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Background: The antioxidant enzymes (manganese- and copper-zinc-containing superoxide dismutases, catalast and glutathione peroxidase) limit cell injury induced by reactive oxygen species. The purpose of the study was to determine whether human oral squamous cell carcinomas have altered antioxidant enzyme levels. This study is the first to undertake this task in human oral mucosa and squamous cell carcinoma. Methods: Semiquantitative immunohistochemistry was used to examine 26 archived oral squamous cell carcinoma biopsies. Fourteen well-differentiated and 12 poorly differentiated tumors were examined, as were 12 specimens of oral mucosa. All sections were reviewed by two oral and maxillofacial pathologists, and image analysis of the immunostained sections was performed using NIH Image. Antioxidant enzyme staining intensities were compared in the different groups by Duncan's multiple range test. Results: In general, mucosal basal cells displayed lower antioxidant enzyme levels than spinous cells, and primary tumor cells displayed lower antioxidant enzyme staining intensities than did their normal cell counterparts. Moreover, poorly differentiated tumor cells showed lower antioxidant enzyme staining intensities than well-differentiated tumor cells. Manganese-containing superoxide dismutase staining intensities were, however, higher in well-differentiated oral squamous cell carcinomas than their normal cells of origin. Conclusions: Detection of antioxidant enzymes may be a useful future marker in the molecular diagnosis of the oral cancer. Moreover, it may be possible to not only monitor the effectiveness of chemopreventitive and therapeutic strategies in oral cancer using these enzymes, but to monitor tumor recurrence.

Manganese superoxide dismutase (Mn SOD), mitochondrial enzyme, defends against the toxic effects of superoxide radical (O2.-) in pathological processes by catalyzing the conversion of O2.- to hydrogen peroxide (H2O2). The activity of another enzyme, NADH diaphorase, forms the basis for a histochemical method used commonly to demonstrate nerve cell bodies in the enteric plexuses. We found identical patterns of localization of Mn SOD immunoreactivity and NADH diaphorase activity in brain, esophagus, stomach, colon, liver, and kidney. NADH diaphorase enzymatic activity co-migrated with complexes of Mn SOD on a non-denaturing gel. This suggests that the NADH diaphorase may in some way be related to Mn SOD.

The antioxidant enzymes (manganese- and copper-zinc-containing superoxide dismutases, catalase and glutathione peroxidase) limit cell injury induced by reactive oxygen species. The purpose of the study was to determine whether human oral squamous cell carcinomas have altered antioxidant enzyme levels. This study is the first to undertake this task in human oral mucosa and squamous cell carcinoma.Semiquantitative immunohistochemistry was used to examine 26 archived oral squamous cell carcinoma biopsies. Fourteen well-differentiated and 12 poorly differentiated tumors were examined, as were 12 specimens of oral mucosa. All sections were reviewed by two oral and maxillofacial pathologists, and image analysis of the immunostained sections was performed using NIH Image. Antioxidant enzyme staining intensities were compared in the different groups by Duncan's multiple range test.In general, mucosal basal cells displayed lower antioxidant enzyme levels than spinous cells, and primary tumor cells displayed lower antioxidant enzyme staining intensities than did their normal cell counterparts. Moreover, poorly differentiated tumor cells showed lower antioxidant enzyme staining intensities than well-differentiated tumor cells. Manganese-containing superoxide dismutase staining intensities were, however, higher in well-differentiated oral squamous cell carcinomas than their normal cells of origin.Detection of antioxidant enzymes may be a useful future marker in the molecular diagnosis of the oral cancer. Moreover, it may be possible to not only monitor the effectiveness of chemopreventive and therapeutic strategies in oral cancer using these enzymes, but to monitor tumor recurrence.

Fibrosis is a common form of normal tissue damage after exposure to a wide variety of insults believed to involve oxidative stress. Plasminogen activator inhibitor-1 (PAI-1) is thought to play a major role in the development of progressive fibrosis via the inhibition of extracellular matrix degradation. Because radiation causes oxidative injury, which has been shown to trigger fibrogenic responses, the present study was designed to test the hypothesis that PAI-1 expression is redox-regulated after irradiation. Irradiating rat kidney tubule epithelial cells (NRK52E) with 1-20 Gy gamma-rays led to dose-dependent increases in steady-state levels of PAI-1 mRNA and immunoreactive protein within 24 and 48 h, respectively. Enhancement of intracellular soluble thiol pools after incubation with N-acetylcysteine (2.5 mM), from 3.27 +/- 0.27 nM/mg protein to 5.34 +/- 0.52 nM/mg protein in cells incubated with N-acetylcysteine 30 min before and assessed 4 h after irradiation, abolished the radiation-induced up-regulation of PAI-1. In addition, overexpression of catalase inhibited radiation-induced increases in PAI-1 expression, suggesting a mechanistic role for hydrogen peroxide (H2O2) in regulating PAI-1 expression after oxidative insult. In support of this notion, incubating NRK52E cells with H2O2 (100 microM) also led to a nearly 3-fold increase in PAI-1 gene expression. These results demonstrate that PAI-1 is redox-regulated after exposure to ionizing radiation or H2O2 and suggest that H2O2 scavenging might represent a fundamental mechanism for modulating fibrogenic disease via inhibition of the induction of profibrogenic mediators after acute or chronic oxidative stress.

