Excessive intake of red meat has been associated with colon carcinogenesis. The effect of hemin and zinc on colon carcinogenesis was investigated in male ICR mice. After acclimation for 1 week, five-week-old mice received three s.c. injections (0-2nd weeks of the experiment) of azoxymethane (AOM; 10 mg/kg b.w.) weekly and were treated by 2% dextran sodium sulfate (DSS) in drinking water for the next 1 week to induce aberrant crypt foci (ACF). Mice were fed on AIN-76A purified rodent diet for 6 weeks. There were three experimental groups; control, hemin, and hemin + zinc groups. The daily oral doses of hemin and zinc were 534 mg/kg and 55 mg/kg b.w., respectively. After staining colonic mucosa with 0.2% methylene blue, the ACF, aberrant crypts (AC), and polyps were counted. Lipid peroxidation in liver was evaluated by thiobarbituric acid-reactive substances (TBARS) assay. The numbers of AC, ACF, large ACF (i.e., ≥4 AC/ACF), and polyps in the hemin group were higher than those in the control group. In hemin + zinc group, the numbers of AC, ACF, large ACF were significantly lower compared to the hemin or control groups (p < 0.05), and the number of polyps was also significantly lower compared to that in the hemin group (p < 0.05). The TBARS level in the livers of the hemin + zinc group was significantly lower than that of the hemin group (p < 0.05). These results suggest that hemin enhances colon carcinogenesis and that co-treatment with zinc can protect against the induction of colon carcinogenesis.
Red meats are important animal foods because of their nutritional aspects, but the over-consumption of red meat produces reactive oxygen species (ROS) caused by heme iron and induces colorectal cancer. The effect of orally administered hemin and calcium provided in drinking water for 6 weeks on colon carcinogenesis was observed in male ICR mice. After the mice were acclimated for 1 week, they received three subcutaneous azoxymethane (AOM, 10 mg/kg b.w.) injections weekly and were provided with 2% dextran sodium sulfate (DSS) via drinking water for the next week. The mice were divided into three groups: the control, hemin, and hemin + calcium groups. The orally administered daily dose of hemin was 2 g/kg b.w., and 0.05% calcium was provided daily via drinking water. Colonic mucosa samples were stained with methylene blue, and then, the numbers of aberrant crypt (AC) and aberrant crypt foci (ACF) were counted. Lipid peroxidation in feces was estimated by thiobarbituric acid-reactive substances (TBARS) assay. The total numbers of AC and ACF per colon in the hemin group were significantly higher than those in the control group. Calcium treatment significantly decreased the numbers of ACF and AC in the colon of mice. The TBARS value in the feces of the hemin + calcium group was significantly lower than that in the feces of the hemin group. These results showed that hemin enhances the formation of pre-neoplastic lesions in the colon of mice and that calcium decreases the risk of colon carcinogenesis.
Colorectal cancer (CRC) is the third most prevalent cancer in the world, and heme iron is known to promote the CRC in an animal model. This study was conducted to investigate the effects of ascorbic acid in the presence of hemin on the formation of pre-neoplastic lesions induced by azoxymethane (AOM)/disodium sulfate (DSS) in mice. After acclimation for 1 week, five-week old mice received three s.c. injections (0-2 weeks of the experiment) of AOM [10 mg/kg body weight (BW)] weekly and were treated with 2% DSS in drinking water for the next week to induce aberrant crypt foci (ACF). All animals were fed the AIN-76A purified rodent diet for experimental period of 6 weeks. Experimental groups were then divided into three groups: carboxymethylcellulose (CMC) alone (control), CMC + Hemin, CMC + Hemin + ascorbic acid (AA). The CMC was used as a solvent for hemin. The daily doses were 534 mg/kg BW hemin and 246 mg/kg BW ascorbic acid administered orally. After the colonic mucosa were stained with methylene blue, aberrant crypt foci (ACF), aberrant crypt (AC) and polyps were counted. Lipid peroxidation in liver was evaluated by the thiobarbituric acid-reactive substances (TBARS) assay. The numbers of ACF, AC and large ACF (≥4 AC/ACF) per colon increased in the hemin group compared to the control group, while they decreased significantly in the hemin + ascorbic acid group compared to the control group or hemin group (p<0.01). The number of polyps/colon in the hemin + AA group was significantly decreased compared to the hemin group (p<0.05). In the liver, the TBARS value of the hemin group was significantly higher than that of the control group (p<0.01). Additionally, the TBARS value of the hemin + AA group decreased slightly compared to that of the hemin group. Taken together, these results suggest that hemin can promote colon carcinogenesis in a mouse model and that ascorbic acid has a protective effect against hemin-promoted colon carcinogenesis.
