In vivo inhibition of nitric oxide synthase gene expression by curcumin, a cancer preventive natural product with anti-inflammatory properties.

Curcumin is a naturally occurring, dietary polyphenolic phytochemical that is under preclinical trial evaluation for cancer preventive drug development and whose working pharmacological actions include anti-inflammation. With respect to inflammation, in vitro, it inhibits the activation of free radical-activated transcription factors, such as nuclear factor kappaB (NFkappaB) and AP-1, and reduces the production of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF alpha), interleukin-1beta (IL-1beta), and interleukin-8. Inducible nitric oxide synthase (iNOS) is an inflammation-induced enzyme that catalyzes the production of nitric oxide (NO), a molecule that may lead to carcinogenesis. Here, we report that in ex vivo cultured BALB/c mouse peritoneal macrophages, 1-20 microM of curcumin reduced the production of iNOS mRNA in a concentration-dependent manner. Furthermore, we demonstrated that, in vivo, two oral treatments of 0.5 mL of a 10-microM solution of curcumin (92 ng/g of body weight) reduced iNOS mRNA expression in the livers of lipopolysaccharide(LPS)-injected mice by 50-70%. Although many hold that curcumin needs to be given at dosages that are unattainable through diet to produce an in vivo effect, we were able to obtain potency at nanomoles per gram of body weight. This efficacy is associated with two modifications in our preparation and feeding regimen: 1) an aqueous solution of curcumin was prepared by initially dissolving the compound in 0.5 N NaOH and then immediately diluting it in PBS; and 2) mice were fed curcumin at dusk after fasting. Inhibition was not observed in mice that were fed ad lib., suggesting that food intake may interfere with the absorption of curcumin.

Effect of exercise on bone mineral metabolism in diabetes.

The authors attempt to assess the relationship between exercise, diabetes, and bone metabolism. An animal model system was developed to study the relationship. Animals given streptozotocin to induce a type I diabetic state show elevated plasma glucose and triglyceride levels and a decrease in body weight. Analysis of femurs revealed alterations in copper, phosphorus, and zinc levels in animals with diabetes compared with controls. Mineral analysis of femurs from diabetic animals placed on an exercise regimen revealed values closer to control levels.

Polyunsaturated fatty acid supplementation in peripheral neuropathy.

This study is an attempt to assess the role of dietary supplementation in the treatment and prevention of diabetic peripheral neuropathy. The authors developed an animal model system to study this problem. Animals given streptozotocin to induce a type I diabetic state showed elevated glucose levels and decreased body weight. Analysis of the sciatic nerve revealed a decrease in nerve conduction velocity and Na(+)-K(+)-ATPase activity. The activity of protein kinase C, another component of the nerve transmission process, was also affected by the diabetic state. The dietary intervention of polyunsaturated fatty acids seemed to revert some of these changes toward normal.

Plasma and cellular zinc levels and membrane lipid composition in streptozotocin diabetic rats.

1. Lipid and zinc analyses were conducted on liver mitochondrial and microsomal membranes as well as erythrocyte ghosts from streptozotocin (STZ) treated animals. 2. In STZ animals, analysis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) fatty acids revealed an increase in palmitic acid and a corresponding decrease in stearic acid. 3. Polyunsaturated fatty acids were also affected, with an increase in 18:2, decrease in 20:4 and an increase in 22:6 in STZ animals compared to controls. 4. The change in fatty acid composition was observed in all three membrane fractions. 5. Plasma zinc levels in STZ animals were elevated while no difference was observed in membrane bound zinc. 6. Thus, while there appears to be both altered trace metal as well as membrane lipid metabolism in STZ treated animals, membrane bound zinc does not seem to be affected.

Effect of a zinc-deficient diet on mitochondrial and microsomal lipid composition in TEPC-183 plasmacytoma.

A zinc-deficient diet caused an increase in microsomal membrane phospholipid levels compared to ad libitum controls. Cholesterol levels were found to be decreased 50% compared to either pair-fed or ad libitum controls, resulting in a sharp decline in the cholesterol/phospholipid ratio. No differences were observed in the distribution of phospholipid classes among all three groups, either in mitochondrial or microsomal membrane fractions. Fatty acid analysis of PC and PE revealed a rise in the 18:2 fraction from zinc-deficient mitochondrial and microsomal membrane fractions. Mitochondrial PE and PC from zinc-deficient animals revealed a rise in the 22:6 fatty acid fraction while microsomal PC also revealed a corresponding decrease in 20:4. None of the zinc-deficient preparations differed significantly from either ad libitum or pair-fed controls in the content of long-chain alk-l-enyl ethers. The results of this study point to an effect of a zinc-deficient diet on lipid metabolism in tumor subcellular membranes which may account for the decreased rate of tumor growth observed in zinc-deficient animals.

Effect of a zinc-deficient diet on lipid peroxidation in liver and tumor subcellular membranes.

The effect of a zinc-deficient diet on lipid peroxidation in liver and tumor mitochondrial and microsomal membrane preparations from BALB/c mice was investigated. Mitochondrial and microsomal membranes from both tissues displayed increased rates of in vitro peroxidation, both enzymatic and nonenzymatic. Measurement of in vivo peroxidation, using diene conjugation as an index of measurement revealed slight increases in tissues from zinc-deficient animals that were not statistically significant. Serum lipoperoxides analyzed from all three groups revealed no significant differences. The results point to an alteration in the peroxidation potential of mitochondrial and microsomal membranes due to zinc deficiency which may be related to an alteration in fatty acid composition.

