Connye N. Kuratko

Econazole and miconazole inhibit steroidogenesis and disrupt steroidogenic acute regulatory (StAR) protein expression post-transcriptionally

The imidazole antifungal drugs econazole and miconazole have previously been shown to disrupt steroidogenesis in Leydig and adrenal cells by inhibiting 17alpha-hydroxylase/17,20-lyase (P450c17) enzyme activity, thus reducing the conversion of progesterone to androstenedione. However, a recent study in Y-1 adrenal cells indicated that these compounds may also reduce the availability of cholesterol to the cytochrome P450 side chain cleavage (P450(scc)) enzyme, the first enzyme in the steroidogenic pathway. Since the steroidogenic acute regulatory protein (StAR) mediates the transfer of cholesterol from the outer to the inner mitochondrial membrane where the P450(scc) enzyme resides, an action which constitutes the rate-limiting and acutely-regulated step in steroidogenesis, we hypothesized that these drugs may also reduce StAR expression and/or activity. Our studies demonstrate that these drugs reversibly inhibited (Bu)(2)cAMP-stimulated progesterone production in a dose- and time-dependent manner in MA-10 cells without affecting total protein synthesis or P450(scc) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) enzyme expression or activity. In contrast, they dramatically decreased (Bu)(2)cAMP-stimulated StAR protein expression post-transcriptionally. This study indicates that StAR protein is susceptible to inhibition by at least some imidazole compounds that inhibit steroidogenesis.

Proliferation of colonic lymphocytes in response to inflammatory cytokines is lower in mice fed fish oil than in mice fed corn oil

The literature supports an association of chronic inflammation with the development of tumors. The colon contains a specialized lymphocyte population that may influence various stages of colon carcinogenesis. Dietary factors are known to affect both inflammation and tumor development in this tissue. Diets high in n-6 fatty acids are considered to be proinflammatory and tumor-promoting whereas n-3 fatty acids are not. This study examined the proliferative response of colonic lymphocytes (CL) from BALB/c mice fed a diet high in either corn oil or menhaden oil when cultured in the presence of proinflammatory cytokines. CL were isolated from mice fed one of the experimental diets and cultured in the presence of anti-CD2, IL-1beta, IL-2, or TNF-alpha. Proliferation of CL in culture was measured by BrdU incorporation. CL from mice fed the high n-3 diet showed lower rates of proliferation following exposure to the inflammatory cytokines than CL from mice fed the high n-6 diet. CL from the high n-3 group showed the same proliferative potential as those from the n-6 diet group following exposure to a combination of anti-CD2 and TNF. Results from this study indicate that diets high in n-3 fatty acids slow the inflammatory response in the colon as compared to diets high in n-6 fatty acids. The n-3 lipids do not appear to compromise overall immune potential, however.

Increasing dietary lipid and iron content decreases manganese superoxide dismutase activity in colonic mucosa

Manganese superoxide dismutase (MnSOD) is an important mitochondrial antioxidant. Alteration in the regulation of MnSOD activity has been proposed to play a critical role in the development of many types of tumors. Colorectal cancer is one of the most common human malignancies and has been shown to be influenced by dietary factors. Two such dietary factors include lipid and iron. Lipid and iron are also potential modulators of MnSOD activity. This study examined lipid and iron influence on MnSOD activity in colonic mucosa. Fischer rats were fed one of eight test diets for six weeks. Four of the diets included AIN-76A-based formulas containing 5% corn oil or 20% lipid from corn oil, menhaden oil, or beef tallow. Four additional diets included identical formulations with iron supplemented to a level of 140 mg/kg. Results showed that iron supplementation decreased MnSOD activity in animals fed the 5% corn oil diet. An increase in dietary lipids from 5% to 20% also decreased MnSOD activity in colonic mucosa. The lipid and iron variables used in this study decreased MnSOD activity without affecting manganese status or other antioxidant mechanisms in this tissue.

Linoleic acid and tumor necrosis factor-α increase manganese superoxide dismutase activity in intestinal cells

Manganese superoxide dismutase (MnSOD) protects mitochondria from oxidative damage. Alterations in the regulation of MnSOD plays an important role in the development of many types of cancer. Activity of this enzyme is induced by inflammatory cytokines and other conditions that increase oxygen radical production. High levels of dietary lipid have been shown to decrease MnSOD activity. This study was designed to define the effect of various type of fatty acids on MnSOD activity and MnSOD induction. IEC-6 cells were treated with 40 micromol/l of either linoleic acid (LA), eicosapentaenoic acid (EPA), or oleic acid (OA) in the presence or absence of 10 ng/ml tumor necrosis factor-alpha (TNF-alpha). Fatty acid supplementation increased MnSOD activity. MnSOD activity was greater in the LA group than in the EPA or OA groups. TNF-alpha induced MnSOD activity equally in all fatty acid-supplemented groups. High levels of MnSOD activity may be an indicator of chronic inflammation resulting from fatty acid, particularly LA, supplementation.