The purpose of this study was to assess heterogeneity of tumor microcirculation determined by dynamic contrast-enhanced magnetic resonance (MR) imaging and its prognostic value for tumor radiosensitivity and long-term tumor control using pixel-by-pixel analysis of the dynamic contrast enhancement. Sixteen patients with advanced cervical cancer were examined with dynamic contrast-enhanced MR imaging at the time of radiation therapy. Pixel-by-pixel statistical analysis of the ratio of post- to precontrast relative signal intensity (RSI) values in the tumor region was performed to generate pixel RSI distributions of dynamic enhancement patterns. Histogram parameters were correlated with subsequent tumor control based on long-term cancer follow-up (median follow-up 4.6 years; range 3.8-5.2 years). The RSI distribution histograms showed a wide spectrum of heterogeneity in the dynamic enhancement pattern within the tumor. The quantity of low-enhancement regions (10th percentile RSI2.5) significantly predicted subsequent tumor recurrence (88% vs. 0%, P = 0.0004). Discriminant analysis based on both 10th percentile RSI and pixel number (reflective of tumor size) further improved the prediction rate (100% correct prediction of subsequent tumor control vs. recurrence). These preliminary results suggest that quantification of the extent of poor vascularity regions within the tumor may be useful in predicting long-term tumor control and treatment outcome in cervical cancer. J. Magn. Reson. Imaging 2000;12:1027-1033.

Manganese-containing superoxide dismutase (MnSOD) is an essential primary antioxidant enzyme that converts superoxide radical to hydrogen peroxide and molecular oxygen within the mitochondrial matrix. Cytosolic glutathione peroxidase (GPX) converts hydrogen peroxide into water. MnSOD is reduced in a variety of tumor types and has been proposed to be a new kind of tumor suppressor gene, but the mechanism(s) by which MnSOD suppresses malignancy is unclear. According to the enzymatic reactions catalyzed by MnSOD and cytosolic GPX, change in the cellular redox status, especially change attributable to accumulation of hydrogen peroxide or other hydroperoxides, is a possible reason to explain the suppression of tumor growth observed in MnSOD-overexpressing cells. To test this possible mechanism, we transfected human cytosolic GPX cDNA into human glioma cells overexpressing MnSOD. The results showed that GPX overexpression not only reversed the tumor cell growth inhibition caused by MnSOD overexpression but also altered the cellular contents of total glutathione, reduced glutathione, oxidized glutathione, and intracellular reactive oxygen species. Overexpression of GPX also inhibited degradation of the inhibitory subunit alpha of nuclear factor-KB. These results suggest that hydrogen peroxide or other hydroperoxides appear to be key reactants in the tumor suppression by MnSOD overexpression, and growth inhibition correlates with the intracellular redox status. This work suggests that manipulations that inhibit peroxide removal should enhance the tumor suppressive effect of MnSOD overexpression.

Oxidative stress results in damage to cellular structures and has been linked to many diseases, including cancer. The authors sought to determine whether the expression of three major antioxidant enzymes, copper-zinc superoxide dismutase (SOD1), manganese superoxide dismutase (SOD2), and catalase, was altered in human prostate carcinoma and its likely precursor, high grade prostatic intraepithelial neoplasia (PIN). The level of reactive oxygen species damage was evaluated by measuring the expression of the DNA adduct 8-hydroxydeoxyguanosine.The authors evaluated the tissue expression of the antioxidant enzymes in prostate carcinoma by immunohistochemistry, immunogold electron microscopy, and enzymatic assay. The polymerase chain reaction was used to amplify and screen tissue specimens for the genes of SOD1, SOD2, and extracellular SOD (SOD3). Matched paraffin embedded tissue sections were evaluated by RNA in situ hybridization for expression of SOD1 and immunohistochemically for the DNA adduct 8-hydroxydeoxyguanosine.All prostatic tissues, including cancer, displayed immunoreactivity for the three antioxidant enzymes in epithelial cells, with no staining of the stroma, inflammatory cells, or endothelial cells. The number of immunoreactive cells was greater in benign epithelium than in PIN and cancer for each enzyme. The mean percentage and intensity of immunoreactive cells was greatest for SOD2, intermediate for SOD1, and lower for catalase. Staining in cancer was heterogeneous. Immunogold ultrasound studies revealed strong mitochondrial labeling for SOD2, which was greater in benign epithelium than in cancer; SOD1 labeling was invariably weaker, with nuclear labeling in benign epithelium and cytoplasmic labeling in cancer cells. There was no difference in enzyme activity for the three antioxidant enzymes between benign epithelium and cancer. No mutations were found in the 5 exons of SOD1, 5 exons of SOD2, and 3 exons of SOD3, except for 3 of 20 cases with polymorphisms for exon 3 of SOD1. Intense nuclear immunoreactivity for 8-hydroxydeoxyguanosine was present in fewer than 3% of epithelial cells, with no apparent differences among benign epithelium, PIN, and cancer.SOD1, SOD2, and catalase had lower expression in PIN and prostate carcinoma than in benign epithelium. The number of immunoreactive cells in PIN was similar to cancer, indicating that these are closely related. Enzyme activities were variable, with no difference between benign epithelial cells and cancer, although this lack of change in enzyme activity could have been due to the presence of contaminating benign cells within the cancer specimens. The results of reactive oxygen species damage were found only in the epithelium and not in the stroma. Expression of the DNA adduct 8-hydroxydeoxyguanosine was present in fewer than 3% of cells, with no apparent differences among benign epithelium, PIN, and cancer. These findings suggest that oxidative stress is an early event in carcinogenesis.

