Calcium exerts antiproliferative effects on cellular targets through the promotion of differentiation and apoptosis. We investigated the influence of calcium on the formation of colonic aberrant crypt foci (ACFs), which were induced by exposure to azoxymethane (AOM) followed by dextran sodium sulfate (DSS), in ICR mice. Six-week-old ICR mice received 3 (weeks 0–2) intraperitoneal injections of AOM (10 mg/kg BW), followed by treatment with 2% DSS via drinking water for a week to induce preneoplastic lesions. The mice were then divided into 3 groups: the control (AOM/DSS), AOM/DSS + 1.0% Ca, and AOM/DSS + 2.0% Ca groups. Calcium (1.0 or 2.0%) was administered via drinking water for 12 weeks. After sacrificing the mice, the total numbers of aberrant crypts (ACs) and ACFs were measured in the colonic mucosa after methylene blue staining. The control group displayed 11.58 ± 2.43 ACFs/colon, which were composed of a total of 30.42 ± 5.18 ACs/colon. The number of ACFs with more than 3 ACs, which are likely to progress to colon cancer, was 2.37 ± 0.68. Compared to the control, 1.0% or 2.0% calcium treatment significantly decreased the number of total ACFs and ACs in a concentration-dependent manner. The decrease in ACFs or ACs after calcium treatment was associated with decreases in cell proliferation and β-catenin expression and an increase in apoptosis in colonic mucosal cells. These results suggest that calcium may exert a protective effect against colon cancer by inhibiting the development of ACFs/ACs in ICR mice.

Copper is an essential micronutrient whose deficiency is often seen to occur in humans. Although many biomedical studies have focused on the use of nanoparticles, the nutritional effects of nano-sized copper oxide particles are not well known. This aim of this study was to investigate the nutritional bioavailability of nano- and micro-sized copper oxide (CuO) particles in copper-deficient (CuD) mice. Copper deficiency was induced in mice by feeding a CuD diet (0.93 mg Cu/kg diet) for 7 weeks. After the induction of copper deficiency, nano- or micro-sized copper oxide particles were administered orally at two different doses (0.8 and 4.0 mg CuO/kg body weight) to mice in the following groups: (1) normal control (NC), (2) CuD, (3) low dose micro-sized CuO, (4) high dose micro-sized CuO, (5) low dose nano-sized CuO, and (6) high dose nano-sized CuO. The hepatic copper concentration in the CuD group was significantly lower than that in the NC group. Compared to the NC group, the CuD group exhibited lower serum ceruloplasmin (CP) activity and CP level. The copper/zinc-superoxide dismutase activity in the CuD group was significantly lower than that in the NC group. Treatment with nano- or micro-sized copper oxide particles for 2 weeks restored the hepatic copper levels and serum CP activities to values similar to those observed in the NC group. The CP levels and copper/zinc-superoxide dismutase activities in all the copper oxide treatment groups also recovered to normal values after 3 weeks of copper oxide treatment. These results show that oral administration of either nano- or micro-sized copper oxide particles for 2–3 weeks restored the normal condition in previously CuD mice.

Flavonoids have a range of biological activities, including anti-allergic, anti-inflammatory, anti-microbial, and anti-cancer activities, as demonstrated by in vitro studies. In this study, we investigated whether luteolin can be applied to suppression of lipopolysaccharide (LPS)-stimulated inflammatory responses in murine macrophages. Luteolin was found to reduce nitric oxide (NO) production in LPS-stimulated Raw 264.7 cells. In addition, expression of inducible nitric oxide synthetase (iNOS), cyclooxygenase-2 (COX-2), and pro-inflammatory cytokine tumor necrotic factor-α (TNF-α) at the mRNA and protein levels were decreased. These inhibitory effects were found to be caused by the blockade of nuclear factor kappa-light- chain-enhancer of activated B cells (NF-κB) activation and phosphorylation of mitogen-activated protein (MAP) kinase family, extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 MAP kinase. In addition, pre-treatment with luteolin resulted in reduced ganglioside expression levels and inhibited expression of GT1b in Raw 264.7 cells. On the basis of these observations, we suggest that luteolin has potential as an anti-inflammatory drug candidate, and ganglioside GT1b may play a role in the inflammatory process.

