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Biochemical Pharmacology (v.76, #3)

Editorial Board (pp. iii).

Ubiquinone and tocopherol: Dissimilar siblings by L. Gille; T. Rosenau; A.V. Kozlov; W. Gregor (pp. 289-302).

Research on antioxidants and their potential health benefits expanded over the last decades from basic science to the medical and nutritional fields. This included supplementation studies of both vitamin E compounds and the endogenous antioxidant ubiquinone, to prevent or alleviate cardiovascular diseases and their pathophysiological consequences. In many of these studies, only one antioxidant or one group of antioxidants was considered, disregarding the pharmacological and toxicological properties of their metabolites as well as possible cooperative and competitive effects on the overall physiological response. There are many – often indirect – effects, especially in gene regulation, observed on administration of both compound groups in cells, which have been assigned to these molecules without identifying the cellular targets. Therefore, this article focuses on direct chemical and biochemical effects of ubiquinone- and α-tocopherol-related compounds, which are evident from direct binding studies or direct interaction leading to chemical modification of the compounds. These groups include para-benzoquinones (ubiquinone and α-tocopheryl quinone) and chroma(e)nols (α-tocopherol and bicyclic ubiquinone derivatives). Their effects as antioxidants, co-antioxidants, and pro-oxidants as well as direct interactions with proteins are considered, pointing out similarities and dissimilarities of the two compound groups in a wider context. The review of the isolated findings about one or a few of these compounds in the literature, disregarding structurally related compounds, suggests that comprehensive structure/activity relationship studies including related compounds would promote the understanding of biological functions and pharmacological effects of ubiquinone- and α-tocopherol-related compounds.

Keywords: Abbreviations; α-CEHC; α-carboxyethyl hydroxychromane; α-CMBHC; carboxymethylbutyl hydroxychromane; cyt; cytochrome; CYP; cytochrome P450; DPPH; diphenyl picryl hydrazyl; dUQ; decyl ubiquinone; LDL; low-density lipoprotein; LPO; lipid peroxidation; LPS; lipopolysaccharide; PBN; phenyl-; tert; -butyl nitrone; Q; quinone; ROS; reactive oxygen species; SMP; submitochondrial particles; SOD; superoxide dismutase; SPF; supernatant protein factor; TAP; α-Toc-associated protein; α-Toc; α-tocopherol (most active component of vitamin E); α-TQ; α-tocopheryl quinone (α-tocopherol quinone, vitamin E quinone); α-TTP; α-Toc transfer protein; UCa; ubichromanol; UCe; ubichromenol; UQ; ubiquinone; UQ; ₁₀; OH; 3′-hydroxy ubiquinone (with 10 side chain isoprenyl units)Ubiquinone; α-Tocopherol; α-Tocopheryl quinone; Ubichromanol; Electron transfer

Different initial steps of apoptosis induced by two types of antineoplastic drugs by Yasumitsu Takagi; Masumi Hidaka; Masayuki Sanada; Hiroki Yoshida; Mutsuo Sekiguchi (pp. 303-311).

O⁶-Methylguanine and O⁶-chloroethylguanine are primary DNA lesions produced by two types of antineoplastic drugs, 8-carbamoyl-3-methylimidazo[5,1- d]-1,2,3,5-tetrazin-4(3H)-one (temozolomide, TMZ) and 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU), respectively. They can be repaired by O⁶-methylguanine-DNA methyltransferase, coded by the Mgmt gene. Otherwise, these two types of lesions induce apoptosis in different ways. O⁶-Chloroethylguanine blocks DNA replication thereby inducing apoptosis. On the other hand, O⁶-methylguanine does not block DNA replication and the resulting O⁶-methylguanine-thymine mispair is recognized by mismatch repair-related proteins, including MLH1, thereby inducing apoptosis. Reflecting this, mouse cells lacking both MGMT and MLH1 are resistant to TMZ, but not to ACNU. The translocation of phosphatidylserine in cell membrane as well as a change of mitochondrial transmembrane potentials occurred in an MLH1-dependent manner after treatment with TMZ, but no such MLH1 dependency was observed in the case of ACNU treatment. By using cell lines defective in both APAF-1 and MGMT, it was revealed that the APAF-1 function is required for execution of apoptosis induced by either TMZ or ACNU. There is almost 12h delay in occurrence of apoptosis-related mitochondrial depolarization in TMZ-treated cells in comparison to those of ACNU-treated cells, reflecting the fact that at least one cycle of DNA replication is required to trigger apoptosis in the former case, but not in the latter.