The risk for prostate cancer seems to be reduced by certain antioxidant compounds (vitamins E and A, and selenium).Antioxidant enzymes and oxidative damage products were localized in normal prostatic epithelium and malignant glands in primary and metastatic prostatic adenocarcinomas, using well-characterized antibodies and immunoperoxidase techniques.Antioxidant enzymes and four markers of oxidative damage were compared in basal and secretory cells of normal prostatic epithelium and prostate adenocarcinoma cells, and each cell type had unique patterns of enzymes and oxidative damage products. One marker of oxidative damage, a fluorophore derived from 4-hydroxy-2-nonenal-lysine adduction, was found in secretory cells of normal but not malignant epithelium, demonstrating a different oxidative metabolism in normal vs. malignant prostate epithelium. Metastatic lesions from primary prostate cancer had higher levels of manganese superoxide dismutase and nuclear oxidative damage products than did primary tumors.Antioxidant enzymes and oxidative damage products are modulated in metastatic compared to primary prostate cancer.

Two polymorphic variants of manganese superoxide dismutase (MnSOD), with either Ile or Thr at amino acid 58, (Ile58MnSOD or Thr58MnSOD), have been found in the human population. The MnSOD activity of these two variants and their effects on the malignant phenotype of human breast cancer MCF-7 cells were compared. It was demonstrated that MnSOD-overexpressing clones obtained from transfection of the two MnSOD cDNAs into MCF-7 cells had increased MnSOD immunoreactive protein and increased MnSOD activity. Cells overexpressing Ile58MnSOD had 3-fold higher MnSOD activity than cells overexpressing Thr58MnSOD in vivo at an equal MnSOD protein level. Tumor-suppressive effects of MnSOD-overexpressing cells were indicated by: (a) decreased plating efficiency; (b) elongated cell population doubling time; (c) lower clonogenic fraction in soft agar; and (d) complete inhibition or delayed onset of tumor formation in nude mice. When compared on the same activity basis, the suppressive effects of Ile58MnSOD were similar to those of Thr58MnSOD. However, far more Thrs58MnSOD protein was required to obtain the same amount of MnSOD activity, making the Thr58MnSOD far less effective. A dose-response suppressive effect was observed when the increase of MnSOD activity was moderate. We conclude that MnSOD is a tumor suppressor in human breast cancer, but the Thr58 form of the protein is a much less effective tumor suppressor than the Ile58 form of the protein.

The oncogenic ras protein controls signal-transduction pathways that are critical for cell proliferation and tumorigenesis. Here, we demonstrate that v-Ha-ras-transduced human keratinocyte HaCaT cells produced significantly larger amounts of superoxide than did control cell lines. The superoxide generation was mediated by the transduced ras protein, because superoxide generation was modified by an inhibitor, lovastatin, that inhibits ras farnesylation during ras protein maturation. Superoxide generation was also inhibited by diphenylene iodonium, an inhibitor of flavoproteins, including NADPH oxidase, but not by rotenone, an inhibitor of the respiratory chain of the mitochondria. Those observations suggested that a phagocytic-like NADPH oxidase exists in keratinocytes that could be activated by the dominant activated v-Ha-ras and produce superoxide. Overexpression of manganese-containing superoxide dismutase and copper and zinc-containing superoxide dismutase cDNA via adenovirus infection also attenuated superoxide generation. Previous work has demonstrated that extracellular superoxide dismutase (SOD) can lower superoxide generation; this is the first report that intracellular SOD could also modify the amount of superoxide production from the cells. This report implies that superoxide radical may act as a second messenger molecule of oncogenic ras.

In order to evaluate the intracellular function of glia maturation factor (GMF), we overexpressed GMF in C6 rat glioma cells using two methods: stable transfection using the pcDNA3 plasmid, and transient transfection using replication-defective human adenovirus. With both methods, C6 cells transfected with GMF and overexpressing the protein exhibit a lower saturation density in culture compared to non-transfected or vector alone controls. Transfected cells also exhibit morphological differentiation as shown by the outgrowth of cell processes. When inoculated into nude mice, transfected cells are less tumorigenic than controls, and express the mature astrocytic marker glial fibrillary acidic protein. In tissue culture, transfected cells show a 3.5-fold increase in CuZn-dependent superoxide dismutase (CuZnSOD) activity. Western blot analysis reveals a 3.5-fold increase in CuZnSOD protein, suggesting an induction of the enzyme. In view of recent findings that reactive oxygen species (ROS) and the antioxidant enzymes are intricately involved in key physiologic processes such as proliferation, differentiation and apoptosis, the study raises the possibility that CuZnSOD may be a mediator of GMF function.

Edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine; ET-18-OCH3), a membrane-targeting anticancer ether lipid drug has been shown previously in vitro to be capable of initiating oxidative processes in cells. Here we study two human leukemia cell lines (HL-60 and K562) that have different sensitivities to edelfosine; HL-60 cells are more sensitive than K562 cells. To determine whether edelfosine alters the sensitivity of these lines to an oxidative stress, cells were subjected to the oxidative stress of iron(II) plus ascorbate and then monitored for free radical formation, membrane integrity, and cytotoxicity. The HL-60 cell was sensitive to the ether lipid drug in clonogenic and dye exclusion assays; a lipid-derived free radical was generated by this sensitive cell in the presence of small amounts of Fe2+ and ascorbate as detected by electron paramagnetic resonance and the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone. There was also simultaneous generation of an ascorbate-free radical, which has been shown to estimate cellular oxidative flux. In contrast, the K562 cell was resistant to edelfosine cytotoxicity in all assays and did not generate either lipid-derived or ascorbate-free radicals. Subcellular homogenates of the HL-60 cell generated both radicals when exposed to the drug, but homogenates of K562 did not generate either, suggesting that differential drug uptake or intracellular drug localization is not the cause of the difference in oxidation. Trypan blue uptake by the HL-60, but not the K562 cells, measured under the same conditions as the oxidation experiments, demonstrated a loss of membrane impermeability with similar time and concentration dependence, suggesting a causal relationship of membrane damage and radical generation. Complementary studies of HL-60 cell membrane integrity with propidium iodide impermeability and light scatter using the flow cytometer showed a concentration dependence that was similar to radical generation. Biochemical studies of the fatty acids of the HL-60 cell revealed more highly polyunsaturated lipids in the cells. Cellular antioxidant enzymes and vitamin E contents of the two cell lines were similar. We conclude that there is a time- and concentration-dependent generation of important oxidations by the sensitive HL-60 cells exposed to the membrane-targeted ether lipid, but the resistant K562 cells are oxidatively silent. This may be due in part to the differences in fatty acid polyunsaturation of the cellular membranes. The difference in oxidative susceptibility could be the basis for drug resistance to this membrane-specific anticancer agent.

