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Maintaining reductive-oxidative (redox) balance is an essential feature in breast cancer cell survival, with cellular metabolism playing an integral role in maintaining redox balance through its supply of reduced NADPH. In the present studies, the effect of 1,25-dihydroxyvitamin D (1,25(OH)2D) on redox balance was investigated in early stages of breast cancer. Treatment with 1,25(OH)2D promoted oxidative stress in MCF10A-ras and MCF10A-ErbB2 breast epithelial cells, as measured by the decreased ratios of NADPH/NADP+ and reduced to oxidized glutathione (GSH/GSSG). The mRNA and protein expression of the enzyme pyruvate carboxylase (PC) was downregulated with 1,25(OH)2D treatment, suggesting a potential mechanism. Genetic depletion of PC in MCF10A-ras cells resulted in a decreased ratio of NADPH/NADP+ and GSH/GSSG, with 1,25(OH)2D treatment having no further effect. Mutation analysis confirmed the presence and functionality of a vitamin D response element in the PC gene promoter region. Collectively, these results provide evidence that 1,25(OH)2D promotes oxidative stress in early breast cancer progression through transcriptional downregulation of PC.

Both increased de novo fatty acid synthesis and higher neutral lipid accumulation are a common phenotype observed in aggressive breast cancer cells, making lipid metabolism a promising target for breast cancer prevention. In the present studies we demonstrate a novel effect of the active metabolite of vitamin D, 1α,25-dihydroxyvitamin D (1,25(OH)2D) on lipid metabolism in malignant breast epithelial cells. Treatment of MCF10CA1a breast epithelial cells with 1,25(OH)2D (10 nM) for five and seven days decreased the level of triacylglycerol, the most abundant form of neutral lipids, by 20%(±3.9) and 50%(±5.9), respectively. In addition, 1,25(OH)2D treatment for five days decreased palmitate synthesis from glucose, the major fatty acid synthesized de novo (48%±5.5 relative to vehicle). We have further identified the anaplerotic enzyme pyruvate carboxylase (PC) as a target of 1,25(OH)2D mediated regulation and hypothesized that 1,25(OH)2D regulates breast cancer cell lipid metabolism through inhibition of PC. PC mRNA expression was downregulated with 1,25(OH)2D treatment at two (73%±6 relative to vehicle) and five (56%±8 relative to vehicle) days. Decrease in mRNA abundance corresponded with a decrease in PC protein expression at five days of treatment (54%±12 relative to vehicle). Constitutive overexpression of PC in MCF10CA1a cells using a pCMV6-PC plasmid inhibited the effect of 1,25(OH)2D on both TAG accumulation and de novo palmitate synthesis from glucose. Together, these studies demonstrate a novel mechanism through which 1,25(OH)2D regulates lipid metabolism in malignant breast epithelial cells.

Advanced glycation end products (AGEs) result from non-enzymatic glycation of proteins and cause cellular oxidative stress in a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent manner. Due to these effects, AGEs are implicated as a causal factor in diabetic complications. Several antioxidants, including vitamin E, improve cell viability and diminish markers of oxidative damage in cells exposed to AGEs. However, vitamin E has been studied in cell culture systems with primary focus on apoptosis and lipid peroxidation, while its influences on AGE-induced protein and DNA oxidation, intracellular antioxidant status and cell morphology remain largely unknown. Here, we verify the suppression of AGE-induced cell death and lipid peroxidation by 200 μM α-tocopherol in SH-SY5Y cells. We report the partial inhibition of DNA oxidation and a decrease in protein carbonyl formation by α-tocopherol with no effects on intracellular GSH concentrations. We observed that 2 mM N-acetyl cysteine (NAC) also had a suppressive effect on DNA and protein oxidation, but unlike α-tocopherol, it caused a marked increase in intracellular GSH. Finally, we compared the ability of both antioxidants to maintain neurites in SH-SY5Y cells and found that α-tocopherol had no effect on neurite loss due to AGEs, while NAC fully maintained cell morphology. Thus, while α-tocopherol suppressed AGE-induced macromolecule damage, it was ineffective against neurite degeneration. These results may implicate thiol oxidation and maintenance as a major regulator of neurite degeneration in this model.

