H2O2 and cGMP may function as an O2 sensor in the pulmonary artery

The effects of O2 tension on force in precontracted isolated pulmonary arterial smooth muscle from calf lungs was characterized to investigate the mechanism of O2 tension sensing. These arteries display a decrease in force with increasing O2 tension that is antagonized via inhibition of soluble guanylate cyclase activation by 10 microM methylene blue or inactivation of catalase by pretreatment with 50 mM 3-amino-1,2,4-triazole for 30 min. O2 tension-dependent relaxation is associated with an increase in intracellular H2O2 metabolism through catalase (detected as the peroxide-dependent inactivation of tissue catalase activity by aminotriazole) and cyclic guanosine 5'-monophosphate (cGMP), known mediators of relaxation in calf pulmonary arteries. Thus a recently reconstructed mechanism of activation of soluble guanylate cyclase involving the metabolism of H2O2 by catalase appears to function as an O2 tension sensor in pulmonary arteries.     

Increased activity of H2O2 in aorta isolated from chronically streptozotocin-diabetic rats: effects of antioxidant enzymes and enzymes inhibitors.

The effects of hydrogen peroxide (H2O2, 1 nM-5 mM) on the tone of the rings of aorta precontracted with phenylephrine (PE) were studied in 4-5 months streptozotocin (STZ)-diabetic rats and their age-matched controls. H2O2 induced brief contraction before relaxation in endothelium-containing rings that was more pronounced in diabetic rats. Removal of the endothelium or pretreatment of rings with N(G)-nitro-L-arginine methyl ester (L-NAME, 100 microM) abolished H2O2-induced immediate and transient increase in tone, but preincubation with indomethacin (10 microM) had no effect on contractions induced by H2O2 in both group of animals. Pretreatment with L-NAME or indomethacin as well as absence of endothelium produced an inhibition of H2O2-induced relaxation that was more pronounced in diabetic rings. Chronically STZ-diabetes resulted in a significant increase in H2O2-induced maximum relaxation that was largely endothelium-dependent. Decreased sensitivity (pD2) of diabetic vessels to vasorelaxant action of H2O2 was normalized by superoxide dismutase (SOD, 80 U/ml). Pretreatment with SOD had no effect on H2O2-induced maximum relaxations in both group of animals but led to an increase in H2O2-induced contractions in control rats. When the rings pretreated with diethyldithiocarbamate (DETCA, 5 mM), H2O2 produced only contraction in control rats, and H2O2-induced relaxations were markedly depressed in diabetic rats. H2O2 did not affect the tone of intact or endothelium-denuded rings in the presence of catalase (2000 U/ml). Aminotriazole (AT, 10 mM) failed to affect H2O2-induced contractions or relaxations in all rings. Our observations suggest that increased production of oxygen-derived free radicals (OFRs) in diabetic state leads to a decrease in SOD activity resulting an increase in endogenous superoxide anions (O2*-), that is limited cytotoxic actions, and an increase in catalase activity resulting a decrease in both H2O2 concentrations and the production of harmful hydroxyl radical (*OH) in diabetic aorta in long-term. Present results indicate that increased vascular activity of H2O2 may be an important factor in the development of vascular disorders associated with chronically diabetes mellitus. Enhanced formation of *OH, that is a product of exogenous H2O2 and excess O2*, seems to be contribute to increased relaxations to exogenously added H2O2 in chronically diabetic vessels.     

Hydrogen peroxide mediates a transient vasorelaxation with tempol during oxidative stress

Tempol catalyzes the formation of H(2)O(2) from superoxide and relaxes blood vessels. We tested the hypothesis that the generation of H(2)O(2) by tempol in vascular smooth muscle cells during oxidative stress contributes to the vasorelaxation. Tempol and nitroblue tetrazolium (NBT) both metabolize superoxide in vascular smooth muscle cells, but only tempol generates H(2)O(2). Rat pressurized mesenteric arteries were exposed for 20 min to the thromboxane-prostanoid receptor agonist, U-46619, or norepinephrine. During U-46619, tempol caused a transient dilation (22 +/- 2%), whereas NBT was ineffective (2 +/- 1%), and neither dilated vessels constricted with norepinephrine, which does not cause vascular oxidative stress. Neither endothelium removal nor blockade of K(+) channels with 40 mM KCl affected the tempol-induced dilation, but catalase blunted the tempol dilation by 53 +/- 7%. Tempol, but not NBT, increased H(2)O(2) in rat mesenteric vessels detected with dichlorofluorescein. To test physiological relevance in vivo, topical application of tempol caused a transient dilation (184 +/- 20%) of mouse cremaster arterioles exposed to angiotensin II for 30 min, which was not seen with NBT (9 +/- 4%). The vasodilation to tempol was reduced by 68 +/- 6% by catalase. We conclude that the transient relaxation of blood vessels by tempol after prolonged exposure to U-46619 or angiotensin II is mediated in part via production of H(2)O(2) and is largely independent of the endothelium and potassium channels.     