An immunosuppressive lipoprotein fraction from TEPC-183 bearing mice.

Lipoprotein (LP) fractions prepared from sera of normal and plasmacytoma (TEPC-183) bearing mice, were compared with respect to their effect on the in vitro anti-2,4,6-trinitrophenyl plaque forming cell response. Very low density lipoprotein (VLDL) and low density lipoprotein (LDL) fractions of sera from the plasmacytoma bearing mice contained immunosuppressive activity that was absent in VLDL and LDL fractions of normal serum LP fractions. The suppressive activity did not correlate with cholesterol or triglyceride content. Crossed-immunoelectrophoresis and polyacrylamide gel electrophoresis revealed components of the suppressive VLDL and LDL fractions which were not present in normal serum LP fractions, suggesting a modification of serum LPs by the plasmacytoma.

The effect of TEPC-183 plasmacytoma growth on beta-glucuronidase activity in serum and tissues of tumor-bearing mice.

beta-Glucuronidase activity increased in the serum of BALB/c mice during the growth of the IgM-secreting plasmacytoma, TEPC-183. The increase appeared to correlate with tumor burden. The beta-glucuronidase activity in tissue homogenates of spleen, liver, and kidney from tumor-bearing mice also increased significantly compared to the levels found in corresponding tissues from normal control mice. Assays of lysosomal and microsomal fractions from livers of TEPC-bearing mice indicated that approximately 70% of the enzyme activity was associated with the lysosomal fraction and the remainder with the microsomal fraction. A similar distribution was found in homogenates prepared from the plasmacytoma itself. In contrast to this the beta-glucuronidase activity in livers from normal BALB/c mice is nearly equally distributed between lysosomal and microsomal fractions.

Changes in serum, liver, and tumor zinc levels during plasmacytoma growth in BALB/c mice.

The effect of a zinc-deficient diet on serum and liver zinc levels was studied in BALB/c mice in the presence and absence of the IgM-secreting plasmacytoma, TEPC-183. While serum zinc levels were significantly decreased in the zinc-deficient (ZD) mice by Week 2, there was no change in the level of zinc in the liver of this group compared to ad libitum (AL) or pair-fed (PF) controls even after 4 weeks on the respective diets. The presence of the tumor itself resulted in a significant decrease in serum zinc levels in mice maintained on a normal-chow diet. This decrease was not seen in the serum from tumor-bearing AL or PF control mice which were fed a synthetic diet containing 50 ppm zinc. There was, however, an increase in the liver weights of mice in these groups in the presence of TEPC-183 which was not seen in the ZD tumor-bearing mice. Although total zinc levels increased in the livers of AL and PF mice reflecting this increase in liver weight, there was no difference among the groups when the amount of zinc was determined per gram of liver tissue. There was also no difference in the amount of zinc per gram of tumor tissue in the tumors obtained from AL, PF, or ZD mice.

Effect of a zinc-deficient diet on the growth of an IgM-secreting plasmacytoma (TEPC-183).

A diet deficient in zinc resulted in a significant decrease in the number of inbred BALB/c female mice developing transplantable plasmacytoma TEPC-183 compared to pair-fed and ad libitum-fed control mice. The size of the tumor relative to body weight was decreased in the zinc-deficient group. Whereas the uptake of [3H]thymidine into tumor cells did not differ between the groups, the incorporation of [3H]thymidine into tumor cells was significantly reduced in the animals maintained on a zinc-deficient diet.

The effect of plasmacytomas on serum immunoglobulin levels of BALB/c mice.

The effect of tumor growth on serum immunoglobulin levels and on the immune response to sheep erythrocytes (SRBC) was studied in BALB/c mice bearing MOPC-315 (IgA), MOPC-460 (IgA), MOPC-173 (IgG2a) and MOPC-104E (IgM) to gain insight into the immunologic competence of the plasmacytoma-bearing host. The initial increase of myeloma protein coincided with the first appearance of the tumor and increased as the tumor progressed. However, at the time of death there was little correlation between spleen weights, tumor size, and myeloma-protein levels. The mean serum concentration of the myeloma proteins reached a higher level in the mice bearing tumors transplanted i.p. compared to those with tumors transferred subcutaneously (s.c.). Non-myeloma immunoglobulin levels in the serum were reduced: IgM was significantly lowered in the presence of MOPC-315 injected i.p. and MOPC-460 injected s.c. and the IgG2 levels were depressed in mice injected i.p. with MOPC-315 and MOPC-104E. The only significant reduction of IgA levels was seen when MOPC-173 was transplanted i.p. The decreases observed in immunoglobulin levels correlated with plasmacytoma growth. They were specific for myeloma and were not due to tumor growth per se since the levels of all immunoglobulins tested increased in the presence of Sarcoma 37, a pleomorphic neoplasm. The primary plaque-forming cell (PFC) response of tumor-bearing animals after the injection of 0.5 ml of 10% SRBC was either similar or enhanced compared to the controls. However, with a lower SRBC dosage (0.5 ml of 2% SRBC) the indirect PFC were reduced with mice bearing MOPC-104E and MOPC-173. Tumor sizes did not seem to correlate with reduction of the PFC response. MOPC-460-bearing mice had a comparable number of PFC per spleen to those of the controls, but reduced numbers when calculated per 10 spleen cells. Consistently, hemagglutination titers were reduced in all tumor-bearing animals. The number of direct and indirect PFC per spleen was increased in mice bearing Sarcoma 37, compared to the controls. The possible implications of these findings are discussed.