Keywords: Apoptosis; DNA repair; MGMT; Temozolomide; ACNU

Induction of G₂/M arrest, endoreduplication, and apoptosis by actin depolymerization agent pextenotoxin-2 in human leukemia cells, involving activation of ERK and JNK by Dong-Oh Moon; Mun-Ock Kim; Sang-Hyuck Kang; Kyeong-Jun Lee; Moon-Soo Heo; Kwang-Sik Choi; Yung Hyun Choi; Gi-Young Kim (pp. 312-321).

Pectenotoxin-2 (PTX-2) is a natural compound from marine sponges and has been known to inhibit cytokinesis through the depolymerization of actin filaments. To investigate the role of actin dysfunction by PTX-2 in human leukemia cells, we analyzed the effect of PTX-2 on the cell cycle and apoptosis. Cell cycle analysis showed that the depolymerization of actin with PTX-2 induces G₂/M phase arrest at 12h and endoreduplication at 24h. Analysis of the cell cycle regulatory proteins demonstrated that PTX-2 increases phosphorylation of cdc25c and decreases the protein levels of cdc2 and cyclin B1. The M phase specific marker protein, phospho-histone 3, was also increased by PTX-2. Furthermore, p21 and CDK2, which are associated with the induction of endoreduplication, were also upregulated. PTX-2 also inhibited the growth of leukemia cells and caused a marked increase in apoptosis, as characterized by annexin-V⁺ cells and caspase-3 activity. Interestingly, we found that induction of G₂/M phase arrest, endoreduplication, and apoptosis by PTX-2 is regulated by the extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) pathway. Inhibitors of ERK and JNK more increased the phosphorylation of cdc25c expression at G₂/M arrest stages, and decreased p21 and CDK2 expression at endoreduplication stages and Bax expression at apoptotic stages in the presence of PTX-2. These molecular mechanisms provide that PTX-2 induces G₂/M phase arrest, endoreduplication, and apoptosis through the ERK and JNK signal pathway via actin depolymerization.

Keywords: Abbreviations; PTX-2; pectenotoxin-2; ERK; extracellular signal-regulated kinase; JNK; c-jun N-terminal kinase; MAPK; mitogen-activated protein kinasePectenotoxin-2; G; ₂; /M phase arrest; Endoreduplication; Apoptosis; Actin depolymerization

Adenoviral-mediated overexpression of human equilibrative nucleoside transporter 1 (hENT1) enhances gemcitabine response in human pancreatic cancer by Sandra Pérez-Torras; José García-Manteiga; Elena Mercadé; F. Javier Casado; Neus Carbó; Marçal Pastor-Anglada; Adela Mazo (pp. 322-329).

Nucleoside-derived anticancer agents must be transported across the plasma membrane as a preliminary step to their conversion into active drugs. Hence, modulation of a specific nucleoside transporter may affect bioavailability and contribute significantly to sensitizing tumor cells to these anticancer agents. We have generated and functionally characterized a new recombinant adenovirus (Ad-hENT1) that has allowed us to overexpress the equilibrative nucleoside transporter hENT1 and to analyze its effects in human pancreatic tumor cells. Overexpression of hENT1 is associated with changes in cell cycle profile, in a variable manner depending on the particular cell type, thus suggesting a metabolic link between hENT1-mediated transport processes and the enzymatic machinery responsible for intracellular nucleoside metabolism. When assayed in vivo in a human pancreatic adenocarcinoma xenograft, intratumoral Ad-hENT1 injection improved the therapeutic response to gemcitabine. In summary, hENT1 overexpression is associated with alterations in nucleoside enzymatic machinery and cell cycle progression in cultured cells and enhances gemcitabine action in vivo.

Keywords: Abbreviations; NT; nucleoside transporters; GE; gemcitabine; NBMPR; nitrobenzylmercaptopurine ribonucleoside; NBTI; nitrobenzylthioinosine; AU; arbitrary unitshENT1; Adenovirus; Nucleoside analogues; Nucleoside transport; Pancreatic cancer

Tumor anti-angiogenic effect and mechanism of action of δ-tocotrienol by Akira Shibata; Kiyotaka Nakagawa; Phumon Sookwong; Tsuyoshi Tsuzuki; Shinichi Oikawa; Teruo Miyazawa (pp. 330-339).

