Modification of mitogen-induced proliferation of murine splenic lymphocytes by in vitro tocopherol.

The effect of alpha-tocopherol on in vitro proliferation of murine splenic lymphocyte cultures supplemented with various concentrations of the vitamin has been measured at sub-optimal, optimal and supra-optimal levels of the T-cell mitogen Concanavalin A (Con A). In the concentration range (1-25 micrograms/ml), tocopherol enhanced proliferation when administered up to 24 hours after exposure to sub-optimal and optimal concentrations of Con A; however, at supra-optimal levels of the mitogen, it appeared to inhibit proliferation. In the concentration range 50-100 micrograms/ml, tocopherol supplementation only enhanced proliferation in response to sub-optimal concentration of Con A. The spontaneous proliferation of lymphocytes in the absence of mitogens was increased by tocopherol supplementation at all concentrations tested. In contrast, there appeared to be only slight stimulation of B-cell proliferation in response to optimal concentration of bacterial lipopolysaccharide (LPS) by lower levels of vitamin E. Tocopherol supplementation of cultures over a broad range of concentrations (0.5-100 micrograms/ml) had no significant effect on cell viability before onset of proliferation at 18 hours after exposure to Con A, nor was there evidence of earlier onset of DNA synthesis in response to mitogen in the presence of 5 micrograms/ml of the vitamin. Although macrophage depletion of cultures impaired proliferation induced by Con A, tocopherol supplementation continued to stimulate proliferation at optimal and sub-optimal levels of mitogen.

Humoral immune response of mice injected with tocopherol after exposure to X-radiation.

Serum haemagglutination (HA) titers have been determined for irradiated and non-irradiated mice responding to injection of two different concentrations of sheep red blood cells (SRBC) 24 to 48 hours after irradiation and immediate intraperitoneal injection of 2.5 mg DL alpha-tocopherol, the emulsifying vehicle, or saline. Mice maintained on tocopherol-deficient diets for 8 weeks post-weaning and those on regular diets exhibited increased IgG titers during peak response when injected with vitamin E. This partially alleviated the radiation-depression of the primary immune response induced by the smaller SRBC injection. This stimulatory effect was most significant in mice maintained on vitamin E-deficient diets. The HA titers of irradiated and non-irradiated mice maintained on normal rations were determined following a 10-fold increase in the SRBC inoculation. Antibody titer was greater following injection of the higher concentration of SRBC but post-irradiation injection of tocopherol immediately or 24 hours after irradiation did not enhance immune response. At the higher SRBC concentration maximum observed HA titers decreased with increasing dose of radiation; however, tocopherol had no significant dose-reducing effect. Tocopherol toxicity as manifested by depressed HA titers was observed occasionally in non-irradiated mice challenged with the higher concentration of SRBC.

Modification of survival and hematopoiesis in mice by tocopherol injection following irradiation.

The LD50/30 of CD-1 female mice increased from 6.6 Gy to 7.0 Gy when 2.5 mg of dl-alpha-tocopherol was injected immediately post irradiation. Increased survival was associated with increased numbers of hematopoietic colony forming units (CFU). Endogenous spleen colonies were found in greater numbers in the tocopherol-treated mice after irradiation. The vitamin, however, must be injected within five hours following irradiation to have this effect. The increased numbers of CFU in tocopherol-treated mice may be due to a stimulation of recovery or repair processes. Split-dose studies suggest that most repair of sublethal damage in hematopoietic stem cells take place within seven and nine hours following irradiation. Tocopherol injection appears to enhance the recovery manifested in the split-dose assay. There is also evidence that tocopherol-treatment caused an earlier onset of mitotic activity in CFU after irradiation. The increased number of spleen colonies in tocopherol-injected mice is not due to an altered CFU seeding efficiency associated with an altered spleen microenvironment. Tocopherol injection did not affect the shoulder of the stem cell survival curve using exogenous spleen colony assays of bone marrow-derived or spleen-derived hematopoietic stem cells. There appears to be a decrease in Do in the higher dose region (4.3 Gy) of the bone marrow exogenous SCA survival curves for the vehicle-injected and the non-injected groups; however, the tocopherol-injected group showed no evidence of change in radiosensitivity up to the highest dose used (5.0 Gy). Data may be interpreted to suggest that the therapeutic effect of tocopherol may involve repair of hematopoietic stem cell damage in the higher dose range of bone marrow syndrome.

Increased haematopoietic stem cell survival in mice injected with tocopherol after X-irradiation.

Tocopherol injection (2.5 mg) immediately after irradiation reduced lethality only during bone-marrow syndrome. Endogenous spleen colony count at 8 days after X-radiation were significantly greater in vitamin-E-injected mice compared to noninjected or vehicle-injected animals; however, 59Fe incorporation into spleen and bone marrow did not suggested enhanced erythropoietic activity in vitamin-E-injected groups at 2,4,8 and 10 days following irradiation. Mitotic index and frequency of micronuclei in marrow at 24 hours post irradiation (3 Gy) were unaffected by tocopherol injection. The uptake of tritium from injected 3H-tocopherol suggests that tocopherol has been accumulated in spleens but not marrows of irradiated animals within a few hours. Also tocopherol has no effect on endogenous spleen colony counts if injected after 5 hours nor is there an effect on the seeding efficiency of exogenous bone-marrow cells injected into recipients receiving tocopherol after irradiation.

Effect of vitamin E on post irradiation death in mice.

The 30-day survival after exposure to 800 Rad 60Co gamma radiation has been compared for female mice maintained on vitamin E deficient, vitamin E supplemented or regular lab rations before and/or after irradiation. Pre- or post-irradiation dietary supplementation had no effect on survival; however, injection of alpha-tocopherol immediately after irradiation significantly reduced radiation lethality.

Erythropoiesis and plasma tocopherol levels in irradiated mice.

Plasma protein and tocopherol concentrations, haematocrit and 59-iron incorporation into erythrocytes have been measured in vitamin E-deficient and supplemented mice before and after exposure to 500 R of 260 kVp X-ray. Supplemented animals had greater haematocrit, plasma tocopherol and protein levels initially. After irradiation plasma tocopherol concentration decreased drastically in the vitamin E-supplemented mice.