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Biochemical Pharmacology (v.81, #2)

Editorial Advisory Board (pp. iii).

Targeting maladaptive glutathione responses in lung disease by Neal S. Gould; Brian J. Day (pp. 187-193).

The lung is unique being exposed directly to the atmospheric environment containing xenobiotics, pathogens, and other agents which are continuously inhaled on a daily basis. Additionally, the lung is exposed to higher ambient oxygen levels which can promote the formation of a complex number of reactive oxygen and nitrogen species. Due to this constant barrage of potential damaging agents, the lung has developed a high degree of plasticity in dealing with ever changing conditions. In the present commentary, we will focus on glutathione (GSH) as a key antioxidant in the lung airways and discuss mechanisms by which the lung uses GSH to adapt to its rapidly changing environment. We will then examine the evidence on how defective and inadequate adaptive responses can lead to lung injury, inflammation and disease. Lastly, we will examine some of the recent attempts to alter lung GSH levels with therapies in a number of human lung diseases and discuss some of the limitations of such approaches.

Keywords: Abbreviations; BSO; l; -buthionine sulfoximine; γ-GCL; γ-glutamylcysteine ligase; GSH; glutathione; MRP; multi-drug resistant protein; ELF; epithelial lining fluid; BALF; bronchoalveolar lavage fluid; SOD; superoxide dismutase; IPF; idiopathic pulmonary fibrosis; ARDS; acute respiratory distress syndrome; CF; cystic fibrosis; COPD; chronic obstructive pulmonary disease; GCL; γ-glutamylcysteine ligase; GGT; γ-glutamyl transpeptidase; ABC; ATP binding cassette proteins; MRP; multidrug resistance protein; CFTR; cystic fibrosis transmembrane conductance regulator; ENaC; epithelial sodium channel; PA; Pseudomonas aeruginosa; BCRP; breast cancer related protein; GSSG; glutathione disulfide; NAC; N-acetylcysteine; ARE; antioxidant response elementKey words; Cystic fibrosis; Chronic obstructive pulmonary disease; Idiopathic pulmonary fibrosis; γ-Glutamylcysteine ligase; Infection and cigarette smoke

Targeting maladaptive glutathione responses in lung disease by Neal S. Gould; Brian J. Day (pp. 187-193).

Poly(ADP-ribose) polymerase signaling of topoisomerase 1-dependent DNA damage in carcinoma cells by Giovanna D’Onofrio; Filomena Tramontano; Annalisa Susanna Dorio; Alessia Muzi; Valeria Maselli; Domenico Fulgione; Grazia Graziani; Maria Malanga; Piera Quesada (pp. 194-202).

A molecular approach to enhance the antitumour activity of topoisomerase 1 (TOP1) inhibitors relies on the use of chemical inhibitors of poly(ADP-ribose)polymerases (PARP). Poly(ADP-ribosyl)ation is involved in the regulation of many cellular processes such as DNA repair, cell cycle progression and cell death. Recent findings showed that poly(ADP-ribosyl)ated PARP-1 and PARP-2 counteract camptothecin action facilitating resealing of DNA strand breaks. Moreover, repair of DNA strand breaks induced by poisoned TOP1 is slower in the presence of PARP inhibitors, leading to increased toxicity.In the present study we compared the effects of the camptothecin derivative topotecan (TPT), and the PARP inhibitor PJ34, in breast (MCF7) and cervix (HeLa) carcinoma cells either PARP-1 proficient or silenced, both BRCA1/2^+/+ and p53^+/+.HeLa and MCF7 cell lines gave similar results: (i) TPT-dependent cell growth inhibition and cell cycle perturbation were incremented by the presence of PJ34 and a 2 fold increase in toxicity was observed in PARP-1 stably silenced HeLa cells; (ii) higher levels of DNA strand breaks were found in cells subjected to TPT+PJ34 combined treatment; (iii) PARP-1 and -2 modification was evident in TPT-treated cells and was reduced by TPT+PJ34 combined treatment; (iv) concomitantly, a reduction of soluble/active TOP1 was observed. Furthermore, TPT-dependent induction of p53, p21 and apoptosis were found 24–72h after treatment and were increased by PJ34 both in PARP-1 proficient and silenced cells. The characterization of such signaling network can be relevant to a strategy aimed at overcoming acquired chemoresistance to TOP1 inhibitors.

Keywords: Topotecan (TPT); PARP-1 and -2; PJ34 Inhibitor; p53; Carcinoma cells

DNA strand breaks and hypoxia response inhibition mediate the radiosensitisation effect of nitric oxide donors on prostate cancer under varying oxygen conditions by Grant D. Stewart; Jyoti Nanda; Elad Katz; Karen J. Bowman; Jill G. Christie; D.J. Gordon Brown; Duncan B. McLaren; Antony C.P. Riddick; James A. Ross; George D.D. Jones; Fouad K. Habib (pp. 203-210).

