Kunihiko Ishii

Prevention of Mammary Tumorigenesis in Acatalasemic Mice by Vitamin E Supplementation

Adult male and female acatalasemic (C3H/AnLCsbCsb), hypocatalasemic (C3H/AnLCscCsc) and normal mice of C3H strain fed on regular laboratory chow for 15 months showed an increased incidence of spontaneous mammary tumor in the decreasing order of female acatalasemic, male acatalasemic, female hypocatalasemic and male hypocatalasemic mice. Normal mice did not develop mammary tumor. We conducted a prospective study with female acatalasemic mice, which showed the highest incidence of mammary tumor, to examine the preventive effect of vitamin E on mammary tumor. Female acatalasemic mice were fed on vitamin E‐deficient (28 animals) and vitamin E‐supplemented diet (25 animals) for 29 months. The incidence of mammary tumor in mice given the vitamin E‐supplemented diet was 47%, while that in mice given vitamin E‐deficient diet was 82% (P<0.002). Mammary tumors were apparent after 9 months of vitamin E deprivation and after 14 months of vitamin E supplementation. Female normal mice did not develop mammary tumor during a comparable period of time. The mean catalase activity of mammary gland in acatalasemic mice was 18.8% of that in normal mice. The results indicate that vitamin E protects acatalasemic mice against the development of mammary tumor.

Enhanced liver injury in acatalasemic mice following exposure to carbon tetrachloride

The hypothtical involvement of hydrogen peroxide (H2O2) in carbon tetrachloride (CCl4)-induced acute liver injury and the potential preventive effect of catalase on hepatotoxicity have been studied in acatalasemic (C3H/AnLCsbC2b) mice and compared with normal (C3H/AnLCsaCsa) mice. A single intraperitoneal injection of CCl4 (20% in olive oil/g body weight) caused increases in serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in both mouse groups, but the extents of increases did not show significant differences between the two mouse groups until 12 h. The variation in increases of serum AST and ALT levels in acatalasemic and normal mice turned to be distinctly different from 12 h. At 18 h (peak point for ALT) and 24 h (peak point for AST), the serum enzyme levels in acatalasemic mice were nearly two-fold higher than those in normal ones, the difference being statistically significant (p <0.01). The liver malondialdehyde (MDA) level in acatalasemic mice was also higher than that in normals at 18 h (p <0.05). The extent of the centrilobular necrosis was histologically more severe in acatalasemic mice. The catalase activity in livers of acatalasemic mice was one-third to one-fifth those of normal mice (p <0.05) before and after treatment. The decreased catalase activity in acatalasemic mice might increase tissue or cellular levels of H2O2 during the later phase of the acute liver injury. From these findings, we conclude that H2O2 breakdown in liver would account for the difference in the later stages of the acute liver damage between the two groups of mice, and catalase is important in inhibiting hepatotoxicity of CCl4 in the later stage.

CHARACTERIZATION OF HYDROGEN PEROXIDE REMOVAL ACTIVITIES IN MOUSE HEMOLYSATES : CATALASE ACTIVITY AND HYDROGEN PEROXIDE REMOVAL ACTIVITY BY HEMOGLOBIN

Hydrogen peroxide removal activities in normal and acatalasemic mouse hemolysates were examined to determine the optimal temperature of catalase. From thermal stability of the removal activities in hemolysates, the removal activities were divided into two activities. The removal activity deactivated at lower temperature was catalase, and the 50% inactivation was observed after 10 min incubation at 47.2 +/- 0.5 degrees C for normal hemolysates and 34.0 +/- 0.8 degrees C for acatalasemic ones. The removal activity deactivated at a higher temperature remained after the addition of sodium azide, and the 50% inactivation was observed at 63.5 +/- 1.4 degrees C. After separation of the removal activities by carboxymethyl-cellulose column chromatography, the removal activity deactivated at higher temperature was attributed to the activity by hemoglobin. From Lineweaver-Burk plot analysis of the removal rates by hemoglobin at 37 degrees C, the Michaelis constant for hydrogen peroxide and the maximum velocity were 201 +/- 53 microM and 5.37 +/- 1.39 micromol/s per g of Hb, respectively. Removal rates by hemoglobin in mouse hemolysates at 37 degrees C in 70 microM hydrogen peroxide were 1.32 +/- 0.12 micromol/s per g of Hb. Catalase activity (k/g Hb: rate constant related to the hemoglobin content) in normal mouse hemolysates was 104 +/- 12 at 25 degrees C and 117 +/- 10 at 37 degrees C, and that in acatalasemic hemolysates was 10.5 +/- 1.7 at 25 degrees C. These results indicate that activity of hydrogen peroxide removal by hemoglobin is substantial and the activity in acatalasemic hemolysates is predominant at low concentration of hydrogen peroxide.

