Michinaga Matsumoto

Cancer screening of upper aerodigestive tract in Japanese alcoholics with reference to drinking and smoking habits and aldehyde dehydrogenase-2 genotype

In this study, 1,000 Japanese male alcoholics were consecutively screened by upper gastrointestinal endoscopy with esophageal iodine staining. Associations among cancer‐detection rates, drinking and smoking habits, and aldehyde dehydrogenase‐2 (ALDH2) genotypes were evaluated. A total of 53 patients (5.3%) had histologically confirmed cancer. Esophageal cancer was diagnosed in 36, gastric cancer in 17, and oropharyngolaryngeal cancer in 9 patients: 8 of the esophageal‐cancer patients were multiple‐cancer patients, with additional cancer(s) in the stomach and/or oropharyngolaryngeal region. Multiple logistic regression revealed that use of stronger alcoholic beverages (whisky or shochu) in contrast with lighter beverages (sake or beer) and smoking of 50 pack‐years or more increased the risks for esophageal (odds ratio 3.2 and 2.8 respectively), oropharyngolaryngeal (4.8 and 5.1 respectively) and multiple cancer (10.5 and 11.8 respectively). The inactive form of ALDH2, encoded by the gene ALDH2*1/2*2 prevalent in Orientals, exposes them to higher blood levels of acetaldehyde, a recognized animal carcinogen, after drinking. This inactive ALDH2 was detected in 19/36 (52.8%) patients with esophageal cancer, in 5/9 (55.6%) patients with oropharyngolaryngeal cancer, and in 7/8 (87.5%) patients with multiple cancer. All of these gene frequencies far exceeded that in a large alcoholic cohort (80/655, 12.2%). The triple combination of the risk factors of the inactive ALDH2, stronger alcoholic beverages and heavy smoking was more commonly associated with multiple‐cancer patients than with patients with esophageal cancer alone (62.5% vs. 7.1%). These results show that the 3 risk factors are important for the development of upper‐aerodigestive‐tract cancer in Japanese alcoholics. For these high‐risk drinkers, regimented screening appears to be indicated.

Simultaneous quantification of retinol, retinal, and retinoic acid isomers by high-performance liquid chromatography with a simple gradiation

A new method of high-performance liquid chromatography (HPLC) analysis to quantify isomers of retinol, retinal and retinoic acid simultaneously was established. The HPLC system consisted of a silica gel absorption column and a linear gradient with two kinds of solvents containing n-Hexane, 2-propanol, and glacial acetic acid in different ratios. It separated six retinoic acid isomers (13-cis, 9-cis, all-trans, all-trans-4-oxo, 9-cis-4-oxo, 13-cis-4-oxo), three retinal isomers (13-cis-, 9-cis-, and all-trans) and two retinol isomers (13-cis- and all-trans). Human serum samples were subjected to this HPLC analysis and at least, all-trans retinol, 13-cis retinol, and all-trans retinoic acid were detectable. This HPLC system is useful for evaluating retinoic acid formation from retinol via a two-step oxidation pathway. Moreover, it could be applied to monitoring the concentrations of various retinoids, including all-trans retinoic acid in human sera.

Effect of chronic ethanol feeding on endotoxin-induced hepatic injury: role of adhesion molecules on leukocytes and hepatic sinusoid

Endotoxin is postulated to be an important aggravating factor for alcoholic liver disease. We have previously reported that rats fed ethanol are more vulnerable to endotoxin-induced liver damage, and hepatic microcirculatory disturbance plays an important role for this liver damage by observation with an intravital microscopy. In this study, we have investigated the role of adhesion molecules in endotoxin-induced microcirculatory disturbance in chronic ethanol-fed rats. Male Wistar rats were pair-fed with ethanol liquid diet (ethanol group) or an isocaloric control diet (control group) for 6 weeks. Leukocyte adherence to the hepatic sinusoid by stimulation with lipopolysaccharides (1 mg/kg of body weight) was observed by an inverted fluorescence microscopy equipped with a silicon-intensified target camera and was found to be enhanced in ethanol-fed rats. Tumor necrosis factor-alpha and GRO/CINC-1 (rat counterpart of interleukin-8) was increased in the blood in these animals. Subsequent expression of adhesion molecules, LFA-1 beta-chain on leukocytes were demonstrated by flow cytometry, which suggests a possible involvement of leukocyte adherence to the hepatic damage in ethanol-fed animals. Preadministration of anti-rat LFA-1 beta-chain monoclonal antibody effectively suppressed leukocyte adherence to the hepatic sinusoid. These results suggest that the enhanced sequestration of neutrophils to the liver with these adhesion molecules may play a significant role in the pathogenesis of alcoholic liver disease.

