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Biochemical Pharmacology (v.79, #8)

Editorial Advisory Board (pp. iii).

Graphical Abstracts Contents (pp. e1-e4).

Emerging structure–function relationships defining monoamine NSS transporter substrate and ligand affinity by Ching-I Anderson Wang; Richard J. Lewis (pp. 1083-1091).

Monoamine transporters are a group of transmembrane neurotransmitter sodium symporter (NSS) transporters that play a crucial role in regulating biogenic monoamine concentrations at peripheral and central synapses. Given the key role played by serotonin, dopamine and noradrenaline in addictive and disease states, structure–function studies have been conducted to help guide the development of improved central nervous system therapeutics. Extensive pharmacological, immunological and biochemical studies, in conjunction with three-dimensional homology modeling, have been performed to structurally and functionally characterise the monoamine transporter substrate permeation pathway, substrate selectivity, and binding sites for ions, substrates and inhibitors at the molecular level. However, only recently has it been possible to start to construct an accurate molecular interaction network for the monoamine transporters and their corresponding substrates and inhibitors. Crystal structures of Aquifex aeolicus leucine transporter (LeuT_Aa), a homologous protein to monoamine transporters that has been experimentally demonstrated to share similar structural folds with monoamine transporters, have been determined in complex with amino acids and inhibitors. The molecular interactions of leucine and tricyclic antidepressants (TCA) has supported many of the predictions based on the mutational studies. Models constructed from LeuT_Aa are now allowing a rational approach to further clarify the molecular determinants of NSS transporter–ligand complexes, and potentially the ability to better manipulate drug specificity and affinity. In this review, we compare the structure–function relationships of other SLC6 NSS family transporters with monoamine transporters, and discuss possible mechanisms involved in substrate binding and transport, and modes of inhibition by TCAs.

Keywords: Abbreviations; NSS; neurotransmitter sodium symporter; DAT; dopamine transporter; NET; noradrenaline transporter; SERT; serotonin transporter; GAT; γ-aminobutyric acid transporter; GlyT; glycine transporter; TCA; tricyclic antidepressant; SSRI; selective serotonin reuptake inhibitor; SNRI; serotonin and noradrenaline reuptake inhibitor; NDRI; noradrenaline and dopamine reuptake inhibitor; MAOI; monoamine oxidase inhibitor; LeuT; _Aa; Aquifex aeolicus; leucine transporter; TMH; transmembrane helix; EL; extracellular loopLeucine transporter; Monoamine transporter; Homology models; Structure–function relationship; Trafficking; Tricyclic antidepressant; Na; ⁺; /Cl; ⁻; binding sites

Inhibition of epidermal growth factor receptor over-expressing cancer cells by the aphorphine-type isoquinoline alkaloid, dicentrine by V. Badireenath Konkimalla; Thomas Efferth (pp. 1092-1099).

Dicentrine exerts preferential cytotoxicity towards EGFR-transfectant U87MG cells. As determined by microarray hybridisations, this was accompanied by activation of BRCA1, p53, AhR signalling and G1/S and G2/M cell cycle regulation.The extraordinary relevance of EGFR in tumour biology makes it an exquisite molecular target for tumour therapy. Despite considerable success with these EGFR tyrosine kinase inhibitors in cancer therapy, resistance against these chemical compounds develops owing to the selection of point-mutated variants of EGFR. Therefore, there is an urgent need for the identification of novel EGFR tyrosine kinase inhibitors for treating tumours with such EGFR mutants. We found a preferential cytotoxicity of dicentrine towards U87MG.ΔEGFR-transduced with a constitutively deletion-activated EGFR expression vector as compared to non-transduced wild-type U87MG cells. As determined by microarray-based mRNA expression profiling, this preferential cytotoxicity was accompanied with an activation of BRCA1-mediated DNA damage response, p53 signalling, G1/S and G2/M cell cycle regulation, and aryl hydrocarbon receptor pathways. The activation of these signalling routes might be explained by the fact that dicentrine intercalates DNA and induces DNA strand break by inhibition of DNA topoisomerases. The cell cycle might be arrested by dicentrine-induced DNA lesions.