This study was conducted in order to investigate the reduction activity of red ginseng extract (RGE; Panax ginseng, C. A. Meyer) on hydroxyl radical (·OH) using an electron spin resonance (ESR) spectrometer and spin-trapping techniques. ·OH generated by a Fenton Reaction System was trapped by 5, 5-dimethyl-l-pyrroline-N oxide (DMPO). The decay rate showed approximately pseudo-firs order kinetics over the period of measurement (by 10 min), and the half lifetime of the DMPO/·OH signal was estimated as approximately 8.15 min. However, the half lifetime of RGE/·OH was estimated as approximately 7.5 min, and the half lifetime of RGE was higher than that of DMPO/·OH adduct only. The order of reduction activities was ascorbic acid > N, Nʹ-dimethylthiourea (DMTU) > RGE > trolox > mannitol in the Fenton Reaction System. Thus, these observations indicate that RGE reaction with ·OH has relative reduction activity. The second-order rate constant of RGE/·OH may be 3.5~4.5 × 109 M-¹ ∙ S-¹.

The objective of this study was to determine the effect of macrophages on growth of human colon cancer cells. The results showed that co-culture of colon cancer cells with macrophages inhibited the growth of colon cancer cells (HCT116 and SW620) depending on the number of macrophages, RAW 264.7 cells, and activated THP-1 cells accompanied by down regulation of pSTAT3 in cancer cells. We also found that expression and release of cancer cell growth inhibitory cytokines, IL-1 receptor antagonist (IL-1ra) and IL-10, was increased in macrophages. Blocking of the STAT3 pathway with specific inhibitor and siRNA of STAT3 abolished the growth of colon cancer cells and expression of IL-1ra and IL-10. In addition, neutralization of IL-1ra and IL-10 with antibodies resulted in reversal of macrophage-induced inhibition of cancer cell growth. These data showed that IL-1ra and IL-10 released from macrophages inhibit growth of colon cancer cells through inhibition of the STAT3 pathway.

Plasma glutathione peroxidase (pGPx) is an extracellular antioxidative selenoenzyme which has been detected in various adult tissues, but little is known about the expression and distribution of pGPx during embryogenesis. To investigate the expression patterns of pGPx during embryogenesis, we performed quantitative real-time PCR, in situ hybridization, Western blot, and immunohistochemistry analyses in whole embryos or each developing organ of mice on embryonic days (E)7.5–18.5. In whole embryos of E7.5–8.5, pGPx mRNA was more typically expressed in extra-embryonic tissues including ectoplacental cone, trophectoderm, and decidual cells than in embryos. However, after E9.5, pGPx mRNA and protein levels were increased in the embryos with differentiation and growth, but trended to gradually decrease in the extra-embryonic tissues until E18.5. In sectioned embryonic tissues on E13.5–18.5, pGPx mRNA and protein were mainly expressed in the developing nervous tissues, the sensory organs, and the epithelia of lung, skin, and intestine, the heart and artery, and the kidney. In particular, pGPx immunoreactivity was very strong in the developing liver. These results indicate that pGPx is spatio-temporally expressed in various embryonic organs as well as extra-embryonic tissues, suggesting that pGPx may function to protect the embryos against endogenous and exogenous reactive oxygen species during organogenesis.

Nicotine, a major teratogen of cigarettes smoke induces embryonic abnormalities during the early stages of organogenesis. In this study, the protective effect of β-carotene against nicotine–induced embryos was evaluated by morphologic scoring, nile blue staining, lipid peroxidation, SOD activity assay and real-time PCR. The embryos exposed to nicotine (1 μM) revealed remarkable morphological anomalies compared to normal control group (p<0.05), but when β-carotene (1×10‒4 μM or 5×10‒4 μM) was added concurrently to the embryos exposed to nicotine, morphological parameters were significantly improved (p<0.05). Nicotine induced oxidative stress by increased lipid peroxidation, expression of proinflammatory cytokines (TNF-α and IL-1β), caspases-3 and decreased SOD activity. However, administration of β-carotene (1×10‒4 μM or 5×10‒4 μM) restored the SOD level and decreased oxidative damage in the embryos. These results indicate that β-carotene effectively counteracts the deleterious effects of nicotine on embryos and attenuates oxidative damage possibly through its antioxidant effects.