Leaky intra-acinar arteries in rat lungs perfused with hydrogen peroxide

We have investigated the effects of H2O2 (150 or 300 microM) on the ultrastructure and permeability of the pulmonary endothelium in rat lungs perfused for 60 min with buffered Hanks' bovine serum albumin medium. In one group of experiments, we examined the effect of H2O2 on the uptake and transport of cationized ferritin (CF) by endothelial cells in intra-acinar arteries, alveolar capillaries, and interlobular veins. The influence of the oxidant on endothelial adsorptive endocytic processes was assessed by measuring the density of ferritin particles in luminal vesicles, multivesicular bodies, and basal lamina. In a second group of experiments, we examined the effects of H2O2 on the fine structure and permeability to electron-dense macromolecules of arterial, microvascular, and venous endothelium. For this purpose, at the end of the 60-min perfusion with H2O2, CF was perfused to identify leaky vessels. We found that H2O2 caused a dose-dependent inhibition of transcytosis of CF in all vascular segments. At the lower dose of H2O2, inhibition of transcytotic activity was not associated with structural injury to the vascular endothelium or with elevation of wet-to-dry ratios. At the higher oxidant dose, inhibition of transcytosis was associated with leaky arterial endothelium and elevation of wet-to-dry ratios (6.44 +/- 0.12 vs. 5.64 +/- 0.16, P less than 0.02). The effects of H2)2 were prevented by adding catalase to the perfusate. The selective loss of structural integrity and leakiness of the arterial endothelium were diminished but not completely abolished by perfusing the oxidant retrograde from the venous side.(ABSTRACT TRUNCATED AT 250 WORDS)     

Parathyroid hormone-related protein-mediated responses in pulmonary arteries and veins of newborn lambs

PTHrP has important roles in lung development and function. Here we determined the vasomotor responses of isolated pulmonary arteries and veins of newborn and adult sheep to PTHrP. In vessels constricted with endothelin-1, PTHrP (PTHrP 1-34) caused greater relaxation of veins than of arteries. In both vessel types, relaxation to the peptide was less in adult than in newborn vessels. In newborn lambs, PTHrP-induced relaxation was not affected by endothelium removal, inhibition of eNOS, or inhibition of adenylyl cyclases by SQ-22536. However, relaxation was attenuated by 4-aminopyridine, inhibitor of voltage-dependent potassium channels, in both arteries and veins, and by charybdotoxin, inhibitor of calcium-activated potassium channels, in veins. When vessels were saturated with 8-BrcAMP (3 x 10(-4) M), to eliminate relaxation mediated by endogenous cAMP, PTHrP-induced relaxation was partially attenuated. In vessels treated with 8-BrcAMP (3 x 10(-4) M), 4-aminopyridine but not charybdotoxin inhibited relaxation induced by PTHrP 1-34 in both arteries and veins. Radioimmunoassay showed that, in the presence of a general phosphodiesterase inhibitor, PTHrP caused a concentration-dependent increase in intracellular cAMP content in arteries and veins, which was largely abolished by SQ-22536. Our results demonstrate that PTHrP is a potent vasodilator of pulmonary vessels, with a greater effect in veins than in arteries. Relaxation induced by the peptide contains both cAMP-dependent and -independent components. In both arteries and veins, voltage-dependent potassium channels mediate the response to PTHrP, at least in part, in a cAMP-independent fashion; and in veins, calcium-activated potassium channels may be stimulated by elevated cAMP levels.     

Is hydrogen peroxide an EDHF in human radial arteries?