Anti-angiogenic therapy mediated by drugs and food components is an established strategy for cancer prevention. Our previous cell-culture studies identified a food-derived anti-angiogenic compound, tocotrienol (T3, an unsaturated vitamin E), as a potential angiogenic inhibitor. Among T3 isomers, δ-T3 is considered as the most potent compound. The purpose of this study was therefore to evaluate the inhibitory effect of δ-T3 on tumor angiogenesis. As growth factors (e.g., vascular endothelial growth factor and fibroblast growth factor) play critical roles in tumor angiogenesis, a conditioned medium rich in these growth factors from human colorectal adenocarcinoma cells (DLD-1-CM) was used as an angiogenic stimulus. δ-T3 (2.5–5μM) significantly suppressed DLD-1-CM-induced tube formation, migration, and adhesion on human umbilical vein endothelial cells. These effects were partly associated with reactive oxygen species generation by δ-T3. Western blot analysis revealed that the anti-angiogenic effect of δ-T3 is attributable to regulation of growth factor-dependent phosphatidylinositol-3 kinase (PI3K)/phosphoinositide-dependent protein kinase (PDK)/Akt signaling as well as to induction stress response in endothelial cells. Moreover, we conducted an in vivo mouse Matrigel plug angiogenesis assay, and found that δ-T3 (10–20μg) exhibits dose-dependent inhibition of DLD-1-induced vessel formation. These results suggest that T3 has potential use as a therapeutic dietary supplement for minimizing tumor angiogenesis.

Keywords: Abbreviations; ASK-1; apoptosis signal-regulating kinase; CM; conditioned medium; DAB; 3,3′-diaminobenzidine; DCDHF; 2,7-dichlorodihydrofluorescein; DHF; 2,7-dichlorofluorescein; DLD-1; human colorectal adenocarcinoma cells; EGF; epidermal growth factor; eNOS; endothelial nitric oxide synthase; ERK 1/2; extracellular signal-regulated kinase 1/2; FBS; fetal bovine serum; FGF; fibroblast growth factor; FPP; farnesylpyrophosphate; GGPP; geranylgeranylpyrophosphate; GSK3 α/β; glycogen synthase kinase 3 α/β; H&E; hematoxylin and eosin; HMG-CoA; 3-hydroxy-3-methylglutaryl-coenzyme A; HUVEC; human umbilical vein endothelial cells; PDK; phosphoinositide-dependent protein kinase; PECAM-1; platelet endothelial cell adhesion molecule-1; PI3K; phosphatidylinositol-3 kinase; PIP2; phosphatidylinositol (4,5)-bisphosphate; PIP3; phosphatidylinositol (3,4,5)-trisphosphate; PTEN; phosphatase and tensin homologue deleted on chromosome 10; ROS; reactive oxygen species; Toc; tocopherol; TOS; tocopheryl succinate; T3; tocotrienol; VCAM-1; vascular cell adhesion molecular-1; VEGF; vascular endothelial growth factor; VEGFR-2; VEGF Receptor 2Anti-angiogenesis; Endothelial cells; Tocotrienol; Tumor; Vitamin E

Development of XPA067.06, a potent high affinity human anti-gastrin monoclonal antibody by Ssucheng J. Hsu; Amita Patel; Paul D. Larsen; David J. Bohmann; Robert J. Bauer; Jeremy K. Ma; Linda Masat; Marina Roell; Susan J. Babuka; Rhonda K. Hansen; Mark White; Mary Haak-Frendscho (pp. 340-352).

The peptide hormone gastrin is a key factor in regulation of gastric acid secretion. It has also been implicated in the development or maintenance of various types of cancer, such as pancreatic and stomach carcinoma. Inhibition of gastrin activity has potential for therapeutic use as a suppressor of acid secretion as well as an inhibitor of gastrin-responsive tumors. XPA067.06 is an affinity matured, 30pM fully human anti-gastrin monoclonal antibody that was generated. The antibody was tested in a mouse gastric pH model to determine its effect on acid secretion. In this model, animals were treated with human gastrin, XPA067.06, and H2R or M1 receptor antagonists. Gastric fluid was collected and acid output was measured as a function of pH. XPA067.06 was shown to significantly inhibit gastrin-17-stimulated acid output for at least 48h. These results demonstrate that XPA067.06 effectively binds and neutralizes human gastrin-17 in vivo with rapid onset and prolonged duration of efficacy.

