Genoprotective pathways. Part I. Extracellular signaling through G(s) protein-coupled adenosine receptors prevents oxidative DNA damage

Adenosine has been previously shown to be a cytoprotective paracrine released by injured cells. However, it is presently unknown whether extracellular adenosine can prevent DNA damage. We show here that the adenosine analog, 2-chloroadenosine (2CA), has a potent (IC(50)=2.04 x 10(-9)M) genoprotective effect in cells subsequently exposed to H(2)O(2). The genoprotective signaling is transduced through adenosine receptors of the A2a and A2b subtypes. Increasing [cAMP](i) by forskolin or by cell permeable 8-br-cAMP produced a similar effect, whereas inhibiting the cAMP-mediated pathway with H-89, or increasing [nitric oxide](i) or [cGMP](i) blocked 2CA effect. Proteasomal inhibitors had no impact on 2CA signaling, while lithium chloride, an inhibitor of glycogen synthase kinase 3beta, completely blocked 2CA-induced genoprotective effect. These data indicate for the first time that extracellular adenosine protects cells against imminent oxidative DNA damage and suggest that prophylactic activation of this pathway prior to genotoxic challenges might reduce mutation rates.     

Transport of monosaccharides in kidney-cortex cells

1. The aerobic transport of d-glucose and d-galactose in rabbit kidney tissue at 25 degrees was studied. 2. In slices forming glucose from added substrates an accumulation of glucose against its concentration gradient was found. The apparent ratio of intracellular ([S](i)) and extracellular ([S](o)) glucose concentrations was increased by 0.4mm-phlorrhizin and 0.3mm-ouabain. 3. Slices and isolated renal tubules actively accumulated glucose from the saline; the apparent [S](i)/[S](o) fell below 1.0 only at [S](o) higher than 0.5mm. 4. The rate of glucose oxidation by slices was characterized by the following parameters: K(m) 1.16mm; V(max.) 4.5mumoles/g. wet wt./hr. 5. The active accumulation of glucose from the saline was decreased by 0.1mm-2,4-dinitrophenol, 0.4mm-phlorrhizin and by the absence of external Na(+). 6. The kinetic parameters of galactose entry into the cells were: K(m) 1.5mm; V(max) 10mumoles/g. wet wt./hr. 7. The efflux kinetics from slices indicated two intracellular compartments for d-galactose. The galactose efflux was greatly diminished at 0 degrees , was inhibited by 0.4mm-phlorrhizin, but was insensitive to ouabain. 8. The following mechanism of glucose and galactose transport in renal tubular cells is suggested: (a) at the tubular membrane, these sugars are actively transported into the cells by a metabolically- and Na(+)-dependent phlorrhizin-sensitive mechanism; (b) at the basal cell membrane, these sugars are transported in accordance with their concentration gradient by a phlorrhizin-sensitive Na(+)-independent facilitated diffusion. The steady-state intracellular sugar concentration is determined by the kinetic parameters of active entry, passive outflow and intracellular utilization.     

Cellular mechanisms underlying nitric oxide-induced vasodilation of descending vasa recta

It has been observed that vasoactivity of explanted descending vasa recta (DVR) is modulated by intrinsic nitric oxide (NO) and superoxide (O(2)(-)) production (Cao C, Edwards A, Sendeski M, Lee-Kwon W, Cui L, Cai CY, Patzak A, Pallone TL. Am J Physiol Renal Physiol 299: F1056-F1064, 2010). To elucidate the cellular mechanisms by which NO, O(2)(-) and hydrogen peroxide (H(2)O(2)) modulate DVR pericyte cytosolic Ca(2+) concentration ([Ca](cyt)) and vasoactivity, we expanded our mathematical model of Ca(2+) signaling in pericytes. We incorporated simulations of the pathways that translate an increase in [Ca](cyt) to the activation of myosin light chain (MLC) kinase and cell contraction, as well as the kinetics of NO and reactive oxygen species formation and their effects on [Ca](cyt) and MLC phosphorylation. The model reproduced experimentally observed trends of DVR vasoactivity that accompany exposure to N(ω)-nitro-L-arginine methyl ester, 8-Br-cGMP, Tempol, and H(2)O(2). Our results suggest that under resting conditions, NO-induced activation of cGMP maintains low levels of [Ca](cyt) and MLC phosphorylation to minimize basal tone. This results from stimulation of Ca(2+) uptake from the cytosol into the SR via SERCA pumps, Ca(2+) efflux into the extracellular space via plasma membrane Ca(2+) pumps, and MLC phosphatase (MLCP) activity. We predict that basal concentrations of O(2)(-) and H(2)O(2) have negligible effects on Ca(2+) signaling and MLC phosphorylation. At concentrations above 1 nM, O(2)(-) is predicted to modulate [Ca(cyt)] and MCLP activity mostly by reducing NO bioavailability. The DVR vasoconstriction that is induced by high concentrations of H(2)O(2) can be explained by H(2)O(2)-mediated downregulation of MLCP and SERCA activity. We conclude that intrinsic generation of NO by the DVR wall may be sufficient to inhibit vasoconstriction by maintaining suppression of MLC phosphorylation.     

