Chandradhar Dwivedi

A simultaneous liquid chromatography/mass spectrometric assay of glutathione, cysteine, homocysteine and their disulfides in biological samples

A liquid chromatography/mass spectrometric (LC/MS) method was developed for simultaneous detection and quantitation of glutathione (GSH), glutathione disulfide (GSSG), cysteine (CysSH), homocysteine (HCysSH) and homocystine in biological samples (rat brain, lung, liver, heart, kidneys, erythrocytes and plasma). Thiols were derivatized with a large excess of Ellmans reagent, a thiol-specific reagent, to ensure an instantaneous and complete derivatization. The derivatization blocked the oxidation of the thiols to disulfides, preventing errors caused by thiol oxidation. The samples were then analyzed by LC/MS. The method provides a highly selective and sensitive assay for these endogenous thiols and their corresponding disulfides. The detection limits for GSH, GSSG, CysSH, HCysSH and homocystine were 3.3, 3.3, 16.5, 29.6 and 14.9 pmol, respectively. An attempt for cystine analysis was unsuccessful due to earlier elution of the compound and strong interferences caused by other endogenous compounds. This method will be a useful tool in the investigation of the roles of these important thiol-containing compounds and their corresponding disulfides in physiological and pathological processes.

Chemopreventive Effects of Dietary Flaxseed on Colon Tumor Development

Abstract: Fatty acid composition of dietary fat plays a vital role in colon tumor development in animal models. Fats containing ω-6 fatty acids (e.g., corn oil) enhanced and ω-3 fatty acids (e.g., flaxseed oil) reduced chemically induced colon tumor development in rats. Lignans have also been shown to prevent colon tumor development in experimental animals. The objective of this investigation is to study the effects of dietary flaxseed meal, a source of both ω-3 fatty acid and lignans, on colon tumor development and compare them with the effects of dietary corn meal. Male Fischer rats, two groups of 24 each, were assigned to the AIN-93M diet supplemented with either 15% corn meal or 15% flaxseed meal, respectively. Carcinogenesis was initiated with subcutaneous injections of azoxymethane (15mg/kg) once a week for 3 consecutive wk. After 35 wk of initiation, rats were anesthetized with ether. Blood was collected by cardiac puncture, and rats were sacrificed. The gastrointestinal tract was isolated. The site, size, and number of tumors were recorded. The fatty acid analysis of the collected serum and colon samples was performed. Expression of cyclooxygenase (COX)-1 and COX-2 was performed by Western blot method. Lignan levels in serum and colon samples were assayed. Colon tumor incidence, multiplicity, and size were found to be 82.6% and 29.4%; 1.3 and 0.3; and 44.4 and 5.3 mm2 in corn and flaxseed meal groups, respectively. Colon and serum samples of the corn meal group showed higher levels of ω-6 fatty acid levels whereas the flaxseed meal group exhibited higher levels of ω-3 fatty acids. COX-1 and COX-2 expression in the flaxseed group was significantly lower (P < 0.05) as compared to the corn group. Dietary flaxseed meal containing high levels of ω-3 fatty acids and lignans is effective in preventing colon tumor development when compared with dietary corn meal possibly by increasing ω-3 fatty acid levels and decreasing COX-1 and COX-2 levels.

Chemoprevention of Chemically Induced Skin Tumor Development by Diallyl Sulfide and Diallyl Disulfide

Garlic and onion oil have been shown to inhibit chemically induced skin tumor development in mice. In the present study, the effects of diallyl sulfide and diallyl disulfide, oil-soluble constituents of garlic and onion, on 7,12-dimethylbenz(a)anthracene-induced and 12, O-tetradecanoylphorbol-13-acetate-promoted skin tumor formation were examined in SENCAR mice. Topical application of diallyl sulfide or diallyl disulfide significantly inhibited skin papilloma formation from the ninth week of promotion and significantly increased the rate of survival in the murine model. Our findings support earlier evidence that these naturally occurring compounds may be useful for the chemoprevention of certain types of tumors.