Keywords: Gastrin; Monoclonal antibody therapy; Affinity maturation; Gastric acid secretion; Antagonist; Cancer

A COX-2 metabolite of the endogenous cannabinoid, 2-arachidonyl glycerol, mediates suppression of IL-2 secretion in activated Jurkat T cells by Cheryl E. Rockwell; Priyadarshini Raman; Barbara L.F. Kaplan; Norbert E. Kaminski (pp. 353-361).

Previous studies from this laboratory have demonstrated that a COX-2 metabolite of the endogenous cannabinoid, 2-arachidonyl glycerol (2-AG), inhibits IL-2 secretion in activated T cells through PPARγ activation independent of the cannabinoid receptors, CB1/CB2. Because numerous cyclooxygenase (COX) products have been shown to activate PPARγ, the primary purpose of the present studies was to determine the role of COX metabolism in the inhibition of IL-2 secretion by 2-AG. Pretreatment with nonselective and COX-2-specific inhibitors completely abrogated 2-AG-mediated suppression of IL-2 secretion. In contrast, pretreatment with COX-1-specific inhibitors had no effect upon 2-AG-mediated inhibition of IL-2 secretion. Interestingly, the current studies also demonstrate that while the potency of 2-AG is comparable between human Jurkat T cells and murine splenocytes, anandamide (AEA) is markedly more potent in suppressing IL-2 production in Jurkat T cells compared to murine splenocytes. Additionally, the present studies also demonstrate that COX-2 protein is readily detectable in resting Jurkat T cells, which is in contrast to resting murine splenocytes in which COX-2 protein is virtually undetectable. Furthermore, COX-2 protein and mRNA levels are significantly increased over basal levels by 2h following activation of Jurkat cells, whereas increases in COX-2 protein in murine splenocytes are not observed until 4h after cellular activation. These studies suggest that the potency of AEA in the suppression of IL-2 secretion may correlate with COX-2 protein levels in different T cell models. The present studies are also significant in that they demonstrate 2-AG-mediated inhibition of IL-2 secretion is dependent upon COX-2 metabolism.

Keywords: 2-Arachidonyl glycerol; 2-AG; 2-AG ether; IL-2; T cell; Cannabinoid; Endocannabinoid; Jurkat; COX; Cyclooxygenase; Anandamide

Neuroprotective role of tripchlorolide on inflammatory neurotoxicity induced by lipopolysaccharide-activated microglia by Xiao-dong Pan; Xiao-chun Chen; Yuan-gui Zhu; Jing Zhang; Tian-wen Huang; Li-min Chen; Qin-yong Ye; Hua-pin Huang (pp. 362-372).

A large body of evidence has suggested a strong association between neuroinflammation and the pathogenesis of many neurodegenerative diseases. Therefore, it is a good target for therapeutic treatment. So far, studies have proven anti-inflammatory herbal medicine and its constituents to be effective in slowing down the neurodegenerative process. The present study tested tripchlorolide, an extract of Tripterygium wilfordii Hook F (TWHF), as a novel agent to suppress inflammatory process in microglia. It showed this novel agent to be cytotoxic at a dose of 20–40nM to primary microglia and BV-2 microglial cells but not to primary cortical neurons and Neuro-2A cells in vitro. Moreover, tripchlorolide protected primary cortical neurons and Neuro-2A cells from neuroinflammatory toxicity induced by the conditioned media from lipopolysaccharide (LPS)-stimulated microglia, which resulted in a significant decrease in their cell survival. The changes of the inflammatory mediators in this process were further investigated. In the LPS-stimulated microglia, the production of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), nitric oxide (NO), prostaglandin E₂ (PGE₂), and intracellular superoxide anion (SOA) was markedly attenuated by tripchlorolide at a dose of 1.25–10nM in a dose-dependent manner. Furthermore, the production of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) was also significantly inhibited by tripchlorolide in both mRNA and protein levels. These results suggest that tripchlorolide can protect neuronal cells via a mechanism involving inhibition of inflammatory responses of microglia to pathological stimulations. Therefore, it is potentially a highly effective therapeutic agent in treating neuroninflammatory diseases.

