The Chemical Transformation of Atrazine in Corn Seedlings

PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY 58, 199–208 (1997) ARTICLE NO. PB972303

The Chemical Transformation of Atrazine in Corn Seedlings M. Raveton, P. Ravanel,* M. Kaouadji,† J. Bastide,‡ and M. Tissut ´ ´ ´ Laboratoire de Physiologie Cellulaire Vegetale and *Laboratoire de Pharmacognosie, Universite Joseph Fourier, ´ ´ BP 53, 38041 Grenoble cedex 09, France; †Departement de Chimie, Universite de Limoges, 123, avenue A. Thomas, 87060, Limoges cedex, France; and ‡Centre de Phytopharmacie, GERAP, ´ Universite de Perpignan, 52, avenue de Villeneuve, 66860, Perpignan cedex, France Received June 5, 1997; accepted November 14, 1997 One of the possible detoxication pathways for atrazine in corn corresponds to a chemical hydroxylation process. The aim of this work was to describe the mechanism of this reaction. Under in vitro experimental conditions, a benzoxazinones mixture (10 mM) extracted from corn plantlets was able to transform 91% of atrazine (6 mM) into 2-hydroxyatrazine during a 24-h period of incubation at 258C. This reaction was shown to be temperature-dependent; the half-life of atrazine was 67 h at 48C, 90 min at 258C, and only 30 min at 508C. However, at this temperature a rapid degradation of the active benzoxazinones occurred. The pH value of the incubation medium was shown to influence greatly the hydroxylation rate of atrazine (no hydroxylation process at pH 9, a relatively low rate at pH 7, and a maximum one at pH 5.5). The presence of an organic solvent (ethanol or acetone) in the reaction medium was responsible for a large decrease in hydroxylation activity. In a water medium, an optimal rate was obtained when the benzoxazinones concentration was close to 10 mM (average rate of hydroxylation: 2 to 3.1024 nmol h21 nmol21 benzoxazinones). For concentration values lower than 1 mM, the rates remained very low. In the presence of 10 mM benzoxazinones, the hydroxylation rate appeared not to be saturable for concentrations in atrazine varying between 6 and 100 mM. The comparison of the hydroxylation rates obtained with purified benzoxazinones (DIMBOA, DIBOA, 2-monoglucosyl DIMBOA 1 2-monoglucosyl DIBOA) suggested that the chemical reactivity of benzoxazinones toward atrazine involved the 4-N-OH of the heterocycle and not the 2-C-OH. This hypothesis was reinforced by the fact that the atrazine hydroxylating activity of DIMBOA or DIBOA remained unchanged even in the presence of AlCl3 (a chelator of the 2-OH group). The natural concentration of benzoxazinones in the vacuolar sap of corn seedlings ($10 mM) and the pH of this solution (close to 5.5) contribute to explain the high rate of atrazine chemical hydroxylation in vivo. q1997 Academic Press

INTRODUCTION

Atrazine is a well-known preemergence herbicide widely used in corn culture. Such a use depends on the fact that most of the weeds growing in this culture are killed by its active ingredient, which is ineffective on corn. In corn, atrazine is readily detoxified through three possible pathways: (a) 2-hydroxylation (1), (b) Ndealkylation (1, 2), and (c) conjugation with glutathione (3–5). Conjugation with glutathione was demonstrated to occur exclusively in the aerial parts of corn seedlings where an active glutathione-s-transferase (GST) was present (6). Shimabukuro et al. (7), studying several corn varieties, demonstrated that the intensity of tolerance to atrazine and GST activity were correlated. Therefore, the role of the 2-hydroxylation reaction seemed ambiguous and needed a

detailed reexamination. The aim of this work was to understand the mechanism of this reaction. MATERIALS AND METHODS

Plant material. Corn seeds (Zea mays L., c.v. Furio) were allowed to germinate with 5 ml water for 10 seeds in petri dishes at 258C in the dark. After a 4- to 10-day culture period, seedlings were collected and roots and shoots isolated. Isolation and characterization of benzoxazinones. The plant material was disrupted at room temperature in a Waring blender in the presence of pure acetone (25 ml for 13 g fresh plant material). The mixture was centrifuged (4000g, 10 min). The supernatant was collected and the pellet reextracted twice with 25 ml acetone/ water (4/1, v/v). The three collected supernatants

199 0048-3575/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.

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were grouped and extracted three times with 10 ml n-hexane to remove lipids. The remaining acetone/water solution containing the benzoxazinones pool was concentrated in vacuo at 308C and separated on silica TLC plates (60F254, Merck) with ethyl acetate/acetic acid/formic acid/water (20/1/1/2, v/v/v/v) as solvent. The benzoxazinones were visualized thanks to their blue–violet color in the presence of FeCl3 sprayed as a 2% solution in acetone. Extracted and purified DIMBOA (2,4-dihydroxy-7methoxy-1,4-benzoxazin-3-one) was identified by comparison with an authentic sample on TLC and by NMR (with the use of a Bruker AC 400). The different UV-absorbing compounds separated on TLC were recovered by scraping off the plates and were solubilized in pure ethanol for the most lipophilic compounds or in ethanol/ water (1/4, v/v) for the others. After centrifugation of the silica gel–solvent mixture, the solutions were dried under a N2 flow. The UV spectra of the different isolated compounds were established and quantitative measurements carried out following the method of Klun and Robinson (8). In order to obtain a great amount of benzoxazinones, corn seedlings (1 kg) were extracted under the same extraction procedure, giving a hydroacetonic solution after partition with nhexane. Acetone was evaporated and the residual water solution was extracted by ethyl acetate and n-butanol afterward. DIMBOA and DIBOA were present in the ethyl acetate extract and their monoglycosides were found in the n-butanol fraction. More hydrophilic glycosides remained in water. The benzoxazinones present in the ethyl acetate fraction were separated on a silica gel column (15 g of silica gel 60 for column chromatography, particle size 0.063–0.100 mm; Merck) successively eluted by tolueneacetic acid (500/1, v/v) and tolueneethyl acetateacetic acid in which the proportion of ethyl acetate was gradually increased. DIMBOA was eluted with tolueneethyl acetateacetic acid (100/100/1, v/v/ v). This compound (700 mg) was obtained in a crystallized form, was identified by TLC and NMR studies, and was then used for hydroxylation experiments.

b-Glucosidase reaction. The polar derivatives of benzoxazinones (i.e., glycosylated benzoxazinones) contained in the n-butanol or water fractions obtained from the main extract (1 kg corn seedlings) were purified by preparative TLC as described above. Then they were separately dissolved in 1 ml of a 0.1 M phosphate buffer (PO4H2K 1 PO4HNa2) at pH 5.5, leading to 5 mM concentrations of benzoxazinone glycosides. b-Glucosidase (EC 3.2.1.21 from Sigma), 5 mg ml21, was added and the mixture was incubated at 308C for 24 h. This aqueous mixture was then extracted twice with 1 ml ethyl acetate. After extraction, organic and water solutions were submitted to TLC analyses as described above. