Comparative investigations of the action of mimosine on nucleic acid synthesis in Ehrlich ascites-tumour cells

Abstract

Mimosine (leucenol; 3 [ 3 -hydroxy-rl-oxo1 (4H)-pyridinyllalanine } is a non-protein amino acid of plant origin (see Fowden et al., 1967). Several studies .have shown that mimosinecontaining plants and mimosine itself are toxic to animals (Matsumoto et al., 1951; Owen, 1958; Hegarty et al., 1964). The toxicity of mimosine has also been studied in cell culture (Tsai & Ling, 1971; Prabhakaran et al., 1973). Exposure of cells in culture for relatively long periods causes inhibition of DNA synthesis (Tsai & Ling, 1971, 1972; Hegarty et al., 1978). In a previous report we showed that mimosine had an inhibitory effect on RNA synthesis in Ehrlich ascites-tumour cells. The inhibition was concentration-dependent in the range of 0.05-1 mwmimosine. It was found also that the inhibition was much more pronounced when the cells were preincubated with mimosine before their capacity for RNA synthesis was determined (Itzhaki & Abdulla, 1982). The work was extended to study the action of mimosine in a wider range of concentration on the synthesis of both RNA and DNA. Ehrlich ascites-tumour cells were grown in mice (Itzhaki, 1972), harvested, washed with 0.9% NaCl and suspended in the incubation medium. RNA synthesis was measured as the rate of [5-3H]uridine incorporation into RNA of cells incubated at 37OC with the labelled precursor for 20min (Harwood & Itzhaki, 1973). In a similar set of determinations DNA synthesis was measured as the rate of incorporation of Irneth~l-~Hlthymidine into DNA. Comparison was made in all cases between cells which were incubated with mimosine for 1 h before the determination of RNA or DNA synthesis and those where the effect of mimosine was tested directly without a period of preincubation. The results show that for RNA synthesis, mimosine has a biphasic action in the preincubated series of experiments. A gradual increase in inhibition of RNA synthesis is observed when the concentration of mimosine is increased from 0 . 0 2 m ~ to about 0.5 mM, reaching a maximum inhibition of 75%. When the concentration of mimosine is raised from 1 to 6 m ~ , the inhibition of RNA synthesis gradually decreased, such that the rate of RNA synthesis returned to almost 80% of the control value. However, when RNA synthesis was measured directly in a 20min period of incubation without a period of preincubation, a slight inhibitory effect was observed at the higher range of mimosine concentrations. The results also confirm the earlier observations (Itzhaki & Abdulla, 1982) that a preincubation period is required to detect the action of mimosine. The effect of mimosine on DNA synthesis was quite different. Here, no noticeable effect was obtained with 0.02-0.2 m ~ mimosine. As the concentration was raised there was a gradual stimulation of DNA synthesis, with a broad peak around 3-4m~-mimosine, when the rate of DNA synthesis reached a value of 40-50% above that of control. Moreover, the pattern of thymidine incorporation into DNA is similar in both the preincubated and the non-preincubated series. The results indicate clearly that the requirement for a period of contact of the cells with mimosine for the latter to act on RNA synthesis may be related to its metabolism inside the cell during the duration of preincubation. The metabolism of mimosine in animal tissues has been studied, and among the products found to be formed are mimosinic acid and mimosinamine (Tang & Ling, 1977). It is possible that the action of mimosine on RNA synthesis observed here is due to a metabolite, or it may be brought about by a combination of effects of more than one metabolite of mimosine. In contrast. mimosine may itself exert the stimulatory action on DNA synthesis observed here; however, we have no indication as to its mechanism of action.