Keywords: Tripchlorolide; Lipopolysaccharide; Neurons; Microglia; Neuroinflammation

Novel iminobenzoxathiolone compound inhibits nuclear factor-κB activation targeting inhibitory κB kinase β and down-regulating interleukin-1β expression in lipopolysaccharide-activated macrophages by Min Hee Kim; Hwa Young Lee; Eunmiri Roh; Byung Hak Kim; Eun Yong Chung; Yong Rok Lee; In Jeong Lee; Heesoon Lee; Chong-Kil Lee; Sang-Bae Han; Youngsoo Kim (pp. 373-381).

Benzoxathiolone derivatives have been reported to show pharmacological potentials in the psoriasis and acne. However, molecular basis for these pharmacological properties is little known. We postulated that the derivatives could mediate some of their pharmacological actions by modulating nuclear factor (NF)-κB activation, which is closely linked to the inflammatory and immune disorders. In this study, a novel iminobenzoxathiolone LYR-71 of 6-methyl-2-propylimino-6,7-dihydro-5 H-benzo[1,3]oxathiol-4-one has been demonstrated to inhibit in vitro catalytic activity of inhibitory κB (IκB) kinase β (IKKβ), a key enzyme required for NF-κB activation, with an IC₅₀ value of 7μM. LYR-71 inhibited IKKβ-mediated phosphorylation of cytoplasmic IκBα in lipopolysaccharide (LPS)-activated macrophages, and sequentially preventing IκBα degradation as well as transcriptional activation of NF-κB. Furthermore, LYR-71 down-regulated LPS-induced transcription of interleukin (IL)-1β or other cytokines in the cells, and inhibited expression vector IKKβ-elicited IL-1β promoter activity. Taken together, LYR-71 was an efficient inhibitor of IKKβ, preventing NF-κB activation in macrophages, and this mechanism of action could contribute its down-regulatory effect on LPS-induced expression of inflammatory cytokines at the transcription level.

Keywords: Iminobenzoxathiolone; NF-κB; IKKβ; Inflammatory cytokine; Macrophages

Metyrapone prevents cortisone-induced preadipocyte differentiation by depleting luminal NADPH of the endoplasmic reticulum by Paola Marcolongo; Silvia Senesi; Barbara Gava; Rosella Fulceri; Vincenzo Sorrentino; Éva Margittai; Beáta Lizák; Miklós Csala; Gábor Bánhegyi; Angelo Benedetti (pp. 382-390).

Preadipocyte differentiation is greatly affected by prereceptorial glucocorticoid activation catalyzed by 11β-hydroxysteroid dehydrogenase type 1 in the lumen of the endoplasmic reticulum. The role of the local NADPH pool in this process was investigated using metyrapone as an NADPH-depleting agent. Metyrapone administered at low micromolar concentrations caused the prompt oxidation of the endogenous NADPH, inhibited the reduction of cortisone and enhanced the oxidation of cortisol in native rat liver microsomal vesicles. However, in permeabilized microsomes, it only slightly decreased both NADPH-dependent cortisone reduction and NADP⁺-dependent cortisol oxidation. Accordingly, metyrapone administration caused a switch in 11β-hydroxysteroid dehydrogenase activity from reductase to dehydrogenase in both 3T3-L1-derived and human stem cell-derived differentiated adipocytes. Metyrapone greatly attenuated the induction of 11β-hydroxysteroid dehydrogenase type 1 and the accumulation of lipid droplets during preadipocyte differentiation when 3T3-L1 cells were stimulated with cortisone, while it was much less effective in case of cortisol or dexamethasone. In conclusion, the positive feedback of glucocorticoid activation during preadipocyte differentiation is interrupted by metyrapone, which depletes NADPH in the endoplasmic reticulum. The results also indicate that the reduced state of luminal pyridine nucleotides in the endoplasmic reticulum is important in the process of adipogenesis.

Keywords: Abbreviations; ER; endoplasmic reticulum; G6PT; glucose-6-phosphate translocase; H6PDH; hexose-6-phosphate dehydrogenase; 11βHSD1; 11β-hydroxysteroid dehydrogenase type 1; IBMX; isobutyl-methylxanthine; MOPS; 4-morpholinepropanesulfonic acid; FBS; fetal bovine serum; ADMSC; adipose-derived mesenchymal stem cell; SVF; stromal vascular fractionEndoplasmic reticulum; NADPH/NADP; ⁺; Metyrapone; 11β-Hydroxysteroid dehydrogenase type 1; Adipogenesis

Fluoxetine-induced proliferation and differentiation of neural progenitor cells isolated from rat postnatal cerebellum by Morena Zusso; Patrizia Debetto; Diego Guidolin; Massimo Barbierato; Hari Manev; Pietro Giusti (pp. 391-403).