Excessive iron can promote the production of free radicals, thereby leading to harmful effects on cancer and aging. Ascorbic acid is not only an antioxidant but also a co-factor of iron absorption. The effect of iron-overload with ascorbic acid on experimental colon carcinogenesis was investigated in male ICR mice. Animals were treated weekly with azoxymethane (AOM, 10 mg/kg b.w.) at 0, 1, and 2 week and then drunk 2% dextran sodium sulfate (DSS)-containing water for the next 1 week. There were four experimental groups: carboxymethylcellulose (CMC) alone (control), CMC + ascorbic acid (AA), CMC + Fe, CMC + Fe + AA. The animals fed on AIN-76A purified rodent diet for six weeks. AA or Fe2O3 at the dose of 450 mg/kg b.w. were daily and orally treated for 6 weeks. The colonic mucosa was stained with methylene blue and then aberrant crypt foci (ACF) and polyps were counted. Thiobarbituric acid-reactive substances (TBARS) in serum and liver were determined. Iron concentration in liver was measured by inductively coupled plasma spectrophotometer. Fe-overload with AA strongly increased liver iron contents compared to control or Fe group (p<0.05). There were no significant differences in the number of ACF or polyps among all groups, although ironoverloaded groups had slightly higher numbers compared with the control or AA group. TBARS values in the liver were increased in the iron-overloaded groups compared to control and AA only group (p<0.05), but serum TBARS values were not changed. These results indicate that the excessive iron treatment did not affect the experimental colon carcinogenesis regardless of presence of AA in mice.
Colorectal cancer is one of the most common types of cancer in men and women who consume a Western diet. We investigated the inhibitory effect of selenium (sodium selenite, Na2SeO3) and selenium nanoparticles (nano-Se) on experimental colon carcinogenesis in ICR mice. After a 1-week acclimation, 6-week-old mice received three intraperitoneal (i.p.) injections (experimental week 0-2) of azoxymethane (AOM, 10 mg/kg body weight, b.w.), followed by 2% dextran sodium sulfate (DSS)-containing drinking water for the next 1 week. The three groups (10 mice/group) were orally administered either distilled water (control), selenium (1.7 ppm), or nano-Se (1.7 ppm) daily for 8 weeks. The numbers of aberrant crypt foci (ACF), aberrant crypt (AC), and tumorous lesions were measured in colonic mucosa. Se and nano-Se treatments significantly decreased the number of ACF, AC, and tumorous lesions compared with the control. However, there was no significant difference between the selenium and nano-Se groups. The glutathione peroxidase (GSH-Px) activity in the liver and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity in serum, were high in the selenium and nano-Se groups, while thiobarbituric acid reactive substance (TBARS) level was low in both Se and nano-Se groups when compared with that in the control group. These findings indicate that selenium and nano-Se showed similar protective effects against colon carcinogenesis by inhibiting the development of ACF and tumorous lesions in mice.
Iron-overload can cause harmful effects such as cancer and aging via promoting the production of free radicals. The effect of orally administered nano-Fe overload with ascorbic acid on colon carcinogenesis was investigated in male ICR mice. After a 1-week acclimation, 5-week-old mice received three intraperitoneal injections (experimental week 0-2) of azoxymethane (AOM, 10 mg/kg body weight) weekly, followed by 2% dextran sodium sulfate (DSS) in drinking water for the next 1 week to induce aberrant crypt foci (ACF). Animals were divided into four groups; carboxymethylcellulose (CMC) alone (control), CMC + ascorbic acid (AA), CMC + nano-Fe (NFe), and CMC + NFe + AA groups. Animals were fed an AIN-76A purified rodent diet and daily administrated oral doses of 450 ppm each of nano-Fe and AA combination for 6 weeks. The colonic mucosa was stained with 0.5% methylene blue, and then the ACF and polyps were counted. Lipid peroxidation in the serum and liver was evaluated using the thiobarbituric acid-reactive substances (TBARS) assay. Iron concentration in the liver was measured using Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES). Iron concentration in the liver of the NFe-overloaded groups was higher than that of the control (p<0.05). AA treatment increased the iron concentration in the liver. The number of ACF was not significantly different among all the groups. The number of polyps in all the NFe-treated groups was slightly higher than that in the control group and AA only-treated group. The serum TBARS was not significantly different among all the groups, but that in the liver was higher in all the NFe-treated groups than it was in the control group (p<0.05). These results indicate that the additional NFe treatment did not affect the experimental colon carcinogenesis in mice regardless of the presence of ascorbic acid.
