Ethanol and age enhances fluoride toxicity through oxidative stress and mitochondrial dysfunctions in rat intestine.

Fluoride toxicity and alcohol abuse are the two serious public health problems in many parts of the world. The current study was an attempt to investigate the effect of alcohol administration and age on fluoride toxicity in rat intestine. Six and 18 months old female Sprague Dawley rats were exposed to sodium fluoride (NaF, 25 mg/kg), 30 % ethanol (EtOH, 1 ml/kg), and NaF+EtOH (25 mg/kg+1 ml/kg) for a period of 20, 40, and 90 days. The levels of lipid peroxidation were increased, while the content of reduced glutathione, total, and protein thiol was decreased with NaF treatment. Under these conditions, animals showed an age-related decline in the activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glutathione-S-transferase which were further aggravated upon NaF or/and EtOH treatment. Mitochondrial respiration rate and the activities of complexes I, II, and IV enzymes of electron transport chain were decreased, while the levels of nitric oxide and citrulline were increased with age and NaF or/and EtOH treatment. Histological examination revealed large reactive lymphoid follicles, excess of lymphocytes in lamina propria of villi, villous edema, focal ileitis, necrosis of villi, and ulceration in NaF- or/and EtOH-treated animals in both the age groups. These findings suggest that fluoride mediate its toxic effects on intestine through oxidative stress and mitochondrial dysfunctions which are further augmented with alcohol consumption and advancing age.

Effects of fluoride and ethanol administration on lipid peroxidation systems in rat brain.

Exposure to fluoride and excessive ethanol consumption has been identified as a serious public health problem in many parts of the world, including India. Thus, the effect of co-exposure to fluoride and ethanol for 3-6 weeks was studied on lipid peroxidation (LPO) and oxidative stress related parameters in the rat brain. After 3 weeks, co-treated animals showed 95% increase in LPO levels compared to control. However, the levels of reduced glutathione, total and protein thiols were decreased. These changes were accompanied by a decrease in the activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione-S-transferase. Rats exposed to fluoride together with ethanol for 6 weeks resulted in 130% increase in LPO and decrease in the reduced glutathione levels. The activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione-S-transferase were reduced under these conditions. Brain histology revealed excessive lymphocytes, edema and spongeosis in the cortical region after six weeks of fluoride and ethanol treatment. These results suggest that exposure to fluoride together with ethanol enhances lipid peroxidation by affecting antioxidant defence systems in the rat brain.

Gestational diabetes affects postnatal development of transport and enzyme functions in rat intestine.

The effect of alloxan-induced gestational diabetes on the postnatal development of brush border disaccharidases and D-glucose transport in rat intestine was studied. Pups born to diabetic mothers showed 92-22% increase in blood sugar levels compared with the controls. Western blot and RT-PCR analyses revealed that the activities of brush border sucrase, lactase and Sodium Glucose Co-transporter 1 (SGLT1) correlates with protein and mRNA levels in intestine of pups born to diabetic rat mothers after 5-45 days of birth. Intestinal histology in pups born to diabetic mothers at day 10 and 45 after birth showed distorted cellular organization of mucosa with a decrease in the number of secretary goblet cells and regression of tubular mass. These findings suggest that the genetic switch in utero regulates the postnatal expression of enzyme and transport functions in intestine of pups born to diabetic rat mothers. This may influence the growth and development of offsprings later in life.

Modulation of intestinal brush border membrane chemical composition during postnatal development in rats: effect of gestational diabetes.

There was a significant increase in fucose (52%), total hexoses (16%) and hexosamine (56%) except sialic acid, which was reduced (77%) in the microvillus membrane of infants born to rat mothers made diabetic by injecting alloxan on day 3 of gestation. Expressed on the protein basis there were a significant increase in membrane, triglyceride, total cholesterol, and phospholipids content of brush border in pups from diabetic group between 5-45 days of postnatal age. Intestinal morphology in diabetic group showed, regression of tubular glands, distorted cellular organization of mucosal cells, reduction in the mucosal cell height and number of secretory goblet cells. These findings suggest that the gestational diabetes affects the sugar and lipid composition of the intestinal brush border membrane in rats during early stages of the postnatal development, which may be associated with compromised tissue functions later in life.

Synergistic antitumor effect of 5-fluorouracil and withaferin-A induces endoplasmic reticulum stress-mediated autophagy and apoptosis in colorectal cancer cells.