Keywords: Microarrays; Natural product; Oncogenes; Pharmacology; Pharmacogenomics; Pharmacognosy; Signal transduction; Systems biology

Moxifloxacin increases anti-tumor and anti-angiogenic activity of irinotecan in human xenograft tumors by Debby Reuveni; Drora Halperin; Ina Fabian; Galia Tsarfaty; Nadir Askenasy; Itamar Shalit (pp. 1100-1107).

Treatment of SCID mice implanted with HT-29 cells with moxifloxacin/irinotecan is more effective than each drug alone (left, A and B), and induces a decrease in tumors blood flow (right).Camptothecins (CPTs) are topoisomerase I inhibitors chemotherapeutic agents used in combination chemotherapy. We showed previously that combination of moxifloxacin (MXF) and CPT induced inhibitory effects on topoisomerase I activity, on proliferation of HT-29 cells in vitro and enhanced apoptosis, compared to CPT alone. Analysis of secretion of the pro-angiogenic factors IL-8 and VEGF showed significant reduction by MXF. Using a murine model of human colon carcinoma xenograft, we compared the effects of MXF/CPT in vitro to MXF/irinotecan combination in vivo. We show that the MXF/CPT inhibitory effects observed in vitro are reflected in the inhibition of the progressive growth of HT-29 cells implanted in SCID mice. Using caliper measurements, Doppler ultrasonography, image analyses and immunohistochemistry of nuclear proteins (Ki-67) and vascular endothelial cells (CD-31) we show that addition of MXF (45mg/kg) to a relatively ineffective dose of irinotecan (20mg/kg), results in a 50% and 30% decrease, respectively, in tumor size and a decrease in Ki-67 staining. Power Doppler Ultrasound showed a significant, pronounced decrease in the number of blood vessels, as did CD-31 staining, indicating decreased blood flow in tumors in mice treated with MXF alone or MXF/irinotecan compared to irinotecan. These results suggest that the combination of MXF/irinotecan may result in enhanced anti-neoplastic/anti-angiogenic activity.

Keywords: Irinotecan; Moxifloxacin; Chemotherapy; Angiogenesis

Increased levels and defective glycosylation of MRPs in ovarian carcinoma cells resistant to oxaliplatin by Giovanni Luca Beretta; Valentina Benedetti; Giacomo Cossa; Yehuda G.A. Assaraf; Eran Bram; Laura Gatti; Elisabetta Corna; Nives Carenini; Donato Colangelo; Stephen B. Howell; Franco Zunino; Paola Perego (pp. 1108-1117).

Pt compounds still represent the mainstay of the treatment of ovarian carcinoma. The aim of the present study was to investigate the molecular bases of resistance to Pt drugs using an oxaliplatin-resistant ovarian carcinoma cell model IGROV-1/OHP. These cells exhibited high levels of resistance to oxaliplatin, cross-resistance to cisplatin and topotecan and displayed a marked accumulation defect of Pt drugs. This feature was associated with increased expression and altered N-linked glycosylation of ATP binding cassette transporters MRP1 and MRP4. Pre-treatment with tunicamycin, which inhibits the biosynthesis of N-linked oligosaccharides, decreased the accumulation of Pt in sensitive cells exposed to oxaliplatin or cisplatin and increased the electrophoretic mobility of MRP1 and MRP4, reproducing the association between decreased glycosylation of MRP1 and MRP4 and decreased Pt accumulation observed in the resistant IGROV-1/OHP cells. The observed N-glycosylation defect of oxaliplatin-resistant cells was linked to reduced levels of N-acetylglucosamine-1-phosphotransferase (GNPTG) and mannosyl (alpha-1,6-)-glycoprotein beta-1,6-N-acetyl-glucosaminyltransferase (MGAT5). This feature, observed in IGROV-1/OHP cells, was associated with decreased retention of Pt drugs. In addition, the overexpression of fully glycosylated MRP1 or MRP4 in tumor cell line of ovarian origin was associated with resistance to oxaliplatin and cisplatin. Our findings, showing that development of resistance to oxaliplatin results in up-regulation of MRPs, support that patients with oxaliplatin-refractory ovarian carcinomas may benefit from non-Pt-based regimens which do not contain MRP1 and MRP4 substrates.

