Reactive oxygen species (ROS) play key roles in many pathogenic processes, including carcinogenesis, inflammation, ischemia-reperfusion injury and signal transduction. Also, reactive nitrogen species (RNS) cause various biological events such as neurodegenerative disorders. Sensitive and specific detection methods for ROS and RNS in biological samples should be useful for elucidation of biological events both in vitro and in vivo. Fluorescent probes based on small organic molecules have become indispensable tools in modern biology because they provide dynamic information concerning the localization and quantity of biological molecules of interest, without the need of genetic engineering of the sample. In this review, we recount some recent achievements in the field of small molecular fluorescent probes. First, the probes for nitric oxide and peroxynitrite as RNS are introduced and the probes of hydroxyl radical, hydrogen peroxide, hypochlorous and singlet oxygen as ROS are discussed, based on the fluorescence off/on switching mechanisms including photoinduced electron transfer and spirocyclization processes, and with some applications for in vitro and in vivo systems.
Non-steroidal anti-inflammatory drugs (NSAIDs), which are used for the treatment of several inflammatory disorders including rheumatoid arthritis, are well known to cause gastroduodenal mucosal lesions as an adverse effect. Recently, the serious problem of NSAID-induced small intestinal damage has become a topic of great interest to gastroenterologists, since capsule endoscopy and double-balloon enteroscopy are available for the detection of small intestinal lesions. Such lesions have been of great concern in clinical settings, and their treatment and prevention must be devised as soon as possible. Proton pump inhibitors (PPI), such as lansoprazole and omeprazole, show a potent anti-secretory effect. PPIs also have a gastroprotective effect, independent of their anti-secretory actions, which is probably mediated by inhibition of neutrophil functions as well as antioxidant actions. Administration of lansoprazole reduced the severity of the intestinal lesions in a dose-dependent manner, but omeprazole had no effect. The amount of heme oxygenase-1 (HO-1) protein in the intestinal mucosa was significantly increased by lansoprazole, but not by omeprazole. These results suggest that lansoprazole, but not omeprazole, ameliorates indomethacin-induced small intestinal ulceration through upregulation of HO-1/carbon monoxide. Therefore, lansoprazole may be useful for preventing the adverse effects of NSAIDs not only in the stomach but also in the small intestine.
We evaluated the effect of cacao procyanidins (CP) on plasma lipid levels in high cholesterol-fed rats. Animals were divided into 4 groups, and each group was fed on either a normal diet, high cholesterol diet (HCD) containing 1% cholesterol (HCD without CP), HCD with 0.5% (HCD with 0.5% CP) or 1.0% CP (HCD with 1.0% CP) for 4 weeks. Plasma cholesterol level was significantly higher in the HCD without CP group than the normal diet group (p<0.01). Supplementation of CP significantly decreased plasma cholesterol (p<0.01) to levels similar to those of the normal diet group. The liver cholesterol and triglyceride levels in all HCD groups were significantly higher (p<0.01), but 1.0% CP feeding significantly reduced this increase. Fecal excretion of neutral sterol and triglyceride was significantly increased in all HCD groups (p<0.01), and the excreted amounts tended to be higher in the HCD with CP groups. The procyanidins dose-dependently reduced micellar solubility of cholesterol and this activity increased with increasing molecular weight. These results suggest that one of the mechanisms of CP to lower plasma cholesterol is inhibition of intestinal absorption of cholesterol.
It has been reported that levels of soluble intercellular adhesion molecule-1 (ICAM-1) in the blood are elevated in hepatocellular carcinoma patients. In the present study, serial observations of the localization of ICAM-1 in the liver were made by light and electron microscopy in rats with carcinogen-induced cancer. Male Fisher rats were given diethylnitrosamine (DEN) orally in their drinking water. Rats were sacrificed at 6, 8, 12, or 14 weeks after the start of DEN administration and the liver tissue was collected. ICAM-1 expression in liver was assessed using indirect immunoperoxidase staining with anti-rat ICAM-1 antibody. Although ICAM-1 expression by endothelial cells in livers of DEN-treated rats was lower than in the control group at 8 weeks, it was higher in the membrane and cytoplasm of hepatocytes. The expression of ICAM-1 in mesenchymal cells was decreased, paralleling development of cellular atypia, whereas in hepatocyte membranes and cytoplasm it was increased in these atypia. ICAM-1 was localized to the cytoplasm of cancer cells, but to the membrane of hepatocytes in the treated livers at 14 weeks. Furthermore, the levels of ICAM-1 in mesenchymal cells tended to be lower in the cancerous area than in the atypical hyperplastic nodule, and were reduced as the density of cell atypia increased, in comparison to cells in areas without cancerous nodules. We concluded that ICAM-1 may be influenced the development of cancer induced in the rat liver by a chemical carcinogen.