Prostate cancer cells can exist in a hypoxic microenvironment, causing radioresistance. Nitric oxide (NO) is a radiosensitiser of mammalian cells. NO-NSAIDs are a potential means of delivering NO to prostate cancer cells. This study aimed to determine the effect and mechanism of action of NO-sulindac and radiation, on prostate cancer cells and stroma, under normoxia (21% oxygen) and chronic hypoxia (0.2% oxygen). Using clonogenic assays, at a surviving fraction of 10% the sensitisation enhancement ratios of radiation plus NO-sulindac over radiation alone on PC-3 cells were 1.22 and 1.42 under normoxia and hypoxia, respectively. 3D culture of PC-3 cells revealed significantly reduced sphere diameter in irradiated spheres treated with NO-sulindac. Neither NO-sulindac nor sulindac radiosensitised prostate stromal cells under normoxia or hypoxia. HIF-1α protein levels were reduced by NO-sulindac exposure and radiation at 21 and 0.2% oxygen. Alkaline Comet assay analysis suggested an increased rate of single strand DNA breaks and slower repair of these lesions in PC-3 cells treated with NO-sulindac prior to irradiation. There was a higher level of γ-H2AX production and hence double strand DNA breaks following irradiation of NO-sulindac treated PC-3 cells. At all radiation doses and oxygen levels tested, treatment of 2D and 3D cultures of PC-3 cells with NO-sulindac prior to irradiation radiosensitised PC-3, with minimal effect on stromal cells. Hypoxia response inhibition and increased DNA double strand breaks are potential mechanisms of action. Neoadjuvent and concurrent use of NO-NSAIDs have the potential to improve radiotherapy treatment of prostate cancer under normoxia and hypoxia.

Keywords: Abbreviations; NO; nitric oxide; NO-NSAIDs; nitric oxide donating non-steroidal anti-inflammatory drugs; Gy; Gray; PE; plating efficiency; SF; surviving fraction; SER; sensitiser enhancement ratio; OER; oxygen enhancement ratios; DSB; DNA double strand break; HIF-1α; hypoxia inducible factor-1α; SSB; single strand DNA breaksHypoxia; Prostate cancer; Nitric oxide donors; Radiation; Radiosensitisation

Thioredoxin reductase 1 knockdown enhances selenazolidine cytotoxicity in human lung cancer cells via mitochondrial dysfunction by Robyn L. Poerschke; Philip J. Moos (pp. 211-221).

Thioredoxin reductase (TR1) is a selenoprotein that is involved in cellular redox status control and deoxyribonucleotide biosynthesis. Many cancers, including lung, overexpress TR1, making it a potential cancer therapy target. Previous work has shown that TR1 knockdown enhances the sensitivity of cancer cells to anticancer treatments, as well as certain selenocompounds. However, it is unknown if TR1 knockdown produces similar effect on the sensitivity of human lung cancer cells. To further elucidate the role of TR1 in the mechanism of selenocompounds in lung cancer, a lentiviral microRNA delivery system to knockdown TR1 expression in A549 human lung adenocarcinoma cells was utilized. Cell viability was assessed after 48 hr treatment with the selenocysteine prodrug selenazolidines 2-butylselenazolidine-4(R)-carboxylic acid (BSCA) and 2-cyclohexylselenazolidine-4-(R)-carboxylic acid (ChSCA), selenocystine (SECY), methylseleninic acid (MSA), 1,4-phenylenebis(methylene)selenocyanate ( p-XSC), and selenomethionine (SEM). TR1 knockdown increased the cytotoxicity of BSCA, ChSCA, and SECY but did not sensitize cells to MSA, SEM, or p-XSC. GSH and TR1 depletion together decreased cell viability, while no change was observed with GSH depletion alone. Reactive oxygen species generation was induced only in TR1 knockdown cells treated with the selenazolidines or SECY. These three compounds also decreased total intracellular glutathione levels and oxidized thioredoxin, but in a TR1 independent manner. TR1 knockdown increased selenazolidine and SECY-induced mitochondrial membrane depolarization, as well as DNA strand breaks and AIF translocation from the mitochondria. These results indicate the ability of TR1 to modulate the cytotoxic effects of BSCA, ChSCA and SECY in human lung cancer cells through mitochondrial dysfunction.