In human radial arteries, a nitric oxide/prostanoid-independent mechanism that has the pharmacological characteristics of an EDHF contributes to endothelium-dependent relaxation. H2O2 can act as an EDHF in some vascular beds. We examined the hypothesis that endogenously produced H2O2 mediated the nitric oxide/prostanoid-independent relaxation to carbachol in radial arteries obtained from patients undergoing coronary artery bypass surgery. Superoxide levels, measured by chemiluminescence, were similar in radial and internal mammary arteries, but immunohistochemical staining for Cu/Zn superoxide dismutase (SOD) was lower in endothelium from radial arteries. In organ chamber studies, neither addition of catalase nor addition of SOD to the bathing fluid modified nitric oxide/prostanoid-independent relaxations to carbachol in radial arteries. However, nitric oxide-dependent vasorelaxation was enhanced in the presence of SOD. Thus the nitric oxide/prostanoid-independent relaxation to carbachol is not due to H2O2 and, unlike nitric oxide-mediated vasorelaxation, is not attenuated by superoxide. Blood vessels showing EDHF-mediated relaxations resistant to oxidative stress may provide favorable outcomes in revascularization surgery.     

Stable H2O2-resistant variants of Chinese hamster fibroblasts demonstrate increases in catalase activity

Hydrogen peroxide (H2O2)-resistant variants of the Chinese hamster ovary HA-1 line have been derived by culturing cells in progressively higher concentrations of H2O2 (greater than 200 days, in 50-800 microM H2O2). The H2O2-resistant phenotype has been stable for over 60 passages (240 days) following removal from the H2O2 stress. The resistant cells demonstrate both increased capacity to deplete exogenously added H2O2 from the growth medium and increased catalase activity. H2O2 resistance correlates well with catalase activity. An increase in chromosome number occurred in the cells adapted to 200-800 microM H2O2, but increases in aneuploidy and tetraploidy were not necessary for resistance. These results suggest that adaptation to chronic oxidative stress mediated by H2O2 in mammalian cells is accompanied by a stable heritable change in expression of catalase activity.     

Increased H(2)O(2) counteracts the vasodilator and natriuretic effects of superoxide dismutation by tempol in renal medulla

A membrane-permeable SOD mimetic, 4-hydroxytetramethyl-piperidine-1-oxyl (tempol), has been used as an antioxidant to prevent hypertension. We recently found that this SOD mimetic could not prevent development of hypertension induced by inhibition of renal medullary SOD with diethyldithiocarbamic acid. The present study tested a hypothesis that increased H2O2 counteracts the effects of tempol on renal medullary blood flow (MBF) and Na+ excretion (UNaV), thereby restraining the antihypertensive effect of this SOD mimetic. By in vivo microdialysis and Amplex red H2O2 microassay, it was found that interstitial H2O2 levels in the renal cortex and medulla in anesthetized rats averaged 55.91 +/- 3.66 and 102.18 +/- 5.16 nM, respectively. Renal medullary interstitial infusion of tempol (30 micromol x min-1x kg-1) significantly increased medullary H2O2 levels by 46%, and coinfusion of catalase (10 mg x min-1x kg-1) completely abolished this increase. Functionally, removal of H2O2 by catalase enhanced the tempol-induced increase in MBF, urine flow, and UNaV by 28, 41, and 30%, respectively. Direct delivery of H2O2 by renal medullary interstitial infusion (7.5-30 nmol x min-1x kg-1) significantly decreased renal MBF, urine flow, and UNaV, and catalase reversed the effects of H2O2. We conclude that tempol produces a renal medullary vasodilator effect and results in diuresis and natriuresis. However, this SOD mimetic increases the formation of H2O2, which constricts medullary vessels and, thereby, counteracts its vasodilator actions. This counteracting effect of H2O2 may limit the use of tempol as an antihypertensive agent under exaggerated oxidative stress in the kidney.     

Calcitonin gene-related peptide vasodilation of human pulmonary vessels

Human calcitonin gene-related peptide (CGRP) is localized to sensory neurons in pulmonary vessels and is a potent vasodilator. We have characterized the effects of CGRP in human pulmonary vessels and localized the receptors for this peptide by autoradiography. Fresh human lung tissue was obtained from eight patients undergoing surgery and small (200-400 microns ID) pulmonary arteries and veins were dissected free of surrounding connective and pulmonary tissue. Pairs of vessels were studied and in one of each pair the endothelium was left intact and from the other of each pair the endothelium was removed by gentle abrasion. For functional studies arteries (n = 9) and veins (n = 9) were suspended in an organ bath, precontracted with 1 microM prostaglandin F2 alpha. CGRP (10 pM to 10 microM) was added in a cumulative manner. CGRP caused a dose-dependent relaxation of endothelium intact human pulmonary arteries and veins with log EC50 values of -8.01 +/- 0.35 and -8.70 +/- 0.40, respectively (not significant). Removal of the endothelium did not diminish the vasodilator potency of CGRP in either vessel. For autoradiographic studies, cryostat sections of the small human pulmonary vessels with or without endothelium were used. 125I-CGRP densely labeled CGRP receptors on vascular smooth muscle and endothelial removal did not have any effect on grain density. We concluded that CGRP is a potent vasodilator of human pulmonary arteries and veins that is not dependent on an intact endothelium. These functional studies correlate with the distribution of CGRP receptors as localized by autoradiography.     