Recent studies suggest that the gene for manganese superoxide dismutase (MnSOD) is a candidate tumor-suppressor gene. The present study was designed to study the effect of overexpression of MnSOD on cultured human prostate carcinoma cells.DU145 human prostate carcinoma cells were transfected with the cDNA for manganese superoxide dismutase (MnSOD), and two clones overexpressing MnSOD activity were subsequently characterized by comparison with parental and plasmid control-transfected cells.One clone overexpressing MnSOD had no change in other antioxidant enzymes (AEs) (nonadapted), while a second clone showed an increase in catalase activity (adapted). Sensitivity of parental, plasmid control-transfected, and MnSOD cDNA-transfected cells to agents that generate oxidative stress correlated with AE profiles. Both clones overexpressing MnSOD activity showed increased reactive oxygen species levels under basal cell culture conditions. Both clones overexpressing MnSOD activity showed inhibition of cell growth in vitro and in vivo compared with parental and plasmid control-transfected cells. Flow cytometry studies using mitochondrial-specific probes showed equal mitochondrial mass in all cell lines, but altered mitochondrial membrane potential in MnSOD-overexpressing clones compared with parental or plasmid control-transfected cells.Our results suggest novel mechanisms by which MnSOD overexpression may modulate the malignant phenotype, with potential applications in developing new therapies for prostate cancer.

NIH/3T3 mouse fibroblasts were transfected with the cDNA for manganese superoxide dismutase (MnSOD), and two clones overexpressing MnSOD activity were subsequently characterized by comparison with parental and control plasmid-transfected cells. One clone with a 1.8-fold increase in MnSOD activity had a 1.5-fold increase in glutathione peroxidase (GPX) activity (increased GPX-adapted clone), while a second clone with a 3-fold increase in MnSOD activity had a 2-fold decrease in copper, zinc superoxide dismutase (CuZnSOD) activity (decreased CuZnSOD-adapted clone). Increased reactive oxygen species (ROS) levels compared with parental or control plasmid-transfected cells were observed in nonsynchronous cells in the increased GPX-adapted clone, but not in the decreased CuZnSOD-adapted clone. The two MnSOD-overexpressing clones showed different sensitivities to agents that generate oxidative stress. Flow cytometry analysis of the cell cycle showed altered cell cycle progression in both MnSOD-overexpressing clones. During logarithmic growth, both MnSOD-overexpressing clones showed increased mitochondrial membrane potential compared with parental and control plasmid-transfected cells. Both MnSOD-overexpressing clones showed a decrease in mitochondrial mass at the postconfluent phase of growth, suggesting that mitochondrial mass may be regulated by MnSOD and/or ROS levels. Our results indicate that adaptation of fibroblasts to overexpression of MnSOD can involve more than one mechanism, with the resultant cell phenotype dependent on the adaptation mechanism utilized by the cell.

Stable transfection of endothelial nitric oxide synthase (eNOS) cDNA in human oral carcinoma SCC-25 cells was performed. Two eNOS-expressing clones were isolated, and both were shown to have increased eNOS expression as assayed by Northern and Western analyses. Furthermore, a nitrite assay indicated that nitric oxide production in eNOS-transfected cells was increased. The growth rate and plating efficiency of eNOS-transfected cells in vitro were lower than that of the wild-type parental or vector control-transfected cells as assayed by growth curves, [3H]thymidine incorporation, and standard plating efficiency assays in L-arginine-rich medium. However, when these cancer cells were inoculated into nude mice, tumor size of eNOS-transfected cells was smaller during the first 25 days but increased later as compared to tumor size of parental and vector control-transfected cells. It is not clear whether the later increase in tumor size was due to an increase in SCC-25 cancer cell proliferation, normal stromal cell proliferation, or both. These results show significant effects of overexpression of eNOS on tumor growth in vitro and in vivo.

Antioxidant enzymes (AEs), which catalyze the conversion of reactive oxygen species (ROS) to water, include catalase (CAT), manganese-containing superoxide dismutase (MnSOD), and copper and zinc-containing superoxide dismutase (CuZnSOD). Previous work has indicated that MnSOD, CAT, and CuZnSOD levels are nearly always low in cancer cells.Formalin-fixed, paraffin-embedded tissue from 31 radical prostatectomy specimens was immunohistochemically stained with polyclonal antibodies to CAT, MnSOD, and CuZnSOD.Malignant glands are typically stained with less intensity than benign/ hyperplastic glands. Marked heterogeneity of staining intensity was seen in the malignant glands for each of the three enzymes. A similar, though less marked, spectrum of heterogeneity of staining intensity was observed in the benign/hyperplastic epithelium contained in the specimens. No statistically significant correlation was found between intensity of staining for any of the three antioxidant enzymes and Gleason score, tumor stage, or preoperative prostate-specific antigen (PSA).Cellular levels of CAT, MnSOD, and CuZnSOD in prostatic adenocarcinoma reveal that many tumors appear to have decreased levels of expression. The finding that malignant prostate epithelium may have lowered expression of AEs suggests that further study of the role of AEs in malignant transformation in the prostate is warranted.