Keywords: Abbreviations; AIF; apoptosis inducing factor; ASK1; apoptosis signaling kinase 1; BSO; L-buthionine-(S,R)-sulfoximine; BSCA; 2-butylselenazolidine-4(R)-carboxylic acid; CDDP; cis; -platinum(II) diammine dichloride; ChSCA; 2-cyclohexylselenazolidine-4(R)-carboxylic acid; Cys; cysteine; DCF; dichlorofluorescein; GSH; glutathione; KEAP1; Kelch-like ECH-associated protein 1; MSA; methylseleninic acid; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NAC; N; -acetyl-; l; -cysteine; NSCLC; non-small cell lung cancer; NRF2; nuclear factor erythroid 2-related factor 2; p; -XSC; 1,4-phenylenebis(methylene)selenocyanate; PI; propidium iodide; ROS; reactive oxygen species; Se; selenium; SECY; selenocystine; SEM; selenomethionine; tet; tetracycline; Trx; thioredoxin; TR; thioredoxin reductaseThioredoxin reductase; Selenazolidine; Selenium; A549; Redox; Apotosis

Thioredoxin reductase 1 knockdown enhances selenazolidine cytotoxicity in human lung cancer cells via mitochondrial dysfunction by Robyn L. Poerschke; Philip J. Moos (pp. 211-221).

The synergistic cytotoxicity of clofarabine, fludarabine and busulfan in AML cells involves ATM pathway activation and chromatin remodeling by Benigno C. Valdez; Yang Li; David Murray; Richard E. Champlin; Borje S. Andersson (pp. 222-232).

Suggested mechanism of synergistic cytotoxicity of clofarabine, fludarabine and busulfan (Bu).DNA alkylating agents alone or with ionizing radiation have been the preferred conditioning treatment in allogeneic hematopoietic stem cell transplantation (allo-HSCT). In search of less toxic alternatives, we hypothesized that combination of busulfan (Bu), fludarabine (Flu) and clofarabine (Clo) would provide superior efficacy. At low concentrations, these drugs show synergistic cytotoxicity in Bu-resistant AML KBM3/Bu250⁶ cells. Similar molecular responses were observed in other AML cell lines and in primary explanted AML cells. The [Clo+Flu+Bu] combination activates an intense DNA damage response through the ATM pathway, leading to cell cycle checkpoint activation and apoptosis. Phosphorylations of SMC1 and SMC3, and methylations of histones 3 and 4, are much more pronounced in cells exposed to [Clo+Flu+Bu] than [Clo+Flu], suggesting their relevance in the efficacy of the triple-drug combination. A possible mechanism for these observed synergistic effects involves the capability of [Clo+Flu] to induce histone methylations and subsequent chromatin remodeling, which may render the genomic DNA more accessible to Bu alkylation. The Bu-mediated DNA cross-linking may provide a feedback loop which perpetuates the DNA damage response initiated by [Clo+Flu] and commits the cells to apoptosis. Our results provide a conceptual mechanistic basis for exploring this triple-drug combination in pretransplant conditioning therapy for allo-HSCT.

Keywords: Clofarabine; Fludarabine; Busulfan; Synergistic cytotoxicity; Chromatin remodeling

Low molecular weight fucoidan prevents intimal hyperplasia in rat injured thoracic aorta through the modulation of matrix metalloproteinase-2 expression by Hanna Hlawaty; Nadine Suffee; Angela Sutton; Olivier Oudar; Oualid Haddad; Veronique Ollivier; Christelle Laguillier-Morizot; Liliane Gattegno; Didier Letourneur; Nathalie Charnaux (pp. 233-243).

The therapeutic potential of low molecular-weight fucoidan (LMWF), a sulfated polysaccharide extracted from brown seaweed was investigated on vascular smooth muscle cell (VSMC) and human vascular endothelial cell (HUV-EC-C) proliferation and migration in vitro and in a rat model of intimal hyperplasia. Sprague–Dawley rats were subjected to balloon injury in the thoracic aorta followed by two weeks’ treatment with either LMWF (5mg/kg/day) or vehicle. Morphological analysis and proliferating cell nuclear antigen immunostaining at day 14 indicated that LMWF prevented intimal hyperplasia in rat thoracic aorta as compared with vehicle (neo-intima area, 3±0.50mm² versus 5±0.30mm², P<0.01). In situ zymography showed that LMWF significantly decreased the activity of matrix metalloproteinase (MMP)-2 in the neo-intima compared to vehicle. The in vitro study demonstrated that 10μg/ml LMWF increased HUV-EC-C migration by 45±5% but reduced VSMC migration by 40±3%. LMWF also increased MMP-2 mRNA expression in HUV-EC-Cs and reduced it in VSMCs. MMP-2 level in the conditioned medium from cells incubated with 10μg/ml LMWF was 5.4-fold higher in HUV-EC-Cs, but 6-fold lower in VSMCs than in untreated control cells. Furthermore, decreasing MMP-2 expression in HUV-EC-Cs or VSMCs by RNA interference resulted in reduced LMWF-induced effects on cell migration.In conclusion, LMWF increased HUV-EC-C migration and decreased VSMC migration in vitro. In vivo, this natural compound reduced the intimal hyperplasia in the rat aortic wall after balloon injury. Therefore, LMWF could be of interest for the prevention of intimal hyperplasia.

