The possible mechanisms of inhibition of benzo(a)pyrene (BaP) binding to DNA in vivo and in vitro by ellagic acid (EA) were investigated. In the in vitro studies BaP was activated by cytochrome P-450-dependent mixed function oxidase system, ascorbate-dependent lipid peroxidation, and peroxidation of linoleic acid. Reactions were performed in the dark at 37°C for 20 to 30min in buffered aqueous solutions with 2mg DNA and 40nmol tritiated BaP. The levels of BaP-DNA adducts were determined by assay of the radioactivity. BaP-DNA adduct levels ranged from 146 to 170fmol/mg DNA in ascorbate-and NADPH-dependent reactions and the level was 8.25±0.425pmol/mg DNA in the hematin-mediated linoleic acid reaction. Addition of EA to the reaction mixtures resulted in a significant inhibition in BaP-DNA adduct formation, depending upon the concentration of EA; e.g., 500μM EA resulted in 42 to 53% inhibition of binding in the three systems of carcinogen activation. Similarly, EA feeding to male NMRI mice at a concentration of 12μg/ml drinking water significantly decreased the levels of carcinogen-DNA adducts in the lungs. Reactions performed in vitro, in which DNA was preincubated overnight at 4°C with EA, revealed a decrease of 20% in adduct formation. MDA formation during the process of lipid peroxidation, stimulated by NADPH and ascorbate in the liver microsomes of mouse and by hematin in linoleic acid, was taken as an index for the free radical reactions. Addition of EA to in vitro systems inhibited the MDA formation with an IC50 (concentration for 50% inhibition) of 480, 400, and 400μM for NADPH, ascorbate, and hematin-mediated free radical reactions, respectively.
Lipid synthesis in monocytes isolated from rabbits fed a high-cholesterol diet and subsequently a low-cholesterol one was examined. Rabbits were fed diet containing 2.5% (w/w) cholesterol for 3 weeks and then transferred to a normal diet for another 6 weeks. The total and free cholesterol levels in the blood increased up to 3 weeks and then steadily decreased. Esterified cholesterol increased only during the first week on the high-cholesterol diet and then decreased throughout the remainder of the experimental period. Lipid synthesis, as assessed by [1-14C]acetate incorporation, was significantly reduced in monocytes isolated from animals on the high-cholesterol diet. This reduction in lipid synthetic activity continued another 3 weeks even after cessation of the high-cholesterol diet and then lipid synthesis was rather increased significantly. Specifically an increase in cholesterol and fatty acid synthesis was observed when animals were fed a high-cholesterol diet for 3 weeks and then allowed to recover for another 6 weeks.
The concentrations of vitamin A (retinol) and retinyl ester in the plasma and liver of normal and diabetic rats were measured by HPLC (high-performance liquid chromatography). Diabetic rats had severe hyperglycemia, induced by a single streptozotocin injection 5 weeks prior to sampling. In the normal rats, plasma retinyl palmitate was very low, and the level was increased 10-fold by diabetes. Detailed time-course studies showed that rats became hyperglycemic within 48h of streptozotocin injection, yet the plasma retinyl palmitate level was not elevated until some three weeks later. Severe diabetes did not significantly influence plasma retinol; however, free retinol in the liver was elevated within 10 days of initiation of the disease and continued to increase for the duration of the study. These results show that streptozotocin-induced diabetes significantly alters the concentrations of hepatic retinol and plasma retinyl ester. The biochemical mechanism(s) of this altered vitamin A homeostasis in diabetes and its possible relationship to tissue pathogenesis are not known at present.
The effects of small peptides on brush-border membrane enzyme activities and on transport carriers for amino acids and dipeptides were investigated by measurement of these enzyme activities and the transmural potential difference in the small intestines of guinea pigs fed elemental diets containing either small peptides (SP) or amino acids (AA) as the nitrogen source. Brush-border membrane aminopeptidase activities were significantly higher in the group fed the diet containing SP. In addition, the transmural potential differences induced by L-leucine and glycyl-L-leucine (Gly-Leu) were significantly higher in the SP group. The maximum potential differences for L-leucine and Gly-Leu were significantly higher in the SP group, whereas the half saturation values did not differ between the two groups. The same effect of a SP diet was seen in rats, when the uptake of L-[U-14C]leucylglycine (L-[14C]Leu-Gly) into intestinal brush-border membrane vesicles was measured. Some 70% of L-[14C]Leu-Gly was not hydrolyzed by aminopeptidase, but was found as a dipeptide in the brush-border membrane vesicles. These results indicate that small peptides as intraluminal substrates increase brush-border membrane aminopeptidase activities and the activity of carriers for amino acids and dipeptides.
