Raymond Toles

Gamma-tocotrienol, a tocol antioxidant as a potent radioprotector

Purpose: To assess the radioprotective potential of gamma-tocotrienol. Materials and methods: To optimise its dose and time regimen, gamma-tocotrienol (GT3) was injected subcutaneously (SC) at different doses into male CD2F1 mice [LD50/30 (lethal radiation dose that results in the mortality of 50% mice in 30 days) radiation dose of 8.6 Gy with vehicle]. The mice were given 10.5, 11 and 11.5 Gy cobalt-60 radiation, and 30-day survival-protection was determined. Time optimisation was done by SC administration of GT3 at different intervals before irradiation. Dose reduction factor (DRF) was determined by probit analysis using mortality as the end point at six radiation doses. Protection from radiation induced pancytopenia was determined by enumerating peripheral blood cells from mice given GT3 and irradiated at 7 Gy. Results: At an optimal dose of 200 mg/kg given SC 24 h before irradiation, GT3 had a DRF of 1.29. GT3 accelerated the recovery of total white blood cells, neutrophils, monocytes, platelets, and reticulocytes in irradiated mice, compared to vehicle-injected, irradiated controls. Conclusion: GT3 is a radioprotectant having a higher DRF than any other tocols. The protection it provides close to the gastro-intestinal range indicate that GT3 can be considered as an ideal radioprotectant meriting further drug development stages for the ultimate use in humans.

Gamma-Tocotrienol Protects Hematopoietic Stem and Progenitor Cells in Mice after Total-Body Irradiation

Abstract We analyzed the radioprotective effects of gamma-tocotrienol (GT3) on hematopoietic stem cells (HSCs) and progenitor cells (HPCs) in sublethally irradiated mice. Flow cytometry analysis indicated that radiation depleted HPCs (c-Kit+, Lin−) to 40% at days 2 and 4 after total-body irradiation (TBI) in all treatment groups. The HPC numbers in GT3-treated mice recovered almost completely (90%) at day 7 but remained depleted in vehicle-treated mice (30%) even at day 13 after TBI. An in vitro colony-forming assay on sorted HSCs (Lin−, Sca1+, c-Kit+) indicated that TBI reduced the number of colonies to 40% and 50% at day 17 and 60, respectively, in vehicle-treated groups compared to unirradiated controls (naïve). GT3-treated irradiated mice maintained higher numbers of colonies (86% and 80% compared to naïve mice), thereby preserving the self-renewable capacity of HSCs. Histopathology of sternal bone marrow indicated more regenerative microfoci for myeloid cells and megakaryocytes and higher overall cellularity in GT3-treated mice compared to vehicle controls at days 7 and 13 after TBI. GT3 treatment also reduced the frequency of micronucleated erythrocytes significantly in irradiated mice. Our results demonstrate that GT3 protected hematopoietic tissue by preserving the HSCs and HPCs and by preventing persistent DNA damage.

Bile acid accumulation in gastric mucosal cells

Abstract Bile acids are one of the components of the gastric contents capable of disrupting the mucosal barrier to diffusion. The mechanism by which bile acids can damage the gastric epithelium is not completely understood. Several studies have emphasized mucosal lipid solubilization by bile acids in the pathogenesis of mucosal injury. Bile acid entry into gastric mucosal cells may be a critical and early step in the genesis of mucosal injury, but this possibility has not yet been investigated. The present study was designed to explore the interaction of bile acids with dispersed gastric mucosal cells isolated from the rabbit and guinea pig stomach. Results showed that both glycocholic and deoxycholic acid rapidly associated with the gastric cells and reached a steady state concentration by 30 min. Glycocholic acid accumulated in the cells to a concentration approximately eight times greater than that in the surrounding medium. The amount of bile acid associated with the cells was greater at an acidic than at a neutral pH, and was a function of the concentration of both the cells and the bile acid. The process did not require cellular energy, was nonsaturable, and was not species specific. Experiments with 86Rb, a cytoplasmic marker, revealed that approximately one half of the cellular glycocholic acid was associated with the cytoplasmic compartment and the rest with the membranes. These findings are consistent with a combination of intracellular entrapment of the bile acids due to intracellular ionization and bile acid binding to cellular membrane components being the mechanisms by which bile acids accumulate in cells. Acid-driven bile acid accumulation may explain how relatively low luminal concentrations of bile acid can be damaging to the gastrointestinal mucosa.