Diabetes mellitus, a disease hallmarked by hyperglycemia, is one of the most common chronic diseases worldwide and new diagnoses are on an upward trajectory. Peripheral neuropathy (PN) affects at least 50% of diabetic patients. Unfortunately, medications available for patients with PN treat the symptoms but not the cellular damage, and come with severe side effects leading many to discontinue their use. The overall objective of this study was to gain a better understanding of the mechanism of action and potential impact of therapeutic substances, such as N-acetylcysteine (NAC), and preventive dietary components, such as the cruciferous vegetable constituent 3H-1,2-dithiole-3-thione (D3T), which is critically important for patient health. Previous studies in our lab have shown that when SH-SY5Y cells, a validated model for PN, are treated with advanced glycation end products (AGE), there is increased reactive oxygen species (ROS) generation, a significant decrease in glutathione (GSH) concentrations, and neurite loss. NAC was shown to protect against AGE-induced cell death, GSH depletion and neurite loss, indicating that maintaining GSH was important for neurite protection. Our lab has also shown that D3T, a potent inducer of nuclear factor (erythroid-derived 2)- like (Nrf2), can significantly increase SH-SY5Y cellular GSH concentrations and protect against H2O2 –induced cell death. Paradoxically, D3T conferred no protection against AGE-induced cell death or neurite degeneration. We report here follow up studies. The first aim of this study was to test whether NAC conferred protection through maintaining GSH, its intrinsic antioxidant properties, or both. Using buthionine sulfoximine (BSO) as a tool to evaluate the mechanistic role of NAC in neurite protection, we found that at high concentrations of NAC (2 mM) both intrinsic and GSH-maintaining properties were observed. At lower concentrations (1 mM), GSH-maintaining properties predominated. The second aim was to elucidate a mechanism for the paradoxical effect of D3T focusing on the role of NADPH-mediated ROS generation. NADPH is an electron donor for the oxidase enzyme that generates superoxide in response to the activation of the receptor for AGE (RAGE). D3T increased ROS generation and depleted GSH only in the presence of AGE, suggesting its role as a NADPH generating agent may promote oxidative stress rather than protect against it under these conditions. Inhibition of NADPH generation via glucose-6-phosphate dehydrogenase (G6PD), the protein product of a Nrf2-responsive gene, with dehydro-epiandrosterone (DHEA) was found to protect against AGE-induced loss of cell viability. These results suggest that the source of ROS is important to consider in evaluating the extent to which adaption to oxidative stress provides protection.

Diabetes is a disease characterized by poor glycemic control for which risk of the type 2 form increases with age. A rise in blood glucose concentration causes increased oxidative stress which contributes to the development and progression of diabetes-associated complications. Studies have shown that primary antioxidants or genetic manipulation of antioxidant defenses can at least partially ameliorate this oxidative stress and consequentially, reduce severity of diabetic complications in animal models. Data from humans is less clear and will be summarized in this review. We highlight results from studies performed to investigate the role of vitamin E in preventing diabetes-induced oxidative damage in cell culture, animal models, and human participants, and summarize evidence testing whether this nutrient has an effect on outcomes related to the diabetic complications of nephropathy, retinopathy, and neuropathy. The most compelling evidence for an effect of vitamin E in diabetes is on protection against lipid peroxidation, whereas effects on protein and DNA oxidation are less pronounced. More studies are required to make definitive conclusions about the effect of vitamin E treatment on diabetes complications in human subjects.

High vitamin D intake is associated with reduced insulin resistance. Expression of extra-renal 1α,25-dihydroxyvitamin D hydroxylase (1α-hydroxylase) has been reported in several tissues and contributes to local synthesis of 1α,25-dihydroxyvitamin D 3 (1,25(OH) 2 D) from the substrate 25-hydroxyvitamin D (25OHD). Expression and dietary regulation of 1α-hydroxylase in tissues associated with energy metabolism, including adipose tissue, has not been assessed. Male Wistar rats were fed a high calcium (1.5%) and high vitamin D (10,000 IU/kg) or a low calcium (0.25%), low vitamin D (400 IU/kg) with either a high fat (40% energy) or high sucrose (66% energy) dietary background for 14 weeks. Expression of 1α-hydroxylase, assessed by real time PCR, was detected in adipose tissue and did not differ with dietary level of calcium and vitamin D. 1α-Hydroxylase mRNA was also detected in 3T3-L1 preadipocytes and 25OHD treatment at 10 nM levels induced 1,25(OH) 2 D responsive gene, CYP24, and this response was reduced in the presence of the p450 inhibitor, ketoconazole. In addition, 3 H 25OHD was converted to 3 H 1,25(OH) 2 D in intact 3T3-L1 preadipocytes. Cumulatively, these results demonstrate that 1α-hydroxylase is expressed in adipose tissue and is functional in cultured adipocytes. Thus, the capacity for local production may play a role in regulating adipocyte growth and metabolism.