The objective of the present study was to assess the ascorbic acid (AA) levels in seminal plasma of the fertile and infertile men and to investigate its relationship with sperm count, motility and normal morphology. Semen samples were provided by fertile [smoker (n = 25), nonsmoker (n = 21)] and infertile men [smoker (n = 23), nonsmoker (n = 32)]. A simplified method of reverse phase high performance liquid chromatography (RP-HPLC) procedure using UV detection was applied for the determination of seminal AA. Fertile subjects, smoker or not, demonstrated significantly higher seminal AA levels than any infertile group (p<0.01). Nonsmokers had high, but no significant, mean AA levels in their seminal plasma compared with smokers. Seminal AA in fertile and infertile (smokers or nonsmokers) males correlated significantly with the percentage of spermatozoa with normal morphology (p<0.01). Seminal AA decreased significantly in infertile men. Decrease of seminal plasma AA is a risk factor for low normal morphology of spermatozoa and idiopathic male infertility. Measurement of seminal AA in the seminal plasma of males with a history of subfertility or idiopathic infertility is necessary and can be helpful in fertility assessment.
Human milk from healthy women contains numerous nutrients such as antioxidants which are necessary for newborns. The aim of this study was to evaluate the changes of total antioxidant capacity (TAC) and free radical scavenging activity in human milk during the first six month period of lactation and also its relationship to maternal plasma. A total of 505 milk samples (colostrum, transitional and mature milks) collected from 115 healthy women with full term newborns. Blood plasma was obtained from 58 women at 3 months postpartum. The TAC of samples were measured by Ferric Reducing/Antioxidant Power assay and free radical scavenging activity were evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals. TAC was obviously higher in colostrums than transitional and mature milks. Similar results were observed for DPPH radical scavenging activity of the samples. There was a high significant correlation between the results of these two methods. The relationship between the antioxidant content of human milk and maternal plasma was also significant. These data suggest that using colostrum, with high antioxidant potential during the first days of life is vital; moreover, reduction in total TAC during the course of lactation may needs more attention about nutritional status.
A palatinose-based liquid formula (palatinose-formula), suppresses postprandial plasma glucose and insulin levels in healthy men. The objective of this study was to investigate the effects of long-term palatinose-formula ingestion on glucose metabolism in patients with impaired glucose tolerance (IGT) or type 2 diabetes. Two patients with IGT and 7 patients with type 2 diabetes participated in the palatinose-formula and dextrin-based liquid formula (dextrin-formula) loading test and long-term palatinose-formula administration study. After a 3-month control period, palatinose-formula (1046 kJ) was ingested daily by patients as a part of breakfast for 5 months. In the loading test, palatinose-formula suppressed postprandial plasma glucose and insulin levels and areas under the curve compared with those after dextrin-formula ingestion. In the long-term study, glycated hemoglobin levels (after 3 months and 5 months of treatment) and serum 8-hydroxydeoxyguanosine levels (after 5 months of treatment) were markedly decreased comparing with those at baseline. Intake of 1046 kJ palatinose-formula as a part of breakfast over a long-term period may be effective for improvement of glucose metabolism in patients with IGT or type 2 diabetes.