Comparison of cimetidine with new H2-antagonists in rabbit and guniea pig gastric cells

We compared the effect of three relatively new H2-antagonists (compounds L-643,411, BL-6341A and SK&F 93479) to cimetidine in two preparations of mucosal cells isolated from rabbit and guinea pig stomachs. The indices for the histamine-stimulated acid secretory response were the changes in [14C]aminopyrine uptake in the rabbit and in cellular cyclic AMP, in the guinea pig. Both functions were mediated by the histamine H2-receptors and hence, can be used to examine antagonist-receptor interaction in vitro. In both rabbit and guinea pig, the new antagonists were highly potent competitive inhibitors of histamine on the H2-receptor, 30- to 200-fold more potent than cimetidine. The Ki values for cimetidine (500-800 nM) and L-643,411 (6-12 nM) were the same in the two animal species, but those for SK&F 93479 and BL-6431A were significantly lower in rabbit than in guinea pig cells. In inhibiting the changes in [14C]aminopyrine uptake in rabbit cells the Ki values for SK&F 93479 and BL-6341A were 2.4-3.5 nM whereas on cyclic AMP in guinea pig cells they were 10-fold higher (25-30 nM). These differences may reflect the structural requirements of the H2-receptors in that in rabbit these antagonists possess higher affinity for the H2-receptors than in guinea pig, or alternatively, uptake or metabolism of histamine by rabbit gastric cells may be responsible for these differences. Furthermore, these preparations appeared to be satisfactory for in vitro assay of gastric acid secretion to test for competitiveness of new H2-receptor antagonists.

Secretion of intrinsic factor from dispersed mucosal cells isolated from guinea pig and rabbit stomach

In dispersed mucosal cells prepared from rabbit and guinea pig stomach, the secretion of intrinsic factor was constant (0.3-0.4%/min) for at least 30 min incubation at 37 degrees C. Histamine or isobutyl methylxanthine increased cyclic AMP and intrinsic factor secretion in both cells preparations. Isobutyl methylxanthine potentiated and cimetidine competitively inhibited (ki = 5.10-7 M) both effects of histamine. Dibutyryl cyclic AMP (1.0 mM), also caused a 3-fold increase in intrinsic factor secretion. These results suggest that in rabbit and guinea pig histamine interacts with H2-receptors to increase cyclic AMP which mediates the rise in the rate of intrinsic factor secretion.

Distortion of H2-Antagonist Equilibrium Constants by Uptake in Rabbit Gastric Mucosal Cells

In rabbit gastric cells the new H2-antagonists, BL-6341A, SK&F 93479 and L-643,441 were highly potent inhibitors of the H2-receptor mediated action of histamine as monitored by 14C-aminopyrine uptake and cyclic AMP formation. BL-6341A and SK&F 93479 acted as competitive antagonists to histamine and dimaprit but they were less potent against dimaprit (Ki, 8.9 nM) than against histamine (Ki, 3.5-4.4 nM). Furthermore, the Schild slope for L-643,441 against histamine was significantly higher than unity (1.69-1.79), which is inconsistent with competitive antagonism, whereas against dimaprit it was close to unity. In contrast to these antagonists, cimetidine was an equally potent competitive antagonist of both histamine and dimaprit. 3H-histamine was taken up by gastric cells as evidenced by the loss of 60-70% of the cell-associated radioactivity upon hypotonic lysis. These results suggest that uptake and possibly metabolism of histamine by rabbit gastric cells is partially responsible for the distortion of the estimated equilibrium constants for these H2-antagonists.