Previous studies have shown that the serotonin-reuptake inhibitor (SSRI) fluoxetine affects neural progenitors derived from postnatal cerebellum or adult hippocampus and stimulates their proliferation. In the human cerebellum, the proliferation of cerebellar granule cells (CGC) continues until the 11th postnatal month and could be influenced in infants by breastfeeding-delivered SSRIs. Current information about fluoxetine effects on postnatal cerebellar neural progenitors is limited. Here we report the characterization of fluoxetine actions on rat postnatal cerebellar neural progenitors. RT-PCR and immunostaining revealed the expression of serotonin transporter (SERT), 5HT_1A receptors, tryptophan hydroxylase (TPH), and serotonin (5HT). Protracted in vitro fluoxetine treatment increased cell proliferation and differentiation. The proliferative effects of fluoxetine, 5HT, and the selective agonist of 5HT_1A receptors trans-8-hydroxy-2-( N- n-propyl- N-3′-iodo-2′-propenyl)aminotetralin (8-OH-PIPAT) were abolished by the selective antagonist of 5HT_1A receptors, N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]- N-(2-pyridinyl)cyclohexanecarboxamide trihydrochloride (WAY-100635). Furthermore, fluoxetine-induced activation of both the cAMP-response element-binding (CREB) protein and extracellular signal-regulated protein kinases (ERK1/2), which was abolished by the selective inhibitor of MAP kinase kinase (MEK) 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126), and increased cyclin D1 expression. All these effects were prevented by WAY-100635. Collectively, our results demonstrate that rat postnatal cerebellum contains neural progenitors capable of proliferating and differentiating in response to fluoxetine exposure, possibly through the activation of 5HT_1A receptors. The relevance of these findings for possible SSRI effects on the developing postnatal/infant human cerebellum should be explored.

Keywords: Postnatal cerebellar neural progenitors; 5HT; _1A; cAMP-response element-binding (CREB) protein; Extracellular signal-regulated protein kinase (ERK1/2); Selective serotonin-reuptake inhibitors (SSRIs)

Characterisation of novel defective thiopurine S-methyltransferase allelic variants by A. Garat; C. Cauffiez; N. Renault; J.M. Lo-Guidice; D. Allorge; D. Chevalier; N. Houdret; P. Chavatte; M.A. Loriot; J.L. Gala; F. Broly (pp. 404-415).

Human thiopurine S-methyltransferase (TPMT, EC 2.1.1.67) is a key enzyme in the detoxification of thiopurine drugs widely used in the treatment of various diseases, such as inflammatory bowel diseases, acute lymphoblastic leukaemia and rheumatic diseases. The TPMT gene is genetically polymorphic and the inverse relationship between TPMT activity and the risk of developing severe hematopoietic toxicity is well known. In this study, the entire coding sequence of TPMT, together with its 5′-flanking promoter region, was analysed in patients with an intermediate phenotype for thiopurine drug methylation. Four polymorphisms were identified, two previously described, c.356A>C (p.Lys¹¹⁹Thr, TPMT*9) and c.205C>G (p.Leu⁶⁹Val, TPMT*21), and two novel missense mutations, c.537G>T (p.Gln¹⁷⁹His, TPMT*24) and c.634T>C (p.Cys²¹²Arg, TPMT*25). Structural investigations, using molecular modeling, were undertaken in an attempt to explain the potential impact of the amino acid substitutions on the structure and activity of the variant proteins. Additionally, in order to determine kinetic parameters ( K_m and V_max) of 6-thioguanine (6-TG) methylation, the four variants were expressed in a recombinant yeast expression system. Assays were performed by HPLC and the results were compared with those of wild-type TPMT. The p.Leu⁶⁹Val and the p.Cys²¹²Arg substitutions encode recombinant enzymes with a significantly decreased intrinsic clearance compared to that of the wild-type protein, and, consequently, characterise non-functional alleles of TPMT. The p.Lys¹¹⁹Thr and the p.Gln¹⁷⁹His substitutions do not affect significantly the catalytic activity of the corresponding variant proteins, which prevents to unambiguously describe these latter alleles as defective TPMT variants.