Keywords: Oxaliplatin; Ovarian carcinoma; ABC transporters; Cellular resistance; MRP4

Identification of small molecule inhibitors of pyruvate kinase M2 by Matthew G. Vander Heiden; Heather R. Christofk; Eli Schuman; Alexander O. Subtelny; Hadar Sharfi; Edward E. Harlow; Jun Xian; Lewis C. Cantley (pp. 1118-1124).

A common feature of tumors arising from diverse tissue types is a reliance on aerobic glycolysis for glucose metabolism. This metabolic difference between cancer cells and normal cells could be exploited for therapeutic benefit in patients. Cancer cells universally express the M2 isoform of the glycolytic enzyme pyruvate kinase (PKM2), and previous work has demonstrated that PKM2 expression is necessary for aerobic glycolysis and cell proliferation in vivo. Because most normal tissues express an isoform of pyruvate kinase other than PKM2, selective targeting of PKM2 provides an opportunity to target cell metabolism for cancer therapy. PKM2 has an identical catalytic site as the related M1 splice variant (PKM1). However, isoform selective inhibition is possible as PKM2 contains a unique region for allosteric regulation. We have screened a library of greater than 1,00,000 small molecules to identify such inhibitors. The inhibitors identified for PKM2 fell primarily into three distinct structural classes. The most potent PKM2 inhibitor resulted in decreased glycolysis and increased cell death following loss of growth factor signaling. At least part of this effect was due to on-target PKM2 inhibition as less cell death was observed in cells engineered to express PKM1. These data suggest that isoform selective inhibition of PKM2 with small molecules is feasible and support the hypothesis that inhibition of glucose metabolism in cancer cells is a viable strategy to treat human malignancy.

Keywords: Abbreviations; PK; pyruvate kinase; FDG-PET; ¹⁸; F-deoxyglucose positron emission tomography; FBP; fructose-1,6-bisphosphate; PEP; phosphoenolpyruvate; LDH; lactate dehydrogenasePyruvate kinase M2; Glycolysis; Cellular metabolism; Small molecule inhibitors; High-throughput screening

Beta tubulin affects the aryl hydrocarbon receptor function via an Arnt-mediated mechanism by Tianmin Zhang; Xiaodong Wang; Annie Shinn; Jingjun Jin; William K. Chan (pp. 1125-1133).

We have been studying the requirement for the aryl hydrocarbon receptor nuclear translocator (Arnt)-dependent DNA complex formation, which precedes the activation of gene transcription. Using DEAE chromatography, we have obtained a Sf9 insect fraction F5 that is highly enriched with β-tubulin. F5 inhibits the formation of the AhR gel shift complex and this inhibition is sensitive to protease, suggesting that proteins that are present in this F5 fraction are responsible for the inhibition. Additional experiments have revealed that this inhibition is less pronounced in the presence of anti-β-tubulin IgG and β-tubulin enriched fraction from pig brain also inhibits the AhR gel shift formation. Sf9 β-tubulin interacts with Arnt and suppresses the binding of the AhR/Arnt heterodimer to its corresponding enhancer. Human β4-tubulin, which shares high sequence identity with Sf9 β-tubulin, suppresses the AhR-dependent luciferase expression by reducing the nuclear Arnt content and retaining Arnt in the cytoplasm. Fluorescence studies using the GFP fusion of human β4-tubulin have revealed that β4-tubulin prevents the localization of Arnt in Sf9 cells. Here we have provided evidence suggesting that β-tubulin may regulate the physiological content of Arnt.

Keywords: Abbreviations; AhR; aryl hydrocarbon receptor; Arnt; Ah receptor nuclear translocator; HIF-1α; hypoxia inducible factor 1 alpha; βME; β-mercaptoethanol; DRE; dioxin response element; HRE; hypoxia response element; βNF; β-napthoflavone; HEDG; 25; mM HEPES, pH 7.4, 1; mM EDTA, 1; mM DTT, 10% glycerol; PBT; pork brain tubulin; 3MC; 3-methylchloranthrene; EPO; erythropoietin; VEGF; vascular endothelial growth factorArnt; β-Tubulin; AhR

Emodin enhances sensitivity of gallbladder cancer cells to platinum drugs via glutathion depletion and MRP1 downregulation by Wei Wang; Yue-ping Sun; Xin-zhi Huang; Min He; Yu-ying Chen; Gui-ying Shi; Hui Li; Jing Yi; Jian Wang (pp. 1134-1140).

Glutathione conjugation and transportation of glutathione conjugates of anticancer drugs out of cells are important for detoxification of many anticancer drugs. Inhibition of this detoxification system has recently been proposed as a strategy to treat drug-resistant solid tumors. Gallbladder carcinoma is resistant to many anticancer drugs, therefore, it is needed to develop a novel strategy for cancer therapy. In the present study, we tested the effect of emodin (1,3,8-trihydroxy-6-methylanthraquinone), a reactive oxygen species (ROS) generator reported by our group previously, in combination with cisplatin (CDDP), carboplatin (CBP) or oxaliplatin in treating the gallbladder carcinoma cell line SGC996. Our results showed that co-treatment with emodin could remarkably enhance chemosensitivity of SGC996 cells in comparison with cisplatin, carboplatin or oxaliplatin treatment alone. We found that the mechanisms may be attributed to reduction of glutathione level, and downregulation of multidrug resistance-related protein 1 (MRP1) expression in SGC996 cells. The experiments on tumor-bearing mice showed that emodin/cisplatin co-treatment inhibited the tumor growth in vivo via increasing tumor cell apoptosis and downregulating MRP1 expression.In conclusion, emodin can work as an adjunct to enhance the anticancer effect of platinum drugs in gallbladder cancer cells via ROS-related mechanisms.

Keywords: Abbreviations; Emodin; 1,3,8-trihydroxy-6-methylanthraquinone; ROS; reactive oxygen species; MDR; multidrug resistance; MRP; multidrug resistance-related protein; HDF; human dermal fibroblasts; CDDP; cisplatin; CBP; carboplatin; DCFH-DA; 2,7-dichlorodihydrofluorescein diacetate; DCF; 2,7-dichlorofluorescein; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; AnnexinV-FITC; AnnexinV-fluorescein isothiocyarate; PI; propidium iodide; GSH; glutathione; GS-Pt; glutathione S–platinum; GS-X pump; ATP-dependent glutathione S-conjugate exportCisplatin; MRP1; Glutathione; Emodin; Gallbladder cancer

The isothiocyanate produced from glucomoringin inhibits NF-kB and reduces myeloma growth in nude mice in vivo by Dario Brunelli; Michele Tavecchio; Cristiano Falcioni; Roberta Frapolli; Eugenio Erba; Renato Iori; Patrick Rollin; Jessica Barillari; Carla Manzotti; Paolo Morazzoni; Maurizio D’Incalci (pp. 1141-1148).

Glucosinolates (GLs), natural compounds extracted from Brassicaceae and precursors of isothiocyanates (ITCs), have been studied in the last decades mostly due to their chemopreventive activity and, more recently, for their potential use as novel chemotherapeutics. The aim of the present study was to investigate the in vitro and in vivo activity of glucomoringin (GMG), an uncommon member of the GLs family, and to compare it with glucoraphanin (GRA), one of the most studied GL. We have evaluated the potency of both compounds in inducing cell death, cell cycle perturbations, apoptosis, NF-kB inhibition and GST-π activity in human carcinoma cells with different GST-π contents as well as in human multiple myeloma and leukaemia cell lines. GMG-derived ITC (GMG-ITC) showed to be more effective compared to GRA-derived ITC (Sulforaphane), especially in inhibiting NF-kB activity and inducing apoptosis through a caspase-dependent pathway; these effects were more pronounced in myeloma cells, in which we could also observe a long lasting growth inhibitory effect, probably due to NF-kB inhibition, which is considered essential for myeloma cell survival. Both GLs were able to induce cell death in the μM range in all tested cell lines but caused cell cycle perturbations only in myeloma cells; they were also able to modulate the GST/GSH pathway by causing a 3-fold increase in GST-π activity in MCF7 cells. In vivo study showed that pure GMG-ITC was only slightly active in a carcinoma mice model, whereas it had significant antitumoral activity in a myeloma model, causing little toxicity.

Keywords: Isothiocyanate; Glucosinolate; Myeloma; NF-kB; Sulforaphane

Anthrax lethal toxin down-regulates type-IIA secreted phospholipase A₂ expression through MAPK/NF-κB inactivation by Benoit Raymond; Lucas Ravaux; Sylvie Mémet; YongZheng Wu; Aude Sturny-Leclère; Dominique Leduc; Chantal Denoyelle; Pierre L. Goossens; Miguel Payá; Michel Raymondjean; Lhousseine Touqui (pp. 1149-1155).

Inflammatory signals (LPS, IL-1β) induce sPLA2-IIA expression in alveolar macrophages via NF-κB and p38/ERK-dependent mechanism. Bacillus anthracis produces lethal toxin (LT) which abrogates this induction by cleaving MAPKK. Bacillus anthracis, the etiological agent of anthrax, produces lethal toxin (LT) that displays a metallo-proteolytic activity toward the N-terminus of the MAPK-kinases. We have previously shown that secreted type-IIA phospholipase A₂ (sPLA₂-IIA) exhibits potent anthracidal activity. In vitro expression of sPLA₂-IIA in guinea pig alveolar macrophages (AMs), the major source of this enzyme in lung tissues, is inhibited by LT. Here, we examined the mechanisms involved in sPLA₂-IIA inhibition by LT. We first showed that chemical inhibitors of p38 and ERK MAPKs reduced sPLA₂-IIA expression in AMs indicating that these kinases play a role in sPLA₂-IIA expression. LT inhibited IL-1β-induced p38 phosphorylation as well as sPLA₂-IIA promoter activity in CHO cells. Inhibition of sPLA₂-IIA promoter activity was mimicked by co-transfection with dominant negative construct of p38 (DN-p38) and reversed by the active form of p38-MAPK (AC-p38). Both LT and DN-p38 decreased IL-1β-induced NF-κB luciferase activity. This contrasted with the effect of AC-p38, which enhanced this activity. However, neither LT nor specific p-38 inhibitor interfered with LPS-induced IκBα degradation or NF-κB nuclear translocation in AMs. Subcutaneous administration of LT to guinea pig before LPS challenge reduced sPLA₂-IIA levels in broncho-alveolar lavages and ears. We conclude that sPLA₂-IIA expression is induced via a sequential MAPK-NF-κB activation and that LT inhibits this expression likely by interfering with the transactivation of NF-κB in the nucleus. This inhibition, which is operating both in vitro and in vivo, may represent a mechanism by which B. anthracis subvert host defense.

Keywords: Anthrax; Phospholipase A2; NF-κB; MAPK kinase; Macrophages

Acetaminophen (paracetamol) inhibits myeloperoxidase-catalyzed oxidant production and biological damage at therapeutically achievable concentrations by Maud Koelsch; Roger Mallak; Garry G. Graham; Tracey Kajer; Marian K. Milligan; Ly Q. Nguyen; Dawn W. Newsham; Jeremy S. Keh; Anthony J. Kettle; Kieran F. Scott; John B. Ziegler; David I. Pattison; Shanlin Fu; Clare L. Hawkins; Martin D. Rees; Michael J. Davies (pp. 1156-1164).

The heme peroxidase enzyme myeloperoxidase (MPO) is released by activated neutrophils and monocytes, where it uses hydrogen peroxide (H₂O₂) to catalyze the production of the potent oxidants hypochlorous acid (HOCl), hypobromous acid (HOBr) and hypothiocyanous acid (HOSCN) from halide and pseudohalide (SCN⁻) ions. These oxidants have been implicated as key mediators of tissue damage in many human inflammatory diseases including atherosclerosis, asthma, rheumatoid arthritis, cystic fibrosis and some cancers. It is shown here that acetaminophen (paracetamol), a phenol-based drug with analgesic and antipyretic actions, is an efficient inhibitor of HOCl and HOBr generation by isolated MPO–H₂O₂–halide systems. With physiological halide concentrations, acetaminophen concentrations required for 50% inhibition of oxidant formation (IC₅₀) were 77±6μM (100mMCl⁻) and 92±2μM (100mMCl⁻ plus 100μMBr⁻), as measured by trapping of oxidants with taurine. The IC₅₀ for inhibition of HOCl generation by human neutrophils was ca. 100μM. These values are lower than the maximal therapeutic plasma concentrations of acetaminophen (≤150μM) resulting from typical dosing regimes. Acetaminophen did not diminish superoxide generation by neutrophils, as measured by lucigenin-dependent chemiluminescence. Inhibition of HOCl production was associated with the generation of fluorescent acetaminophen oxidation products, consistent with acetaminophen acting as a competitive substrate of MPO. Inhibition by acetaminophen was maintained in the presence of heparan sulfate and extracellular matrix, materials implicated in the sequestration of MPO at sites of inflammation in vivo. Overall, these data indicate that acetaminophen may be an important modulator of MPO activity in vivo.

Keywords: Paracetamol; Acetaminophen; Myeloperoxidase; Oxidation; Neutrophil

N-Glycosylation plays a role in protein folding of human UGT1A9 by Miki Nakajima; Toshihisa Koga; Haruko Sakai; Hiroyuki Yamanaka; Ryoichi Fujiwara; Tsuyoshi Yokoi (pp. 1165-1172).

UDP-glucuronosyltransferases (UGTs) catalyze the glucuronidation of a variety of xeno/endobiotics. UGTs are type I membrane proteins of the endoplasmic reticulum (ER) with a glycosylated luminal domain. In the present study, we investigated the role of N-glycosylation in the function of human UGT1A9. Mutation analysis at the potential N-glycosylation sites at residues 71, 292, and 344 (from asparagine to glutamine) revealed that all of them were glycosylated, but the extent of glycosylation and/or size of the glycan differed. In comparison with the wild-type, these mutants showed decreased enzyme activities in parallel with the extent of the band shift in Western blot analysis. To evaluate the role of glycosylation in the enzyme activity, we produced unglycosylated UGT1A9 by treating HEK293 cells transiently transfected with expression plasmid with tunicamycin. The unglycosylated UGT1A9 was almost inactive, which was not an indirect effect of ER stress. To the contrary, the deglycosylated UGT1A9, which was produced by the treatment with Endo H under the non-denaturing condition, showed the same enzyme kinetics as the control. These results suggest that the glycosylation that occurs during translation is important for the folding of UGT1A9. The thermal stability analysis of the mutated and deglycosylated UGT1A9 proteins supported the findings. In conclusion, we found that the N-glycosylation has an important role in the folding of UGT1A9.

Keywords: UGT1A9; N; -Glycosylation; Enzyme activity; Protein folding

Role of serum and glucocorticoid-regulated kinase-1 in the protective effects of erythropoietin during renal ischemia/reperfusion injury by Krisztina Rusai; Ágnes Prókai; Beáta Szebeni; Andrea Fekete; András Treszl; Ádám Vannay; Veronika Müller; György Reusz; Uwe Heemann; Jens Lutz; Tivadar Tulassay; Attila J. Szabó (pp. 1173-1181).