The [Ru(Hedta)NO](0.1-) system: structure, chemical reactivity and biological assays

The [Ru(II)(Hedta)NO(+)] complex is a diamagnetic species crystallizing in a distorted octahedral geometry, with the Ru-N(O) length 1.756(4) A and the RuNO angle 172.3(4) degrees . The complex contains one protonated carboxylate (pK(a)=2.7+/-0.1). The [Ru(II)(Hedta)NO(+)] complex undergoes a nitrosyl-centered one-electron reduction (chemical or electrochemical), with E(NO+/NO)=-0.31 V vs SCE (I=0.2 M, pH 1), yielding [Ru(II)(Hedta)NO](-), which aquates slowly: k(-NO)=2.1+/-0.4x10(-3) s(-1) (pH 1.0, I=0.2 M, CF(3)COOH/NaCF(3)COO, 25 degrees C). At pHs>12, the predominant species, [Ru(II)(edta)NO](-), reacts according to [Ru(II)(edta)NO](-)+2OH(-)-->[Ru(II)(edta)NO(2)](3-), with K(eq)=1.0+/-0.4 x 10(3) M(-2) (I=1.0 M, NaCl; T=25.0+/-0.1 degrees C). The rate-law is first order in each of the reactants for most reaction conditions, with k(OH(-))=4.35+/-0.02 M(-1)s(-1) (25.0 degrees C), assignable mechanistically to the elementary step comprising the attack of one OH(-) on [Ru(II)(edta)NO](-), with subsequent fast deprotonation of the [Ru(II)(edta)NO(2)H](2-) intermediate. The activation parameters were DeltaH(#)=60+/-1 kJ/mol, DeltaS(#)=-31+/-3 J/Kmol, consistent with a nucleophilic addition process between likely charged ions. In the toxicity up-and-down tests performed with Swiss mice, no death was observed in all the doses administered (3-9.08 x 10(-5) mol/kg). The biodistribution tests performed with Wistar male rats showed metal in the liver, kidney, urine and plasma. Eight hours after the injection no metal was detected in the samples. The vasodilator effect of [Ru(II)(edta)NO](-) was studied in aortic rings without endothelium, and was compared with sodium nitroprusside (SNP). The times of maximal effects of [Ru(II)(edta)NO](-) and SNP were 2 h and 12 min, respectively, suggesting that [Ru(II)(edta)NO](-) releases NO slowly to the medium in comparison with SNP.     

Susceptibility of protein kinase C to oxidative inactivation: loss of both phosphotransferase activity and phorbol diester binding

Exposure of protein kinase C to low concentrations of either N-chlorosuccinimide or H2O2 resulted in rapid and parallel loss of phosphotransferase activity and phorbol ester binding. This oxidative inactivation of protein kinase C also occurred in intact cells exposed to a low concentration of H2O2. With H2O2 treatment the rate of inactivation of protein kinase C in the cytosol of MCF-7 cells was rather slower than that which occurred in the cytosol of PYS cells. However, in both cell types, the oxidative inactivation of membrane-associated protein kinase C occurred rapidly in comparison to the enzyme in the cytosol. Prior treatment of cells with phorbol ester to induce membrane association (stabilization) of protein kinase C, followed by exposure to H2O2, resulted in increased inactivation of protein kinase C, suggesting that membrane association of protein kinase C increases its susceptibility to oxidative inactivation.     