Effects of dietary fat and 1,2-dimethylhydrazine on microsomal lipid peroxidation

Abstract An increased intake of polyunsaturated fatty acids, especially those of the omega-3 series, has shown beneficial effects in prevention of chronic diseases, including cancer. It is not known if at high levels, incorporation of these fatty acids into membrane phospholipids will cause adverse effects due to increased tissue susceptibility to peroxidation. This study was designed to determine whether diets high in polyunsaturated fatty acids would increase liver and colon susceptibility to peroxidation. Secondly, the study examined the effect of diet and the colon carcinogen, 1,2-diemethylhydrazine, on potential peroxidation in these tissues. Rats were fed diets of varied fatty acid composition and effects were compared over time and as the result of injection of 1,2-dimethylhydrazine. Test diets consisted of a low fat corn oil diet or high fat diets of either corn oil, beef tallow, or menhaden oil. Fatty acid composition of colon microsomes was determined. Lipid peroxidation products were measured as thiobarbituric acid-reacting substances in liver and colon microsomes before and after induction with iron/ADP/ascorbate. Content of polyunsaturated fatty acids in colon microsomes was shown to vary according to diet. Induced peroxidation of liver microsomes was highest in the menhaden oil group but only in young animals. Older animals showed greater levels of induced peroxidation in the liver and less effect of diet. Dimethylhydrazine increased inducible peroxidation in the liver without a clear pattern of difference by diet. Tissue differences were also apparent. Colon microsomes showed resistance to peroxide induction by iron/ADP/ascorbate and no difference in peroxide content as the result of DMH. Increases in lipid peroxidation do not appear to be associated with this model of colon carcinogenesis.

Dietary lipids alter fatty acid composition and PGE2 production in colonic lymphocytes

The immune system can influence the development and growth of tumors, including those of the colon. Most studies of immune function utilize immune cells from the blood. Colonic mucosa, however, contains independently functioning lymphocytes that may have significant impact on colon tumor formation. Dietary lipids are known to influence the development of colon tumors and the function of peripheral immune cells. This study was designed to determine dietary lipid effect colonic lymphocytes (CL). CL were isolated from rats fed a diet high in corn oil or menhaden oil and cultured for 72 hours in the presence of absence of mitogen stimulation. Fatty acid composition of CL was measured by gas chromatography. Prostaglandin E2 levels were measured in conditioned media by enzyme-linked immunosorbent assay. CL proliferation rates were determined by colorimetric assay and thymidine incorporation. Results showed that CL from rats fed a diet high in corn oil had greater membrane concentrations of linoleic and arachidonic acids and higher levels of prostaglandin E2 production than those from rats fed a menhaden oil diet. Mitogen stimulation did not increase CL proliferation. Antitumor effects of n-3 fatty acids in the colon may involve anti-inflammatory responses by colonic lymphocytes.

Effects of dietary calcium and vitamin D3 on tumor promotion in mouse skin

The effects of low, adequate, and supplemental intake of calcium and vitamin D3 on 12-O-tetradecanoylphorbol-13-acetate (TPA) skin tumor promotion were examined. Administration of the experimental diets was started one week before the first TPA application to the 7,12-dimethylbenz[a]anthracene-initiated dorsal skin of female Sencar mice. Neither dietary calcium in a range from 0.15% to 2.0% of the diet as calcium carbonate nor vitamin D3 ranging from 200 to 4,000 IU/kg diet resulted in modulation of the skin papilloma response in terms of incidence, number per mouse, or size distribution of tumors. There were also no effects of the varied levels of calcium or vitamin D3 on mouse body weights, serum calcium, or TPA induction of epidermal ornithine decarboxylase activity. These results indicate that dietary administration of a wide range of doses of calcium or vitamin D does not alter the serum calcium levels and, therefore, does not appear capable of altering skin tumor promotion. These results are in contrast to reports that demonstrate antineoplastic activity for both calcium ion and active hormonal vitamin D, either in control of epidermal cell proliferation and/or differentiation or inhibition of carcinogenesis.