Acetaldehyde inhibits the formation of retinoic acid from retinal in the rat esophagus

Objective. It has already been demonstrated that the rat esophagus produces retinoic acid from retinol. In this study, this process is further characterized and the effect of acetaldehyde examined to elucidate the possible mechanisms behind the epidemiological evidence that the incidence of esophageal cancer is higher in alcoholics. Material and methods. Rat esophageal samples were incubated with all-trans retinal and newly formed all-trans retinoic acid (ATRA) was quantified using high-performance liquid chromatography (HPLC). Furthermore, β-nicotinamide adenine dinucleotide (NAD)-dependent acetaldehyde oxidation by the rat esophagus was examined by tracing NAD reduction using a spectrophotometer. Results. Rat esophageal samples produced ATRA from all-trans retinal in a NAD-dependent manner and the potential was significantly attenuated by phenetyl isothiocynate, an ALDH inhibitor, or acetaldehyde depending on the concentration used. Rat esophageal samples also oxidized acetaldehyde of various concentrations NAD dependently. The ATRA formation potential that was temporarily inhibited by acetaldehyde was recovered to the control level by dialysis when the specimen was incubated with up to 50 µM of acetaldehyde. Conclusions. The rat esophagus produces retinoic acid from retinal. An ALDH isoform(s) is responsible for this process and physiological concentration of acetaldehyde hampers the process, probably in a competitive manner. Since the disturbance of retinoic acid supply has been implicated in carcinogenicity, this finding may, at least in part, explain the high incidence of esophageal cancer in alcoholics, especially in those with inactive ALDH 2 whose blood acetaldehyde levels become higher than those with active ALDH 2.

Alcohol Dehydrogenase Activities in the Human Gastric Mucosa: Effects of Helicobacter pylori Infection, Sex, Age, and the Part of the Stomach

BACKGROUND Human gastric mucosa contains three alcohol dehydrogenase (ADH) isozymes (classes I, III, and IV). Various factors such as Helicobacter pylori infection, sex, age, and the part of the stomach involved have been suggested to affect alcohol dehydrogenase activities, although these views are controversial. In this study, these unsettled issues were reexamined. METHODS Activities of class I and IV ADHs were evaluated in the cytosolic fraction of human gastric mucosa samples by reduction of their preferred substrates, namely acetaldehyde and m-nitrobenzaldehyde, and activities of class III were evaluated by oxidation of its preferred substrate, formaldehyde. Then, effects of Helicobacter pylori infection, sex, age, and the part of the stomach involved were examined. RESULTS Class I, III, and IV ADH activities were 17.5 +/- 8.4, 4.2 +/- 2.5, and 8.9 +/- 3.9 nmol of nicotinamide adenine dinucleotide oxidation per minute per milligram of protein, respectively, for the entire population. Helicobacter pylori infection significantly reduced class I and IV ADH activities but did not affect activity of class III. In the samples without Helicobacter pylori infection and severe gastritis, sex did not affect class I, III, or IV ADH activities. In the same series, class IV ADH activity significantly decreased with age (p = 0.006), whereas no correlation was found between age and ADH activity of class I and III ADHs. The level of class IV ADH activity was significantly higher in the upper body than in the lower regions, whereas no such heterogeneity was observed in class I and III ADH. CONCLUSIONS Various factors affect human gastric ADH activities, such that careful interpretation of their significance is necessary.

