RESUMO
Two sublines of L5178Y (LY) murine lymphoma, differing in sensitivity to hydrogen peroxide, served as a cellular model for examination of the antioxidant defense system. The contribution of catalase, glutathione peroxidase (G-Px) and glutathione were evaluated. Sensitivity to 3-amino-1,2,4-triazole (AMT), inhibitor of catalase, was higher in LY-R (hydrogen peroxide sensitive) than in LY-S (hydrogen peroxide resistant) cells. Accordingly, activity of catalase was twofold lower in LY-R than in LY-S cells. G-Px activity was about two times higher in LY-R than in LY-S cells. After induction with selenium it increased 15.6 times in LY-R cells and 50.3 times in LY-S cells. Reduced glutathione (GSH) content (and possibly other monobromobimane-reactive thiols) were determined fluorimetrically with monobromobimane and fluorescence found 54% higher in LY-S than in LY-R cells. Inhibition of catalase caused GSH decrease in LY-S cells; this decrease was abrogated by inducing G-Px by selenium treatment. On the contrary, in LY-R cells inhibition of catalase decreased GSH content only slightly and selenium treatment did not further change the GSH level. DNA damage (estimated by "comet" assay) was the same in hydrogen peroxide-treated cells in the presence or absence of AMT; however, after induction of G-Px by selenium, DNA damage was considerably lowered. This sparing effect of selenium was accompanied by decreased growth inhibition in selenium pretreated, hydrogen peroxide-treated cell cultures.
Assuntos
Antioxidantes/metabolismo , Peróxido de Hidrogênio/farmacologia , Leucemia L5178/metabolismo , Amitrol (Herbicida)/farmacologia , Animais , Catalase/antagonistas & inibidores , Catalase/metabolismo , Dano ao DNA , Resistência a Medicamentos , Indução Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Radicais Livres/metabolismo , Glutationa/metabolismo , Glutationa Peroxidase/biossíntese , Glutationa Peroxidase/metabolismo , Camundongos , Selênio/farmacologia , Células Tumorais CultivadasRESUMO
A new single-step Extrelut column procedure for obtaining the 25-hydroxyvitamin D extract, suitable for a direct radiocompetitive test, is described. Separation of 0.1 ml of serum, on a small Extrelut column and radioassay allows a rapid, sensitive and accurate determination of 25-hydroxyvitamin D concentration. The detection limit of the determination was 2 nmol/l (0.8 microgram/l) of serum. The recovery of 250HD3 added to serum was 99-108%. Intra-assay and interassay CV values were 10 and 14%, respectively. Although the method does not permit separation of dihydroxylated metabolites of vitamin D from 25-hydroxyvitamin D, it is useful in clinical practice.