Human breast cancer cells (MCF-7) overexpressing manganese-containing superoxide dismutase (MnSOD) by stable or transient transfection were challenged with the cytotoxicity of tumor necrosis factor alpha (TNF-alpha), hyperthermia, and a combination of both. In contrast to the vector control and wild-type MCF-7 cells, the stable MnSOD transfectants showed significant resistance to the cytotoxicity of TNF-alpha (100 units/ml), heat at 43 or 45 degrees C, and a combination of TNF-alpha and heat at 43 degrees C. To probe the correlation of MnSOD levels with cell survival, cell sensitivity to TNF-alpha, heat at 43 degrees C, or a combination of both was also measured after MnSOD cDNA transient transfection. The data showed that the level of cell resistance was proportional to the exogenous MnSOD gene expression. These results suggest that superoxide free radicals or their reaction products are responsible for much of the synergistic cytotoxicity of TNF-alpha and hyperthermia.

Normal cells are protected by antioxidant enzymes from the toxic effects of high concentrations of reactive oxygen species generated during cellular metabolism. Even though cancer cells generate reactive oxygen species, it has been demonstrated biochemically that antioxidant enzyme levels are low in most animal and human cancers. However, a few cancer types have been found to have elevated levels of antioxidant enzymes, particularly manganese superoxide dismutase. Morphologic studies of animal and human cancer have confirmed that although the majority of tumor cell types from several organ systems have low antioxidant enzymes, adenocarcinomas may have elevated manganese superoxide dismutase and catalase levels. However, all cancers examined to date have some imbalance in antioxidant enzyme levels compared with the cell of origin. Antioxidant enzyme importance in cancer genesis has been difficult to evaluate in early cancerous lesions using biochemical techniques because such lesions are small and therefore below the level of detection. Using immunohistochemical techniques, early lesions of human and animal cancers were demonstrated to have low antioxidant enzymes, thus suggesting a role for these enzymes both in the genesis of cancer and the malignant phenotype. All but one human cancer cell type (the granular cell variant of human renal adenocarcinoma) examined showed both low catalase and glutathione peroxidase levels, suggesting that most cancer cell types cannot detoxify hydrogen peroxide. Our results to date are used to propose new cancer therapies based on modulation of cellular redox state.

Samples of normal human lung and six major types of human lung carcinomas were immunostained for antioxidant enzymes (manganese and copper, zinc superoxide dismutases, catalase, and glutathione peroxidase) and six isoenzymes of glutathione S-transferase staining was generally low in tumor cells compared with the high level of staining noted in respiratory epithelium. A notable exception was heterogeneity in immunostaining for manganese superoxide dismutase in lung adenocarcinoma, which showed both positive and negative cells in the same tumor. Tumor stromal cells (fibroblast-appearing cells) often showed strong immunostaining for manganese superoxide dismutase, while stromal cells were negative for other antioxidant and glutathione S-transferase enzymes. None of the carcinomas studied had significant levels of catalase or glutathione peroxidase; this finding has potential clinical relevance since it indicates that these tumors cannot detoxify hydrogen peroxide. The low levels of antioxidant and glutathione S-transferase enzymes in tumor cells is consistent with the hypothesis that these enzymes are markers of cell differentiation.

The effects of overexpression of human manganese superoxide dismutase (MnSOD) on cell proliferation and response to oxidative stress in rat glioma cells were studied. MnSOD-overexpressing cells had a 2- to 14-fold increase in MnSOD activity, but did not have consistent changes in the activities of CuZnSOD, catalase, or glutathione peroxidase. Cells with more than a 5-fold increase in MnSOD activity became more sensitive to radiation, 1,3-bis(2-chloroethyl)-1-nitrosourea, and buthionine sulfoximine and had a lower growth rate than parental and vector control cells. The sensitivity to 1,3-bis(2-chloroethyl)-1-nitrosourea was partially reduced by pyruvate, a H2O2 scavenger. Our results suggest that overexpression of MnSOD can cause an imbalance of antioxidant enzymes, which we hypothesize results in an elevation of intracellular H2O2. Overexpression of MnSOD can either inhibit cell proliferation or increase cell death by oxidative agents, depending on the levels of peroxide-removing enzymes.

Manganese superoxide dismutase (MnSOD) has been found to be low in a wide range of tumor cells as well as in vitro-transformed cell lines and has been implicated as a new type of tumor suppressor gene. The relationship between MnSOD activity and the malignant phenotype was studied by transfection of MnSOD cDNA into the SV40-transformed human fibroblast cell line WI-38 VA13 subline 2RA. The integration and expression of the exogenous MnSOD cDNA was confirmed in three selected clones with a 2-3.5-fold increase in MnSOD activity. The effect of elevated expression of MnSOD on the cell phenotype was determined by observing growth characteristics. Compared with the parental and neo control cells, the MnSOD-overexpressing clones had a slower growth rate, lower plating efficiency, increased anchorage dependence, and morphological differences. These changes were correlated strongly with the level of MnSOD activity. The results suggest that an increase of MnSOD activity can reverse part of the malignant phenotype in SV40-transformed human fibroblast cells. A possible mechanism is that overexpression of MnSOD might alter the intracellular redox state by modulation of the balance of reactive oxygen species.