The serum and glucocorticoid-regulated kinase 1 (SGK1) is an anti-apoptotic protein kinase activated through phosphorylation and might contribute to the mediation of EPO effects under renal ischemia/reperfusion (I/R) conditions.Erythropoietin (EPO) protects the kidneys from ischemia/reperfusion (I/R) injury; however, the exact signalling mechanisms are not fully understood. The serum and glucocorticoid-regulated kinase 1 (SGK1) is an anti-apoptotic protein kinase regulated through the phosphatidylinositol 3-kinase (PI3-kinase) pathway by cellular stimuli, hormones and growth factors. The objective of the present study was to examine the role of SGK1 in the renoprotective effects of EPO in renal I/R injury. In vitro, cultures of HEK293 cells were exposed to 16h hypoxia. Incubation with EPO at a doses of 400U/ml exerted a protective effect on cell death assessed by LDH release and Annexin V FACS analysis. This was paralleled by up-regulation of SGK1 expression, as well as phosphorylation. Downregulation of SGK1 expression by small interfering RNA technique ameliorated the anti-apoptotic effect of EPO treatment.In an in vivo rat model of unilateral renal I/R injury, rats were treated with 500U/kg EPO 24h prior to ischemia. EPO resulted in less severe tissue injury and ameliorated the elevation in creatinine and urea nitrogen levels 24h after reperfusion. Furthermore, SGK1 expression and phosphorylation were higher in EPO compared to vehicle-treated rats as demonstrated by real-time PCR, Western blot and immunofluorescence technique.We conclude that EPO protects from renal I/R injury and SGK1 might contribute to the mediation of EPO effects under ischemic conditions.

Keywords: EPO; Apoptosis; Hypoxia; Ischemia/reperfusion; Kidney; SGK1

Polycyclic aromatic hydrocarbon metabolizing cytochrome P450s in freshly prepared uncultured rat blood lymphocytes by Kumar Saurabh; Amit Sharma; Sanjay Yadav; Devendra Parmar (pp. 1182-1188).

In an attempt to develop blood lymphocyte cytochrome P450 expression profile as a surrogate to monitor tissue enzyme, the present study aimed to identify the expression and regulation of polycyclic aromatic hydrocarbons (PAHs) responsive CYPs in freshly prepared rat blood lymphocytes. Semi-quantitative and RT-PCR studies demonstrated constitutive and inducible mRNA expression of CYP1A1, 1A2, 1B1 isoenzymes and the associated transcription factors, aryl hydrocarbon receptor (AhR) and AhR translocator (ARNT) in blood lymphocytes. Absolute quantification using RT-PCR revealed several fold lower basal expression of CYP1A1, 1A2 and 1B1 in lymphocytes when compared to the liver. However, significant increase in the mRNA expression of these isoenzymes as well as AhR and ARNT in lymphocytes following pretreatment with 3-methylcholanthrene (MC) have demonstrated that responsiveness is retained in the blood lymphocytes, though the magnitude of increase is several fold lower when compared to liver. This increase in the mRNA expression was found to be associated with an increase in the protein expression of CYP1A1 and 1A2 in blood lymphocytes. Further, CYPs expressed in blood lymphocytes catalysed the O-dealkylation of 7-ethoxy- and 7-methoxyresorufins (ER or MR), though the reactivity was several fold lower in lymphocytes when compared to the liver enzyme. Our data providing quantitative evidence for similarities in the regulation of PAH-regulated CYP in uncultured and non-mitogen stimulated blood lymphocytes with the liver enzyme has led us to suggest that blood lymphocytes could be used as a surrogate to monitor tissue expression of CYPs.

Keywords: Lymphocytes; Tissue; Cytochrome P450; Expression; Enzyme; Surrogate

The UV-filter benzophenone-1 inhibits 17β-hydroxysteroid dehydrogenase type 3: Virtual screening as a strategy to identify potential endocrine disrupting chemicals by Lyubomir G. Nashev; Daniela Schuster; Christian Laggner; Seloni Sodha; Thierry Langer; Gerhard Wolber; Alex Odermatt (pp. 1189-1199).