Objective To investigate the effect of supplementation with different sources of oils rich in long chain fatty acids, ie, fish oil (FO) and safflower oil (SO), on the production of leukotriene B 4 (LTB 4 ) by polymorphonuclear leukocytes (PMNLs) in hemodialysis patients and the consequent effects on the symptoms of pruritus. Design Randomized, prospective, double-blind study for 2 treatment groups. Setting Three Medical Center—affiliated units. Patients Twenty-two patients on maintenance hemodialysis, of both sexes, age ≥ 20 years with complaint of dry and/or itchy skin. Intervention Two groups of patients receiving daily supplements of 6 g ethyl ester of FO or SO for 16 weeks. Main outcome measures Red blood cell (RBC) fatty acid profile, LTB 4 production by PMNLs, and pruritus symptoms at baseline and after supplementation. Results After supplementation, the FO group had a higher RBC 22:6n3, total n-3 fatty acids, and ratio of total n-3 to total n-6 fatty acids ( P 4 production (pg/mL) from baseline to week 16 was 240.7 ± 200.2 to 29.2 ± 14.6 in the FO group and from 171.1 ± 121.7 to 31.9 ± 14.7 in the SO group. The overall pruritus score change was 16.7 ± 11.4 to 8.9 ± 9.2 in the FO group and from 17.5 ± 8.8 to 13.1 ± 5.6 in the SO group. FO supplementation did not result in a significant specific effect on LTB 4 production by the PMNLs. There was a nonsignificant decrease in the pruritus scores that could be clinically significant and important to patients suffering with this condition. Conclusion Supplementation with FO results in significant incorporation of n-3 fatty acids in the RBCs. Intervention with both FO and SO resulted in a nonsignificant improvement of clinical symptoms of pruritus and a nonsignificant reduction in LTB 4 production by PMNLs in the hemodialysis patients. The percent decrease in total puritus score was greater for the FO group compared with the SO group.

In these studies, prostaglandin H synthase activity was increased in stably Harvey-ras transfected C3H10T1/2 cells. The level of total prostaglandin H synthase protein was two-fold higher in stably Harvey-ras transfected C3H10T1/2 cells than in control cells with no difference in prostaglandin H synthase-2 level. Prostaglandin H synthase-1 mRNA level was two-fold higher in transfected than in control cells, while prostaglandin H synthase-2 was not significantly different. Thus, prostaglandin H synthase-1, but not prostaglandin H synthase-2, expression was increased in Harvey-ras transfected C3H10T1/2 cells.

Several lines of evidence suggest that phospholipase A2 may play a role in the activated ras-mediated transformation process. In the present study, phospholipase A2 activity and expression were assessed in a murine fibroblast cell line (C3H10T1/2 cells) that was stably transfected with the Harvey ras oncogene, a cellular model used for studying multistage carcinogenesis. Reduced levels of fatty acids were released from the ras-transfected cells compared to untransfected controls. The in vitro phospholipase A2 activity apparent in the C3H10T1/2 showed preference for sn-2 arachidonyl phosphatidylcholine compared to dipalmitoyl phosphatidylcholine. The activity, as well as the cytosolic phospholipase A2 immunoreactive protein, was reduced by 50% in the ras-transfected cells compared to control cells. These results suggest that the cytosolic phospholipase A2 is the predominant form of this enzyme family expressed in C3H10T1/2 cells and that the activity and protein amount is reduced by 50% in these cells when stably transfected with the Harvey ras oncogene.