Chemopreventive effects of dietary mustard oil on colon tumor development

Fatty acid composition of dietary fat is one of the detrimental factors in colon cancer development. Fats containing omega 6-polyunsaturated fatty acids (e.g. corn oil) enhance and omega 3-polyunsaturated fatty acids (e.g. fish oil) reduce chemically-induced colon cancer in animal studies. The purpose of this study is to investigate the effects of dietary mustard oil (containing omega 3-polyunsaturated fatty acid) on azoxymethane-induced colon cancer in rats and compare with corn and fish oil treated groups. Colon tumor incidence and multiplicity were found to be 90, 75, and 50% and 1.7, 0.8, and 0.4 tumors/rat in corn, fish and mustard oil treated groups respectively. Omega-3 polyunsaturated fatty acid levels were highest in serum and colon microsomal fractions of the fish oil group followed by the mustard oil group. Corn oil group had the highest level of omega 6-polyunsaturated fatty acid levels in serum and colon microsomal fractions. The results indicate that dietary mustard oil is more effective in preventing colon cancer in rats than dietary fish oil.

Effects of Dietary Flaxseed on Intestinal Tumorigenesis in Apc Min Mouse

Dietary flaxseed has been shown to prevent azoxymethane (AOM)-induced colorectal cancers in male Fisher rats. The present study was designed to investigate the chemopreventive effects of dietary flaxseed on the development of intestinal tumors in Apc Min mice. Apc Min mice were divided into five different groups, fed with control (AIN-93M meal), corn meal, flaxseed meal, corn oil, and flaxseed oil supplemented diets. Results showed that dietary flaxseed significantly decreased ( P < 0.05) tumor multiplicity and size in the small intestine and colon as compared to control, corn-treated groups. Intestine, colon, and serum samples of corn-treated groups showed higher levels of ω -6 fatty acids, whereas the flaxseed treated groups exhibited higher levels of ω -3 fatty acids. Lignans were detected in the serum, intestine, and colon samples for flaxseed meal group. COX-1 and COX-2 expression in the colon samples from the flaxseed meal group were significantly lower ( P < 0.05) as compared to the corn meal group. Dietary flaxseed may be chemopreventive for intestinal tumor development in Apc Min mice possibly by increasing ω -3 fatty acid levels, lignans, and decreasing COX-1 and COX-2 levels.

Effects of oil-soluble organosulfur compounds from garlic on doxorubicin-induced lipid peroxidation.

Clinical efficacy of doxorubicin is compromised due to free radical generation leading to cardiac toxicity. Oil-soluble organosulfur compounds, diallyl sulfide (DAS), diallyl disulfide (DADS), dipropyl sulfide (DPS) and dipropyl disulfide (DPDS), present in garlic were examined for their antiperoxidant effects. DADS inhibited liver microsomal lipid peroxidation induced by NADPH, ascorbate and doxorubicin. DAS, DPS and DPDS were ineffective inhibitors of liver microsomal lipid peroxidation. DADS could be used in combination with doxorubicin to protect oxidative injuries to improve the clinical efficacy of doxorubicin.

Rheological, sensorial, and chemopreventive properties of milk fermented with exopolysaccharide-producing lactic cultures.

The objective of this research was to evaluate the rheological, sensorial, and chemopreventive properties of milk fermented with different exopolysaccharide (EPS)-producing lactic cultures. Reconstituted skim milk (11% wt/vol) was fermented with single strains of EPS-producing and non-EPS-producing cultures. Whey that collected on the surface of undisturbed fermented milks and after cutting was measured. All EPS-producing cultures reduced the amount of whey present on the surface of the undisturbed samples, whereas only 3 out of 5 strains reduced syneresis measured after cutting. All EPS-producing cultures except a strain of Lactobacillus delbrueckii ssp. bulgaricus reduced viscoelastic moduli in fermented milk. There was a linear correlation between ropiness and smoothness. In the chemoprevention study, 140 male Fisher rats were divided into 7 groups of 20 each. Rats in 6 groups were fed diets supplemented with fermented milks each made with a single strain of EPS-producing or non-producing cultures, whereas rats in group 7 (control) were fed a diet supplemented with milk acidified with glucono-delta-lactone (GDL). All rats were injected with azoxymethane (15 mg/kg, subcutaneous) at wk 7 and 8 of age to induce tumors and fed their respective diets ad libitum throughout the study. After 30 wk of initiation, all rats were anesthetized with ether, and their intestinal tissues were isolated and washed with cold normal saline. The number and size of tumors in the colon and small intestine were recorded. Rats fed diets supplemented with fermented milk made with 2 EPS-positive and 1 EPS-negative strains had significantly lowered incidence of colon tumor and colon tumor multiplicity. Cyclooxygenase-2 enzyme activity (the enzyme implicated in colon tumor development) was significantly lower in the colon tissue of rats fed diets containing milk fermented with 4 EPS-producing and 1 non-producing cultures than that in rats fed diets supplemented with GDL-acidified milk. Different EPS-positive cultures produced fermented milks with distinct rheological characteristics and levels of ropiness. No relationship was found between rheological properties or level of ropiness of fermented milk and its chemopreventive effect.