Hypertension alters role of iNOS, COX-2, and oxidative stress in bradykinin relaxation impairment after LPS in rat cerebral arteries

This study was performed to investigate the role of reactive oxygen species and inducible nitric oxide (NO) synthase (iNOS) and cyclooxygenase-2 (COX-2) metabolites in the lipopolysaccharide effect on bradykinin-induced relaxation in middle cerebral arteries from normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs). LPS exposure (10 microg/ml for 1-5 h) reduced bradykinin relaxation; this effect appeared earlier and was greater in arteries from SHR than WKY rats. LPS also reduced the relaxation to the NO donor diethylamine (DEA)-NO; however, LPS modified neither the bradykinin relaxation after inhibiting NO synthesis with N(G)-monomethyl-L-arginine (0.1 mM) nor endothelial NOS expression. In arteries from WKY rats, the respective iNOS and COX-2 inhibitors aminoguanidine (0.1 mM) and NS-398 (10 microM) and the superoxide anion scavenger SOD (100 U/ml) reduced the LPS effect on bradykinin relaxation; however, the thromboxane A(2) (TxA(2))PGH(2) receptor antagonist SQ-29548 (1 microM) and the H(2)O(2) scavenger catalase (1,000 U/ml) did not modify the LPS effect. In arteries from SHR, all of these drugs reduced the LPS effect. LPS exposure (5 h) increased superoxide anion levels in arteries from both strains and TxA(2) levels only in SHR. COX-2 expression rose to a similar level in arteries from both strains after 1 and 5 h of LPS incubation, whereas expression of Cu/Zn- and Mn-SOD only increased after 5 h. In conclusion, in segments from WKY rats, LPS reduced bradykinin-induced relaxation through increased production of NO (from iNOS) and superoxide anion. The greater LPS effect observed in arteries from SHR seems to be related to higher participation of reactive oxygen species and contractile prostanoids (probably TxA(2)).     

The mechanism of EDHF-mediated responses in subcutaneous small arteries from healthy pregnant women

We studied the importance of endothelium-derived hyperpolarizing factor (EDHF) vs. nitric oxide (NO) and prostacyclin (PGI(2)) in bradykinin (BK)-induced relaxation in isolated small subcutaneous arteries from normal pregnant women. We also explored the contribution of cytochrome P-450 (CYP450) product of arachidonic acid (AA) metabolism, hydrogen peroxide (H(2)O(2)), and gap junctions that have been suggested to be involved in EDHF-mediated responses. Isolated arteries obtained from subcutaneous fat biopsies of normal pregnant women (n = 30) undergoing planned cesarean section were mounted in a wire-myography system. In norepinephrine-constricted vessels, incubation with N(G)-nitro-L-arginine methyl ester (L-NAME) resulted in a significant reduction in relaxation to BK. Simultaneous incubation with L-NAME and indomethacin failed to modify this response further. BK-mediated dilatation in the presence of K(+)-modified solution was decreased to similar level as obtained after incubation with L-NAME. Incubation with L-NAME abolished BK-induced responses in K(+)-modified solution. Sulfaphenazole, a specific inhibitor of CYP450 epoxygenase, and catalase (an enzyme that decomposes H(2)O(2)) did not affect the EDHF-mediated relaxation because concentration-response curves to BK were similar in arteries after incubation with L-NAME vs. L-NAME + sulfaphenazole and L-NAME + catalase. The inhibitor of gap junctions, 18 alpha-glycyrrhetinic acid, significantly reduced BK-mediated relaxation both without and with incubation with L-NAME. We found that both NO and EDHF, but not PGI(2), are involved in the endothelium-dependent dilatation to BK. BK-induced relaxation is almost equally mediated by NO and EDHF. CYP450 epoxygenase metabolites of AA or H(2)O(2) do not account for EDHF-mediated response; however, gap junctions are involved in the EDHF-mediated responses to BK in subcutaneous small arteries in normal pregnancy.     