Cyclothiazide binding to functionally active AMPA receptor reveals genuine allosteric interaction with agonist binding sites

The agonist, [3H](-)[S]-1-(2-amino-2-carboxyethyl)-5-fluoro-pyrimidine-2,4-dione ([3H](S)F-Willardiine) binding to functional alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors of resealed plasma membrane vesicles and nerve endings freshly isolated from the rat cerebral cortex displayed two binding sites (K(D1)=33+/-7 nM, B(MAX1)=1.6+/-0.3 pmol/mg protein, K(D2)=720+/-250 nM and B(MAX2)=7.8+/-4.0 pmol/mg protein). The drug which impairs AMPA receptor desensitisation, 6-chloro-3,4-dihydro-3-(2-norbornene-5-yl)-2H-1,2,4-benzothiadiazine-7-sulphonamide-1,1-dioxide (cyclothiazide, CTZ) fully displaced the [3H](S)F-Willardiine binding at a concentration of 500 microM. In the presence of 100 microM CTZ (K(I(CTZ))=60+/-6 microM), both the antagonist [3H]-1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo(F)quinoxaline-7-sulfonamide ([3H]NBQX: K(D)=24+/-4 nM, B(MAX)=12.0+/-0.1 pmol/mg protein) and the high-affinity agonist binding showed similar affinity reduction ([3H](S)F-Willardiine: K(D)=140+/-19 nM, B(MAX)=2.9+/-0.5 pmol/mg protein; [3H]NBQX: K(D)=111+/-34 nM, B(MAX)=12+/-3 pmol/mg protein). To disclose structural correlates underlying genuine allosteric binding interactions, molecular mechanics calculations of CTZ-induced structural changes were performed with the use of PDB data on extracellular GluR2 binding domain dimeric crystals available by now. Hydrogen-bonding and root mean square (rms) values of amino acid residues recognising receptor agonists showed minor alterations in the agonist binding sites itself. Moreover, CTZ binding did not affect dimeric subunit structures significantly. These findings indicated that the structural changes featuring the non-desensitised state could possibly occur to a further site of the extracellular GluR2 binding domain. The increase of agonist efficacy on allosteric CTZ binding may be interpreted in terms of a mechanism involving AMPA receptor desensitisation sequential to activation.     

Biological activity of complexes derived from pyridine-2-carbaldehyde thiosemicarbazone. Structure of

Biological studies on [Fe(L)2](NO3).0.5H2O (1), [Fe(L)2][PF6] (2), [Co(L)2](NCS) (3), [Ni(HL)2]Cl2.3H2O (4) and Cu(L)(NO3) (5), where HL=C7H8N4S, pyridine-2-carbaldehyde thiosemicarbazone, have been carried out. The crystal structure of compound 3 has been solved. It consists of discrete monomeric cationic entities containing cobalt(III) ions in a distorted octahedral environment. The metal ion is bonded to one sulfur and two nitrogen atoms of each thiosemicarbazone molecule. The thiocyanate molecules act as counterions. The copper(II) and iron(III) complexes react with reduced glutathione and 2-mercaptoethanol. The reaction of compound 1 with the above thiols causes the reduction of the metal ion and bis(thiosemicarbazonato)iron(II) species are obtained. The redox activity, and in particular the reaction with cell thiols, seems to be related to the cytotoxicity of these complexes against Friend erithroleukemia cells and melanoma B16F10 cells.     

Impact of electrostatics in redox modulation of oxidative stress by Mn porphyrins: protection of SOD-deficient Escherichia coli via alternative mechanism where Mn porphyrin acts as a Mn carrier