Keywords: Abbreviations; LMWF; low molecular weight fucoidan; MMP; matrix metalloproteinase; SDF-1; stromal cell-derived factor-1; VEGF; vascular endothelial growth factor; VSMC; vascular smooth muscle cellFucoidan; Intimal hyperplasia; Smooth muscle cell; Endothelial cell; MMP-2

Acetaminophen-induced stimulation of MDR1 expression and activity in rat intestine and in LS 174T human intestinal cell line by Carolina I. Ghanem; Agostina Arias; Analia Novak; Griselda Delli Carpini; Silvina Villanueva; Alba G. Blazquez; Jose J.G. Marin; Aldo D. Mottino; Modesto C. Rubio (pp. 244-250).

The well-known analgesic and antipyretic drug N-acetyl- p-aminophenol (acetaminophen; APAP) has been previously reported to affect MDR1 expression in rat liver. In this study, we have investigated the effect of subtoxic doses of APAP on MDR1 expression and activity in rat intestine and human intestinal cells. Administration of APAP at increasing doses of 0.2, 0.3, and 0.6g/kg b.w., i.p. over three consecutive days, induced MDR1 expression in rat duodenum (+240%) and ileum (+160%) as detected by western blotting. This was accompanied by preserved localization of the protein at the surface of the villus, as detected by confocal immunofluorescence microscopy. MDR1 activity was increased by 50% in APAP treated rats, as evaluated by serosal to mucosal secretion of rhodamine 123 in everted intestinal sacs. Treatment with APAP also decreased by 65% the portal vein concentrations of digoxin found in anesthetized rats after intraduodenal administration of this drug, which is consistent with an APAP-induced increased efficacy of intestinal barrier for digoxin net absorption. Exposure of LS 174T human colon adenocarcinoma cells to subtoxic APAP concentration (5mM) induced an increase in MDR1 mRNA expression (+46%), which was accompanied with an enhanced ability (+78%) to reduce intracellular content of rhodamine 123. Taken together these data suggest the existence of APAP-induced stimulation of MDR1 transcription in the intestinal epithelium. These findings are of clinical relevance, as co-administration of APAP with other MDR1 substrates could indirectly inhibit the net intestinal absorption of these drugs, leading to changes in their pharmacokinetics and therapeutic efficacy.

Keywords: Acetaminophen; P-glycoprotein; MDR1; Intestine; LS 174T; Digoxin

The anti-tumoral drug enzastaurin inhibits natural killer cell cytotoxicity via activation of glycogen synthase kinase-3β by Henry Ogbomo; Tsigereda Biru; Martin Michaelis; Nadine Loeschmann; Hans Wilhelm Doerr; Jindrich Cinatl Jr. (pp. 251-258).

Enzastaurin is a selective protein kinase Cβ inhibitor which is shown to have direct antitumor effect as well as suppress glycogen synthase kinase-3β (GSK-3β) phosphorylation (resulting in its activation) in both tumor tissues and peripheral blood mononuclear cells (PBMC). It is currently used in phase II trials for the treatment of colon cancer, refractory glioblastoma and diffuse large B cell lymphoma. In this study, the direct effect of enzastaurin on effector function of human natural killer (NK) cells was investigated. The results obtained showed that enzastaurin suppressed both natural and antibody-dependent cellular cytotoxicity (ADCC) of NK cells against different tumor targets. This inhibition was associated with a specific down-regulation of surface expression of NK cell activating receptor NKG2D and CD16 involved in natural cytotoxicity and ADCC respectively, as well as the inhibition of perforin release. Analysis of signal transduction revealed that enzastaurin activated GSK-3β by inhibition of GSK-3β phosphorylation. Treatment of NK cells with GSK-3β-specific inhibitor TDZD-8 prevented enzastaurin-induced inhibition of NK cell cytotoxicity. Apart from the known antitumor and antiangiogenic effects, these results demonstrate that enzastaurin suppresses NK cell activity and may therefore interfere with NK cell-mediated tumor control in enzastaurin-treated cancer patients.