Long-Evans hooded rats maintained on vitamin E and selenium-deficient diets for 6 weeks from weaning exhibited a pattern of enhancement of calcium-independent phospholipase A2 activity in spleen cytosol similar to that previously reported for the lung and liver. The spleen cytosols from vitamin E and selenium deficient rats had approximately 5 fold higher activity than the samples from animals maintained on diets sufficient in these two nutrients (control) or deficient in either nutrient alone. The calcium-dependent PLA2 activity was about 6 fold higher in spleen cytosol of vitamin E and selenium deficient rats compared with cytosolic samples from animals on the other three diets. Time course studies indicated that in rats the calcium-independent phospholipase A2 activity in lung, liver, and spleen increased initially between 4 and 6 weeks of consuming the diets deficient in both vitamin E and selenium, and increased even further again at 7 weeks. This bi-phasic response to the deficiency occurred 1 week after indicators of vitamin E and Se status had reached minimum levels. When animals that were maintained on the deficient diet for 6 weeks consumed the control diet for 1 week the phospholipase A2 activity of lung, liver, and spleen was not different than the activity of the control animals. The malondialdehyde concentration of lung and spleen measured at 6 and 7 weeks correlated positively with the calcium-independent phospholipase A2 activity. These results indicate that deficiency of vitamin E and selenium in the rat leads to a bi-phasic increase in calcium-independent PLA2 activity in rat lung, liver, and spleen, and that the initial increase can be reversed by partial repletion of the two antioxidant nutrients.

Attention-deficit hyperactivity disorder (ADHD) is the term used to describe children who are inattentive, impulsive, and hyperactive. The cause is unknown and is thought to be multifactorial. Based on the work of others, we hypothesized that some children with ADHD have altered fatty acid metabolism. The present study found that 53 subjects with ADHD had significantly lower concentrations of key fatty acids in the plasma polar lipids (20:4n-6, 20:5n-3, and 22:6n-3) and in red blood cell total lipids (20:4n-6 and 22:4n-6) than did the 43 control subjects. Also, a subgroup of 21 subjects with ADHD exhibiting many symptoms of essential fatty acid (EFA) deficiency had significantly lower plasma concentrations of 20:4n-6 and 22:6n-3 than did 32 subjects with ADHD with few EFA-deficiency symptoms. The data are discussed with respect to cause, but the precise reason for lower fatty acid concentrations in some children with ADHD is not clear.

We have reported previously that a dietary deficiency in selenium results in an increase in glutathione S-transferase (GST) activity of various rat tissues. In order to verify that the increased GST activity observed in a selenium deficiency results from increased synthesis of GST protein, cytosolic fractions of livers obtained from rats fed selenium-deficient and selenium-supplemented diets were analyzed by Western (protein) blots. Antisera raised against purified individual GST subunits (Ya, Yb, and Yc) were used to detect the corresponding subunits on the blots. The Ya subunit was induced 2.5-fold in the selenium-deficient state. The amount of Yc subunit also increased significantly (p less than 0.05) in selenium deficiency but not to the extent of the Ya subunit. The Yb subunit was not significantly affected by altered selenium nutritional status. A corresponding increase in poly(A) RNAs coding for the Ya and Yc subunits was also observed by Northern blot analysis. Transcriptional activity of GST YaYc genes was elevated by approximately 2-fold in purified nuclei isolated from selenium-deficient rat livers, which is sufficient to account for the increase in YaYc mRNA levels. Therefore, it appears that transcriptional activation of rat liver YaYc genes is the primary cause for the elevation of the corresponding gene products in the selenium-deficient state. Since the GSTs, especially the isozymes containing Ya subunit, have been implicated in the formation of prostaglandin (PG) F2 alpha, we investigated the effect of selenium deficiency on the PGF2 alpha-forming activity using a specific inhibitor of GSTs, S-decyl-GSH. In rats fed a nutritionally adequate diet, the activity inhibited by S-decyl-GSH accounted for at least half of the conversion of PGH2 to PGF2 alpha. During selenium deficiency, this GST-catalyzed activity was approximately doubled with no change in PGF2 alpha formation by other pathways, resulting in a 2-fold increase in overall synthesis of PGF2 alpha. These data strongly support a role of GSTs, especially those composed of the Ya size subunit, in the synthesis of PGF2 alpha from PGH2.