Nicotine, a major toxic component in tobacco smoke, leads to severe embryonic damages on organogenesis. We investigated if resveratrol can inhibit the nicotine–induced teratogenesis in the cultured mouse embryos (embryonic day 8.5) for 48 hours using a whole embryo culture system. The embryos exposed to nicotine (1 μM) revealed severe morphological anomalies, the increased levels of caspase-3 mRNA and lipid peroxidation, and further the lowered levels of mitochondrial manganese superoxide dismutase (SOD), cytosolic glutathione peroxidase (GPx), phospholipid hydroperoxide GPx, hypoxia-inducible factor 1α, and sirtuin mRNAs and SOD activity significantly compared to normal control group (p<0.05). However, whenre sveratrol(1×10‒5 μMor1 ×10‒4 μM) was added concurrently to the embryos exposed to nicotine, these all parameters were significantly improved (p<0.05).These findings indicate that resveratrol has a protective effect against nicotine-induced teratogenesis in mouse embryos throughout antioxidative and anti-apoptotic activities.

Zinc oxide nanoparticles (nZnO) are used in a various range, including ceramic manufacture, photocatalysis, UV filters, and the food industry. However, little is known about the effects of micro- and nano-particles during mouse embryo organogenesis. To determine whether ZnO affects size-dependent anomalies during embryonic organogenesis, mouse embryos were cultured for two days with 300 ug/ml micro ZnO (mZnO;80±25 μm) and nZnO (< 100 nm) and the developmental changes were then investigated. Quantity of Zn by inductively coupled plasma mass spectrometry analysis, and expression patterns of various antioxidant enzymes in the embryos were investigated. Embryos exposed to mZnO or nZnO exhibited severe retardation of growth and development. In embryos exposed to mZnO and nZnO, yolk sac diameter, crown-rump length, and head length were significantly diminished. The morphological parameters, including yolk sac circulation, allantois, flexion, heart, hindbrain, midbrain, forebrain, otic system, optic system, branchial bars, maxillary process, mandibular process, olfactory system, caudal neural tube, forelimb, hindlimb, and somites in mZnO and nZnO-treated groups were significantly decreased. Zn absorption of the nZnO-treated group was significantly higher than that of the mZnO-treated group. Significantly decreased levels of CuZn-SOD, Mn-SOD, cGPx, and PHGPx mRNA were observed in the ZnO-treated group. In addition, antioxidant enzyme mRNA expressions of the nZnO group were significantly diminished, less than those of the mZnO treated group. These findings indicate that 300 ug/ml ZnO showed abnormality and nZnO may have a more severe effect than mZnO in developing embryos.