Understanding the factors that determine the ability of Mn porphyrins to scavenge reactive species is essential for tuning their in vivo efficacy. We present herein the revised structure-activity relationships accounting for the critical importance of electrostatics in the Mn porphyrin-based redox modulation systems and show that the design of effective SOD mimics (per se) based on anionic porphyrins is greatly hindered by inappropriate electrostatics. A new strategy for the beta-octabromination of the prototypical anionic Mn porphyrins Mn(III) meso-tetrakis(p-carboxylatophenyl)porphyrin ([Mn(III)TCPP](3-) or MnTBAP(3-)) and Mn(III) meso-tetrakis(p-sulfonatophenyl)porphyrin ([Mn(III)TSPP](3-)), to yield the corresponding anionic analogues [Mn(III)Br(8)TCPP](3-) and [Mn(III)Br(8)TSPP](3-), respectively, is described along with characterization data, stability studies, and their ability to substitute for SOD in SOD-deficient Escherichia coli. Despite the Mn(III)/Mn(II) reduction potential of [Mn(III)Br(8)TCPP](3-) and [Mn(III)Br(8)TSPP](3-) being close to the SOD-enzyme optimum and nearly identical to that of the cationic Mn(III) meso-tetrakis(N-methylpyridinium-2-yl)porphyrin (Mn(III)TM-2-PyP(5+)), the SOD activity of both anionic brominated porphyrins ([Mn(III)Br(8)TCPP](3-), E(1/2)=+213 mV vs NHE, log k(cat)=5.07; [Mn(III)Br(8)TSPP](3-), E(1/2)=+209 mV, log k(cat)=5.56) is considerably lower than that of Mn(III)TM-2-PyP(5+) (E(1/2)=+220 mV, log k(cat)=7.79). This illustrates the impact of electrostatic guidance of O(2)(-) toward the metal center of the mimic. With low k(cat), the [Mn(III)TCPP](3-), [Mn(III)TSPP](3-), and [Mn(III)Br(8)TCPP](3-) did not rescue SOD-deficient E. coli. The striking ability of [Mn(III)Br(8)TSPP](3-) to substitute for the SOD enzymes in the E. coli model does not correlate with its log k(cat). In fact, the protectiveness of [Mn(III)Br(8)TSPP](3-) is comparable to or better than that of the potent SOD mimic Mn(III)TM-2-PyP(5+), even though the dismutation rate constant of the anionic complex is 170-fold smaller. Analyses of the medium and E. coli cell extract revealed that the major species in the [Mn(III)Br(8)TSPP](3-) system is not the Mn complex, but the free-base porphyrin [H(2)Br(8)TSPP](4-) instead. Control experiments with extracellular MnCl(2) showed the lack of E. coli protection, indicating that "free" Mn(2+) cannot enter the cell to a significant extent. We proposed herein the alternative mechanism where a labile Mn porphyrin [Mn(III)Br(8)TSPP](3-) is not an SOD mimic per se but carries Mn into the E. coli cell.     

Mitochondria play an important role in 17beta-estradiol attenuation of H(2)O(2)-induced rat endothelial cell apoptosis

Studies have shown salutary effects of 17beta-estradiol following trauma-hemorrhage on different cell types. 17beta-Estradiol also induces improved circulation via relaxation of the aorta and has an anti-apoptotic effect on endothelial cells. Because mitochondria play a pivotal role in apoptosis, we hypothesized that 17beta-estradiol will maintain mitochondrial function and will have protective effects against H(2)O(2)-induced apoptosis in endothelial cells. Endothelial cells were isolated from rats' aorta and cultured in the presence or absence of H(2)O(2), a potent inducer of apoptosis. In additional studies, endothelial cells were pretreated with 17beta-estradiol. Flow cytometry analysis revealed H(2)O(2)-induced apoptosis in 80.9% of endothelial cells; however, prior treatment of endothelial cells with 17beta-estradiol resulted in an approximately 40% reduction in apoptosis. This protective effect of 17beta-estradiol was abrogated when endothelial cells were cultured in the presence ICI-182780, indicating the involvement of estrogen receptor (ER). Fluorescence microscopy revealed a 17beta-estradiol-mediated attenuation of H(2)O(2)-induced mitochondrial condensation. Western blot analysis demonstrated that H(2)O(2)-induced cytochrome c release from mitochondrion to cytosol and the activation of caspase-9 and -3 were decreased by 17beta-estradiol. These findings suggest that 17beta-estradiol attenuated H(2)O(2)-induced apoptosis via ER-dependent activation of caspase-9 and -3 in rat endothelial cells through mitochondria.     

Salidroside inhibits H2O2-induced apoptosis in PC12 cells by preventing cytochrome c release and inactivating of caspase cascade

We used a rat pheochromocytoma (PC12) cell line to study the effects of salidroside on hydrogen peroxide (H(2)O(2))-induced apoptosis. In PC12 cells, H(2)O(2)-induced apoptosis was accompanied by the down-regulation of Bcl-2, the up-regulation of Bax, the release of mitochondrial cytochrome c to cytosol, and the activation of caspase-3, -8 and -9. However, salidroside suppressed the down-regulation of Bcl-2, the up-regulation of Bax and the release of mitochondrial cytochrome c to cytosol. Moreover, salidroside attenuated caspase-3, -8 and -9 activation, and eventually protected cells against H(2)O(2)-induced apoptosis. Taken together, these results suggest that treatment of PC12 cells with salidroside can block H(2)O(2)-induced apoptosis by regulating Bcl-2 family members and by suppressing cytochrome c release and caspase cascade activation.     