Subjects were fifty children with clinically diagnosed Attention-Deficit/Hyperactivity Disorder (ADHD) that reported frequent symptoms of essential fatty acid deficiency (EFA) and twenty-four children without AD/HD and reporting few EFA deficiency symptoms. Subjects were required to do three-day food records that were analyzed for nutrient content using the Minnesota Nutrient Database System. The intake records were analyzed to determine 1) whether intake of EFAs was sufficient, 2) whether intake of LCPUFA differed between groups, and 3) whether nutrient intake in the diets differed between ADHD children and the control group. The results of the diet record analysis indicated that the intake of total fat, total protein, sodium, and mono-unsaturated fatty acids was significantly higher in the ADHD group as compared to the control group. Conversely, caffeine intake was greater in the control group. No differences between ^the two groups were observed with respect to intake of EFA and LCPUFAs, indicating that primary deficiency of EFA was not the cause for their reported higher frequency of symptoms.

When prostaglandin H 2 (PGH 2 ) was incubated with a mixture of glutathione S -transferases (GSTs) obtained from S -hexylglutathione affinity chromatography, as much as 40% of it was transformed into a prostanoid whose R f value corresponded to that of the standard PGF 2α . The reaction product was identified as PGF 2α by cochromatography with a standard on TLC and HPLC. The stereochemistry of the hydroxyl groups on C-9 and C-11 of the cyclopentane ring was confirmed by mass-spectral analysis of the butylboronate derivative of the reaction product. Neither PGE 2 nor PGD 2 could substitute for PGH 2 in the reaction mixture, indicating that the mechanism of formation of PGF 2α is a direct two-electron reduction of the endoperoxide moiety and not through a reduction of the keto group on PGE 2 or PGD 2 . Individual GST isozymes exhibited distinct differences in their catalytic rates of formation of PGF 2α from PGH 2 . Among various GSTs, isozyme IV, a homodimer of Ya size subunit showed the highest activity with a V max value of approximately 6000 nmol · min −1 · mg −1 . In general, the isozymes containing Ya and Yc subunits exhibited relatively high activity toward PGH 2 , indicating that it is the non-selenium-dependent glutathione peroxidase activity associated with the GSTs that might be responsible for the reduction of PGH 2 to PGF 2α . Interestingly, isozyme IV also exhibited the highest PGE 2 forming activity with a V max value of ~3000 nmol · min −1 · mg −1 followed by isozyme I, a homodimer of Yb subunit, which had a V max value of 420 nmol · min −1 · mg −1 . Based on these results, it appears that the GSTs play an important role in the biosynthesis of classical PGs. Therefore, it is conceivable that the tissue-specific formation of PGF 2α and PGE 2 might, in part, be due to the relative distribution of these enzyme activities in a given tissue. Our results have not only confirmed the previously published reports (E. Christ-Hazelhof et al. (1976) Biochim. Biophys. Acta 450 , 450–461), but also have characterized the specificity of GST isozymes in the formation of PGF 2α .

The glutathione S-transferases (EC 2.5.1.18) have been purified to electrophoretic homogeneity from 105,000g supernatant of sheep liver homogenate by employing a combination of gel filtration on Sephadex G-150 and affinity chromatography on S-hexylglutathione-linked Sepharose-6B columns. Approximately 70% of the original glutathione S-transferase activity toward 1-chloro-2,4-dinitrobenzene and glutathione peroxidase activity toward cumene hydroperoxide could be recovered by this purification method. Of particular importance in developing this procedure was the fact that the enzyme preparation obtained after affinity column chromatography represented all the isozymes of sheep liver glutathione S-transferases. Further purification by CM-cellulose and DEAE-cellulose column chromatography resolved the glutathione S-transferases into seven distinct cationic isozymes designated C-1, C-2, C-3, C-4, C-5, C-6, and C-7 and five overlapping anionic transferases designated A-1, A-2, A-3, A-4, and A-5, respectively, in the order of their elution from the ion-exchange columns. The sodium dodecyl sulfate SDS-gel electrophoretic data on subunit composition revealed that cationic enzymes are composed of two subunits with an identical Mr of 24,000 whereas a predominant subunit with Mr of 26,000 was observed in all anionic isozyme peaks except A-1. Cationic isozymes accounted for approximately 98% of the total peroxidase activity associated with the glutathione S-transferase whereas only A-1 of the anionic isozymes displayed some peroxidase activity. Isozyme C-4 was found to be the most abundant glutathione S-transferase in the sheep liver. Characterization of the individual transferases by their specificity toward a number of selected substrates, subunit composition, and isoelectric points showed some similarities to those patterns for human liver glutathione S-transferases.