The development of chemo-resistance against 5-fluorouracil (5-FU) in tumor cells is one of the main debacles in colorectal cancer (CRC) patients. A recent combination of 5-FU with oxaliplatin or cetuximab drastically improves the survival rate in CRC patients; however, the toxicity issue cannot be evaded completely. Thus, searching for novel drug combinations with high specificity and low toxicity is seemingly important. Owing to the less undesirable effects of natural products on normal cells, here we investigated the synergistic antitumor effect of withaferin-A (WA) in combination with 5-FU. Our results demonstrate that the combination of WA and 5-FU induces a significant antiproliferative effect and modulates endoplasmic reticulum (ER) stress in favor of cell death in colorectal cancer (CRC) cells. Mechanistically, the combination upregulates the expression of ER stress sensors (BiP, PERK, CHOP, ATF-4, and eIF2α) and executes PERK axis mediated apoptosis in CRC cells. Additionally, the combined treatment of WA and 5-FU mediated ER stress induces autophagy and apoptosis, which were confirmed by immunoblotting, acridine orange (AO) staining and annexin-V FITC by flow cytometry. In contrast, inhibition of ER stress with salubrinal significantly decreases both autophagic and apoptotic cell populations. Moreover, pharmacological inhibition of either autophagy or apoptosis by their respective inhibitors 3-methyladenine (3-MA) or carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoro-methyl ketone (Z-VAD-FMK) decreases their respective population of cells but could not affect either of the population significantly. Finally, the combination attenuates the expression of ß-catenin pathway associated proteins and arrests cell cycle at the G2M phase in CRC cells. In summary, the combination of WA and 5-FU decreases cell viability by inducing ER stress-mediated induction of autophagy and apoptosis, inhibiting the ß-catenin pathway and arresting the cell cycle at a G2M phase in CRC cells.

Growth inhibition and apoptosis induction by (+)-Cyanidan-3-ol in hepatocellular carcinoma.

The objective of this study was to evaluate the cytotoxicity of (+)-cyanidan-3-ol (CD-3) in human hepatocellular carcinoma cell line (HepG2) and chemopreventive potential against hepatocellular carcinoma (HCC) in Balb/c mice. The HepG2 cell line was treated with CD-3 at various concentrations and the proliferation of the HepG2 cells was measure by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT), sulforhodamine B (SRB) and lactate dehydrogenase (LDH) assays. Cell apoptosis was detected by Hoechst 33258 (HO), Acridine orange/ethylene dibromide (AO/EB) staining, DNA fragmentation analysis and the apoptosis rate was detected by flow cytometry. The HCC tumor model was established in mice by injecting N-nitrosodiethylamine/carbon tetrachloride (NDEA/CCl4) and the effect of CD-3 on tumor growth in-vivo was studied. The levels of liver injury markers, tumor markers, and oxidative stress were measured. The expression levels of apoptosis-related genes in in-vitro and in vivo models were determined by RT-PCR and ELISA. The CD-3 induced cell death was considered to be apoptotic by observing the typical apoptotic morphological changes under fluorescent microscopy and DNA fragmentation analysis. Annexin V/PI assay demonstrated that apoptosis increased with increase in the concentration of CD-3. The expression levels of apoptosis-related genes that belong to bcl-2 and caspase family were increased and AP-1 and NF-κB activities were significantly suppressed by CD-3. Immunohistochemistry data revealed less localization of p53, p65 and c-jun in CD-3 treated tumors as compared to localization in NDEA/CCl4 treated tumors. Taken together, our data demonstrated that CD-3 could significantly inhibit the proliferation of HepG2 cells in-vitro and suppress HCC tumor growth in-vivo by apoptosis induction.

Modulation of lipid peroxidation and antioxidant defense systems in rat intestine by subchronic fluoride and ethanol administration.

Excessive consumption of fluoride and ethanol has been identified as injurious to human health. Fluoride and ethanol co-exposures are commonly seen among the alcoholics residing in endemic fluoride areas worldwide. This study was undertaken to examine the modulation of lipid peroxidation and antioxidant defense systems in rat intestine by subchronic fluoride and ethanol administration. Female Sprague-Dawley rats were divided into four groups: group I (control), group II (fluoride was given orally at a dose of 25 mg/kg body weight), group III (30% ethanol was given orally at a dose of 1 mL/kg body weight), and group IV (a combination of fluoride and ethanol was administered orally at the dose described for groups II and III). Lipid peroxidation was elevated (P<.05) in intestine of rats by fluoride or ethanol treatments for 20 or 40 days. However, glutathione content was reduced by fluoride (32 and 44%) and ethanol (21 and 40%) treatments after 20 and 40 days, respectively. Fluoride-exposed animals showed reduction (P<.05) in the activities of superoxide dismutase (22 and 42%), catalase (30 and 37%), glutathione peroxidase (22 and 35%), glutathione reductase (32 and 34%), and glutathione-S-transferase (24 and 30%) after 20 and 40 days. A similar decrease (P<.05) in the activities of these enzymes was also noticed in animals exposed to ethanol for 20 or 40 days. The observed changes in lipid peroxidation, reduced glutathione levels, and enzyme systems were further augmented in intestine of rats exposed to fluoride and ethanol together. Intestinal histology showed large reactive lymphoid follicles along with mild excess of lymphocytes in lamina propria of villi, villous edema, focal ileitis, and necrosis of villi in animals exposed to fluoride and ethanol for 40 days. These findings suggest that fluoride and ethanol exposure induces considerable changes in lipid peroxidation, antioxidant defense, and morphology of rat intestine, which may affect its functions.