Keywords: Antibody-dependent cellular cytotoxicity; Natural cytotoxicity; NKG2D; Protein kinase Cβ; Glycogen synthase kinase-3β

Arzanol, a prenylated heterodimeric phloroglucinyl pyrone, inhibits eicosanoid biosynthesis and exhibits anti-inflammatory efficacy in vivo by Julia Bauer; Andreas Koeberle; Friederike Dehm; Federica Pollastro; Giovanni Appendino; Hinnak Northoff; Antonietta Rossi; Lidia Sautebin; Oliver Werz (pp. 259-268).

Arzanol causes reduced levels of leukotrienes and PGE2 in vivo, connected to anti-inflammatory effects.Based on its capacity to inhibit in vitro HIV-1 replication in T cells and the release of pro-inflammatory cytokines in monocytes, the prenylated heterodimeric phloroglucinyl α-pyrone arzanol was identified as the major anti-inflammatory and anti-viral constituent from Helichrysum italicum. We have now investigated the activity of arzanol on the biosynthesis of pro-inflammatory eicosanoids, evaluating its anti-inflammatory efficacy in vitro and in vivo. Arzanol inhibited 5-lipoxygenase (EC 7.13.11.34) activity and related leukotriene formation in neutrophils, as well as the activity of cyclooxygenase (COX)-1 (EC 1.14.99.1) and the formation of COX-2-derived prostaglandin (PG)E₂ in vitro (IC₅₀=2.3–9μM). Detailed studies revealed that arzanol primarily inhibits microsomal PGE₂ synthase (mPGES)-1 (EC 5.3.99.3, IC₅₀=0.4μM) rather than COX-2. In fact, arzanol could block COX-2/mPGES-1-mediated PGE₂ biosynthesis in lipopolysaccharide-stimulated human monocytes and human whole blood, but not the concomitant COX-2-derived biosynthesis of thromboxane B₂ or of 6-keto PGF_1α, and the expression of COX-2 or mPGES-1 protein was not affected. Arzanol potently suppressed the inflammatory response of the carrageenan-induced pleurisy in rats (3.6mg/kg, i.p.), with significantly reduced levels of PGE₂ in the pleural exudates. Taken together, our data show that arzanol potently inhibits the biosynthesis of pro-inflammatory lipid mediators like PGE₂ in vitro and in vivo, providing a mechanistic rationale for the anti-inflammatory activity of H. italicum, and a rationale for further pre-clinical evaluation of this novel anti-inflammatory lead.

Keywords: Abbreviations; AA; arachidonic acid; COX; cyclooxygenase; cPL; cytosolic phospholipase; DMSO; dimethyl sulfoxide; DPPH; 2,2-diphenyl-1-picrylhydrazyl; FCS; fetal calf serum; FLAP; 5-lipoxygenase-activating protein; 12-HHT; 12(; S; )-hydroxy-5-; cis; -8,10-; trans; -heptadecatrienoic acid; IL; interleukin; 5-LO; 5-lipoxygenase; LPS; lipopolysaccharide; LT; leukotriene; mPGES; microsomal prostaglandin E; ₂; synthase; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NFκB; nuclear factor kappa beta; NSAIDs; non steroidal anti-inflammatory drugs; PBS; phosphate-buffered saline; PBMC; peripheral blood mononuclear cells; PG; prostaglandin; PGC buffer; PBS pH 7.4 containing 1; mg/ml glucose and 1; mM CaCl; ₂; TBH; tert; -butyl hydroperoxide5-Lipoxygenase; Leukotriene; Prostaglandin; Inflammation; Phloroglucinol

Signaling mechanisms of inhibition of phospholipase D activation by CHS-111 in formyl peptide-stimulated neutrophils by Ling-Chu Chang; Tai-Hung Huang; Chi-Sen Chang; Ya-Ru Tsai; Ruey-Hseng Lin; Pin-Wen Lee; Mei-Feng Hsu; Li-Jiau Huang; Jih-Pyang Wang (pp. 269-278).