Liver Function Tests of Workers Exposed to Toluene and Toluene/Dimethylformamide at Low Concentrations

Liver Function Tests of Workers Exposed to Toluene and Toluene/Dimethylformamide at Low Concentrations: Da‐Hong Wang, et al. Department of Public Health, Okayama University Medical School—The purpose of our study was to analyse the results of liver function tests of workers exposed to toluene or to toluene/dimethylformamide (DMF) at levels below 100 ppm of toluene [(previous Occupational Exposure Limits of the Japan Society for Occupational Health); 10 ppm of DMF] in comparison with those of the reference group in order to see whether these solvents had any effect on the liver at levels generally considered to be safe. Our results suggested that occupational exposure to toluene below previous Occupational Exposure Limits of the Japan Society for Occupational Health lowered serum glutamic‐pyruvic transaminase (GPT) and gamma‐glutamyl transpeptidase (7‐GTP) activities significantly in males. On the other hand, serum levels of GPT and 7‐GTP in the female toluene‐ exposed group were significantly higher than those in the control group. Median Body Mass Index (BMI) levels and alcohol consumption did not show any significant difference between toluene‐exposed subjects and the controls, suggesting that toluene exposure or other unknown life‐style factors were likely to have more effects on the difference of serum GPT and 7‐GTP levels between toluene‐exposed and control subjects. Serum GPT activity in the DMF/tolu‐ ene‐exposed group was significantly higher than that in the toluene‐exposed group, and DMF exposure was considered to play a role in increasing the serum GPT activity in the DMF/toluene‐exposed male group. The present results that the decreased serum GPT and 7‐GTP levels in the toluene‐exposed male group necessitate performing further epidemiological and experimental studies to investigate the possible hepatic effect of toluene exposures at levels generally considered to be safe.

Inhibition of carbon tetrachloride-induced liver injury in acatalasemic mice by hepatic iron deprivation

Abstract We have demonstrated an enhanced liver injury in the later phases (after 12 h) of carbon tetrachloride (CCl 4 ) intoxication in acatalasemic mice, which was considered to be attributed to the increased formation of hydrogen peroxide (H 2 O 2 ) or hydroxyl radicals ( OH) in the absence of catalase. The present study was attempted to investigate the effects of hepatic iron deprivation on CCl 4 -induced hepatotoxicity in acatalasemic mice. Hepatic iron deprivation was induced by phlebotomy, subcutaneous injection of erythropoietin, and intraperitoneal injection of the iron-chelating agent DFO. CCl 4 was injected intraperitoneally on the fourth day of the treatment. By the iron deprivation, the hepatic iron concentrations (HICs) of both acatalasemic and normal mice were decreased below half the iron-undeprived levels. In the iron-deficient CCl 4 -treated mice, the increases of serum alanine aminotransferase (ALT) activity and the liver thiobarbituric acid reactive substance (TBARS) level 18 h after CCl 4 treatment were suppressed in both acatalasemic and normal mice, the difference in serum ALT level between acatalasemic and normal mice became insignificant, and the extent of the centrilobular necrosis was also histologically attenuated, resulting in the disappearance of the difference in the extent of hepatocellular necrosis between them. Analysis of the results by ANOVA indicated that both the catalase levels and iron levels were related to the difference in the extent of CCl 4 -induced liver damage. We conclude that CCl 4 -induced hepatotoxicity in the later phases is likely to be produced through the formation of H 2 O 2 or OH formed from H 2 O 2 , in which iron is important in mediating its toxicity, and catalase plays a critical role in the prevention of CCl 4 -induced secondary hepatotoxicity.

An enhanced liver injury induced by carbon tetrachloride in acatalasemic mice.

Carbon tetrachloride (CC14) had been widely used as a lolvent to some organic compounds, such as oil, resin or tar. CC14 had also been used as a raw material of halo-fluorocarbon for coolant or as an extinguish agent of fire. Since some cases of acute toxic injury caused by CC14 used as a solvent have been reported, the above-mentioned use of CC14 is now uncommon. However, it is still widely used in animal models to induce acute and chronic liver injuries for investingating the mechanisms of CCl4induced liver injury.

Prevention of Breast Cancer in Acatalasemic Mice by Vitamin E Supplementation

The preventive effect of vitamin E on spontaneous mammary tumor was studied on acatalasemic mice. Mammary tumors developed in 34.3% of acatalasemic mice (C3H/AnLCsbCsb) maintained on laboratory chow for 15 months, in 15.1% of homozygous hypocatalasemic mice (C3H/AnLCscCsc), and in none of the normal mice (C3H/AnLCsaCsa). Dietary supplementation of vitamin E reduced the incidence of spontaneous mammary tumors in acatalasemic mice to 47%, compared with 82% in acatalasemic mice maintained on a vitamin E-deprived diet for 29 months.