Reactivity of human placental chorionic plate vessels from pregnancies complicated by intrauterine growth restriction (IUGR)

A successful pregnancy is dependent on liberal placental perfusion via the maternal and fetal circulations. Doppler waveform analyses of umbilical arteries suggest increased resistance to flow in the fetoplacental circulation of pregnancies complicated by intrauterine growth restriction (IUGR). Neither the site nor the mediators responsible for this altered vascular reactivity are known, to date. In placentas in normal pregnancy, reduced oxygenation promotes contraction of the in vitro-perfused placental cotyledon and modulates agonist-induced contraction of chorionic plate arteries and veins. Placental oxygenation has also been suggested to be reduced in IUGR. We tested the hypothesis that oxygen tension could directly modify placental chorionic plate vessel vasoreactivity in IUGR. Small arteries and veins from the chorionic plate were dissected from biopsies from placentas of pregnancies complicated by IUGR and were studied using parallel wire myography. Vasoconstriction at 20%, 7%, and 2% oxygen was assessed utilizing the thromboxane mimetic U46619. Experiments were also performed in the presence of 4-aminopyridine (4AP), a blocker of voltage-gated potassium channels. Increased oxygenation reduced venous vasoconstriction but did not modify arterial vasoconstriction. 4AP increased basal tone in arteries and veins. We suggest that venoconstriction in response to hypoxia may provide a mechanism for increased fetoplacental vascular resistance associated with IUGR.     

Prostacyclin producing activity of human umbilical, placental and uterine vessels

Prostacyclin-like material producing activity of umbilical, placental and uterine vessels was studied. Umbilical arteries and veins were separated at sites 10-15cm and 1-2cm from insertion of the umbilical cord to the placenta. Placental arteries and veins were prepared from the first, second and third branches on the chorionic plate. Uterine vessels were obtained at abdominal hysterectomy. After incubation of each specimen in Tris buffer 1 ml (pH8.5, 0.5M) for 30 min at room temperature, the inhibitory effect of the medium on ADP induced platelet aggregation was measured and the prostacyclin-like material was quantified. These procedures were repeated consecutively four times in total for each specimen. Prostacyclin-like material production rate and its total production were calculated. In total prostacyclin-like material production, umbilical arteries and veins were much higher than placental arteries and veins respectively (p less than 0.001), but there was no significant difference between placental and uterine vessels. These results showed that prostacyclin-like material producing activity of blood vessels declined remarkably at the transitive region from umbilical to placental vessels. It seems that this distribution of vascular prostacyclin-like material producibility in the fetoplacental vascular system correlates with that of vascular reactivity to prostacyclin.     

Role of glutathione in the adaptive tolerance to H2O2

Endogenous antioxidant defense systems are enhanced by various physiological stimuli including sublethal oxidative challenges, which induce tolerance to subsequent lethal oxidative injuries. We sought to evaluate the contributions of catalase and the glutathione system to the adaptive tolerance to H2O2. For this purpose, H9c2 cells were stimulated with 100 microM H2O2, which was the maximal dose at which no significant acute cell damage was observed. Twenty-four hours after stimulation, control and pretreated cells were challenged with a lethal concentration of H2O2 (300 microM). Compared with the control cells, pretreated cells were significantly tolerant of H2O2, with reduced cell lysis and improved survival rate. In pretreated cells, glutathione content increased to 48.20 +/- 6.38 nmol/mg protein versus 27.59 +/- 2.55 nmol/mg protein in control cells, and catalase activity also increased to 30.82 +/- 2.64 versus 15.46 +/- 1.29 units/mg protein in control cells, whereas glutathione peroxidase activity was not affected. Increased glutathione content was attributed to increased gamma-glutamylcysteine synthetase activity, which is known as the rate-limiting enzyme of glutathione synthesis. To elucidate the relative contribution of the glutathione system and catalase to tolerance of H2O2, control and pretreated cells were incubated with specific inhibitors of gamma-glutamyl cysteine synthetase (L-buthionine sulfoximine) or catalase (3-amino-1,2,4-triazole), and challenged with H2O2. Cytoprotection by the low-dose H2O2 pretreatment was almost completely abolished by L-buthionine sulfoximine, while it was preserved after 3-amino-1,2,4-triazole treatment. From these results, it is concluded that both the glutathione system and catalase can be enhanced by H2O2 stimulation, but increased glutathione content rather than catalase activity was operative in the tolerance of lethal oxidative stress.     

