Lowered superoxide dismutase activity in distant organs of tumor-bearing mice.

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 activity in 1,2-dimethylhydrazine-induced rat colon adenocarcinoma.

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.

Cyclic nucleotide concentrations in 7.12-dimethylbenz[a]anthracene-induced pancreatic cancer in rats.

Pancreas cancer was induced in noninbred male Holtzman rats by the implantation of beeswax containing 7.12-dimethylbenz[a]anthracene (DMBA) into the "head" of the pancreas. The tumors that developed 4--6 months later were examined for their cyclic AMP and cyclic GMP levels. The lesions could be considered in one of two categories according to their cyclic nucleotide contents: lesions with significantly smaller amounts and those with greater amounts, compared with levels measured in the pancreas tissues of the control rats. The existence of two biochemically distinct groups may indicate different growth patterns of the DMBA-induced pancreatic neoplasia.

Superoxide dismutase levels in Chinese hamster ovary cells and ovarian carcinoma cells after hyperthermia or exposure to cycloheximide.

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.

Superoxide dismutase activity in the intestine of the streptozotocin-diabetic rat.

Insulin stimulates the production of superoxide and hydrogen peroxide in various tissues. Hydrogen peroxide has been proposed to be an intracellular second messenger for insulin and a moderator of cellular proliferation and differentiation. We previously found that cell proliferation is increased in small intestinal mucosa of streptozotocin-diabetic rats. The current study was undertaken to determine if superoxide dismutase (SOD), the enzyme that converts superoxide to hydrogen peroxide, is altered in the mucosa of the alimentary tract and renal cortex of the diabetic rat, and if so, whether SOD responds to insulin treatment. Total SOD and cyanide-insensitive [manganese-containing SOD (Mn SOD)] SOD were measured by the nitroblue tetrazolium inhibition assay. We studied ad libitum fed animals, where diabetics are hyperphagic and pair-fed animals, where hyperphagia is not present. Since cyclic nucleotides appear to control cell proliferation in some tissues, we also measured cAMP and cGMP in mucosa of the small intestine. In ad libitum fed animals, total SOD was depressed in the mucosa of duodenum, jejunum, and ileum, but not in the cecum or colon of the streptozotocin-diabetic rats. The level of Mn-SOD was not affected by diabetes or insulin treatment, but the cyanide-sensitive [copper- and zinc containing SOD (Cu-Zn SOD] SOD was depressed in the small intestine and colon of diabetic rats. Insulin treatment restored total and Cu-Zn SOD activity in the small intestine to normal and increased Cu-Zn SOD activity in the colon to normal. Pair-fed animals showed the same changes in the SOD activity of jejunal mucosa that were found in ad libitum fed animals. In renal cortex, diabetes did not alter total SOD, but increased Mn SOD and decreased Cu-Zn SOD. Both responses were reversed by insulin treatment. Cyclic nucleotide concentrations were not affected by diabetes. We conclude that SOD enzymes re altered in diabetes, at least in proliferating tissues. Responses are tissue specific. The mucosa of the small intestine and colon show decreased Cu-Zn SOD, the SOD of the cecum is unaffected, and the kidney shows increased Mn SOD and decreased Cu-Zn SOD. The SOD responses of diabetics are reversed by insulin treatment.

Adaptation of the duodenum and ileum of the rat to mid-gut resection: enzyme activity and trace metal status.

Activities of the enzymes lactase, sucrase, maltase, alkaline phosphatase, and superoxide dismutase (SOD) were measured in mucosa of duodenum and ileum of the rat after 70% resection of mid-small intestine or sham operation (transection). We also measured the concentrations of zinc, copper, and manganese in several tissues to assess trace metal homeostasis postresection. Resection resulted in decreased specific activities of disaccharidases and alkaline phosphatase in duodenum, while specific activities remained unchanged in ileum. Specific activity of total SOD (the sum of Cu-Zn and Mn SOD) and Mn SOD was the same in duodenum after resection but was markedly increased in ileum. Tissue trace metal concentrations changed minimally. Because of postresection mucosal growth, total segmental activity of disaccharidases and alkaline phosphatase was the same in duodenum and increased in ileum of resected compared to transected rats. Segmental activity of total SOD and Mn SOD doubled in duodenum and trebled in ileum of resected as compared to transected rats. Thus, total segmental enzyme activity is maintained or increased postresection by increased enterocyte proliferation rate and mucosal growth.

Marrow antioxidant enzyme activity in tumor-bearing and non-tumor-bearing mice following vincristine treatment.

