Characterization of reactive oxygen species induced effects on human spermatozoa movement and energy metabolism

Reactive oxygen species (ROS) inhibit sperm movement and have been implicated in male infertility. In this study, we determined the effects of specific ROS produced by activated leukocytes on human spermatozoa and investigated their metabolic site of action. We used chemiluminescence and electron paramagnetic resonance (EPR) to characterize the ROS generated by both blood and seminal leukocytes. We also determined the effects of these ROS on sperm energy metabolism using biochemical analyses and flow cytometry. Both blood and seminal leukocytes produced the same characteristic ROS which were determined to be hydrogen peroxide (H2O2) and superoxide radicals (O2*-). EPR using the spin trapping technique indicated that superoxide radical-dependent hydroxyl radicals (HO.) were also generated. ROS generated by PMA-stimulated blood leukocytes (2-5 x 10(6)/ml) caused inhibition of sperm movement in 2 h (p < .01). Using the hypoxanthine/ xanthine oxidase (0.5 U/ml) system to generate ROS, we determined that spermatozoa ATP levels, after ROS treatment, were reduced approximately eight-fold in 30 min (0.10 x 10(10) moles/10(6) sperm cells) compared to control (0.84 X 10(-10) moles/10(6) sperm cells) (p < .01). Sperm ATP reduction paralleled the inhibition of sperm forward progression. Neither superoxide dismutase (100 U/ml) nor dimethyl sulfoxide (100 mM) reversed these effects; however, protection was observed with catalase (4 X 10(3) U/ml). Flow cytometric analyses of sperm treated with various doses of H2O2 (0.3 mM-20.0 mM) showed a dose-dependent decrease in sperm mitochondrial membrane potential (MMP); however, at low concentrations of H2O2, sperm MMP was not significantly inhibited. Also, sperm MMP uncoupling with CCClP had no effect on either sperm ATP levels or forward progression. These results indicate that H2O2 is the toxic ROS produced by activated leukocytes causing the inhibition of both sperm movement and ATP production. O2*- and HO. do not play a significant role in these processes. Low concentrations of H2O2 causing complete inhibition of sperm movement and ATP levels inhibit sperm energy metabolism at a site independent of mitochondrial oxidative phosphorylation.     

Synoviocytes, not chondrocytes, release free radicals after cycles of anoxia/re-oxygenation

By oxymetry and electron paramagnetic resonance (EPR), we investigated the effects of repeated anoxia/re-oxygenation (A/R) periods on the respiration and production of free radicals by synoviocytes (rabbit HIG-82 cell line and primary equine synoviocytes) and equine articular chondrocytes. Three periods of 20 min anoxia followed by re-oxygenation were applied to 10(7)cells; O(2) consumption was measured before anoxia and after each re-oxygenation. After the last A/R, cellular free radical formation was investigated by EPR spectroscopy with spin trapping technique (n=3 for each cell line). Both types of synoviocytes showed a high O(2) consumption, which was slowered after anoxia. By EPR with the spin trap POBN, we proved a free radical formation. Results were similar for equine and rabbit synoviocytes. For chondrocytes, we observed a low O(2) consumption, unchanged by anoxia, and no free radical production. These observations suggest an oxidant activity of synoviocytes, potentially important for the onset of osteoarthritis.     

Comparison of the antinociceptive effect of celecoxib, diclofenac and resveratrol in the formalin test

The peripheral antinociceptive effect of the selective COX-2 inhibitor celecoxib in the formalin-induced inflammatory pain was compared with that of resveratrol (COX-1 inhibitor) and diclofenac (non-selective COX inhibitor). Rats received local pretreatment with saline, celecoxib, diclofenac or resveratrol followed by 50 microl of either 1% or 5% formalin. Peripheral administration of celecoxib did not produce antinociception at either formalin concentration. In contrast, diclofenac and resveratrol produced a dose-dependent antinociceptive effect in the second phase of both 1% and 5% formalin test. The peripheral antinociception produced by diclofenac or resveratrol was due to a local action, as drug administration in the contralateral paw was ineffective. Results indicate that the selective COX-2 inhibitor celecoxib does not produce peripheral antinociception in formalin-induced inflammatory pain. In contrast, selective COX-1 and non-selective COX inhibitors (resveratrol and diclofenac, respectively) are effective drugs in this model of pain.     

