1-OHP levels in bus drivers were significantly higher than that in the control group. Significant difference in 1-OHP was found between smokers and non-smokers, in only bus drivers. Significantly increasing of 1-OHP levels were observed in bus drivers with CYP1A1 MspI and exon 7 variants. Whereas, bus drivers with GSTP1 Val and GSTM1 null genotypes showed decreasing in excretion of 1-OHP. No association between 1-OHP and polymorphisms of GSTT1 was found. This study indicated that 1-OHP concentrations were associated with exposure to air pollution, cigarette smoking and polymorphisms of CYP1A1, GSTM1 and GSTP1 genes.

Influence of caffeine on aspirin pharmacokinetics

SummaryThe effects of caffeine on the pharmacokinetics and bioavailability of aspirin were studied in 12 healthy adult male volunteers. The subjects received 650 mg of aspirin or 650 mg of aspirin with 120 mg of caffeine citrate orally. It was found that caffeine significantly increased the rate of appearance as well as the maximum concentration of salicylate in the plasma by about 25% and 17%, respectively. Moreover, area under the plasma concentration-time curve of salicylate was significantly higher in the subjects given the drug combination as compared to those given aspirin alone. There was no change in the plasma half-life, volume of distribution and clearance of salicylate.

Effects of amino acid substitutions at positions 33 and 37 on UDP-glucuronosyltransferase 1A9 (UGT1A9) activity and substrate selectivity.

UGT1A9 contributes to the glucuronidation of numerous drugs and xenobiotics. There is evidence to suggest that the Met33Thr substitution, as occurs in the polymorphic variant UGT1A9*3, variably affects xenobiotic glucuronidation. The equivalent position in UGT1A4 is also known to influence enzyme activity, whilst an N-terminal domain histidine (His37 in UGT1A9) is believed to function as the catalytic base in most UGT enzymes. To elucidate the roles of key amino acids and characterise structure-function relationships, we determined the effects of amino acid substitutions at positions 33 and 37 of UGT1A9 on the kinetics of 4-methylumbelliferone (4-MU), mycophenolic acid (MPA), propofol (PRO), sulfinpyrazone (SFZ), frusemide (FSM), (S)-naproxen (NAP) and retigabine (RTB) glucuronidation, compounds that undergo glucuronidation at either a phenolic (4-MU, MPA, PRO), carboxylate (FSM, NAP), acidic carbon (SFZ) or amine (RTB) function. Substitution of Met33 with Val, Ile, Thr, and Gln, as occur in UGT1A1, UGT1A3, UGT1A4 and UGT1A6 respectively, variably affected kinetics and catalytic efficiency. Whilst K(m) values were generally higher and V(max) and CL(int) values were generally lower than for wild-type UGT1A9 with most substrate-mutant pairs, the pattern and the magnitude of the changes in each parameter differed substantially. Moreover, exceptions occurred; CL(int) values for MPA and FSM glucuronidation by the position-33 mutants were the same as or higher than that of UGT1A9. Mutation of His37 abolished activity towards all substrates, except RTB N-glucuronidation. The data confirm the importance of single amino acids for UGT enzyme activity and substrate selectivity, and support a pivotal role for residue-33 in facilitating substrate binding to UGT1A9.

Cytotoxic and inflammatory responses of TiO2 nanoparticles on human peripheral blood mononuclear cells

Titanium dioxide nanoparticles (TiO2‐NPs) have been widely used in many applications. Owing to their nanoscale size, interactions between cells and NPs have been expansively investigated. With the health concerns raised regarding the adverse effects of these interactions, closer examination of whether TiO2‐NPs can induce toxicity towards human cells is greatly needed. Therefore, in this study, we investigated the cytotoxicity of TiO2‐NPs towards human blood cells (peripheral blood mononuclear cells [PBMCs]) in serum‐free medium, for which there is little information regarding the cytotoxic effects of TiO2‐NPs. Our results provide evidence that PBMCs treated with TiO2‐NPs (at concentrations ≥25 μg ml−1) for 24 h significantly reduced cell viability and significantly increased production of toxic mediators such as reactive oxygen species and inflammatory response cytokines such as interleukin‐6 and tumor necrosis factor‐α (P < 0.05). Cell apoptosis induction also occurred at these concentrations. Significant expressions of cyclooxygenase‐2 and interleukin‐1β were also observed in PBMCs treated with TiO2‐NPs at concentrations ≥125 μg ml−1. Our data presented here clearly indicate that the concentration of TiO2‐NPs (at size ~26.4 ± 1.2 nm) applied to human blood cells has a strong impact on cytotoxic induction. Copyright

Environmental Lead Exposure, Catalase Gene, and Markers of Antioxidant and Oxidative Stress Relation to Hypertension: An Analysis Based on the EGAT Study

Lead has been linked to the development of hypertension via oxidative stress. Catalase plays an important role in the disposal of hydrogen peroxide in erythrocyte and its activity was determined by CAT gene. The aims of this study were to investigate (1) the association between blood levels of antioxidant markers such as catalase, superoxide dismutase, glutathione, glutathione peroxidase, oxidative stress-marker (malondialdehyde), and blood lead level and (2) the influence of genetic polymorphism of CAT gene (rs769217) on change in blood pressure in general population of EGAT study project. This is a cross-sectional study of 332 normotensive, 432 prehypertensive, and 222 hypertensive male subjects. Hypertensive subjects had significantly higher blood lead level (5.28 μg/dL) compared to normotensive (4.41 μg/dL) and prehypertensive (4.55 μg/dL) subjects (P < 0.05). These significant findings are also found in MDA levels. Moreover, individuals with TT genotype in hypertensive group had significantly higher blood lead and MDA levels (6.06 μg/dL and 9.67 μmol/L) than those with CC genotype (5.32 μg/dL and 8.31 μmol/L, P < 0.05). Our findings suggested that decreased blood catalase activity in this polymorphism together with low level lead exposure induced lipid peroxidation may be responsible for hypertension.

Biphasic effect of methadone on hepatic drug metabolism.

When methadone HC1 (30 mg/kg, po) was given acutely to mice, it was found to inhibit drug metabolism as evidenced by a prolongation of hexobarbital sleeping time and zoxazolamine paralysis time. Pharmacokinetic studies revealed that this acute dose of the narcotic analgesic could also prolong the plasma half-life of aminopyrine without any change in its volume of distribution. When added to the incubation mixture containing 10,000 g mouse liver supernatant fraction and a complete system for measuring aminopyrine .N-demethylase or aniline hydroxylase, metadone showed a dose-dependent inhibition of the enzymes; the former enzyme was inhibited to a greater extent than the latter one. However, subacute treatment of mice with methadone HC1 (30 mg/kg, po, twice daily for 3 days) resulted in increases in liver weight, microsomal protein, and cytochrome P-450 content in consonant with the increased activities of four hepatic drug-metabolizing enzymes: aminopyrine N-demethylase, aniline hydroxylase, p-nitroanisole, O-demethylase, and benzphetamine N-demethylase. Moreover, both hexobarbital sleeping time and zoxazolamine paralysis time were shortened. The plasma half-life of aminopyrine was decreased. These changes were prevented by simultaneous administration of puromycin diHCl (80 mg/kg, ip). Methadone thus seems to act in a manner very similar to that of propoxyphene or SKF-525A, acting as a potent inhibitor of hepatic drug metabolism when given acutely and as an inducer when given subacutely.