Formation of superoxide anion in the hepatic sinusoid after lipopolysaccharide challenge

Using the cytochrome c method, superoxide anion that is released into the hepatic sinusoid was measured after a lipopolysaccharide challenge in a liver perfusion system. Moreover, damages of epithelial cells of the hepatic sinusoid were estimated with scanning electron microscopic analysis and levels of purine nucleoside phosphorylase/GPT ratio. Lipopolysaccharide administration increased the conversion of oxidized cytochrome c into reduced cytochrome c in the perfusate, indicating that superoxide anion was formed in the hepatic sinusoid. This change was associated with increase in levels of portal tumor necrosis factor-alpha and attenuated by the simultaneous administration of superoxide dismutase. Scanning electron microscope analysis revealed that diameters of sinusoidal fenestrae increased in rats treated with lipopolysaccharide, compared with controls. Moreover, levels of purine nucleoside phosphorylase/GPT ratio was significantly increased in the liver perfusate in lipopolysaccharide-treated rats, compared with controls. Superoxide anion in hepatic sinusoid may be one of the pathogenic factors behind damages of epithelial cells of the hepatic sinusoid caused by lipopolysaccharide.

Induction of NADPH Cytochrome P-450 Reductase in Kupffer Cells After Chronic Ethanol Consumption Associated with Increase of Superoxide Anion Formation

Using native polyacrylamide gel electrophoresis (PAGE) and diaphorase staining, NADPH cytochrome c P-450 reductase was examined in the Kupffer cells of rats fed ethanol chronically as well as in controls. Formation of superoxide anion released from Kupffer cells into the hepatic sinusoid was estimated using the cytochrome c method, which was applied to a liver perfusion model in both groups after an additional acute ethanol challenge. Kupffer cells were found to carry NADPH cytochrome c P-450 reductase, and chronic ethanol consumption resulted in its induction being doubled. This change was associated with increase of superoxide anion release from Kupffer cells into the hepatic sinusoid after an acute ethanol challenge (0.020 ± 0.03 O.D./g liver versus 0.012 ± 0.002 O.D./g liver; P < .05). In conclusion, release of superoxide anion from Kupffer cells into the hepatic sinusoid increases in rats chronically fed ethanol. Induction of NADPH reductase in Kupffer cells caused by chronic ethanol consumption may, at least in part, be involved in this mechanism.

Ethanol, acetaldehyde and H2 blockers inhibit NAD dependent retinoic acid (RA) formation from retinol (Vitamin A) in human gastric mucosa

BACKGROUND AND AIMS Retinoic acid is formed from retinol (Vitamin A) via a two step oxidation conducted by some dehydrogenase families. Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) isozymes have recently been suggested to participate in this process in NAD dependent manners. Since human gastric mucosa contains several isozymes of AOH and ALDH, it is conceivable that human gastric mucosa can produce RA from retinol via the two step oxidation and disturbance of responsible enzymes may reduce the production of RA. The aims of this study were to investigate effects of inhibltors for ADH and ALDH on RA formation from retinol in human gastric mucosa. MATERIALS AND METHODS To that effect, biopsy samples were endoscopically obtained from patients who agreed to their sample donation. Gastric cytosolic samples were prepared and incubated with 3000 no of a/I traos retinol in the buffer (pH=7.4) containing 2 mM NAD at 37 OC for 20 minutes. After the incubation, retinoids were extracted and applied to HPLC to determine the quantity of all trans RA formation. RESULTS When all-trans retinol were incubated with gastric mucosal sample, considerable amount of all trans RA was formed in the presence of NAD, whereas the formation was hardly observed when NAD was removed. Ten mM of ethanol tended to hamper the RA formation and ethanol more than 100 mM significantly attenuated its formation. Moreover, acetaldehyde also attenuated the formation in a concentration dependent manner. H2 blockers including 2 mM of ranitidine and 2 mM of cimetidine, which are known as inhibitors for ADH, significantly attenuated RA formation, whereas 2 mM of famotidine, which has no inhibitory effect on ADH, failed to suppress it. CONCLUSION There is an NAD dependent pathway by which all trans RA is produced from all-trans retinol in human gastric mucoso. This process was disturbed by inhibitors for ADH or those for ALDH, including ethanol, acetaldehyde and some H2 blockers. Impairment of this pathway due to ethanol or acetaldehyde may stand behind the pathogenesis of various lesions in human gastric mucosa in alcoholics. Although significance of effects of H2 blockers on RA formation remains unknown, our present findings imply that overuse of some H2 blockers may be harmful for human gastric mocoso,