Effects of nimesulide, a preferential cyclooxygenase-2 inhibitor, on carrageenan-induced pleurisy and stress-induced gastric lesions in rats

Intrapleural injection of carrageenan in rats increased prostaglandin E₂ (PGE₂) production and induced newly synthesized cyclooxygenase-2 (COX-2) in pleural exudate cells without affecting COX-1 levels. Nimesulide, a preferential inhibitor of COX-2, reduced pleural PGE₂ production and was almost as active as indomethacin and 10 times more active than ibuprofen. Only COX-1, and no COX-2, was detected in gastric mucosal cells, and PGE₂ concentration of gastric mucosa was significantly decreased by indomethacin and ibuprofen. The decrease in gastric PGE₂ production induced by indomethacin and ibuprofen was enhanced in stressed rats, resulting in aggravation of stress-induced gastric lesions at anti-inflammatory doses. However, nimesulide did not produce stress-induced gastric lesions even at 30 times the anti-inflammatory dose. This supports the hypothesis that inhibition of COX-1 causes unwanted side effects and inhibition of COX-2 produces anti-inflammatory effects.     

Hypoxic constriction and reactive oxygen species in porcine distal pulmonary arteries

To determine whether reactive oxygen species (ROS) play an essential role in hypoxic pulmonary vasoconstriction (HPV) and the cellular locus of ROS production and action during HPV, we measured internal diameter (ID) at constant transmural pressure, lucigenin-derived chemiluminescence (LDCL), and electron paramagnetic resonance (EPR) spin adduct spectra in small distal porcine pulmonary arteries, and dichlorofluorescein (DCF) fluorescence in myocytes isolated from these arteries. Hypoxia (4% O2) decreased ID, increased DCF fluorescence, tended to increase LDCL, and in some preparations produced EPR spectra consistent with hydroxyl and alkyl radicals. Superoxide dismutase (SOD, 150 U/ml) or SOD + catalase (CAT, 200 U/ml) did not alter ID during normoxia but reduced or abolished the constriction induced by hypoxia. SOD also blocked HPV in endothelium-denuded arteries after restoration of the response by exposure to 10-10 M endothelin-1. Confocal fluorescence microscopy demonstrated that labeled SOD and CAT entered pulmonary arterial myocytes. SOD, SOD + CAT, and CAT blocked the increase in DCF fluorescence induced by hypoxia, but SOD + CAT and CAT also caused a stable increase in fluorescence during normoxia, suggesting that CAT diminished efflux of DCF from cells or oxidized the dye directly. We conclude that HPV required increased concentrations of ROS produced by and acting on pulmonary arterial smooth muscle rather than endothelium.     

Oxidation of tetrahydrobiopterin by peroxynitrite or oxoferryl species occurs by a radical pathway.

The molecular mechanisms of tetrahydrobiopterin (BH4) oxidation by peroxynitrite (ONOO-) was studied using ultra-weak chemiluminescence, electron paramagnetic resonance (EPR) and UV-visible diode-array spectrophotometry, and compared to BH4 oxidation by oxoferryl species produced by the myoglobin/hydrogen peroxide (Mb/H2O2) system. The oxidation of BH4 by ONOO- produced a weak chemiluminescence, which was altered by addition of 50 mM of the spin trap alpha-(4-pyridyl-1-oxide)-N-tert butylnitrone (POBN). EPR spin trapping demonstrated that the reaction occurred at least in part by a radical pathway. A mixture of two spectra composed by an intense six-line spectrum and a fleeting weak nine-line one was observed when using ONOO-. Mb/H2O2 produced a short-living light emission that was suppressed by the addition of BH4. Simultaneous addition of POBN, BH4 and Mb/H2O2 produced the same six-line EPR spectrum, with a signal intensity depending on BH4 concentration. Spectrophotometric studies confirmed the rapid disappearance of the characteristic peak of ONOO- (302 nm) as well as substantial modifications of the initial BH4 spectrum with both oxidant systems. These data demonstrated that BH4 oxidation, either by ONOO- or by Mb/H2O2, occurred with the production of activated species and by radical pathways.     