A selective phospholipase D (PLD) inhibitor 5-fluoro-2-indolyl des-chlorohalopemide (FIPI) inhibited the O₂⁻ generation and cell migration but not degranulation in formyl-Met-Leu-Phe (fMLP)-stimulated rat neutrophils. A novel benzyl indazole compound 2-benzyl-3-(4-hydroxymethylphenyl)indazole (CHS-111), which inhibited O₂⁻ generation and cell migration, also reduced the fMLP- but not phorbol ester-stimulated PLD activity (IC₅₀ 3.9±1.2μM). CHS-111 inhibited the interaction of PLD1 with ADP-ribosylation factor (Arf) 6 and Ras homology (Rho) A, and reduced the membrane recruitment of RhoA in fMLP-stimulated cells but not in GTPγS-stimulated cell-free system. CHS-111 reduced the cellular levels of GTP-bound RhoA, membrane recruitment of Rho-associated protein kinase 1 and the downstream myosin light chain 2 phosphorylation, and attenuated the interaction between phosphatidylinositol 4-phosphate 5-kinase (PIP5K) and Arf6, whereas it only slightly inhibited the guanine nucleotide exchange activity of human Dbs (DH/PH) protein and did not affect the arfaptin binding to Arf6. CHS-111 inhibited the interaction of RhoA with Vav, the membrane association and the phosphorylation of Vav. CHS-111 had no effect on the phosphorylation of Src family kinases (SFK) but attenuated the interaction of Vav with Lck, Hck, Fgr and Lyn. CHS-111 also inhibited the interaction of PLD1 with protein kinase C (PKC) α, βI and βII isoenzymes, and the phosphorylation of PLD1. These results indicate that inhibition of fMLP-stimulated PLD activity by CHS-111 is attributable to the blockade of RhoA activation via the interference with SFK-mediated Vav activation, attenuation of the interaction of Arf6 with PLD1 and PIP5K, and the activation of Ca²⁺-dependent PKC in rat neutrophils.

Keywords: CHS-111; Phospholipase D; RhoA; Arf6; Protein kinase C; Vav; Neutrophils

Differential sensitivity to adrenergic stimulation underlies the sexual dimorphism in the development of diabetes caused by Irs-2 deficiency by Maria Jose Garcia-Barrado; Maria Carmen Iglesias-Osma; Veronica Moreno-Viedma; Maria Francisca Pastor Mansilla; Silvia Sanz Gonzalez; Jose Carretero; Julio Moratinos; Deborah J. Burks (pp. 279-288).

The diabetic phenotype caused by the deletion of insulin receptor substrate-2 (Irs-2) in mice displays a sexual dimorphism. Whereas the majority of male Irs-2^−/− mice are overtly diabetic by 12 weeks of age, female Irs-2^−/− animals develop mild obesity and progress less rapidly to diabetes. Here we investigated β-cell function and lipolysis as potential explanations for the gender-related differences in this model. Glucose-stimulated insulin secretion was enhanced in islets from male null mice as compared to male WT whereas this response in female Irs-2^−/− islets was identical to that of female controls. The ability of α₂-adrenoceptor (α₂-AR) agonists to inhibit insulin secretion was attenuated in male Irs2 null mice. Consistent with this, the expression of the α_2A-AR was reduced in male Irs-2^−/− islets. The response of male Irs-2^−/− islets to forskolin was enhanced, owing to increased production of cAMP. Basal lipolysis was increased in male Irs-2^−/− but decreased in female Irs-2^−/− mice, concordant with the observation that adipose tissue is sparse in males whereas female Irs2 null mice are mildly obese. Adipocytes from both male and female Irs-2^−/− were resistant to the anti-lipolytic effects of insulin but female Irs-2^−/− fat cells were additionally resistant to the catabolic effects of beta-adrenergic agonists. This catecholamine resistance was associated with impaired generation of cAMP. Consequently, targets of cAMP-dependent protein kinase (PKA) which mediate lipolysis were not phosphorylated in adipose tissue of female Irs-2^−/− mice. Our findings suggest that IRS-2 deficiency in mice alters the expression and/or sensitivity of components of adrenergic signaling.

Keywords: Insulin; Diabetes; Adrenergic receptors; Lipolysis; cAMP

Human CYP2A6 is regulated by nuclear factor-erythroid 2 related factor 2 by Shin-ichi Yokota; Eriko Higashi; Tatsuki Fukami; Tsuyoshi Yokoi; Miki Nakajima (pp. 289-294).

Human CYP2A6 is responsible for the metabolism of nicotine and coumarin as well as the metabolic activation of tobacco-related nitrosamines. Earlier studies revealed that CYP2A6 activity was increased by dietary cadmium or cruciferous vegetables, but the underlying mechanisms remain to be clarified. In the present study, we investigated the possibility that Nrf2 might be involved in the regulation of CYP2A6. Real-time RT-PCR analysis revealed that the CYP2A6 mRNA level in human hepatocytes was significantly ( P<0.01, 1.4-fold) induced by 10μM sulforaphane (SFN), a typical activator of Nrf2. A computer-based search identified three putative antioxidant response elements (AREs) in the 5′-flanking region of the CYP2A6 gene at positions −1212, −2444, and −3441, termed ARE1, ARE2, and ARE3, respectively. Electrophoretic mobility shift assays demonstrated that Nrf2 bound only to ARE1. Luciferase assays using HepG2 cells revealed that the overexpression of Nrf2 significantly increased the reporter activities of the constructs containing a 30-bp fragment that included ARE1. However, the activity of the construct containing the intact 5′-flanking region (−1 to −1395) including ARE1 was not increased by the overexpression of Nrf2. In contrast, when the reporter construct was injected into mice via the tail vein, the reporter activity in the liver was significantly ( P<0.05, 1.9-fold) increased by SFN (1mg/head) administration. In conclusion, we found that human CYP2A6 is regulated via Nrf2, suggesting that CYP2A6 is induced under oxidative stress.