D-mannose transport and metabolism in isolated enterocytes

D-mannose transport and metabolism has been studied in enterocytes isolated from chicken small intestine. In the presence of Na(+), the mannose taken up by the cells either remains free, is phosphorylated, is catabolized to H(2)O, or becomes part of membrane components. The mannose remaining free in the cytosol is released when the cells are transferred to an ice bath. The Na(+)-dependent D-mannose transport is electrogenic and inhibited by ouabain and dinitrophenol; its substrate specificity differs from SGLT-1 transporter. The Glut2 transporter inhibitors phloretin and cytochalasin B added following 30-min mannose uptake reduced the previously accumulated D-mannose, whereas these two agents increased the cell to external medium 3-O-methyl-glucose (3-OMG) concentration ratio. D-mannose efflux rate from preloaded D-[2-(3)H]-mannose enterocytes is Na(+)-independent. Phloretin did not affect D-mannose efflux rate, whereas it inhibited that of 3-OMG. Neither mannose uptake nor efflux rate were affected by fructose. It is concluded that part of the mannose taken up by the enterocytes is rapidly metabolized and that enterocytes have two D- mannose transport systems: one is concentrative and Na(+)-dependent and the other is Na(+)-independent and passive.     

重组nkx2.5腺病毒抑制过氧化氢诱导的H9c2细胞凋亡

为研究心脏发育关键基因nkx2.5的功能及应用价值,构建Ad-Nkx2.5重组腺病毒,并检测nkx2.5过表达拮抗氧化应激损伤的效应及机制。采用AdEasy腺病毒表达系统构建Ad-Nkx2.5重组腺病毒,建立H2O2诱导H9c2心肌细胞凋亡模型,分别用Ad-Nkx2.5重组病毒或对照病毒感染细胞,采用Hoechst33342染色观察细胞形态变化、MTT法检测细胞存活率,免疫印迹检测caspase-3活化、细胞色素C的胞浆含量。并通过Real-timePCR检测凋亡相关基因bcl-2和bax表达。结果发现,nkx2.5过表达促进H9c2细胞存活,抑制H2O2诱导的caspase-3活化及线粒体细胞色素C的释放。Nkx2.5过表达上调bcl-2表达,显著下调H2O2诱导的bax表达。并发现H2O2对Nkx2.5核定位无明显影响。结果显示重组腺病毒介导的Nkx2.5过表达可通过调控凋亡相关基因表达,抑制线粒体凋亡途径,保护心肌细胞抗氧化损伤。     

Unprecedented forms of vanadium observed within the blood cells of Phallusia nigra using K-edge X-ray absorption spectroscopy

Fits to the vanadium K-edge X-ray absorption spectra (XAS) of five whole blood cell samples from the tunicate Phallusia nigra revealed unprecedented forms of intracellular vanadium. Endogenous vanadium was divided between the V(III) ion (74.2+/-5.1% of total V) and the vanadyl ion [V(IV)=O](2+) (25.2+/-5.4% of total V). The V(III) fraction included both [V(H(2)O)(6)](3+) (36.7+/-5.5%) modeled as VCl(3) in 1 M HCl, and three previously unprecedented chelated V(III) forms (37.5+/-4.6%). Two of these could be represented by the model ligand environments V(acetylacetonate)(3) (17.9+/-3.2%) and K(3)V(catecholate)(3) (13.1+/-4.7%), implying DOPA-like complexation. The third chelated form was represented by the 7-coordinate N(2)O(5) complex Na[V(edta)(H(2)O)] (8.0+/-1.8%). This coordination array, suggestive of a novel mononuclear V(III) protein site, contributed only to fits to samples 1, 2, 3 and 5, which were prepared in the presence of DTT. Endogenous V(IV) (25.2+/-5.4%) was principally modeled as VOCl(2) in 1 M HCl. EPR spectra (averages: A(parallel)=(1.842+/-0.006)x10(-2) cm(-1); A( perpendicular)=(0.718+/-0.007)x10(-2) cm(-1); g(parallel)=1.936+/-0.002; g( perpendicular)=1.990+/-0.001) confirmed the predominance of the aquated vanadyl ion. Blood cell sample five uniquely required the XAS spectrum of VOSO(4) in 0.1 M H(2)SO(4) solution (13.0%) and of [OV(V)(pivalate)(3)] (3.1%) to successfully fit the XAS pre-edge energy region. This endogenous V(V) signal is also unprecedented. These results are compared with those of analogous fits to the blood cells of Ascidia ceratodes and may support assignment of P. nigra to a different genus.     