Molecular dynamics simulations of arachidonic acid-derived pentadienyl radical intermediate complexes with COX-1 and COX-2: insights into oxygenation regio- and stereoselectivity

The two cyclooxygenase enzymes, COX-1 and COX-2, are responsible for the committed step in prostaglandin biosynthesis and are the targets of the nonsteroidal antiinflammatory drugs aspirin and ibuprofen and the COX-2 selective inhibitors, Celebrex, Vioxx, and Bextra. The enzymes are remarkable in that they catalyze two dioxygenations and two cyclizations of the native substrate, arachidonic acid, with near absolute regio- and stereoselectivity. Several theories have been advanced to explain the nature of enzymatic control over this series of reactions, including suggestions of steric shielding and oxygen channeling. As proposed here, selective radical trapping and spin localization in the substrate-derived pentadienyl radical intermediate can also be envisioned. Herein we describe the results of explicit, 10 ns molecular dynamics simulations of both COX-1 and COX-2 with the substrate-derived pentadienyl radical intermediate bound in the active site. The enzymes' influence on the conformation of the pentadienyl radical was investigated, along with the accessible space above and below the radical plane and the width of several channels to the active site that could function as access routes for molecular oxygen. Additional simulations demonstrated the extent of molecular oxygen mobility within the active site. The results suggest that spin localization is unlikely to play a role in enzymatic control of this reaction. Instead, a combination of oxygen channeling, steric shielding, and selective radical trapping appears to be responsible. This work adds a dynamic perspective to the strong foundation of static structural data available for these enzymes.     

Oxygen consumption and electron spin resonance studies of free radical production by alveolar cells exposed to anoxia: inhibiting effects of the antibiotic ceftazidime

By EPR spectroscopy, we investigated free radical production by cultured human alveolar cells subjected to anoxia/re-oxygenation (A/R), and tested the effects of ceftazidime, an antibiotic previously demonstrated to possess antioxidant properties. Two A/R models were performed on type II pneumocytes (A549 cell line), either on cells attached to culture dishes (monolayer A/R model; 3.5 h of anoxia, 30 min of re-oxygenation) or after cell detachment (suspension A/R model; 1 h of anoxia, 10 min of re-oxygenation). Ceftazidime and selective inhibitors (SOD, Tiron, L-NMMA) were added before anoxia. Free radical production was assessed by the EPR spin trapping technique. Oxygen consumption was monitored, in parallel with EPR studies, in the suspension A/R model. The production of free radical species was demonstrated by the generation of PBN-radical adducts: (a(N) = 15.2 G) in the monolayer A/R model and a six-line EPR spectrum (a(N) = 15.7 G and a(H) = 2.7 G) in the suspension A/R model. A kinetic study performed by oximetry, in parallel with EPR spectroscopy, demonstrated marked alterations of the cell respiratory function and that the free radical production started during anoxia and increased during re-oxygenation. In the suspension A/R model, the amplitude of EPR spectra were decreased upon the addition of 200 U/ml SOD (37% inhibition), 0.1 mM Tiron (67% inhibition) and 1 mM L-NMMA (43% inhibition). Addition of 1 mM ceftazidime decreased the amplitude of EPR spectra (37% inhibition) in both A/R models. Complementary in vitro EPR studies demonstrated that CAZ scavenged the hydroxyl radical (produced by the Fenton reaction). The protective effect of ceftazidime in the cell model could thus be linked to its ability to scavenge superoxide anions, nitrogen-derived species and hydroxyl radicals.     