Neurotoxicity and oxidative injury induced by glutamate cause neuronal degeneration related to various central nervous system diseases. Resveratrol, a polyphenolic compound, is known to have antioxidative and anti-inflammatory effects. The aim of this study was to investigate the question of whether resveratrol has a neuroprotective effect against glutamate-induced toxicity in cultured cortical neurons. Following exposure to glutamate for 15 min, cortical neurons originating from ICR mouse fetuses on embryonic days 15-16 were then treated with resveratrol for 24 h in the post-treatment paradigm. Glutamate induced a significant reduction in cell viability; however, resveratrol induced a significant increase in cell viability. Glutamate induced generation of ROS and apoptotic neuronal death; however, these were decreased by exposure to resveratrol. mRNA expression in antioxidant enzymes, cytoplasmic glutathione peroxidase, copper/zinc superoxide dismutase (SOD), and manganese SOD, and anti-apoptotic regulator Bcl-xL were decreased by exposure to glutamate, however, exposure to resveratrol resulted in a significant increase in their mRNA levels. In addition, mRNA expression of pro-inflammatory cytokines, interleukin-1β and tumor necosis factor-α, was increased by glutamate insult, but significantly reduced by resveratrol. These findings indicate that resveratrol is neuroprotective against glutamate-induced toxicity, suggesting a useful therapeutic application in treatment of neurodegenerative disorders.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a unique antioxidant enzyme involved in reduction of peroxidized phospholipids within biomembranes. To investigate the expression pattern of the PHGPx gene during fetal development, in situ hybridization analyses were performed using mouse FITC-labeled PHGPx cRNA probes in fetal tissues on embryonic days (Ed) 13.5-18.5. During these periods, PHGPx mRNA appeared in the developing telencephalon, diencephalon, spinal cord, and spinal ganglion. In particular, PHGPx mRNA was strongly expressed in pyramidal cells of the cerebral cortex. On Eds 17.5-18.5, PHGPx mRNA was detected in various tissues including liver, intestinal villi and crypt, pancreas, lung, and olfactory epithelium of the nasal cavity. In addition, PHGPx mRNA was highly expressed in the inner ear on Eds 14.5-18.5, brown fat on Ed 17.5, and adrenal gland on Ed 18.5. It is conceivable that PHGPx may act as an important antioxidant against fetal oxidative stress during mouse organogenesis.

Animal crude drugs (natural medicines derived from animal organs) have been widely used in various Chinese medicine for the therapeutic effects and for enhancement of immunologic functions. We found the specific identification methods using DNA sequencing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analyses for mitochondrial DNA (mt DNA) in order to discriminate between the animal species and organs as well as the placenta of humans. Species-specific PCR bands of D-loop mt DNA for equine, bovine, porcine, and human were 133 bp, 137 bp, 231 bp, and 240 bp, respectively. Porcine organs were identified using restriction enzyme, HphI cut into two subfragments, 36 bp and 195 bp bands in the heart, spleen, and liver, except for kidney. The heart and liver of porcine were identified using restriction enzyme, SpeI cut into two subfragments, 84 bp and 147 bp bands, except for kidney and spleen. Bovine organs were cut into 68 bp and 69 bp bands in the liver, kidney, and spleen using NalIV, except heart and placenta. Placentas of bovine and humans were easily identified using each primer. Our results suggest that sequencing of mt DNA and its PCR-RFLP methods are useful for identification and discrimination of inter- and intra-specific variations in equine, bovine, porcine, and human by routine analysis.

Chronic inflammatory diseases such as Crohn′s disease and ulcerative colitis are associated with increased risk of colon adenocarcinoma. Apoptic induction of colon cancer cells by cytokines and death receptors is an important anti-cancer therapy. We observed that co-administration of TNFα and IFNγ in human colon cancer cell line, HCT116, resulted in cell death and expression of IL-32. Cleavage forms of caspase-3, caspase-9, and PARP were increased in TNFα / IFNγ-treated HCT116. mRNA expression of death receptors, including TNFR1 and Fas were not changed and NO generation was not induced by combination of TNFα and IFNγ. However, mRNA expression of IL-32α, β, and γ was increased in TNFα / IFNγ-treated HCT116. To determine the effect of IL-32 in HCT116 cell apoptosis by TNFα / IFNγ stimulation, IL-32 siRNA-transfected HCT116 cells were cultured with TNFα / IFNγ and cell proliferation was measured. IL-32 siRNA induced slight recovery of cell viability of TNFα / IFNγ-stimulated HCT116. These results suggest that IL-32 is not directly related to apoptosis of HCT116 by TNFα / IFNγ stimulation. However, IL-32 expression by TNFα or TNFα / IFNγ in a colon cancer cell line is very interesting because of the unknown effect of IL-32 in colon cancer. Our study will contribute to development of studies for IL-32 function in human colon cancer and anti-cancer therapies using cytokines.