Pretreatment or "priming" with vincristine (VcR) has been documented to radioprotect animals from whole body irradiation by accelerating recovery of hematopoietic marrow. The mechanisms underlying this phenomenon are unclear, but the marked similarities between priming with VcR and with immune stimulants such as endotoxin and glucan have led to speculation that VcR may be inducing such radioprotective immunoregulators as interleukin 1 (IL-1) and tumor necrosis factor (TNF). The radioprotective ability of these cytokines, in turn, has been linked to an induction of the antioxidant enzyme manganese superoxide dismutase (Mn SOD). To establish whether priming with VcR is associated with induction of antioxidant enzymes, the activities of Mn SOD, copper-zinc (Cu-Zn) SOD, catalase (CAT), and glutathione peroxidase (GPX) were measured in the marrow of both LLca tumor-bearing and non-tumor-bearing mice given a priming dose of VcR. Results in non-tumor-bearing mice indicate that, similar to IL-1 and TNF administration, VcR treatment increases Mn-SOD activity, but not Cu-Zn SOD, CAT, or GPX activity. Furthermore, this increase occurs at the time VcR priming has been demonstrated previously to exhibit maximal radioprotection, suggesting that it may be contributing factor. However, VcR priming has been demonstrated to radioprotect both tumor-bearing and non-tumor-bearing animals, and no increase in Mn SOD activity (or the other enzymes monitored) was found in the tumor-bearing group. Rather, the presence of tumor significantly suppressed antioxidant enzyme activity. Collectively, the present data suggest that it is unlikely that increased antioxidant enzyme activity is directly involved in the VcR priming response.

Intracellular adenosine and guanosine 3',5'-cyclic monophosphate concentrations in rat small and large bowel following single and multiple exposures to 1,2-dimethylhydrazine.

The adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) levels were determined in the small and large intestinal tissue of rats that had been exposed to single and chronic administration of the colon carcinogen 1,2-dimethylhydrazine (DMH). A single subcutaneous injection of DMH resulted in a decrease in the intracellular concentration of cAMP and increase in cGMP beyond the levels which had been measured in the unexposed intestinal tissue and DMH induced intestinal adenocarcinomas. Recovery to normal concentrations of the cyclic nucleotides occurred within 30 days. Multiple exposures resulted in maintaining reduced levels of cAMP while cGMP was also found to be lowered upon the chronic administration. A possible explanation for these observations is the expansion of the crypt cell population consisting of replicating intestinal cells that occurs upon exposure to the carcinogen. These findings suggest that cyclic nucleotide alterations may represent a characteristic of the precancerous state of intestinal tissue and indicates further studies are warranted to determine whether these changes may serve as a useful marker in a screening program for colon cancer.

In utero effects of 1,2-dimethyl hydrazine on adenosine and guanosine 3',5'-cyclic monophosphate content in rat fetal intestine.

Sperm positive 12-14-day pregnant Holtzman rats were exposed to a single subcutaneous injection (20 mg/kg body wt) of 1,2-dimethylhydrazine (DMH). Two days post-exposure, the fetal intestinal tissues were examined for their content and metabolic activities for cyclic 3',5'-adenosine monophosphate (cAMP) and cyclic 3',5'-guanosine monophosphate (cGMP). The in utero exposure to the carcinogen resulted in lowering the intracellular content of cAMP and increasing cGMP. The decreased levels of cAMP may be accounted for the finding of a corresponding increase in its breakdown by its phosphodiesterases; however, associated with the increase in cGMP was correspondingly an increase in its phosphodiesterases, suggesting the activities for synthesis of this cyclic nucleotide may be the major factor for its elevated concentration. These observations indicate that DMH may cross the placental barrier and can effect the intracellular cyclic nucleotide concentrations in the fetal tissues as well as acting as a colorectal carcinogen in the adult rat. The changes in the cyclic nucleotide levels were toward the direction expected for an increased cell proliferation; consequently, further investigations are suggested to determine whether a hyperproliferative state is induced by the DMH in the already rapidly dividing fetal intestinal tissue.

Adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate phosphodiesterase activities in 1,2-dimethylhydrazine induced colon adenocarcinoma.

The intracellular concentrations of adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) in 1,2-dimethylhydrazine (DMH) induced rat colon tumors were previously reported to be one-half and twice, respectively, that measured in normal rat colon tissue. The results of this present study indicate that the total amount of cyclic nucleotide hydrolysis by the phosphodiesterase enzymes (PDE) could account for the lower cAMP and elevated cGMP noted in cancer tissue. PDE activities in homogenates prepared from normal and neoplastic tissue showed general similarities in both their hydrogen and metal ion dependencies with optimum degradation of nucleotides occurring at pH 7.2-7.8 in the presence of either Mg2+ or Mn2+ ions. The enzymes from both tissue types were inhibited to a similar extent by papaverine and theophylline. Distribution studies indicated PDE activities were similar throughout the entire length of the normal colon; consequently, the anomalous activities measured in the tumors were attributable to the specific cell population and not to the particular site at which malignancy arose. Thus, the findings of this study suggest that one cause for lowered cAMP and elevated cGMP levels in DMH induced colon adenocarcinomas was the amount of cyclic nucleotide hydrolysis by PDE in the malignant tissue.

