Glutathione Conjugation

Abstract

Glutathione conjugation (GS-atrazine) of the herbicide, 2-chloro-4-ethylami no-6-isopropylamino-s-triazine (atra-zine) is another major detoxication mechanism in leaf tissue of corn (Zea mays, L.). The identification of GS-atrazine is the first example of glutathione conjugation as a biotrans-formation mechanism of a pesticide in plants. Recovery of atrazine-inhibited photosynthesis was accompanied by a rapid conversion of atrazine to GS-atrazine when the herbicide was introduced directly into leaf tissue. N-De-alkylation pathway is relatively inactive in both roots and shoots. The nonenzymatic detoxication of atrazine to hydroxyatrazine is negligible in leaf tissue. The hydroxyla-tion pathway contributed significantly to the total detoxi-cation of atrazine only when the herbicide was introduced into the plant through the roots. The metabolism of atra-zine to GS-atrazine may be the primary factor in the resistance of corn to atrazine. The selective herbicides 2-chloro-4-ethylamino-6-isopropyl-amino-s-triazine (atrazine) and 2-chloro-4,6-bis(ethylamino)-s-triazine (simazine) are used extensively to control annual weeds in fields of corn and sorghum. These compounds are effective inhibitors of the Hill reaction in photosynthesis (7) and also reduce the rate of "4CO2 fixation in plants (1, 16). The rate of atrazine metabolism in higher plants is an important factor in herbicidal selectivity (9). Detoxication of atrazine was reported to occur by the 2-hydroxylation and N-dealkylation pathways in higher plants (9, 10). In sorghum a rapid conversion of atrazine to a water-soluble compound (metabolite B) resulted in a recovery of atrazine-inhibited photosynthesis (14). The subsequent identification of metabolite B as a mixture of two closely related compounds, GS-atrazinel and y-glutamyl-S-(4-ethylamino-6-isopropylamino-2-s-triazino)cysteine, indicated the presence of a third detoxication pathway present in higher plants (6). In this paper, the mixture will be referred to as GS-' Abbreviations: GS-atrazine: S-(4-ethylamino-6-isopropylamino-2-s-triazino)glutathione; hydroxyatrazine: 2-hydroxy-4-ethylamino-6-isopropylamino-s-triazine; hydroxysimazine: 2-hydroxy-4,6-bis(ethyl-amino-s-triazine; compound I: 2-chloro4-amino-6-isopropylamino-s-triazine; compound II: 2-chloro4-amino-6-ethylamino-s-triazine; hydroxycompound I: 2-hydroxy4-amino-6-isopropylamino-s-triazine; hydroxycompound It: 2-hydroxy-4-amino-6-ethylamino-s-triazine; benzoxazinone: 2,4-dihydroxy-3-keto-7-methoxy-1,4-benzoxazine; TLC: thin layer chromatography. atrazine only. A possible precursor-product relationship between the two metabolites was discussed by Lamoureux et al. (6). This investigation was undertaken to determine the significance of the glutathione-atrazine conjugation pathway in corn. Corn is reported to be resistant to atrazine and simazine largely because of its ability to convert the two herbicides to nonphytotoxic hydroxyatrazine and hydroxysimazine (2-4, 8). The hydroxyla-tion reaction is catalyzed nonenzymatically by the cyclic hy-droxamate, benzoxazinone, present in corn tissue (2-4, 8). However, an unknown metabolite subsequently identified as GS-atrazine (6) was also detected in the shoots of intact corn plants treated with atrazine through their roots (9). MATERIALS AND METHODS Plant Material. Corn seeds (Zea mays L. North Dakota KE 47101) were germinated in vermiculite. A group of seedlings were transferred to continuously aerated, half-strength Ho-agland's solution after 6 days of germination. Other seedlings were left in vermiculite and intermittently watered with Hoagland's solution. All plants were grown in the greenhouse. Atrazine Metabolism in Plants. Uniformly ring-labeled atra-zine-'4C (specific radioactivity 7.8 Ac/mg) was purified as previously described (11) and used for treatment of corn. Plants were selected and treated with atrazine in a controlled environment room under conditions described previously (10). Plants grown in nutrient solution were used for root treatment with atrazine-'4C. Plants grown in vermiculite were used to study atrazine-14C metabolism in excised leaves and leaf discs. The fourth leaf (30-40 cm) of 24-day-old corn plants was excised under water. Two leaves each were treated by immersing their cut ends into a test tube containing 3 ml of atrazine-14C solution (301,180 dpm). Distilled water was added intermittently to compensate for water loss due to uptake and transpiration. The metabolism of surface-absorbed atrazine-'4C was determined by leaf surface application of atrazine-14C in 10%c oil in water (v/v) (Sun Superior oil 11 N containing 1 %c [v/v] Triton X-207) as described previously (14). Assay for Atrazine-'4C and Its Metabolites. Atrazine-'4C-treated tissues were extracted with 80%7, methanol. The methanol in the extract was removed under vacuum to give an aqueous solution. Further purification of the aqueous extract and qualitative and quantitative assay of atrazine-14C and its radioactive metabolites were performed as described previously (9, 11, 12). The 'IC activity in chloroform-and water-soluble compounds was determined by liquid scintillation counting (12). Water-soluble radioactive metabolites of atrazine-14C were purified by cation exchange chromatography and separated by TLC for detection and identification as reported (9). Radioactive compounds were determined quantitatively from TLC plates by carefully removing the compounds from the plate and counting by gel scintillation techniques (11).