T-2 toxin extracted from a strain of Fusarium tricinctum has a structure similar to that of the Fusarium mycotoxin found in grains in Kaschin-Beck Disease endemic areas. By scanning and freeze-fracture transmission electron microscope observations, we found that 0.01ppm T-2 toxin caused a significant decrease of collagen microfibrils in the extracellular matrix and intramembrane particles on the protoplasmic face of the plasma membrane of cultured chicken embryonic chondrocytes. In addition, T-2 toxin obviously reduced the activities of mitochondrial cytochrome c oxidase and H+-ATPase, and the sensitivity of the latter to oligomycin, of chondrocytes. In our experiment we found that, when 0.01ppm T-2 toxin was added in the presence of 1ppm Na2SeO3 in the culture, the decreases in collagen microfibril and intramembrane particle numbers could not be seen and that the activities of cytochrome c oxidase and H+-ATPase and the sensitivity of the latter to oligomycin were less decreased. Furthermore, the experimental result showed that the effect of Se on chondrocytes is concentration dependent and that 1ppm of Na2SeO3 is the optimal concentration. Thus we conclude that Se deficiency may be an important factor for the pathogenesis of Kaschin-Beck disease.
Isolated human fetal hepatocytes could be successfully cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with placental cord serum, which is a rich source of various growth factors. These cultured hepatocytes were found to maintain their viability, morphological integrity, and detoxifying capacity. The expression of two urea-cycle enzymes, i.e., carbamyl phosphate synthetase (CPS) and argininosuccinate lyase (ASL), was studied in isolated and cultured hepatocytes from human fetuses of different gestational age classified as Group I: 17-20 weeks, Group II: 21-24 weeks, Group III: 25-28 weeks, Group IV: 29-32 weeks, and Group V: 33-36 weeks. We found that on culture the activities of the two enzymes increased for a period of 4-6 days. Hepatocytes of Group I and Group II showed maximum expression of CPS by the 6th day of culture, whereas those of Groups III, IV, and V showed theirs by the 5th day of culture. In the case of ASL the maximum levels were seen by the 4th day of culture, after which the activities of both enzymes decreased. CPS activity in cultured hepatocytes was about 1.4-4.7 times higher whereas that of ASL was about 1.2-1.6 times higher, when compared with that of freshly isolated cells. This study indicates that isolated hepatocytes undergo reversible changes and that on culture for 4-6 days, liver cell functions recover with enhanced enzyme activities. Cultured human fetal hepatocytes of Group III gestation would appear to be more suitable for transplantation than isolated hepatocytes.
A factor activating brain nitric oxide synthase was detected in rat urine and partially purified by reversed-phase high-performance liquid chromatography (HPLC). Its intracerebroventricular injection reduced the blood pressure and heart rate of rats. A similar activating factor was detected in rat brain, heart, kidney, liver, and the adrenal. Since its activity per wet weight was the highest in the adrenal, we decided to purify it from bovine adrenals and obtained a partially purified preparation by FPLC MonoQ column chromatography. The active fraction stimulated cyclic GMP (cGMP) production in primary cultures of neuronal cells from fetal rat brain. These results suggest that an activator of nitric oxide synthase is present in bovine adrenals as well as in the urine and various tissues of rats. This factor showed hypotensive and bradycardiac actions when injected into the brain.
The chemopreventive action of four dietary flavonoidal compounds, quercetin, rutin, luteolin, and (+)catechin, on 7, 12-dimethylbenz(a)anthracene (DMBA)-induced skin carcinogenesis was studied in male Swiss albino mice. Topical application of DMBA (0.24%) as initiator for 2 weeks and croton oil as promoter for 4 weeks produced 100% incidence of skin tumor in control animals. Prior topical treatment with quercetin or luteolin reduced the mean papilloma formation, while rutin and (+)catechin were found to exert a minimal effect against DMBA-induced papilloma formation. By continuous application of flavonoidal compound along with initiator and promoter, the papilloma incidence was found to be 32% for quercetin, 40% for luteolin, 58% for rutin, and 71% for (+)catechin. The mean number of papillomas per mouse was found to be 2.52 in the DMBA control, whereas it was significantly decreased in quercetin (0.45, p<0.001)-, luteolin (0.52, p<0.001)-, rutin (1.58, p<0.05)-, and (+)catechin (1.67, p<0.05)-treated mice. Increased levels of glutathione and glutathione-S-transferase and decreased levels of lipid peroxides and cytochrome P-450 were observed when the flavonoid treatment was given along with the DMBA. The possible mode of action of the flavonoidal compounds may be their influence on the activating enzymes of the carcinogens in mouse skin.
The effect of vitamin E (Vit E), both α-tocopherol (α-T) and γ-tocotrienol (γ-T3), supplementation on splenocyte proliferation and phagocytic activity of peritoneal macrophages in rats treated with diethylnitrosamine (DEN) and 2-acetylaminofluorene (AAF) was investigated. Splenocyte proliferation in response to concanavalin A or phytohemagglutinin and phagocytic activity of peritoneal macrophages in the DEN/AAF-treated rats were significantly reduced compared with the control levels. In contrast, mitogenesis and phagocytic activity of peritoneal macrophages were increased significantly in the DEN/AAF-treated rats supplemented with Vit E; and the vitamin effect was dose dependent. However, the increases were not so great as those observed in the controls. The highest doses of α-T and γ-T3 used effected the highest phagocytic activity, with α-T generally showing a higher activity than γ-T3. Although long-term Vit E supplementation at half the optimum dose significantly increased mitogenesis, phagocytic activity in the DEN/AAF-treated and control rats was only marginally increased.
Vacuolation of gastric parietal cells was studied in guinea pigs chronically given high doses of omeprazole (OPZ) with or without the antioxidant α-tocopherol. Animals were divided into two groups: one group was treated with OPZ alone (20mg/kg); and the other, with OPZ (20mg/kg) plus α-tocopherol (50mg/kg). After 4 weeks of administration, the histological changes in the parietal cells and lipid peroxide formation in the gastric mucosa were assessed. The α-tocopherol concentration of the gastric mucosa after α-tocopherol administration was also measured. Parietal cell vacuolation was completely inhibited in the group given OPZ and α-tocopherol concomitantly, and lipid peroxidation levels were also significantly lower than in the group given OPZ alone. The α-tocopherol concentration of the gastric mucosa was increased significantly by α-tocopherol. These findings suggest that lipid peroxidation by OPZ is involved in parietal cell vacuolar degeneration when high doses of OPZ are administered for long periods.
Effects of dietary fats consisting of different fatty acids on the acetyl CoA-carboxylase activity in liver was studied in rats. Sprague-Dawley male rats were meal-fed an isoenergetic diet based on either beef tallow or safflower oil for 8 weeks. The acetyl CoA-carboxylase activity in liver was lower in the safflower oil diet group than in the beef tallow diet group. Body fat accumulation was less in rats fed the safflower oil diet, however, their norepinephrine turnover rate in liver was higher. α1-Adrenergic receptor binding was determined with [3H] prazosin. Binding affinity of α1-adrenergic receptor in liver was higher in the safflower oil diet group, presumably resulting from higher membrane fluidity in that group. These results suggest that the intake of the safflower oil diet promoted less body fat accumulation by reducing the acetyl CoA-carboxylase activity, which may be a result of higher sympathetic nervous system activity in the liver.