Keywords: TPMT; genetic polymorphism; Thiopurine drugs; Heterologous expression; HPLC; Molecular modeling

Glucuronidation of flavonoids by recombinant UGT1A3 and UGT1A9 by Yakun Chen; Shenggu Xie; Shuqing Chen; Su Zeng (pp. 416-425).

Flavonoids are highlighted for their potential roles in the prevention of oxidative stress-associated diseases. Their metabolisms in vivo, such as glucuronidation, are the key points to determine their health beneficial properties. In this paper, we tested the glucuronidation of nineteen flavonoids by both recombinant human UGT1A3 and UGT1A9. Eleven compounds could be catalyzed by both enzymes. In general, both enzymes showed moderate to high catalyzing activity to most flavonoid aglycones, while the catalyzing efficiency changed with structures. Each flavonoid produced more than one monoglucuronide with no diglucuronide detected by liquid chromatography–mass spectrometry (LC–MS). Enzymatic kinetic analysis indicated that the catalyzing efficiency ( V_max/ K_m) of UGT1A9 was higher than that of UGT1A3, suggesting its important role in flavonoid glucuronidation. Both human UGT1A3 and UGT1A9 preferred flavonoid aglycone to flavonoid glycoside, and their metabolism to arabinoside was stronger than to other glycosides. Of the flavonoids studied, it is the first time to report isorhamnetin, morin, silybin, kaempferol, daidzein, quercetin-3′,4′-OCHO-, quercetin xylopyranoside and avicularin as substrates of UGT1A3. Apigenin, morin, daidzein, quercetin-3′,4′-OCHO-, quercetin xylopyranoside and avicularin were the newly reported substrates of UGT1A9.

Keywords: Abbreviations; UGTs; UDP-glucuronosyltransferases; UDPGA; UDP-glucuronic acid; HPLC; high performance liquid chromatography; LC–MS; liquid chromatography–mass spectrometry; NMR; nuclear magnetic resonance; FBS; fetal bovine serum; LC/ESI–MS/MS; liquid chromatography–electrospray ionization–tandem mass spectrometry; TIC; total ion chromatograms; m; /; z; mass-to-charge ratio; MOI; multiplicity of infectionUGT1A3; UGT1A9; Flavonoids; Glucuronidation; Monoglucuronide

An in vitro method of alcoholic liver injury using precision-cut liver slices from rats by Lynell W. Klassen; Geoffrey M. Thiele; Michael J. Duryee; Courtney S. Schaffert; Amy L. DeVeney; Carlos D. Hunter; Peter Olinga; Dean J. Tuma (pp. 426-436).

Alcohol abuse results in liver injury, but investigations into the mechanism(s) for this injury have been hampered by the lack of appropriate in vitro culture models in which to conduct in depth and specific studies. In order to overcome these shortcomings, we have developed the use of precision-cut liver slices (PCLS) as an in vitro culture model in which to investigate how ethanol causes alcohol-induced liver injury. In these studies, it was shown that the PCLS retained excellent viability as determined by lactate dehydrogenase and adenosine triphosphate (ATP) levels over a 96-h period of incubation. More importantly, the major enzymes of ethanol detoxification; alcohol dehydrogenase, aldehyde dehydrogenase, and cytochrome P4502E1, remained active and PCLS readily metabolized ethanol and produced acetaldehyde. Within 24h and continuing up to 96h the PCLS developed fatty livers and demonstrated an increase in the redox state. These PCLS secreted albumin, and albumin secretion was decreased by ethanol treatment. All of these impairments were reversed following the addition of 4-methylpyrazole, which is an inhibitor of ethanol metabolism. Therefore, this model system appears to mimic the ethanol-induced changes in the liver that have been previously reported in human and animal studies, and may be a useful model for the study of alcoholic liver disease.

Keywords: Abbreviations; ATP; adenosine triphosphate; LDH; lactate dehydrogenase; 4-MP; 4-methylpyrazole; AA; acetaldehyde; ALD; alcohol liver disease; ADH; alcohol dehydrogenase; ALDH; aldehyde dehydrogenase; CYP2E1; cytochrome P450 2E1; PCLS; precision-cut liver slicesPrecision-cut liver slice; Alcoholic liver disease; Fatty liver; Acetaldehyde; Ethanol metabolism

Acknowledgement (pp. 437-442).

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Biochemical Pharmacology (v.76, #3)