Changes in placental adipocytokine gene expression associated with gestational diabetes mellitus

Leptin and adiponectin, two adipocytokines, may work together in regulating energy homeostasis and insulin action. Leptin gene expression has been investigated in term placental tissue complicated by gestational diabetes mellitus (GDM), but never in conjunction with all isoforms of the leptin receptor (LEPR A-D), or with adiponectin receptors (ADIPOR1 and 2). In this study we examined the association between changes in expression of these genes in placental tissue and GDM risk. We assessed placental gene expression of leptin, LEPR A-D and ADIPOR1 and 2 by real time PCR using mRNA from maternal and fetal biopsies. Tissues were collected from uncomplicated pregnancies (n=28) and those complicated by GDM (n=19). Gene expression was normalized to three endogenous housekeeping genes. Relative gene expression values were reported as fold change between groups. Adiponectin gene expression was out of the sensitive range of our assay. There were increases in leptin mRNA expression in GDM cases compared with controls for maternal-side (p=0.06), and fetal-side (p=0.09) placental biopsies. No significant changes were seen in GDM cases compared with controls in LEPR A-D or ADIPOR1 and 2. mRNA derived from maternal-side tissue was positively correlated with tissue from the fetal side for all genes studied (all p<0.01). Finally, we noted that absence or presence of GDM was a major factor in leptin mRNA expression after adjusting for maternal age, mode of delivery, parity and smoking status. In conclusion, increases in leptin mRNA expression in term placenta, but not that of its receptors, are associated with the diagnosis of GDM. Changes seen in the ligand, but not the receptor, of the leptin pathway in GDM-complicated pregnancies may also apply to the adiponectin pathway, as the ADIPOR1 and 2 mRNAs do not change with GDM diagnosis.     

H(2)O(2) mediates Ca(2+)- and MLC(20) phosphorylation-independent contraction in intact and permeabilized vascular muscle

One purpose of the current study was to establish whether vasoconstriction occurs in all vessel types in response to H(2)O(2). Isometric force was measured in pulmonary venous and arterial rings, and isobaric contractions were measured in mesenteric arteries and veins in response to H(2)O(2). A second purpose was to determine whether H(2)O(2)-induced contraction is calcium independent. The addition of H(2)O(2) to calcium-depleted (using the Ca(2+) ionophore ionomycin in zero calcium EGTA buffer) muscle caused contraction. Furthermore, permeabilized muscle contracted in response to H(2)O(2) even in zero Ca(2+). The final purpose was to determine whether the 20-kDa regulatory myosin light chain (MLC(20)) phosphorylation plays a role in H(2)O(2)-induced contraction. Pulmonary arterial strips were freeze-clamped at various time points during H(2)O(2)-induced contractions, and the relative amounts of phosphorylated MLC(20) were measured. H(2)O(2) caused dose-dependent contractions that were independent of MLC(20) phosphorylation. ML-9, a myosin light chain kinase inhibitor, had no effect on the H(2)O(2) contractile response. In conclusion, H(2)O(2) induces Ca(2+)- and MLC(20) phosphorylation-independent contraction in pulmonary and systemic arterial and venous smooth muscle.     

Maintenance of higher H(2)O(2) levels, and its mechanism of action to induce growth in breast cancer cells: Important roles of bioactive catalase and PP2A

We assessed the catalase bioactivity and hydrogen peroxide (H(2)O(2)) production rate in human breast cancer (HBC) cell lines and compared these with normal human breast epithelial (HBE) cells. We observed that the bioactivity of catalase was decreased in HBC cells when compared with HBE cells. This was also accompanied by an increase in H(2)O(2) steady-state levels in HBC cells. Silencing the catalase gene led to a further increase in the steady-state level of H(2)O(2) which was also accompanied by an increase in growth rate of HBC cells. Catalase activity was up regulated on treatment with superoxide (O(2)(-)) scavengers such as pegylated SOD (PEG-SOD, indicating inhibition of catalase by the increased O(2)(-) produced by HBC cells. Transfection of either catalase or glutathione peroxidase to HBC cells decreased intracellular H(2)O(2) levels and led to apoptosis of these cells. The H(2)O(2) produced by HBC cells inhibited PP2A activity accompanied by increased phosphorylation of Akt and ERK1/2. The importance of catalase bioactivity in breast cancer was further confirmed as its bioactivity was also decreased in human breast cancer tissues when compared to normal breast tissues. We conclude that inhibition of catalase bioactivity by O(2)(-) leads to an increase in steady-state levels of H(2)O(2) in HBC cells, which in turn inhibits PP2A activity, leading to phosphorylation of ERK 1/2 and Akt and resulting in HBC cell proliferation.