Superoxide radical (O2-.) combines with nitric oxide (NO) to form peroxynitrite, thereby nullifying the biological activity of NO. Superoxide dismutase (SOD) prevents this reaction by converting O2-. to H2O2. We tested the hypotheses that the antioxidant enzymes catalase (CAT), Mn SOD, and Cu/Zn SOD are present in enteric neurons of the opossum esophagus, and that O2-. alters esophageal motor function. Immunostaining demonstrated CAT, Mn SOD, and Cu/Zn SOD immunoreactivity in interganglionic nerve bundles and ganglia of the myenteric and submucosal plexuses. Western blot analysis confirmed the presence of these enzymes in homogenates of esophageal muscularis propria, and enzyme assays demonstrated Cu/Zn SOD and Mn SOD activities of 262 and 73 U/mg protein, respectively. Both diethyldithiocarbamic acid, an inhibitor of Cu/Zn SOD, and xanthine (X) with xanthine oxidase (XO), which generate O2-., shortened the latency of the nerve-mediated contraction of circular esophageal muscle, the off response, by 20.2 and 23.4%, respectively. SOD alone did not affect the latency, but it inhibited the effect of X with XO on the latency. Antioxidant enzymes found in intramural esophageal nerves may play a role in regulating NO-mediated neuromuscular communication in the esophagus.

Immunogold studies of normal human kidney and common human kidney cancers were performed using polyclonal antibodies to antioxidant enzymes, including antibodies to copper, zinc and manganese superoxide dismutases, catalase, glutathione peroxidase, and glutathione S-transferases and their subunits. Normal tissue adjacent to human renal tumors had the same antioxidant enzyme immunoreactive protein profiles as normal human kidney, thus establishing that the presence of tumor does not alter the levels of antioxidant enzyme immunoreactive proteins in adjacent kidney tissue. Levels of immunoreactive protein for antioxidant enzymes were determined in four common types of malignant renal cancer. In general, tumors had low levels of antioxidant enzymes; however, certain histologic types of renal tumors had high levels of immunoreactive protein for glutathione S-transferase subunits, which could affect their susceptibility to chemotherapy. Studies of transitional carcinoma of the renal pelvis were especially informative since it was possible to compare levels of antioxidant enzyme immunoreactive protein with adjacent normal transitional epithelium; the majority of antibodies resulted in lower levels of immunoreactive protein in transitional cell carcinoma than in adjacent normal transitional epithelium. Our results are discussed in relation to the response of renal tumors to therapy.

Immunolocalization studies of hamster kidney development were performed using polyclonal antibodies to antioxidant enzymes, including antibodies to copper, zinc and manganese superoxide dismutases, catalase, glutathione peroxidase and glutathione S-transferases and their subunits. Antibodies to extracellular matrix proteins were also studied to determine the temporal sequence between expression of immunoreactive protein for basement membrane proteins, which serve as markers of embryonic induction of nephron development, and antioxidant enzyme expression in kidney development. Immunoreactive proteins for antioxidant enzymes were not detectable in the developing kidney until after extracellular matrix proteins had been deposited. However, immunoreactive proteins for the antioxidant enzymes copper, zinc and manganese superoxide dismutases, catalase, and alpha class glutathione S-transferase Ya subunit were detected in renal tubules before birth. mu class glutathione S-transferase subunits Yb1 and Yb2 stained transitional epithelium at high levels before birth. Our results indicate: (1) each type of kidney cell has a unique antioxidant enzyme profile, (2) antioxidant enzymes are expressed in different types of cell at different times during development, but antioxidant enzyme immunoreactive protein was not present until after immunoreactive proteins for extracellular matrix molecules were detected, and (3) certain antioxidant enzymes are present before birth, indicating that high oxygen tension present at birth is not crucial for induction of immunoreactive protein.

The model of toxic liver injury was used to examine the role of manganese superoxide dismutase (MnSOD) expression in cellular resistance to tumor necrosis factor (TNF)-alpha toxicity. The effects of the hepatotoxin D-galactosamine (GalN) and lipopolysaccharide (LPS) on hepatic and splenic TNF-alpha and MnSOD expression were studied. Treatment with GalN and LPS alone or in combination led to equivalent increases in hepatic and splenic TNF-alpha gene expression. Hepatic MnSOD mRNA levels were not affected by GalN or GalN with LPS but were increased 13-fold by LPS alone. Splenic MnSOD mRNA levels were increased twofold by GalN and 12-fold by either LPS alone or GalN plus LPS. The determination of MnSOD protein content, however, revealed no changes in hepatic or splenic steady-state levels of the protein with any of the treatments, despite the marked increases in MnSOD gene expression. Hepatic MnSOD enzyme activity was also unchanged by LPS or GalN plus LPS administration. Biosynthesis of MnSOD protein in rat hepatocytes isolated from an in vivo LPS-treated rat was unchanged compared with control. MnSOD mRNA levels were increased when GalN treatment was combined with uridine rescue, but again no change in protein was seen. The lack of any increase in MnSOD protein after GalN or LPS administration indicates that MnSOD upregulation is not involved in cellular resistance against TNF-alpha cytotoxicity in the liver in vivo.