Effects of vitamin E and/or selenium (Se) deficiency on the secretion of arachidonic acid metabolites by zymosan-stimulated pulmonary alveolar macrophages (AM) were examined using cells from male Long-Evans hooded rats fed torula-yeast based diets with or without the supplementation of vitamin E (150 IU/kg) or Se (0.5 mg/kg). Alveolar macrophages obtained by lavage were purified by adherence and cultured for 4 h in Hankś balanced salt solution containing bovine serum albumin (0.1%) and zymosan (300 μg/ml). The arachidonic acid metabolites present in the culture supernatant were measured by radioimmunoassay. Altered vitamin E and Se nutrition had no effect on the number of cells or cell types recovered from the pulmonary airways. Alveolar macrophages derived from animals fed on diets deficient in vitamin E or Se or both nutrients secreted higher levels of prostaglandins E 2 and thromboxane B 2 . Levels of both 5-hydroxyeicosatetraenoic acid and leukotriene B 4 were significantly increased only in the group fed the diet adequate in Se but deficient in vitamin E. Our data suggest that vitamin E and Se might play an important role to control the levels of several physiologically and pathologically important arachidonic acid metabolites.

An anionic glutathione S-transferase representing approximately 20% of the total glutathione S-transferase protein and 10% of the total transferase activity toward 1-chloro 2,4-dinitrobenzene has been purified to homogeneity from the 105,000 x g supernatant of rat liver homogenate. The SDS gel electrophoretic data on subunit composition revealed that the anionic isozyme is composed of two subunits with an identical Mr of 26,000. The Km values for 1-chloro 2,4-dinitrobenzene and reduced glutathione were determined to be 0.94 mM and 0.23 mM respectively. A significant amount of glutathione peroxidase activity toward cumene hydroperoxide is associated with the new isozyme.

In recent years, growing evidence suggests that glutathione peroxidases (GSH-Pxs), both selenium-dependent GSH-Px (Se-GSH-Px) and selenium-independent GSH-Px (non-Se-GSH-Px) play an important role in the biosynthesis of prostaglandins and leukotrienes and in the regulation of key enzymes associated with the arachidonic acid cascade. The precise nature of their involvement in eicosanoid metabolism, however, is not yet completely understood. In the study reported here, we have systematically determined the catalytic efficiencies of Se-GSH-Px and non-Se-GSH-Px toward prostaglandin (PG) G2 (PGG2) and PGH2. Se-GSH-Px exhibited high catalytic activity for the reduction of PGG2 as indicated by Km and Vmax values of 12 microM and 78 mumol/min/mg, respectively, whereas PGH2 was found to be a poor substrate, an indication that Se-GSH-Px reduces the hydroperoxide moiety but not the endoperoxide moiety of PGG2. The kinetic constants of Se-GSH-Px toward PGG2 were comparable to those determined for such classical substrates as H2O2 and cumene hydroperoxide. In contrast to Se-GSH-Px, non-Se-GSH-Px associated with cationic isozyme II of glutathione S-transferases (GSTs) from sheep lung cytosol was very active in the conversion of PGH2 to PGF2 alpha with a Vmax of 960 nmol/min/mg and a Km of 77 microM. This study shows that PGF2 alpha formation by non-Se-GSH-Px occurred in a GSH-dependent reduction of either PGG2 or PGH2. When PGG2 was used as the substrate for non-Se-GSH-Px, a novel intermediate compound appeared and was later identified by several methods of structural analysis as 15-hydroperoxy PGF2 alpha. Thus, the reductive cleavage of the endoperoxide occurs faster than the 15-hydroperoxide reduction allowing 15-hydroperoxy PGF2 alpha to accumulate briefly. A study of GSTs from several different tissues and species indicated that the transformation of PG endoperoxides to PGF2 alpha is catalyzed specifically by GST isozymes, which contain Ya size subunits. This specificity of GST isozymes in PG biosynthesis, coupled with their tissue-specific expression, may be a mechanism by which the body modulates the type of PGs produced in these tissues. Also, these results suggest a possible interaction of Se-GSH-Px and non-Se-GSH-Px in the biosynthesis of PGF2 alpha.