"Distant spin trapping": a method for expanding the availability of spin trapping measurements

The technique of spin trapping is used to study a wide range of free radicals in various systems, including those generated in vitro and in vivo. But unfortunately, EPR spectrometers are not always immediately accessible at the site of experimentation, and therefore it is important to find a method that can preserve a radical adduct over longer periods of time. We describe here an alternative method in which the samples can be frozen and transported for EPR measurements at another site. Various spin adducts of DEPMPO were frozen and measured at 0 degrees C at various intervals after freezing to determine their stability in the frozen state. The radical adducts were generated by established methods and stored at two different temperatures; -196 degrees C (liquid nitrogen) and -80 degrees C (dry ice). The experiments were carried out in an aqueous solution with and without a model of reducing environment (2 mM ascorbate). The results indicate that it is feasible to store and transport spin adducts for subsequent analysis. We conclude that this approach, which we term "distant spin trapping", makes it feasible to transport samples to another site for EPR measurements. This should significantly expand the ability to use spin trapping in biology and medicine.     

Inhibition of platelet-derived growth factor-induced mesangial cell proliferation by cyclooxygenase-2 overexpression is abolished through reactive oxygen species

Proliferation of mesangial cells (MC) is an early event in many forms of glomerulonephritis. We have previously shown that platelet-derived growth factor (PDGF)-induced proliferation of MC was inhibited by the overexpression of cyclooxygenase-2 (COX-2). Since reactive oxygen species (ROS) are important mediators of mitogenic signaling, we evaluated the role of ROS in the COX-2 induced growth arrest in MC. We demonstrate that ROS are reduced in COX-2 overexpressing MC. Intracellular elevation of ROS restored PDGF-induced proliferation, while the expression of the cyclin-dependent kinase inhibitors p21(cip1) and p27(kip1) were decreased in these cells. The data suggest that COX-2 decreases ROS formation which consequently leads to the PDGF-induced inhibition of MC proliferation.     

Safety, efficacy and anti-inflammatory activity of rho iso-alpha-acids from hops

A defined mixture of rho iso-alpha-acids (RIAA), a modified hop extract, was evaluated for anti-inflammatory efficacy and safety. RIAA inhibited LPS-stimulated PGE(2) formation with >200-fold selectivity of COX-2 (IC(50)=1.3 microg/ml) over COX-1 (IC(50)>289 microg/ml). This occurred only when RIAA was added prior to, but not post, LPS stimulation. Consistent with this observation, RIAA produced no physiologically relevant, direct inhibition of COX-1 or COX-2 peroxidase activity. This suggests that RIAA inhibits inducible but not constitutive COX-2. In support, we found RIAA showed minimal PGE(2) inhibition (IC(50)=21mug/ml) relative to celecoxib (IC(50)=0.024 microg/ml), aspirin (IC(50)=0.52 microg/ml) or ibuprofen (IC(50)=0.57 microg/ml) in the AGS gastric mucosal model, where COX-1 and -2 are expressed constitutively. Taken together these results predict RIAA may have lower potential for gastrointestinal and cardiovascular toxicity observed with COX enzyme inhibitors. Following confirmation of bioavailable RIAA administered orally, gastrointestinal safety was assessed using the fecal calprotectin biomarker in a 14-day human clinical study; RIAA (900 mg/day) produced no change compared to naproxen (1000 mg/day), which increased fecal calprotectin 200%. Cardiovascular safety was addressed by PGI-M measurements where RIAA (1000 mg) did not reduce PGI-M or affect the urinary PGI-M/TXB(2) ratio. Drug interaction potential was evaluated against six major CYPs; of relevance, RIAA inhibited CYP2C9. Toxicity was assessed in a 21-day oral, mouse subchronic toxicity study where no dose dependent histopathological effects were noted. Clinically, RIAA (1000 mg/day) produced a 54% reduction in WOMAC Global scores in a 6-week, open-label trial of human subjects exhibiting knee osteoarthritis.     