Effect of variable glutathione peroxidase activity on H2O2-related cytotoxicity in cultured aortic endothelial cells

Primary cultures of porcine aortic endothelial cells were used to assess the effects of O2 intermediates produced by 10-40 mU/ml xanthine oxidase (XO; +2 mM hypoxanthine) or 25-100 mU/ml glucose oxidase (GO; +5 mM glucose). A 60-min incubation in the presence of the enzyme systems resulted in a dose-dependent toxic effect with evidence of cytolysis (increased LDH release) and cell loss (decrease in DNA and protein content), when these indexes were measured 24 hr after completion of the enzyme reaction. Decreased [3H]thymidine incorporation into DNA was the most sensitive index of cell dysfunction for both enzyme systems. The effects of various scavengers and enzymes indicated that H2O2 was the main O2 intermediate involved in the cytotoxicity resulting from the XO-hypoxanthine reaction. Increased glutathione peroxidase activity associated with the addition of 2 X 10(-7) M selenomethionine to culture medium had a partial protective effect which could be related to an increased rate of H2O2 degradation. Evidence for increased DNA synthesis after injury was found in cells previously exposed to XO-hypoxanthine, the degree of increase in [3H]thymidine incorporation being dependent on the intensity of the initial cytotoxicity. Cultured endothelial cells provide a useful tool to evaluate the role of O2 intermediates in endothelial cell injury, to test the effects of protective agents, and to study the repair process.     

Rapid intracellular acidification and cell death by H2O2 and alloxan in pancreatic beta cells

Pancreatic beta-cell death induced by oxidative stress plays an important role in the pathogenesis of diabetes mellitus. We studied the relation between rapid intracellular acidification and cell death of pancreatic beta-cell line NIT-1 cells exposed to H2O2 or alloxan. Intracellular pH was measured by a pH-sensitive dye, and cell damage by double staining with Annexin-V and propidium iodide using flow cytometry. H2O2 and alloxan caused a rapid fall in intracellular pH and suppressed Na+/H+ exchanger activity in the NH4Cl prepulse method. H2O2 induced necrotic cell death, which shifted to apoptotic cell death when initial acidification was prevented by pH clamping to 7.4 using nigericin (unclamped cells vs clamped cells, necrosis 43.8 +/- 5.8% vs 21.1 +/- 10.6%, P < 0.05; apoptosis 8.0 +/- 1.9% vs 44.5 +/- 5.0%, P < 0.01). pH-clamped cells showed enhanced caspase 3 activity and proapoptotic Bax expression. On the other hand, NIT-1 cells were resistant to alloxan toxicity, but treatment with alloxan and nigericin strikingly enhanced the cytotoxicity. Antioxidants partly prevented cell death, although intracellular pH remained similarly acidic. The rapid intracellular acidification was not the cause of cell death but a significant determinant of the mode of death of H2O2 -treated beta cells, whereas no link between cell death and acidification was demonstrated in alloxan toxicity.     

Overexpression of catalase in cytosolic or mitochondrial compartment protects HepG2 cells against oxidative injury.

HepG2 cells were transfected with vectors containing human catalase cDNA and catalase cDNA with a mitochondrial leader sequence to allow comparison of the effectiveness of catalase overexpressed in the cytosolic or mitochondrial compartments to protect against oxidant-induced injury. Overexpression of catalase in cytosol and in mitochondria was confirmed by Western blot, and activity measurement and stable cell lines were established. The intracellular level of H(2)O(2) induced by exogenously added H(2)O(2) or antimycin A was lower in C33 cell lines overexpressing catalase in the cytosol and mC5 cell lines overexpressing catalase in the mitochondria as compared with Hp cell lines transfected with empty vector. Cell death caused by H(2)O(2), antimycin A, and menadione was considerably suppressed in both the mC5 and C33 cell lines. C33 and mC5 cells were also more resistant to apoptosis induced by H(2)O(2) and to the loss of mitochondrial membrane potential induced by H(2)O(2) and antimycin A. In view of the comparable protection by catalase overexpressed in the cytosol versus the mitochondria, catalase produced in both cellular compartments might act as a sink to decompose H(2)O(2) and move diffusable H(2)O(2) down its concentration gradient. The present study suggests that catalase in cytosol and catalase in mitochondria are capable of protecting HepG2 cells against cytotoxicity or apoptosis induced by oxidative stress.     