Keywords: Key words; Cytochrome P450; Nuclear factor-erythroid 2 related factor 2; Transcriptional regulation

Human CYP2A6 is regulated by nuclear factor-erythroid 2 related factor 2 by Shin-ichi Yokota; Eriko Higashi; Tatsuki Fukami; Tsuyoshi Yokoi; Miki Nakajima (pp. 289-294).

Keywords: Key words; Cytochrome P450; Nuclear factor-erythroid 2 related factor 2; Transcriptional regulation

Influence of renal compensatory hypertrophy on mitochondrial energetics and redox status by Bavneet Benipal; Lawrence H. Lash (pp. 295-303).

A reduction in functional renal mass is common in numerous renal diseases and aging. The remaining functional renal tissue undergoes compensatory growth primarily due to hypertrophy. This is associated with a series of physiological, morphological and biochemical changes similar to those observed after uninephrectomy. Previous work showed that compensatory renal cellular hypertrophy resulted in an increase in susceptibility to several drugs and environmental chemicals and appeared to be associated with oxidative stress. Compensatory renal cellular hypertrophy was also associated with increases in mitochondrial metabolic activity, uptake of glutathione (GSH) across renal plasma and mitochondrial inner membranes, and intracellular GSH concentrations. Based on these observations, we hypothesize that the morphological, physiological and biochemical changes in the hypertrophied kidney are associated with marked alterations in renal cellular energetics, redox status and renal function in vivo. In this study, we used a uninephrectomized (NPX) rat model to induce compensatory renal growth. Our results show alterations in renal physiological parameters consistent with modest renal injury, altered renal cellular energetics, upregulation of certain renal plasma membrane transporters, including some that have been observed to transport GSH, and evidence of increased oxidative stress in mitochondria from the remnant kidney of NPX rats. These studies provide additional insight into the molecular changes that occur in compensatory renal hypertrophy and should help in the development of novel therapeutic approaches for patients with reduced renal mass.

Keywords: Abbreviations; Acivicin; L-(αS,5S)-α-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid; BLM; basolateral plasma membrane; BBM; brush-border plasma membrane; DIC; dicarboxylate carrier; GAPDH; glyceraldehyde 3-phosphate dehydrogenase; GCS; L-γ-glutamyl-L-cysteine synthetase; GGT; γ-glutamyltransferase; GPX; glutathione peroxidase; GRD; glutathione reductase; GSH; glutathione; GSSG; glutathione disulfide; GST; glutathione; S; -transferase; HNE; 4-hydroxy-2-nonenal; MDA; malondialdehyde; MDH; malic dehydrogenase; Mrp; multidrug resistance associated protein; NaC3; sodium-dicarboxylate 3 carrier; NAG; N; -acetyl-β-D-glucosaminidase; 3-NT; 3-nitrotyrosine; Oat; organic anion transporter; OGC; 2-oxoglutarate carrier; OPT; o; -phthalaldehyde; PT; proximal tubular; SDH; succinate: cytochrome c oxidoreductase; Sod2; superoxide dismutase 2; tBH; tert-butyl hydroperoxide; Trx2; thioredoxin 2; NPX; uninephrectomized; VDAC; voltage-dependent anion channel (porin)Oxidative stress; Mitochondria; Glutathione; Membrane transporters; Uninephrectomy

Influence of renal compensatory hypertrophy on mitochondrial energetics and redox status by Bavneet Benipal; Lawrence H. Lash (pp. 295-303).

Induction of the Ras activator Son of Sevenless 1 by environmental pollutants mediates their effects on cellular proliferation by Stéphane Pierre; Anne-Sophie Bats; Aline Chevallier; Linh-Chi Bui; Ariane Ambolet-Camoit; Michèle Garlatti; Martine Aggerbeck; Robert Barouki; Xavier Coumoul (pp. 304-313).