Involvement of MAPK activation in chemokine or COX-2 productions by Toxoplasma gondii

This experiment focused on MAPK activation in host cell invasion and replication of T. gondii, as well as the expression of CC chemokines, MCP-1 and MIP-1 alpha , and enzyme, COX-2/prostaglandin E2 (PGE2) in infected cells via western blot, [3H]-uracil incorporation assay, ELISA and RT-PCR. The phosphorylation of ERK1/2 and p38 in infected HeLa cells was detected at 1 hr and/or 6 hr postinfection (PI). Tachyzoite proliferation was reduced by p38 or JNK MAPK inhibitors. MCP-1 secretion was enhanced in infected peritoneal macrophages at 6 hr PI. MIP-1 alpha mRNA was increased in macrophages at 18 hr PI. MCP-1 and MIP-1 alpha were reduced after treatment with inhibitors of ERK1/2 and JNK MAPKs. COX-2 mRNA gradually increased in infected RAW 264.7 cells and the secretion of COX-2 peaked at 6 hr PI. The inhibitor of JNK suppressed COX-2 expression. PGE2 from infected RAW 264.7 cells was increased and synthesis was suppressed by PD98059, SB203580, and SP600125. In this study, the activation of p38, JNK and/or ERK1/2 MAPKs occurred during the invasion and proliferation of T. gondii tachyzoites in HeLa cells. Also, increased secretion and expression of MCP-1, MIP-1 alpha , COX-2 and PGE2 were detected in infected macrophages, and appeared to occur via MAPK signaling pathways.     

The expression of COX-2 in VEGF-treated endothelial cells is mediated through protein tyrosine kinase

Cyclooxygenase (COX), existing as the COX-1 and COX-2 isoforms, converts arachidonic acid to prostaglandin H2, which is then further metabolized to various prostaglandins. Vascular endothelial growth factor (VEGF) has been shown to play important roles in inflammation and is upregulated by the prostaglandin E series through COX-2 in several cell types. Here, we have investigated the effects of VEGF on the COX isoform expressed in human umbilical vein endothelial cells (HUVEC). The signalling mechanism of the COX isoform expressed in endothelial cells activated with VEGF will be also investigated using the tyrosine kinase inhibitor, genistein, and protein kinase C inhibitor, staurosporine. The activity of COX-2 was assessed by measuring the production of 6-keto-prostaglandin F1alpha in the presence of exogenous arachidonic acids (10 microM, 10 min) by enzyme immunoassay. The expression of COX isoform protein was detected by immunoblot using specific antibodies. Untreated HUVEC contained no COX-2 protein. In HUVEC treated with VEGF (0.01-50 ng/ml), COX-2 protein, but not COX-1, and COX activity were increased in a dose-dependent manner. Interestingly, the increased COX-2 protein and activity in response to VEGF (10 ng/ml) was inhibited by the tyrosine kinase inhibitor, genistein (0.05-5 microg/ml), but not by the protein kinase C inhibitor, staurosporine (0.1-10 ng/ml). Thus, the induction of COX-2 by VEGF in endothelial cells was mediated through protein tyrosine kinase, and the uses of specific COX-2 inhibitors in these conditions, in which VEGF was involved, might have a role.     

Formation of superoxide radicals in isolated cardiac mitochondria: Effect of low oxygen concentration

Formation of superoxide radical in isolated rat heart mitochondria under controlled oxygenation has been studied by spin trapping and EPR oxymetry. Lithium phthalocyanine and perdeuterated Tempone-D-¹⁵ N ₁₆ were used to determine the oxygen concentration. Tiron was used as a spin trap. By varying the oxygen content in the reaction medium, we have shown that isolated heart mitochondria can produce superoxide even at an oxygen partial pressure of 17.5 mmHg, though at a rate considerably lower than under normal conditions. Raising the oxygen concentration increases the rate of superoxide generation.     