When 10 weeks old male BALB/c mice received whole-body irradiation with an 8-Gy single dose of 60Co γ-ray, the survival rate 30 days after the irradiation was only 5%, while the survival rate was increased to 70% when the mice were subcutaneously injected with catecholestrogen 2-hydroxyestradiol (2-OHE2) 3h before and 3h after the irradiation. The survival rates of the mice given other test compounds were as follows: 2-hydroxyestrone, 20%; 2-hydroxyestriol, 20%; 4-hydroxyestradiol, 0%; 2-methoxyestrone, 0%; 2-methoxyestradiol, 0%; 2-methoxyestriol, 0%; estrone, 0%; estradiol, 5%; and estriol, 20%. Upon the irradiation, lipid peroxide levels in the liver of the animals markedly increased 4 days after the irradiation. This increase in the level was significantly suppressed by the subcutaneous injection of 2-OHE2. These results indicate that 2-OHE2 has a protective effect against radiation injury.
This study was designed to detect whether the plasma level of lipid peroxide and of erythrocyte ghost (EG) would be affected by treadmill exercise testing carried out by the modified Bruce method in patients with hypertension or hypercholesterolemia. A total of 27 patients were studied for changes in their ECG. On exercise testing, 10 patients responded positively and 17 negatively. Before testing, plasma levels of lipid peroxide were 3.9±0.9nmol/ml (mean±SE) in the positive group and 1.6±0.4nmol/ml in the negative group, a significant difference (p<0.05). The lipid peroxide (LPO) level of the EG was 3.7±0.8nmol/mg protein in the positive group and 3.2±0.7nmol/mg protein in the negative group. Exercise testing tended to induce a rise in the lipid peroxide values in the EG of the positive group (11.3±5.0nmol/mg protein vs. 3.7±0.8nmol/mg protein, p<0.1). The fold-increase in the level of erythrocyte membrane lipid peroxide was significantly higher in the positive responders (2.3±0.7 vs. 0.8±0.1, p<0.05). There was no significant change in the plasma level of lipid peroxide in either group. Other clinical parameters including blood pressure, heart rate, obesity, smoking, medication, exercise intensity, and plasma lipid levels showed no differences between the positive and the negative responders. We conclude that the erythrocyte membranes of the positive responders to treadmill exercising are easily peroxidized by various stimuli, which may contribute in part to the high plasma lipid peroxide level before testing. A stimulus such as treadmill testing may accelerate the lipid peroxidation of the erythrocyte membranes significantly and independently.
Employing a monoclonal antibody-based two-site enzyme immunoassay method for basic fibroblast growth factor, we measured immunoreactive bFGF levels in sera of patients with breast cancer, and found that the level of immunoreactive bFGF was significantly increased in sera of most of the patients. Two immunoreactive peaks were observed in the fractions of sera of the normal subjects and the patients with breast cancer by HPLC size-exclusion chromatography: one peak eluted at the void volume; and the other, at the same position as that of standard recombinant bFGF. This result suggests that bFGF-like immunoreactive substance of high molecular (HMW bFGF-LI) together with normal bFGF exists in sera of normal subject and patients with breast cancer. The amount of HMW bFGF-LI of the patient was considerably larger than that of the normal subject, and the former decreased to the normal level after surgical resection of the tumor region.
The implication of biotin depletion in metabolic abnormalities occurring in 30 patients with sternocostoclavicular hyperostosis was studied, because the patients had low serum biotin levels. Levels of serum amino acids and fatty acids, fasting blood glucose level, plasma ascorbic acid level, and urinary excretion of hydroxyproline were measured in the patients before and after their treatment with biotin, which was done as 9mg daily in three divided doses. Before the treatment, the levels of serum branched-chain amino acids, threonine, serine, glutamic acid, glutamine, proline, and taurine were elevated in the patients. Fatty acids in the serum phospholipids from the patients contained higher levels of 18: 2n-6 and 18: 3n-3 but lower levels of 20: 4n-6 and 20: 5n-3. Hyperglycemia and abnormal glucose tolerance were observed in the patients. Plasma ascorbic acid level tended to be low, while urinary excretion of hydroxyproline was high in the patients. The biotin treatment corrected these abnormalities, resulting in clinical improvement. The results suggest that the metabolic abnormalities resulting from biotin depletion may have an important role in the development of sternocostoclavicular hyperostosis.