Although anti-cancer effects of cyclooxygenase 2 (COX2) inhibitors have been reported, most studies focused on the direct effects of COX2 inhibiters on colon cancer cells. On the other hand, several types of cancers express Fas ligand (FasL) and/or TRAIL and mediate apoptosis of T cells in vitro. The “counter-attack” machinery may account for the mechanisms by which tumors evade host immune surveillance. In this study we determined if COX2 inhibitor could modulate effector molecules of cell death on colon cancer cells changing their effects on cytotoxic T lymphocytes. Colon adenocarcinoma cells, HCA7 and HCT116, the former COX2-positive and the latter COX2-negative, were pre-incubated with/without a COX2 inhibitor, NS398. Subsequently, the cells were co-cultured with Jurkat T cell leukemia cells and damage to Jurkat cells was determined. Treatment with NS398 resulted in reduction of expression of FasL and TRAIL in HCA7 cells, whereas NS398 did not affect the expression of FasL and TRAIL in HCT116 cells. The number of viable Jurkat cells was diminished when cells were co-cultured with naive, non-pretreated HCA7 or HCA116 cells. Preincubation of HCA7 cells with NS398 before co-culture blunted the HCA7 cell-induced cell toxicity on Jurkat cells. In contrast, pretreatment with NS398 failed to inhibit the HCT116-induced Jurkat cell killing. Our results suggest that COX2 regulates the expression of FasL and TRAIL on COX2-positive colon cancer cells thereby evoking a counter-attack against cytotoxic T cells, which may lead to compromised host immune responses.
Epidemiologic investigations indicate a close relationship between colorectal cancer and fat intake. However, to date the effects of lipid peroxidation-derived products that are formed from fat (especially free or esterified unsaturated fatty acids) on the initiation or progression of colorectal cancer have not been investigated extensively. Therefore, in the present study, we examined the effects of fatty acids, fatty acid hydroperoxides and aldehydes on the growth of human colorectal cancer cell line HT-29. At concentrations of 1 and 10 μM, linoleic, arachidonic and eicosapentaenoic acids, and 13-hydroperoxyoctadecadienoic and 15-hydroperoxyeicosapentaenoic acids had no significant effects on the growth of HT-29 cells. 4-Hydroxynonenal and 4-hydroxyhexenal had no significant effects on the growth of HT-29 cells up to 10 μM, whereas 4-oxononenal potently inhibited HT-29 cell growth (1–10 μM, 16–85% inhibition). Further experiments concerning DNA fragmentation, expression levels of Bax and Bcl-2 mRNA, expression levels of pro-caspase-3 and caspase-3 proteins, and activity of caspase-3 suggested that 4-oxononenal may increase the sensitivity of HT-29 cells to apoptosis through a decreased expression level of Bcl-2 and then increased formation of caspase-3 from pro-caspase-3.
Retinoic acid (RA) has been effective for improving wrinkles. However, it has also been reported that RA induces skin irritation. In this study, we explored new botanical compounds that show RA-like activity, but do not induce inflammation in vitro. Keratinocytes were maintained in a confluent condition and induced differentiation. Under this condition keratinocytes were treated with many botanical extracts and their morphological change were observed and compared with RA-treated. We found that silybin, which is a major flavonolignan from Silybum Marianum seeds, induced RA-like morphological change and prevented differentiation. We showed that silybin, like RA, reduced the expression of keratinocyte terminal differentiation markers and stimulated the expression of basement membrane component proteins. In contrast, silybin, unlike RA, did not stimulate the secretion of IL-1α, which is a skin irritation mediator. These results suggest that silybin has RA-like activity on keratinocytes and has the potential to improve winkle without inducing skin irritation.
Whether the speed of body mass (BM) reduction influences the body composition is uncertain. To investigate the effects of rapid vs slow body mass reduction on body composition, rats were divided into three groups; fed ad libitum for 16-day (Control, C); received restricted food intake during 16-day to decrease BM slowly (Slow, S); or fed ad libitum for 13-days and fasted for the last 3 days to rapidly reach a BM comparable to that of S (Rapid, R). Drinking water was restricted for R on day 16 to rapidly decrease their BM. All rats trained during the study. Final BM and adipose tissues mass were similar for R and S, and both were lesser than C. The skeletal muscle mass did not decrease in R and S. The liver mass was lower in R and S than C, and the decrease tended to be greater in R than S. Both the stomach and small intestine masses were significantly lower in R than C, but did not differ between S and C. In conclusion, differences of the speed of BM reduction affect the splanchnic tissues, and the decrease in splanchnic tissue mass was greater with rapid than slow BM reduction.
