Date of Award:

5-1974

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Animal, Dairy, and Veterinary Sciences

Department name when degree awarded

Interdepartmental Curriculum in Toxicology

Committee Chair(s)

D. K. Salunkhe

Committee

D. K. Salunkhe

Committee

W. A. Brindley

Committee

W. F. Campbell

Committee

R. K. Olsen

Committee

J. L. Shupe

Abstract

Germination of mung bean, Phaseolus mungo L. , and wheat, Triticum aestivum L., seeds was used for bioassay to demonstrate the toxic effects of selected pesticides--menazon, disulfoton, and GS-14254-- and allergens-- alantolactone and usnic acid. The ability of gibberellic acid to counteract the toxic effects of these chemicals on germination and seedling growth was studied. Chemical composition of the treated and untreated seeds was made with special attention to starch and protein degradation. Effect of these toxicants on the synthesis of amylase, ATPase, and protease enzymes during germination was studied since these enzymes are synthesized de novo during germination. To ascertain their effect on protein synthesis in storage tissue of the germinating seeds, uptake and incorporation of 14C-L-leucine into protein was studied in potato tuber slices and germinating mung beans.

Correlation of biochemical data and histochemical changes in the treated and untreated seeds of mung bean was obtained with menazon and usnic acid. Furthermore, ultrastructural changes were studied in order to relate functional and structural changes in the seeds .in conjunction with phytotoxic actions of these chemicals.

Among the insecticides, menazon (250 ppm) was found to be more toxic to both species than was disulfoton. GS-14254 (100 ppm) also was equally inhibitory to seed germination and seedling growth of mung bean and wheat seeds. When a solution of the herbicide GS-14254 (100 ppm) was added to either of the insecticides at their maximum concentrations the inhibitory effect of the combined pesticides on seed germination and seedling growth was more pronounced, especially with wheat.

Usnic acid (50 to 250 mg/1) and alantolactone (100 mg/1) significantly inhibited germination and root and shoot growth in both mung bean and wheat seeds. These two compounds appeared to be more phytotoxic than the pesticides.

Gibberellic acid partially counteracted the inhibitory effects of the pesticides and allergens, thus these chemicals showed no antiauxin activity.

Before any growth is observed there is a marked increase in respiration during germination that releases energy from food materials already present in usable form in the cells. At their maximum concentrations, menazon, disulfoton, GS-14254, alantolactone, and usnic acid significantly blocked the respiration of the germinating seeds at the end of 72 h after treatment. In all cases except alantolactone respiration of wheat seeds was considerably more affected than that of the mung beans.

Compared to control seeds, pesticide chemicals as well as allergenic compounds caused significant reduction in the amounts of soluble reducing sugars and free amino acids after 72 h germination period. Similarly, starch degradation was less in the treated seeds. Among the species of seeds, considerably less amounts of reducing sugars and amino acids were formed in the pesticide-treated wheat seeds than in the mung beans as compared to their respective controls. Such differences in the inhibitory effects were not observed in seeds treated with allergenic compounds.

The development of amylase and ATPase activity in the seeds treated with maximum concentrations of pesticides tended to be lower than that in the control seeds. In case of menazon, inhibition of amylase activity was more pronounced than that of disulfoton or GS-14254. Proteoiytic activity in control and disulfoton- and menazon-treated seeds was not significantly different during germination period, but in case of GS-14254, it was considerably lower.

Usnic acid at highest concentration tested completely inhibited the development of amylase activity in mung beans whereas it was significantly lower in seeds treated with the maximum concentration of alantolactone. The inhibition of amylase activity in wheat seeds treated with these compounds was more or less similar. ATPase inhibition in seeds treated with usnic acid was more severe than that in alantolactone treated seeds. However, proteolytic activity in control and treated seeds showed almost the same trend during the germination period.

The activity per se of amylase isolated from mung bean and wheat seeds germinated for 3 days was not significantly inhibited by the presence of the pesticides or allergens in the reaction mixture indicating that these chemicals do not inhibit already synthesized amylase enzyme.

Observations with potato tissue and germinating mung beans indicated that both total uptake and incorporation of 14c-L-leucine into protein were significantly inhibited by menazon, disulfoton, GS-14254, and alantolactone. On the other hand, the uptake in germinating mung bean treated with usnic acid was not affected although both uptake and incorporation were inhibited in potato tissue.

Menazon and usnic acid were then selected as the representative chemicals for pesticides and allergens, respectively, and their toxic effects were studied histochemically in 3-day germinating mung beans. It was observed that total nucleic acid content and RNA content in seeds treated with these chemicals were considerably less than that in the control seeds. Similarly, treated seeds showed more starch grains and protein bodies indicating less metabolic activity in these seeds.

At the ultrastructural level, menazon- or usnic acid-treated mung bean cotyledons at day 3 of germination contained no vacuoles but many undigested protein bodies were observed. In contrast, fully developed mitochondria, endoplasmic reticulum with ribosomes, and vacuoles were seen in control cells indicating protein (enzyme) synthesis and digestion of the food reserves.

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