Maternal hypoxia induced by a variety of exogenous oxidative stresses such as ethanol intake, diabetes, and cigarette during pregnancy provokes the impaired embryonic gene expression and developmental malformations. We investigated the gene expression patterns of the representative selenium containing antioxidant enzymes (selenoproteins) such as cytosolic GPx (cGPx), gestrointestinal GPx (GI-GPx), plasma GPx (pGPx), phospholipid hydroperoxide GPx (PHGPx), and selenoprotein P (SePP) in the cultured mouse embryos under normal or hypoxic (low oxygen state, 5% O₂) condition at embryonic day 8.5 for 2 days using real-time PCR analysis. cGPx, pGPx, and SePP mRNAs were significantly decreased, but GI-GPx and PHGPx mRNAs were remarkably increased in the hypoxic state compared to normal gassing state (p<0.05). These findings indicate that hypoxic condition leads to the unusual expressions of selenoproteins during mouse organogenesis.

A level of dietary iron may play a role in colon carcinogenesis. The effect of dietary iron on colon carcinogenesis was investigated in male ICR mice. Five-week old mice were acclimated for one week and fed on iron-normal diet (35 ppm Fe), iron-deficient diet (3 ppm), or iron-overloaded diet (350 ppm Fe) for 8 weeks. Animals received three (0-2^nd weeks after starting experiment) injections of azoxymethane (AOM; 10 mg/kg b.w.) to induce colonic aberrant crypt foci (ACF). There were five experimental groups including normal control without AOM, AOM+iron-normal diet (AOM+NFe), AOM+iron-deficient (AOM+LFe), AOM+ironoverloaded diet (AOM+HFe) groups. The total numbers of ACF and aberrant crypt (AC) were measured in the colonic mucosa after staining with methylene blue. The blood and serum were analyzed with a blood cell differential counter and an automatic serum analyzer. The hepatic iron levels were significantly dependent on the presence of iron in the diets. Iron-deficient diet significantly decreased the several hematological values. The values of glutamic oxaloacetic transaminase (GOT) and glutamic pyruvate transaminase (GPT) were also significantly decreased in iron-overloaded or iron-deficient diet groups, compared with normal iron diet group. Dietary iron-deficiency decreased the numbers of ACF (64.9) and AC (79.8) per colon by 20.6 and 21.8%, respectively, compared with AOM+NFe group (72.4 ACF/colon and 90.3 AC/colon). However, ironoverloaded diet increased ACF (82.9) and AC (96.0) induction by AOM, compared with normal iron diet. These results suggest that dietary iron can affect the colon carcinogenesis in the animal model of mice.

The Maillard Reaction Products (MRPs) such as Glucose-tyrosine (Glu-Tyr) and Xylose-arginine (Xyl-Arg) have antioxidant, antimutagenic, and antibacterial effects. However, to date, still little is known about the other biological effects of the MRPs. In this study, we investigated whether the fructose-tyrosine MRP, 2,4-bis(p-hydroxyphenyl)-2-butenal (Fru-Tyr), could modulate cell cycle progression and NF-κB activity, and thereby induce apoptotic cell death of colon cancer cells. Treatment with different concentrations (10-40 μg/ ml) of Fru-Tyr for 24 h inhibited colon cancer cell (SW620 and HCT116) growth followed by induction of G₂/M phase cell cycle arrest and apoptosis in a dose-dependent manner. We also found that Fru-Tyr suppressed tumor necrosis factor-alpha (TNF-α)-induced NF-κB transcriptional activity. Moreover, Fru-Tyr induced the expression of apoptotic gene, cleaved caspse-3. These results suggest that Fru-Tyr inhibited colon cancer cell growth through induction of G₂/M phase cell cycle arrest and apoptotic cell death by modulating of NF-κB.

Flavonoids are a large family of polyphenolic compounds synthesized by plants that have a common chemical structure. Flavonoids may be further divided into subclasses, Anthocyanidins, Flavan-3- ols, Flavanones, Flavonols, Flavones, Isoflavones. Flavonoids have antioxidant and antiradical activities. The antiradical efficacy of flavonoids have established structure-activity relationships (SAR) of the antioxidant activity. Reactivity of flavonoids is highly dependent on the configuration of OH groups on the flavonoid B and C rings, there being little contribution from A ring to antioxidant effectiveness. This short article presents the current knowledge on structural aspects on their free radical reactions of flavonoids.