This study was conducted to evaluate the accumulation and distribution of hydrophobically modified glycol chitosan (HGC) as a degradable nanoparticle in the body. To determine the movement of degradable HGC nanoparticles in the body, 20 mg/kg of lutetium177-labeled HGC (Lu177-HGC) with the size ranging from 320 to 400 nm was injected intravenously into ICR mice, and the amount of radioactivity remaining in blood and several organs was measured at various time points during the period of 5 days. In the pharmacokinetics analysis using the Lu177 radioisotope, the free Lu177 was mainly distributed and accumulated in the order of kidney>liver>lung at 1 day after the injection of the radioisotope. However, the Lu177-HGC showed a high distribution of nanoparticles in the order of liver>spleen>kidney during the experimental period of 5 days. These results would provide a basic pharmacokinetics for the use of HGC as a drug carrier in drug delivery system.
The ultimate goal of this study is to assess the accumulation and distribution of hydrophobically modified glycol chitosan (HGC) as a degradable nanoparticle in the body. To understand the movement of degradable nanoparticle HGC in the body, we intravenously injected a dose of 20 mg/kg of Cy5.5-labeled HGC with size ranging from 320 to 400 nm into ICR mice, and measured the amount of fluorescence remaining in blood and several organs at various time intervals. In blood, the level of Cy5.5-labeled HGC was the highest at 15 min, then after 30 min it decreased rapidly and reached a plateau form 30 min to 28 days. In the tissue we confirmed the presence of nanoparticles at high levels in the order of kidney>liver>submandibular gland until 28 days after injection. However, we did not find the presence of the particles in the brain or testes. These results will provide basic information on HGC as a drug delivery agent.
Iron is an essential trace element for normal functions of the body. Restriction of iron availability directly limits erythropoiesis. The objective of this experiment was to compare the bioavailability of iron nanoparticles (Fe-NPs) with iron-microparticles (Fe-MPs) in anemic mice. There were four experimental groups, including the normal control group, iron-deficiency anemia (IDA) group, Fe-NPs group, and Fe-MPs group. Animals in the normal group fed on an adequate iron-containing diet (45 ppm Fe). Meanwhile, animals in the other three groups fed on a low Fe diet (4.5 ppm Fe) for seven weeks. Double deionized water was supplied as drinking water ad libitum. After feeding for three weeks with the low Fe diet, animals in the Fe-NPs and Fe-MPs groups received oral administration of Fe-NPs or Fe-MPs at a daily dose of 40 mg/kg for four weeks. The IDA group showed markedly decreased red blood cell (RBC) count, hematocrit (Hct), and hemoglobin (Hb) values compared with the normal group throughout the experimental periods. Treatments with Fe-NPs or Fe-MPs for four weeks resulted in restoration of the decreased RBC count and hematological values similar to normal values. The Fe-NPs group showed faster restoration in values than Fe-MPs with passage of time. The iron contents of the upper small intestine in the Fe-NPs and Fe-MPs groups were higher than in the normal group at weeks 2 and 4. Treatment with Fe-NPs and Fe-MPs resulted in a significant increase in hepatic iron contents and lipid peroxidation, compared with the IDA group with passage of time. The iron contents in liver and ferritin deposits in spleen were identified in the Fe-NPs and Fe-MPs groups, similar to the normal group. These results indicate that oral administration of both Fe-NPs and Fe-MPs can result in recovery from anemia and Fe-NPs is more absorbable and available in the body than Fe-MPs.