Cyclic nucleotide concentrations in 1,2-dimethylhydrazine induced rat colon adenocarcinoma.

The intracellular concentrations of adenosine 3',5'-cyclic monophosphate (cAMP) in colonic tumors induced in adult male Holtzman rats by 1,2-dimethylhydrazine (DMH) were found to be 1/2 the concentration found in normal large bowel tissue. Intracellular concentrations of guanosine 3'-5'-cyclic monophosphate (cGMP) in the neoplastic cells were twice the normal colon level. The concentrations of these two cyclic nucleotides were relatively constant throughout the normal colon. Thus, the anomalous tumor cyclic nucleotide concentrations are attributed to the specific cell population of the lesion and not to the site of development within the colon.

Cyclic nucleotide concentrations in 1,2-dimethylhydrazine and x-ray induced rat small intestinal cancer.

The intracellular concentrations of adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) were determined in adult male Holtzman rat small intestinal tumors induced by subcutaneous administration of 1,2-dimethylhydrazine (DMH) or X-irradiation of only the hypoxic ileum and jejunum. The levels of cAMP and cGMP in the cancer cells from DMH-treated animals were 50% and 200%, respectively, of the values measured for intestinal tissue. The amounts of cGMP in the lesion of the X-irradiation induced rat small bowel adenocarcinoma determined in the present investigation and of cAMP measured previously were observed to be only 50% of the value for unirradiated intestine. It has thus been shown that in the DMH-induced colon and small intestinal tumors, the X-irradiation induced rat small bowel adenocarcinoma and the human colon adenocarcinoma, the cAMP concentrations are consistently about 50% of the levels measured in comparable normal tissues. These findings of diminished intracellular cAMP concentrations imply a serious cellular defect mechanism occurring in intestinal cancer. However, there appears to be no similar pattern of change for the intracellular concentrations of cGMP, which suggests that different biochemical pathways exist in these malignancies.

Evidence for a defective mitochondrial membrane in 1,2-dimethylhydrazine-induced colon adenocarcinoma in rat: enhanced lipid peroxidation potential in vitro.

Enhanced lipid peroxidation potential was measured in Holtzman rat colon tumors induced by chronic subcutaneous injection of 1,2-dimethyl-hydrazine as compared with normal colonic tissue. The peroxidation potentials were determined in the mitochondrial cellular components by measuring the ferrous-ascorbate induced formation of malondialdehyde. The tumor mitochondria were found to peroxidize at a rate 8-10-fold higher than the comparable normal tissue components. In addition, we found that the mitochondria from the cancer cells exhibited reduced NADH-cytochrome c reductase activity. These observations suggest an involvement of non-enzymatic free radical flux in DMH-induced carcinogenesis, which may be the result of structurally altered mitochondrial membranes.

Antioxidant enzyme activity in murine hematopoietic bone marrow following treatment with interleukin 1 alpha: influence of tumor.

To determine whether non-hematologic tumors influence the bone marrow's antioxidant enzyme response to the radioprotective cytokine interleukin 1 alpha (IL-1), studies were undertaken using BDF1 and Balb/c mice bearing small, medium or large Lewis lung carcinoma (LLCa) or EMT6 mammary carcinoma tumors, respectively. Results demonstrated that, similar to nontumor-bearing mice, treatment of tumor-bearing animals with IL-1 was associated with a significant increase in marrow MnSOD activity. However, the duration of this elevated activity was reduced as tumor burden increased, and this reduction may have an impact on IL-1's ability to radioprotect tumor bearing animals, especially when tumor burden is large. In addition to cytokine-mediated responses, significant tumor-related influences on the marrow's antioxidant enzyme status were seen. Notably, it was observed that the presence of tumor was correlated with a marked suppression of antioxidant enzyme activity. Surprisingly, however, the pattern of enzyme suppression was found to differ between the two tumor models studied both in temporal onset and in the number of enzymes involved. In conclusion, the data obtained from these studies on tumor-bearing animals demonstrate that there are both cytokine-related and tumor-related influences which can effect the antioxidant enzyme status of the hematopoietic marrow-influences which may have the potential to alter the marrow's ability to tolerate free radical-generating events, both endogenous (i.e inflammation, infection) and exogenous (i.e. radiation, certain chemotherapeutic drugs) in origin.