Characterization of the Ah receptor mediating aryl hydrocarbon hydroxylase induction in the human liver cell line Hep G2

The Ah receptor, a soluble cytoplasmic receptor that regulates induction of cytochrome P450IA1 and mediates toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), was detected and characterized in the continuous human liver cell line Hep G2. The mean concentration of specific binding sites for TCDD was 112 +/- 26 (SEM) fmol/mg cytosol protein as determined in eight separate cytosol preparations in the presence of sodium molybdate. This is equivalent to 14,000 binding sites per cell, approximately 40% of the sites per cell found in the mouse hepatoma line Hepa-1. The cytosolic Ah receptor from Hep G2 cells sedimented at 9 S and was specific for those halogenated and nonhalogenated aromatic compounds known to be agonists for the Ah receptor in rodent tissues and cells. Specific binding in the 9 S region was detected with both [3H]TCDD and 3-[3H]methylcholanthrene. 3-[3H]Methylcholanthrene did not bind to any component besides that at approximately 9 S. Phenobarbital, dexamethasone, and estradiol did not compete with [3H]TCDD for binding to the Hep G2 Ah receptor. Specific binding of [3H]triamcinolone acetonide to glucocorticoid receptor could also be demonstrated in Hep G2 cytosol. The apparent equilibrium dissociation constant (Kd) for binding of [3H]TCDD to Hep G2 Ah receptor was 9 nM by Woolf plot analysis, about an order of magnitude weaker than the affinity of [3H]TCDD for the mouse Hepa-1 Ah receptor or for the C57BL/6 murine hepatic Ah receptor. [3H]TCDD.Ah receptor complex, which was extracted from nuclei of Hep G2 cells incubated with [3H]TCDD at 37 degrees C in culture, sedimented at approximately 6 S under conditions of high ionic strength. Aryl hydrocarbon hydroxylase (AHH) activity was significantly induced after 24 h of incubation with polycyclic aromatic hydrocarbons: the EC50 for AHH induction was 5.3 microM for benz(a)anthracene and 1.3 microM for 3-methylcholanthrene. Modification of the preparative technique for cell cytosol, especially inclusion of 20 mM sodium molybdate in homogenizing and other buffers, was necessary to detect cytosolic Hep G2 Ah receptor. Hep G2 cells appear to conserve drug-metabolizing activity associated with cytochrome P450IA1 as well as the receptor mechanism which regulates its induction.     

DNA damage and apoptosis in hydrogen peroxide-exposed Jurkat cells: bolus addition versus continuous generation of H(2)O(2)

Aspects of the molecular mechanism(s) of hydrogen peroxide-induced DNA damage and cell death were studied in the present investigation. Jurkat T-cells in culture were exposed either to low rates of continuously generated H(2)O(2) by the action of glucose oxidase or to a bolus addition of the same agent. In the first case, steady state conditions were prevailing, while in the latter, H(2)O(2) was removed by the cellular defense systems following first order kinetics. By using single-cell gel electrophoresis (also called comet assay), an initial increase in the formation of DNA single-strand breaks was observed in cells exposed to a bolus of 150 microM H(2)O(2). As the H(2)O(2) was exhausted, a gradual decrease in DNA damage was apparent, indicating the existence of an effective repair of single-strand breaks. Addition of 10 ng glucose oxidase in 100 microl growth medium (containing 1.5 x 10(5) cells) generated 2.0 +/- 0.2 microM H(2)O(2) per min. This treatment induced an increase in the level of single-strand breaks reaching the upper limit of detection by the methodology used and continued to be high for the following 6 h. However, when a variety of markers for apoptotic cell death (DNA cell content, DNA laddering, activation of caspases, PARP cleavage) were examined, only bolus additions of H(2)O(2) were able to induce apoptosis, while the continuous presence of this agent inhibited the execution of the apoptotic process no matter whether the inducer was H(2)O(2) itself or an anti-Fas antibody. These observations stress that, apart from the apparent genotoxic and proapoptotic effects of H(2)O(2), it can also exert antiapoptotic actions when present, even at low concentrations, during the execution of apoptosis.