Antineoplastic properties of Maharishi-4 against DMBA-induced mammary tumors in rats

Maharishi-4 (M-4), an ayurvedic food supplement, was tested for anticarcinogenic and anticancer properties against 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumors in rats. The 6% M-4-supplemented diet protected DMBA-induced carcinogenesis by reducing both tumor incidence and multiplicity during initiation and promotion phases. The control animals who developed tumors when supplemented with M-4 diet for four weeks showed tumor regression in 60% of cases. There was no significant difference in the food intake or weight gain in rats who were on M-4-supplemented diet compared to control group. Possible mechanisms of action of M-4 are discussed.

Effects of magnolol on UVB-induced skin cancer development in mice and its possible mechanism of action

BackgroundMagnolol, a plant lignan isolated from the bark and seed cones of Magnolia officinalis, has been shown to have chemopreventive effects on chemically-induced skin cancer development. The objectives of this investigation are to study the anticarcinogenic effects of magnolol on UVB-induced skin tumor development in SKH-1 mice, a model relevant to humans, and determine the possible role of apoptosis and cell cycle arrest involved in the skin tumor development.MethodsUVB-induced skin carcinogenesis model in SKH-1 mice was used for determining the preventive effects of magnolol on skin cancer development. Western blottings and flow cytometric analysis were used to study the effects of magnolol on apoptosis and cell cycle.ResultsMagnolol pretreated groups (30, 60 μ g) before UVB treatments (30 mJ/cm2, 5 days/week) resulted in 27-55% reduction in tumor multiplicity as compared to control group in SKH-1 mice. Magnolol pretreatment increased the cleavage of caspase-8 and poly-(-ADP-ribose) polymerase (PARP), increased the expression of p21, a cell cycle inhibitor, and decreased the expression of proteins involved in the G2/M phase of cell cycle in skin samples from SKH-1 mice.Treatment of A431 cells with magnolol decreased cell viability and cell proliferation in a concentration dependent manner. Magnolol induced G2/M phase cell cycle arrest in A431 cells at 12 h with a decreased expression of cell cycle proteins such as cyclin B1, cyclin A, CDK4, Cdc2 and simultaneous increase in the expression of Cip/p21, a cyclin-dependent kinase inhibitor. Magnolol induced apoptosis in vivo and in vitro with an increased cleavage of caspase-8 and PARP. Phospho-signal transducers and activators of transcription 3 (Tyr705), B-Raf, p-MEK, and p-AKT were down-regulated, whereas phosphorylation of ERK was induced by magnolol in A431 cells.ConclusionsMagnolol pretreatments prevent UVB-induced skin cancer development by enhancing apoptosis, causing cell cycle arrest at G2/M phase, and affecting various signaling pathways. Magnolol could be a potentially safe and potent anticarcinogenic agent against skin cancer.

Effects of diallyl sulfide and diallyl disulfide on cisplatin-induced changes in glutathione and glutathione-S-transferase activity.

The effects of diallyl sulfide (DAS) and diallyl disulfide (DADS) on cisplatin-induced changes in glutathione (GSH) and glutathione-S-transferase (GST) activity in rat liver and kidney was investigated. Cisplatin treatment significantly (p < 0.05) decreased GSH and GST activity in both liver and kidney. DADS treatment significantly (p < 0.05) enhanced GSH and GST activity in rat liver and kidney. Furthermore, DADS treatment reversed the effect of cisplatin on GSH and GST activity both in liver and kidney. Administration of DADS with cisplatin could enhance GSH and GST activity and lower cisplatin-induced nephrotoxicity.