Altered levels of superoxide dismutase (SOD), the enzyme that scavenges toxic superoxide anion produced during normal metabolism or after oxidative insult, have been implicated in multistage carcinogenesis of both rodents and humans. Using a mouse liver cell model, we report here that after cellular immortalization, both copper- and zinc-containing superoxide dismutase (Cu,ZnSOD) and manganese superoxide dismutase (MnSOD) activities decreased dramatically and that cellular transformation further decreased MnSOD but not Cu,ZnSOD activity. Decreased enzyme activities seen in transformed cells (Tx) were due to decreased amounts of immunoreactive enzyme protein that results from decreased superoxide dismutase mRNA expression. This downregulation of gene expression may occur at the transcriptional level, as suggested by results with cycloheximide (Chx) and actinomycin D (AcD) treatments.

Tissues from adult Syrian hamsters were studied with immunoperoxidase techniques using polyclonal antibodies to glutathione-S-transferase (rat liver and human placental enzymes) and human erythrocyte glutathione peroxidase. Most tissues immunostained similarly with these antibodies. Most notable was the cytoplasmic staining of mesenchyme tissues, especially smooth muscle, by all three antibodies. Epithelial cells stained distinctively, but usually less intensely than mesenchyme. Epithelial cells from all levels of the gastrointestinal tract, respiratory epithelium, transitional epithelium, and epidermis all showed strong staining with these antibodies. Other epithelial cell types were usually positive but showed less dramatic staining. Most epithelial tissues showed both nuclear and cytoplasmic staining; some also showed cell-surface (eg, cilia) staining. The role of these enzymes in cell differentiation of a stable organ was studied by immunostaining the kidney during its development. Early stroma (13- and 15-day fetuses) of the kidney (metanephric mesenchyme) showed strong cell-surface staining for glutathione transferases and moderate staining for glutathione peroxidase; renal tubules (which are epithelial cells) at this stage were negative for these markers. As renal tubules differentiated, first cytoplasm and then nuclei stained moderately, suggesting that glutathione-S-transferases and glutathione peroxidase are markers of both mesenchymal cells, including embryonic mesenchyme, and terminal differentiation of at least some epithelial cells.

Syrian hamsters were treated with diethylstilbestrol (DES), a potent estrogen and kidney carcinogen, or ethinyl estradiol (EE), a strong estrogen but weak carcinogen, for 1-9 months. At monthly intervals their kidneys were studied using light, immunoperoxidase, and electron microscopic techniques. At 5 months, DES-treated animals exhibited interstitial lesions composed of small round cells with a high nuclear:cytoplasmic ratio. Immunoperoxidase and ultrastructural studies showed these cells to be similar to cells in fully formed tumors at 9 months. Early lesions in EE-treated animals (seen as early as 1 month) were dissimilar, these lesions appeared in the deep cortex adjacent to the renal pelvis, where proximal tubules underwent hyperplastic changes, showing columnar cells with large nuclei, occasional mitoses, and sloughing of apical cytoplasm. Cells in early lesions of EE-treated animals did not resemble the fully developed tumor in either immunoperoxidase or ultrastructural features; although with longer treatment these tubular lesions progressed to dysplasia (3-5 months) and severe dysplasia/carcinoma in situ (7 months), they did not form grossly visible tumors during the 9-month study. Both early lesions identified were specific, inasmuch as they were not observed in control animals and animals treated with beta-dienestrol and 17 alpha-estradiol (noncarcinogenic weak estrogens). Animals given a combination of DES and EE showed tubular hyperplasia but not interstitial lesions; this finding was of particular interest because hamsters given this combination of estrogens do not develop gross renal tumors. These results strongly implicate the primitive interstitial cell in the hamster kidney as the cell of origin of the DES-induced neoplasm.

The activities of superoxide dismutase (SOD), catalase, glutathione peroxidase, glutathione S-transferases, GSSG reductase, thiol transferases, gamma glutamyl-cysteine synthetase, and glucose-6-phosphate dehydrogenase, and the concentrations of H2O2 and reduced and oxidized glutathione were determined in the various developmental stages of houseflies. Housefly development was correlated with a progressive increase of cellular oxidizing equivalents and a loss of cellular reducing capacity. The loss of reducing equivalents appeared to result from a decrease in the activity of enzymes involved in glutathione and NADPH synthesis and a concomitant increase in glutathione-oxidizing enzymes. Relatively little change was observed in SOD activity during housefly development; however, the electrophoretic pattern of MnSOD varied in a manner specific to developmental stage. A striking increase in H2O2 concentration occurred prior to pupation possibly due to changes in substrate catabolism. These results support the hypothesis that the cellular environment becomes progressively more oxidizing during development.

A crude extract of Ehrlich ascites tumor cell homogenate was found to contain three distinct bands of superoxide dismutase activity by polyacrylamide gel electrophoresis. Activity bands migrated approximately the same distance and were inhibited by cyanide ions. Isolated mitochondria produced two bands of activity that were also inhibited by cyanid. Ethanol-chloroform treatment of the homogenate had no observable effect on these bands of activity, which suggested that the cyanide-insensitive mitochondrial superoxide dismutase activity in these malignant cells was either present in concentrations below detectable levels or completely absent. Normal liver was used as a control for the detection system.

The effect of glutathione and a glutathione reductase inhibitor on the antitumor effect of Cu(II)(3,5-diisopropylsalicylate)2 (CuDIPS) was studied. CuDIPS is a low-molecular-weight copper coordination compound that exhibits superoxide dismutase-like activity. CuDIPS had antitumor activity against intraperitoneal Ehrlich ascites carcinoma in Swiss mice. A single ip injection of glutathione partially eliminated the antitumor effect of CuDIPS, whereas a single ip injection of 1,3-bis(2-chloroethyl)-1-nitrosourea enhanced the antitumor effect of CuDIPS. These results are consistent with the hypothesis that CuDIPS exerts part of its antitumor effect by producing H2O2.