TCDD (2,3,7,8-tetrachlorodibenzodioxin), a highly persistent environmental pollutant and a human carcinogen, is the ligand with the highest affinity for the Aryl Hydrocarbon Receptor (AhR) that induces via the AhR, xenobiotic metabolizing enzyme genes as well as several other genes. This pollutant elicits a variety of systemic toxic effects, which include cancer promotion and diverse cellular alterations that modify cell cycle progression and cell proliferation. Large-scale studies have shown that the expression of Son of Sevenless 1 (SOS1), the main mediator of Ras activation, is one of the targets of dioxin in human cultured cells. In this study, we investigated the regulation of the previously uncharacterized SOS1 gene promoter by the AhR and its ligands in the human hepatocarcinoma cell line, HepG2. We found that several environmental pollutants (AhR ligands) induce SOS1 gene expression by increasing its transcription. Chromatin immunoprecipitation experiments demonstrated that the AhR binds directly and activates the SOS1 gene promoter. We also showed that dioxin treatment leads to an activated Ras-GTP state, to ERK activation and to accelerated cellular proliferation. All these effects were mediated by SOS1 induction as shown by knock down experiments. Our data indicate that dioxin-induced cellular proliferation is mediated, at least partially, by SOS1 induction. Remarkably, our studies also suggest that SOS1 induction leads to functional effects similar to those elicited by the well-characterized oncogenic Ras mutations.

Keywords: Dioxin; AhR; SOS1; Ras; Cell proliferation

α-Elapitoxin-Aa2a, a long-chain snake α-neurotoxin with potent actions on muscle (α1)₂βγδ nicotinic receptors, lacks the classical high affinity for neuronal α7 nicotinic receptors by Benjamin Blacklow; Rachelle Kornhauser; Peter G. Hains; Richard Loiacono; Pierre Escoubas; Andis Graudins; Graham M. Nicholson (pp. 314-325).

In contrast to all classical long-chain α-neurotoxins possessing the critical fifth disulfide bond, α-elapitoxin-Aa2a (α-EPTX-Aa2a), a novel long-chain α-neurotoxin from the common death adder Acanthophis antarcticus, lacks affinity for neuronal α7-type nicotinic acetylcholine receptors (nAChRs). α-EPTX-Aa2a (8850Da; 0.1–1μM) caused a concentration-dependent inhibition of indirect twitches, and blocked contractures to cholinergic agonists in the isolated chick biventer cervicis nerve-muscle preparation, consistent with a postsynaptic curaremimetic mode of action. α-EPTX-Aa2a (1–10nM) produced a potent pseudo-irreversible antagonism of chick muscle nAChRs, with an estimated p A₂ value of 8.311±0.031, which was not reversed by monovalent death adder antivenom. This is only 2.5-fold less potent than the prototypical long-chain α-neurotoxin, α-bungarotoxin. In contrast, α-EPTX-Aa2a produced complete, but weak, inhibition of¹²⁵I-α-bungarotoxin binding to rat hippocampal α7 nAChRs (p K_I=3.670), despite high sequence homology and similar mass to a wide range of long-chain α-neurotoxins. The mostly likely cause for the loss of α7 binding affinity is a leucine substitution, in loop II of α-EPTX-Aa2a, for the highly conserved Arg³³ in long-chain α-neurotoxins. Arg³³ has been shown to be critical for both neuronal and muscle activity. Despite this substitution, α-EPTX-Aa2a retains high affinity for muscle (α1)₂βγδ nAChRs. This is probably as a result of an Arg²⁹ residue, previously shown to be critical for muscle (α1)₂βγδ nAChR affinity, and highly conserved across all short-chain, but not long-chain, α-neurotoxins. α-EPTX-Aa2a therefore represents a novel atypical long-chain α-neurotoxin that includes a fifth disulfide but exhibits differential affinity for nAChR subtypes.

Keywords: Abbreviations; ACh; acetylcholine; ANOVA; analysis of variance; CBCNM; chick biventer cervicis nerve-muscle; CCh; carbachol; ESI-QTOF; electrospray ionization quadrupole time-of-flight; MALDI-TOF; matrix-assisted laser desorption/ionization time-of-flight; nAChR; nicotinic acetylcholine receptor; NSW; New South Wales; EPTX; elapitoxin; RP-HPLC; reversed-phase high-pressure liquid chromatography; TFA; trifluoroacetic acid; t; ₉₀; time to 90% neuromuscular blockade; V; _e; elution volume; V; _o; void volumeSnake toxin; Long-chain α-neurotoxin; α-Elapitoxin-Aa2a; Acanthophis antarcticus; Neuronal α7 receptor

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Biochemical Pharmacology (v.81, #2)

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Biochemical Pharmacology (v.81, #2)