Iron increases manganese superoxide dismutase activity in intestinal epithelial cells

Manganese superoxide dismutase (MnSOD) is an enzyme that functions in the mitochondria to detoxify superoxide radicals. MnSOD is low or absent in tumor cells and may act as a tumor suppressor. MnSOD is induced by inflammatory cytokines such as tumor necrosis factor alpha (TNF), potentially as a protective measure for host cells. Previous studies have shown that MnSOD activity decreases during early stages of colon carcinogenesis. Studies using a rodent model showed that diets high in iron also decreased the activity of this enzyme. The present study was designed to use a cell culture model to further investigate the effect of supplemental iron on MnSOD production and activity. Rat intestinal epithelial cells (IEC-6) were cultured in media supplemented with either linoleic acid or eicosapentaenoic acid and iron. The effect of iron on lipid peroxidation products and rate of proliferation was determined. MnSOD protein levels and activity were determined in the lipid and iron treated cells following treatment with TNF. Results showed that lipid peroxidation products increased with iron supplementation. Iron supplementation did not alter cell proliferation. Iron supplementation increased MnSOD protein levels and activity in the non-TNF treated cells but did not influence the ability of TNF to induce MnSOD in IEC-6 cells. Findings indicate that supplemental iron may increase MnSOD due to increased oxidative stress but does not compromise the ability of inflammatory mediators to further increase the activity of this enzyme.

Dietary lipid and iron modify normal colonic mucosa without affecting phospholipase A2 activity

Phospholipase A2 (PLA2) functions as the rate-limiting step in arachidonic acid metabolism and in the removal of damaged or peroxidized membrane lipids. It is elevated in some human tumors and may be involved with mechanisms of tumor promotion. In vitro systems have shown PLA2 activity to be altered by variations in fatty acid and antioxidant components. This study encompassed two objectives. First, PLA2, activity in colon tumors produced using azoxymethane (AOM) in Fischer-344 rats was examined. Secondly, this study tested the effect of iron supplementation as a potential pro-oxidant in diets of varied fatty acid composition on PLA2 activity. Diets included 35 or 140 mg/kg or iron in AIN-76A based diets high in corn oil, menhaden oil, or beef tallow. Results for the first objective showed PLA2 activity to be significantly higher in colon tumors than in normal mucosa with the increase due primarily to an increase in activity within a particulate subcellular fraction. In the second objective, fatty acid composition of colon mucosa was altered by both dietary fat and iron. Animals fed beef tallow had the highest level of oleic acid and corn oil-fed animals had the highest level of linoleic acid. Animals fed menhaden oil had the lowest level of arachidonic acid and highest level of alpha-linolenic, eicosapentaenoic, docosapentaenoic, and docosahexaenoic acids. Iron supplementation in diets high in corn oil resulted in decreased membrane composition of palmitoleic and delta-linolenic acid. In spite of these changes in membrane composition, there were no changes in PLA2 activity. These results show that PLA2 activity is increased in AOM-induced tumors but that diet alone does not influence PLA2 activity in this model.

Mitochondrial lipid peroxidation is influenced by dietary factors in early colon carcinogenesis

Abstract Oxidative damage to mitochondrial proteins, lipids, and DNA seem to influence the promotion and progression of tumors. High-fat diets and diets high in iron decrease manganese superoxide dismutase activity, a mitochondrial antioxidant, in colon mucosa. Lipid peroxidation products are low in microsomal preparations from colonic mucosa even under peroxide-inducing conditions. However, damage specific to mitochondrial membranes is unknown. This study was designed to investigate dietary lipid and iron effects on fatty acid incorporation and lipid peroxide formation in mitochondrial membranes of colonic mucosa. Male Fischer rats were fed high-fat diets containing either corn oil or menhaden oil with an iron level of either 35 or 535 mg/kg diet. Animals were given two injections of the colon carcinogen, azoxymethane, or saline. Colon tissue was collected 1 and 6 weeks after injections. Mitochondrial and microsomal fractions were prepared for fatty acid analysis and quantitation of lipid peroxidation products. Results showed that lipid composition of both subcellular fractions were influenced by diet. Fatty acid composition of mitochondria differed from microsomes, but overall saturation remained constant. Peroxidation products in mitochondrial membranes were significantly greater than in microsomal membranes. Dietary treatment significantly affected mitochondrial peroxidation in carcinogen-treated animals. Therefore, mitochondria from colon mucosa are more susceptible to peroxidation than are microsomes, dietary factors influence the degree of peroxidation, and the resulting damage may be important in early colon carcinogenesis.