The growth of a solid tumor induced by im implantation of Ehrlich ascites tumor cells in inbred CBA/J mice was retarded by treatment with an iron chelator, picolinic acid (PLA). Survival of the mice was also significantly increased after PLA treatment. However, the iron chelator deferoxamine had no such effects; tumor growth was slightly enhanced, and survival was decreased.

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.

X-ray-induced adenocarcinomas in the small bowels of outbred Lewis Brown Norway and Holtzman rats contained significantly lower concentrations of adenosine 3',5'-cyclic monophosphate than did normal rat intestinal tissue. No significant differences were observed between the intracellular concentrations of the nucleotide in the normal rat small bowel and those occurring in normal-appearing intestinal tissue exposed to tumor induction conditions.

1,2-Dimethylhydrazine-induced large bowel tumors in adult male noninbred Holtzman rats contained greatly elevated levels of copper-zinc-containing superoxide dismutase (Cu-Zn SD) activity when compared to levels in normal intestinal tissue but nearly equal levels of manganese-containing superoxide dismutase (Mn SD) activity. Inasmuch as normal intestinal tissue contains many cell types, SD activities were also measured and compared in isolated intestinal epithelial cells. When compared to the activities in villus cells, Cu-Zn SD activity was greatly increased, whereas Mn SD activity was greatly reduced in tumor cells. The SD activity of tumor cells most nearly resembled that of isolated intestinal crypt cells.

This assay for superoxide dismutase (SOD, EC 1.15.1.1) activity involves inhibition of nitroblue tetrazolium reduction, with xanthine-xanthine oxidase used as a superoxide generator. By using a reaction terminator, we can determine 40 samples within 55 min. One unit of activity of pure bovine liver Cu,ZnSOD and chicken liver MnSOD was expressed by 30 ng and 500 ng of protein, respectively. The mean concentrations of Cu,ZnSOD as measured by this method in blood from normal adults were 242 (SEM 4) mg/L in erythrocytes, 548 (SEM 20) micrograms/L in serum, and 173 (SEM 11) micrograms/L in plasma. The Cu,ZnSOD concentrations in serum and plasma of patients with cancer of the large intestine tended to be less and greater than these values, respectively, but not statistically significantly so.

By means of both direct assay and gel electrophoresis, normal A/J mouse liver was shown to possess both Cu-Zn and Mn superoxide dismutase (SD) activity. H6 hepatoma cells contained Cu-Zn SD activity, but no Mn SD activity was detectable. Isolated mitochondria from normal liver contained both forms of the enzyme, but isolated mitochondria from H6 hepatoma cells contained no SD activity. To ascertain whether this loss of Mn SD activity was characteristic of these tumor cells or was simply a property of rapidly dividing cells, SD activity was measured in regenerating liver. Mn SD activity was present in the regenerating liver at all times after surgery. Hence loss of the Mn SD activity seemed to be a characteristic of some tumor cells but not of corresponding rapidly dividing normal cells.

Solid tumors were induced by implantation of 5 X 10(6) Ehrlich carcinoma cells im into the right flank of 8- to 12-week-old female CBA/J mice. Tumor-bearing mice were killed at 0, 2, 4, 10, or 24 days after im implantation of the tumor cells, and superoxide dismutase (SOD) activities were determined in liver, spleen, kidneys, lungs, and leg muscle. Depressed SOD activities were seen in all organs studied. In liver, spleen, and kidneys, the manganese SOD (MnSOD) activities were depressed at some point after implantation, even though microscopic examination revealed no evidence of metastases in these organs. Cytochrome c oxidase activity was not diminished in any of the tissues studied, indicating that the decline in MnSOD was not due to a decline in the number of mitochondria or to a general decline in mitochondrial enzymes. When the tumor cells were dialyzed against 0.9% saline, the dialysate contained a factor that when injected im also inhibited SOD activity.

Superoxide dismutase (SOD) activity in Chinese hamster ovary (CHO) and ovarian carcinoma (OvCa) cells was measured after exposure to hyperthermia and correlated with the development of thermotolerance. The SOD activity of each cell type was largely copper- and zinc-containing SOD activity. Both cell types had similar but low levels of SOD activity when the cells were grown at 37 degrees C. After exposure for 2 h at 41.5 degrees C, SOD activity of OvCa cells, but not of CHO cells, was increased. After exposure to 45 degrees C for 15 min, SOD activity was also increased in the OvCa cells, but not in CHO cells. After 15 min at 45 degrees C followed by 1 h incubation at 37 degrees C, SOD activity was increased in OvCa and CHO cells; after an 8-h incubation at 37 degrees C, SOD activity doubled in each cell type. Thermotolerance is maximal after 2 to 3 h of exposure at 41.5 degrees C and after 8 to 10 h incubation at 37 degrees C following exposure to 15 min at 45 degrees C. The turnover of SOD activity in OvCa cells was estimated by the rate at which activity was lost following addition of cycloheximide (10 micrograms/ml). Twenty-four % of the activity was lost with a half-life of 10 min, and 76% was lost with a half-life of 4.5 h. Despite restriction of general protein synthesis 3 to 4 h after 45 degrees C hyperthermia, SOD activity was increased at 1 and 8 h after exposure, presumably coincidental with heat shock protein synthesis and development of thermotolerance. These data suggest that SOD activity may be important in protecting cells exposed to heat and that it may play a role in the development of thermotolerance.