We have developed a simple ESR spin trapping based method for hydroxyl (OH) radical scavenging-capacity determination, using iron-free OH radical source. Instead of the widely used Fenton reaction, a short (typically 5 seconds) in situ UV-photolysis of a dilute hydrogen peroxide aqueous solution was employed to generate reproducible amounts of OH radicals. ESR spin trapping was applied to quantify OH radicals; the decrease in the OH radical level due to the specimen’s scavenging activity was converted into the OH radical scavenging capacity (rate). The validity of the method was confirmed in pure antioxidants, and the agreement with the previous data was satisfactory. In the second half of this work, the new method was applied to the sera of chronic renal failure (CRF) patients. We show for the first time that after hemodialysis, OH radical scavenging capacity of the CRF serum was restored to the level of healthy control. This method is simple and rapid, and the low concentration hydrogen peroxide is the only chemical added to the system, that could eliminate the complexity of iron-involved Fenton reactions or the use of the pulse-radiolysis system.
The present study was conducted in order to determine whether oxidative stress during aging involves dysfunction of the hypothalamic-pituitary-adrenal (HPA) axis in association with the emergence of cognitive deficits. When young rats were subjected to oxidative stress in the form of hyperoxia, thiobarbituric acid reactive substances, conjugated diene and lipid hydroperoxides increased markedly in the HPA axis. Vitamin E inhibited such increases in lipid peroxides in each organ. Levels of corticotrophin-releasing hormone in the hypothalamus and plasma levels of adrenocorticotrophic hormone and corticosterone were markedly elevated in young rats exposed to hyperoxia. However, young rats fed vitamin E-supplemented diets showed no abnormal hormone secretion, even after being subjected to hyperoxia. Furthermore, glucocorticosteroid receptors (GR) in pyramidal cells in the Cornus ammonis 1 region of the hippocampus in young rats were markedly decreased by oxidative stress. Similar phenomena were also observed in normal aged rats and young rats fed vitamin E-deficient diet kept in a normal atmosphere. Vitamin E supplementation prevented the decrease in GR in the hippocampus and the increase in corticosterone secretion caused by hyperoxia. These results suggest that oxidative stress induces oxidative damage in the hippocampus and the HPA axis during aging, resulting in a cognitive deficit in rats, and that negative-feedback inhibition on HPA activity was markedly dampened due to an increase in corticosterone levels caused by loss of GR.
It is well known that hydroxyl radicals are generated by ultrasound in water. This study with an electron spin resonance spin-trapping technique showed that hydroxyl radical generation was positively correlated with ultrasound duration and water temperature. The clear fungicidal action against Trichophyton spp. evident by studying cultured cells and the degradation of cytoplasmic and surface structures observed by transmission and scanning electron microscopy suggest that ultrasound in hot water is effective for sterilization of dermatophyte contamination and could be effective for the treatment of tinea infection.
We previously reported that low-dose X-irradiation alleviates ischemia-reperfusion injury such as mouse paw edema. In this study, we examined active changes in the biological function of mouse liver grafts in cold storage after low-dose X-irradiation. Mouse livers were sham-irradiated or were irradiated with 0.25, 0.5, 1.0, or 5.0 Gy of X-ray and stored for 4, 8, 24, or 48 h in preservation or saline solution. The results show that storage for 24 h in saline solution after 0.5 Gy irradiation significantly increased the activity of superoxide dismutase (SOD) and catalase. Following storage for 4, 8, or 48 h in preservation solution, lipid peroxide levels of the 0.5 Gy irradiated group were significantly lower than those of the sham irradiated group. Following storage for 24 h in preservation solution, the activity of SOD and catalase of the 1.0 Gy irradiated group were significantly higher than those of the sham irradiated group. Hepatocytes stored in saline solution were vacuolated. However, no vacuole formation was observed in hepatocytes stored in preservation solution. These findings suggest that low-dose irradiation significantly activates antioxidative functions of liver grafts. Moreover, the dose at which enhancement of antioxidative function occurs in livers stored in preservation solution, which contains glutathione, is significantly higher than that in saline solution.