Both iron-deficient and zinc-sufficient diets have been known to be associated with a decreased risk of colon cancer. We investigated that effect of dietary zinc on the formation of colonic aberrant crypt foci (ACF) induced by azoxymethane (AOM) followed by dextran sodium sulfate in iron-deficient mice. Five-week old ICR mice were acclimated for 1 week and fed on iron-deficient diet (4.50 ppm iron) with three different zinc levels (0.01, 0.1, and 1.0 ppm) for 12 weeks. The total number of aberrant crypt (AC) and ACF was measured in the colonic mucosa after methylene blue staining. The total ACF numbers of low Zn (LZn), medium Zn (MZn) and high Zn (HZn) diet groups were 10.00 ± 2.67, 8.78 ± 3.12, and 7.96 ± 2.44, respectively and there were no significant differences among the groups. However, the total AC numbers of HZn (27.07 ± 3.88) and MZn (26.39 ± 5.59) diet groups were significantly low compared to LZn (22.57 ± 5.09) diet group (p<0.01). Cytosolic SOD activity was the highest in LZn diet group. But thiobarbituric acid-reactive substances level in liver was also the highest in LZn diet group compared to other groups. There is no difference in cell proliferation in mucous membrane among the groups, while apoptotic positive cells were increased in the HZn diet group. The high zinc diet exhibited decreased β-catenin-stained areas on the mucous membrane of colon compared to the LZn or MZn diet group. These findings indicate that dietary zinc might exert a modulating effect on development of ACF/AC in the mice with iron-deficient status.
Iron nanoparticles (Fe-NPs) have recently been used for cancer diagnosis and therapy for imaging contrast and drug delivery. However, no study on nutritional bioavailability of Fe-NPs in the body has been reported. Ascorbic acid (AA) is known to aid in absorption of iron in the stomach by reducing Fe (III) to Fe (II). In this study, we investigated the bioavailability of Fe-NPs with AA in iron-deficiency-anemic mice in comparison with non-nano iron particles. Iron-deficient anemia was induced by feeding an iron-deficient diet (4.5 mg Fe/kg) and ocular bleeding from retro-orbital venous plexus for four weeks. Normal control mice were given a normal diet (45 mg Fe/ kg). After induction of anemia in mice, anemic mice received daily oral administration of Fe (40 mg/kg B.W.) + AA (5 g/kg B.W) and Fe-NPs (40 mg/kg B.W) + AA (5 g/kg B.W). After sacrifice, liver and spleen tissues were observed by haematoxylin & eosin stain. Amount of trace iron in liver and upper small intestine was investigated using an inductively coupled plasma-atomic emission spectrometer. Red blood cells (RBC), hematocrit (Hct), hemoglobin (Hb), and total iron binding capacity were also measured. The concentrations of iron in the Fe-NPs + AA group were significantly higher in liver and in upper small intestine than that in the Fe + AA group. The values of RBC, Hct, and Hb in the Fe-NPs + AA group were more rapidly increased to normal values compared with the Fe + AA group with increasing time. These results suggest that Fe-NPs in the presence of AA may be more bioavailable than non-nano Fe in Fe-deficient anemic mice.
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.
The current study was conducted in order to investigate promotional effects of herbal extracts on hair growth in an animal model of mice. There were four experimental groups, including distilled water (DW) as a negative control (NC), 3% minoxidil (MXD) as a positive control (PC), 50% ethanol (EtOH) as a vehicle control (VC), and herbal extract (HE) as the experimental treatment (E). The HE was extracted with ethanol from plants, including Gardenia, Mentha arvensis, Rosemary, and Lavender. Six-week-old C57BL/6 male mice were shaved with an electric clipper and the test materials were topically treated with 0.2 ml per mouse daily for three weeks. Photographic evaluation of hair re-growth was performed weekly during a period of three weeks. The number of mast cells was counted on the dorsal skin section of mice. The enzymes, alkaline phosphatase (ALP) and γ-glutamyl transpeptidase (γ-GT), were determined using a biochemical autoanalyzer. No clinical signs were observed in any of the experimental groups. As a result of photometric analysis, topical application of HE to dorsal skin for two weeks resulted in significantly faster acceleration of hair regrowth, compared with that of the NC or VC group (P<0.05). The PC and E groups showed a significant decrease in mast cell population, compared to the NC group. Activities of ALP and γ-GT were significantly increased in the PC and E groups, compared to the NC or VC group (P<0.05). Taken together, these results suggest that the herbal extract may have hair-growth promoting activity equal to that of MXD.