Adenosine 3',5'-cyclic monophosphate dependent and independent protein kinase activities in 1,2-dimethylhydrazine induced rat colon cancer.

The adenosine 3',5'-cyclic monophosphate (cAMP)-dependent and cAMP-independent kinase activities were measured in the 1,2-dimethylhydrazine (DMH) induced rat colon cancer and in untreated colon. Previous studies had shown that intestinal tumors induced by chronic exposure to DMH contained 2-fold less intracellular cAMP. The present findings indicate that reduction in cAMP-dependent protein kinase activities also occur in colon cancer cells. Similar hydrogen ion dependence (pH 6-7) and approximate association constants (Ka approximately 0.1 microM) were observed for the enzymes existing in both normal and tumor tissues, while the cAMP-dependent tumor protein kinase was found to phosphorylate phosvitin and casein to a greater degree. These recent findings are consistent with the concept that the concentrations of cAMP and activities of its associated enzyme system are inversely related to the cell proliferation state.

A role for reduced oxygen species in heat induced cell killing and the induction of thermotolerance.

A model suggesting a role for superoxide (O2-) and hydrogen peroxide (H2O2) in heat induced cytotoxicity and development of thermotolerance is proposed: (1) Heat shock increases cellular generation of O2- and H2O2 in proportion to the severity of the heat shock. (2) Heat induced generation of O2- or H2O2 in excess of the ability of the antioxidant enzymes to remove these toxic species causes heat induced cell injury and cytotoxicity. This damage is caused by lipid peroxidation, leading to disruption of the cytoskeleton and calcium metabolism. (3) The flux of O2- and H2O2 generated by heat shock induces the synthesis of additional antioxidant enzymes. Other treatments which induce thermotolerance also cause oxidative stress and induce the antioxidant enzymes. The ability of various agents to modify heat induced cytotoxicity and development of thermotolerance is reviewed in light of this model.

Transformation affects superoxide dismutase activity.

A human, aging lung fibroblast cell strain manifested both copper-zinc superoxide dismutase activity (CuZn-SOD) and manganese-containing superoxide dismutase activity (MnSOD), and these activities were found to be modulated by physiological concentrations of thyroid hormone. The immortal cell lines examined, whether normal or malignant, had no MnSOD activity. The immortal normal cell lines examined had CuZnSOD activity which was modulated by thyroid hormone, whereas the immortal malignant cell lines had CuZnSOD activity which was not affected, or not as strongly affected, by the presence of thyroid hormone.

Adenosine and guanosine 3',5' cyclic monophosphate phosphodiesterase activities in rat small and large bowel following single and multiple exposure to 1,2-dimethylhydrazine.

The hydrolytic activities of adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) phosphodiesterases (PDE) in rat large and small bowel tissue were determined after exposure to the colon carcinogen 1,2-dimethylhydrazine (DMH). Immediate increases in the cAMP-PDE activities were found in the tissues after a single exposure to the chemical which returned towards normal while chronic exposure resulted in no visible changes. The increased cAMP-PDE activities may be the factor responsible for the diminished intracellular cAMP concentrations found after exposure to the carcinogen. In contrast, the cGMP-PDE activities changed little in the colon and increased in the small bowel, suggesting that the determinant for the increases in the concentration cGMP was the activation of guanylate cyclase enzymes following exposure to DMH. Ratios of cAMP to cGMP and cAMP-PDE to cGMP-PDE in the colon showed major changes after carcinogenic insult by the DMH, suggesting that such measurements might serve as useful markers for exposure to environmental toxicants.

Altered radiosensitivity of hematopoietic stem cells by vincristine pretreatment: superoxide dismutase activity as a possible mechanism.

The effect of vincristine (VCR) on hematopoietic stem cell and progenitor compartments and its ability to induce transient periods of radioresistance was investigated so that we could ascertain the drug-radiation intertreatment interval affording optimal radioprotection and determine if its ability to induce increased levels of superoxide dismutase (SOD) is a potential mechanism for this radioprotection. Measurement of marrow stem cell and progenitor compartments demonstrated that these subsets displayed differential sensitivity to VCR and that this sensitivity appeared to be proportional to how "primitive" the subset was. Treatment with VCR prior to irradiation was observed to enhance significantly both 8- and 12-day spleen colony-forming unit recovery with maximal radioprotection occurring for a drug-radiation interval of 12-48 hours. Monitoring of copper-zinc SOD levels demonstrated an increase in activity following VCR that was localized in a fraction of the bone marrow enriched for stem cells and progenitors. The temporal pattern of this increase, however, did not correlate with the drug-radiation schedules affording optimal radioprotection, which indicates that other factors appear to be operative in this radioprotection as well.