The prevalence of male reproductive disorders and testicular cancer is steadily increasing. Because the exposure to chemicals disrupting natural hormone action has been associated with these diseases, it is important to identify endocrine disrupting chemicals (EDCs) and their targets of action. Here, a 3D-structural database that can be applied for virtual screening approaches to facilitate the identification of EDCs was constructed. The database was screened using pharmacophores of 17β-hydroxysteroid dehydrogenase type 3 (17β-HSD3), which catalyzes the last step of testosterone synthesis in testicular Leydig cells and plays an essential role during male sexual development. Among other chemicals, benzophenone (BP) UV-filters were predicted as potential 17β-HSD3 inhibitors. Biological analyses revealed (2,4-dihydroxyphenyl)-phenylmethanone (also known as benzophenone-1, BP-1) as an inhibitor of human 17β-HSD3 (IC₅₀ 1.05μM). BP-1 also efficiently blocked conversion of androstenedione to testosterone by mouse and rat 17β-HSD3 in whole-organ enzyme assays. Moreover, BP-1 antagonized the testosterone-dependent activation of androgen receptors (IC₅₀ 5.7μM), suggesting synergistic anti-androgenic effects of BP-1 by preventing testosterone formation and blocking receptor activation. In addition, analyses of several commonly used UV-filters on estrogen- and androgen-metabolizing 17β-HSD enzymes revealed 3-benzylidene camphor (3-BC) and 4-methylbenzylidene camphor (4-MBC) as low micromolar 17β-HSD2 inhibitors. In conclusion, screening of virtual chemical structure libraries can facilitate the identification of compounds interfering with hormone action. The potential disruption of 17β-HSD enzyme function by the UV-filters BP-1, 3-BC and 4-MBC requires further investigation and should be considered for safety assessment of these chemicals.

Keywords: 17β-Hydroxysteroid dehydrogenase; Androgen; Testosterone; Endocrine disruptor; Pharmacophore; Virtual screening; UV-filter

Effect of simvastatin on cholesterol metabolism in C2C12 myotubes and HepG2 cells, and consequences for statin-induced myopathy by Peter James Mullen; Barbara Lüscher; Hubert Scharnagl; Stephan Krähenbühl; Karin Brecht (pp. 1200-1209).

Simvastatin reduces protein prenylation and N-linked glycosylation in C2C12 myotubes, whereas the total cholesterol and ubiquinone levels are not altered.The mechanism of statin-induced skeletal muscle myopathy is poorly understood. We investigated how simvastatin affects cholesterol metabolism, ubiquinone levels, and the prenylation and N-linked glycosylation of proteins in C2C12 myotubes. We used liver HepG2 cells for comparison, as their responses to statins are well-characterized in terms of their cholesterol metabolism (in contrast to muscle cells), and statins are well-tolerated in the liver. Differences between the two cell lines could indicate the mechanism behind statin-induced myopathy. Simvastatin reduced de novo cholesterol production in C2C12 myotubes by 95% after 18h treatment. The reduction was 82% in the HepG2 cells. Total cholesterol pools, however, remained constant in both cell lines. Simvastatin treatment similarly did not affect total ubiquinone levels in the myotubes, unlike in HepG2 cells (22% reduction in CoQ10). Statin treatment reduced levels of Ras and Rap1 prenylation in both cell lines, whereas N-linked glycosylation was only affected in C2C12 myotubes (21% reduction in rate). From these observations, we conclude that total cholesterol and ubiquinone levels are unlikely to be involved in statin-mediated myopathy, but reductions in protein prenylation and especially N-linked glycosylation may play a role. This first comparison of the responses to simvastatin between liver and skeletal muscle cell lines may be important for future research directions concerning statin-induced myopathy.

Keywords: Statins; Prenylation; Ubiquinone; Cholesterol; N; -linked glycosylation

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Biochemical Pharmacology (v.79, #8)

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Biochemical Pharmacology (v.79, #8)