Phosphatidylcholine (PC) and its hydrolysates are considered to stimulate intestinal lipid absorption, however, their exact effects on lipoproteins and apolipoprotein (apo) metabolism remain ambiguous. This study aimed to further differentiate the effects of them using fully differentiated enterocyte-like Caco-2 cells. Lipid micelles (oleic acid 0.6, cholesterol 0.05, monooleylglycerol 0.2, taurocholate 2 in mmol/l) with or without choline, PC, and lysoPC (0.2 mmol/l each) were applied apically to Caco-2 cells. 3H-oleic acid and 14C-cholesterol were added to the micelles when necessary. Secreted lipoproteins were analyzed by a HPLC method. LysoPC had the most potent promoting effect on lipid uptake, and lipoprotein and apolipoprotein B-48 secretion among the molecules tested. LysoPC doubled the output of cholesterol and triglyceride as the lipoprotein component, but PC did not. On the other hand, PC only increased the secretion of apoA-IV in the presence of lipid micelles. These findings confirm that the alteration of PC by PLA2 hydrolysis is intrinsically involved in the intestinal lipid absorption process and suggest that PC and its hydrolysis are coordinately associated with not only lipid absorption efficiency but also lipoprotein output and metabolism.
Altered inflammatory immune responses have been shown to be associated with functional gastro intestinal disorder. We aimed to clarify the effect of functional promoter polymorphism of RANTES, which is a potent chemoattractant peptide for memory T lymphocytes and eosinophils, on the risk of functional dyspepsia in a Japanese population. RANTES promoter C-28G polymorphism was genotyped in 246 subjects including 134 FD patients according to Roma III criteria and 112 non-symptomatic healthy controls. Although frequency of RANTES promoter polymorphisms in overall dyspeptic patients and non-symptomatic healthy controls did not show any significant differences, a significant association was found between G carrier and reduced risk of PDS according to Roma III criteria (age, sex, H. pylori infection adjusted OR = 0.23, 95% CI = 0.06–0.80). We also found that the same genotype held a lower risk of PDS in H. pylori positive PDS subjects (age, sex adjusted OR = 0.11, 95% CI = 0.01–0.94). Our data suggest that RANTES promoter -28G carriers is associate with a reduced risk of PDS especially in H. pylori positive subjects.
The present study was undertaken to evaluate the effects of lansoprazole (LPZ) on lipopolysaccharide (LPS)-stimulated toll-like receptor 4 (TLR4) signal transduction systems using the 293hTLR4/MD2-CD14 cells. The cells were incubated and then divided into the following groups: (a) untreated group, (b) non-LPZ treated (1h) group, (c) LPZ-treated (1h) plus non LPS-stimulated (1h) group, (d) LPZ-treated (1h) plus non LPS-stimulated (6h) group, (e) LPZ-treated (1h) plus LPS-stimulated (1h) group, (f) LPZ-treated (1h) plus LPS-stimulated (6h) group, (g) non LPZ-treated (1h) plus LPS-stimulated (1h) group and (h) non LPZ-treated (1h) plus LPS-stimulated (6h) group. Samples from each group were subjected to western blotting for analysis of IkB phosphorylation, intranuclear transfer of NF-kB, phosphorylation of MAP kinase (MAPK), intranuclear transfer of interferon regulatory factor 5 (IRF5), and expression of suppressor of cytokine signaling-1 (SOCS1). In the LPZ-treated groups, neither phosphorylation of MAPK nor intranuclear transfer of IRF5 was suppressed under stimulation with LPS, and enhanced intranuclear transfer of NF-kB and increased expression of SOCS1 were noted by comparison with the group treated with LPS alone. These results suggest that LPZ stimulates the expression of SOCS1 and regulates protein phosphorylation through its activity on TLR4 signal transduction under LPS stimulation.
Although low-dose aspirin is widely used, since it is a cheap and effective means of prevention of cardiovascular events, it can cause hemorrhagic gastrointestinal complications. The aim of this study was to evaluate the efficacy of rebamipide in preventing low-dose aspirin-induced gastric injury. A randomized, double-blind, placebo-controlled, crossover trial was performed in twenty healthy volunteers. Aspirin 81 mg was administered with placebo or rebamipide 300 mg three times daily for 7 consecutive days. The rebamipide group exhibited significant prevention of erythema in the antrum compared with the placebo group (p = 0.0393, respectively). Results for the body and fornix did not differ significantly between the placebo and rebamipide groups. In conclusion, short-term administration of low-dose aspirin induced slight gastric mucosal injury in the antrum, but not in the body or fornix. Rebamipide may be useful for preventing low-dose aspirin-induced gastric mucosal injury, especially which confined to the antrum.