Selective cyclooxygenase-2 inhibitors show a differential ability to inhibit proliferation and induce apoptosis of colon adenocarcinoma cells

Although the influence of selective cyclooxygenase (COX)-2 inhibitors on the proliferation of colon adenocarcinoma cells have been the subject of much investigation, relatively little research has compared the effects of different COX-2 inhibitors. Celecoxib strongly suppressed the proliferation of COX-2 expressing HT-29 cells at 10-40 microM. NS-398 and nimesulide also inhibited cell proliferation, whereas rofecoxib, meloxicam, and etodolac did not. Only celecoxib induced apoptosis of HT-29 cells, as detected on the basis of DNA fragmentation, TUNEL positivity, and caspase-3/7 activation. DNA fragmentation was also increasd in COX-2 non-expressing cell lines (SW-480 and HCT-116) by exposure to celecoxib for 6-24 h. All six COX-2 inhibitors suppressed the production of prostaglandin E(2) by HT-29 cells, suggesting that the pro-apoptotic effect of celecoxib was unrelated to inhibition of COX-2. Inactivation of Akt might explain the differential pro-apoptotic effect of these selective COX-2 inhibitors on colon adenocarcinoma cells.     

Protein oxidative damage and redox imbalance induced by ionising radiation in CHO cells

Reactive oxygen species (ROS) are important mediators of the cytotoxicity induced by the direct reaction of ionising radiation (IR) with all critical cellular components, such as proteins, lipids, and nucleic acids. The derived oxidative damage may propagate in exposed tissues in a dose- and spatiotemporal dependent manner to other cell compartments, affecting intracellular signalling, and cell fate. To understand how cell damage is induced, we studied the oxidative events occurring immediately after cell irradiation by analysing the fate of IR-derived ROS, the intracellular oxidative damage, and the modification of redox environment accumulating in Chinese hamster ovary (CHO) within 1?h after cell irradiation (dose range 0–10?Gy). By using the immuno-spin trapping technique (IST), spectrophotometric methods, and electron paramagnetic resonance (EPR) spectroscopy, we showed that IR-derived ROS (i) induced an IST-detectable, antioxidant-inhibitable one-electron oxidation of specific intracellular proteins; (ii) altered the glutathione (GSH) content (which was found to increase below 2?Gy, and decrease at higher doses, leading to a redox imbalance); (iii) decreased glutathione peroxidase and glutaredoxin activity; (iv) modified neither glutathione reductase nor thioredoxin reductase activity; (v) were detected by spin trapping technique, but adduct intensity decreased due to cell competition for ROS; and (vi) induced no EPR-detectable radicals assignable to oxidised cellular components. In conclusion, our results showed that IR generated an early high oxidising potential (protein radical intermediates, redox imbalance, modified redox enzyme activity) in irradiated cells potentially able to propagate the damage and induce oxidative modification of secondary targets.     

Reciprocal regulation of NADPH oxidases and the cyclooxygenase-2 pathway

The objective of this work was to analyze the possible association between cyclooxygenase-2 (COX-2) and NADPH oxidases (NOX) in liver cells, in response to various proinflammatory and toxic insults. First, we observed that treatment of Chang liver (CHL) cells with various COX-2 inducers increased reactive oxygen species (ROS) production concomitant with GSH depletion, phorbol 12-myristate 13-acetate (PMA) being the most effective treatment. Moreover, early changes in the oxidative status induced by PMA were inhibited by glutathione ethyl ester, which also impeded COX-2 induction. In fact, CHL cells expressed NOX1 and NOX4, although only NOX4 expression was up-regulated in the presence of PMA. Knock-down experiments suggested that PMA initiated a pathway in which NOX1 activation controlled COX-2 expression and activity, which, in turn, induced NOX4 expression by activation of the prostaglandin receptor EP4. In addition, CHL cells overexpressing COX-2 showed higher NOX4 expression and ROS content, which were decreased in the presence of the COX-2 inhibitor DFU. Interestingly, we found that addition of prostaglandin E(2) (PGE(2)) also induced NOX4 expression and ROS production, which might promote cell adhesion. Finally, we determined that NOX4 induction by PGE(2) was dependent on ERK1/2 signaling. Taken together, these results indicate that NOX proteins and COX-2 are reciprocally regulated in liver cells.     

Synthetic pathways of gallbladder mucosal prostanoids: the role of cyclooxygenase-1 and 2.

Acute cholecystitis is associated with increased gallbladder prostanoid formation and the inflammatory changes and prostanoid increases can be inhibited by nonsteroidal anti-inflammatory agents. Recent information indicates that prostanoids are produced by two cyclooxygenase (COX) enzymes, COX-1 and COX-2. The purpose of this study was to determine the COX enzymatic pathway in gallbladder mucosal cells involved in the production of prostanoids stimulated by inflammatory agents. Human gallbladder mucosal cells were isolated from cholecystectomy specimens and maintained in cell culture and studied in comparison with cells from a well differentiated gallbladder mucosal carcinoma cell line. COX enzymes were evaluated by Western immunoblotting and prostanoids were measured by ELISA. Unstimulated and stimulated cells were exposed to specific COX-1 and COX-2 inhibitors. In both normal and transformed cells constitutive COX-1 was evident and in gallbladder cancer cells lysophosphatidyl choline (LPC) induced the formation of constitutive COX-1 enzyme. While not detected in unstimulated normal mucosal cells and cancer cells, COX-2 protein was induced by both lipopolysaccharide (LPS) and LPC. Unstimulated gallbladder mucosal cells and cancer cells produced prostaglandin E2 (PGE2) and prostacyclin (6-keto prostaglandin F1alpha, 6-keto PGF1alpha) continuously. In freshly isolated normal gallbladder mucosal cells, continuously produced 6 keto PGF1alpha was inhibited by both COX-1 and COX-2 inhibitors while PGE2 levels were not affected. Both LPS and LPC stimulated PGE2 and 6 keto PGF1alpha formation were blocked by COX-2 inhibitors in freshly isolated, normal human gallbladder mucosal cells and in the gallbladder cancer cells. The prostanoid response of gallbladder cells stimulated by proinflammatory agents is inhibited by COX-2 inhibitors suggesting that these agents may be effective in treating the pain and inflammation of gallbladder disease.     

Catalase ameliorates polychlorinated biphenyl-induced cytotoxicity in nonmalignant human breast epithelial cells

Polychlorinated biphenyls (PCBs) are environmental chemical contaminants believed to adversely affect cellular processes. We investigated the hypothesis that PCB-induced changes in the levels of cellular reactive oxygen species (ROS) induce DNA damage resulting in cytotoxicity. Exponentially growing cultures of human nonmalignant breast epithelial cells (MCF10A) were incubated with PCBs for 3 days and assayed for cell number, ROS levels, DNA damage, and cytotoxicity. Exposure to 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153) or 2-(4-chlorophenyl)benzo-1,4-quinone (4-Cl-BQ), a metabolite of 4-chlorobiphenyl (PCB3), significantly decreased cell number and MTS reduction and increased the percentage of cells with sub-G1 DNA content. Results from electron paramagnetic resonance (EPR) spectroscopy showed a 4-fold increase in the steady-state levels of ROS, which was suppressed in cells pretreated with catalase. EPR measurements in cells treated with 4-Cl-BQ detected the presence of a semiquinone radical, suggesting that the increased levels of ROS could be due to the redox cycling of 4-Cl-BQ. A dose-dependent increase in micronuclei frequency was observed in PCB-treated cells, consistent with an increase in histone 2AX phosphorylation. Treatment of cells with catalase blunted the PCB-induced increase in micronuclei frequency and H2AX phosphorylation that was consistent with an increase in cell survival. Our results demonstrate a PCB-induced increase in cellular levels of ROS causing DNA damage, resulting in cell killing.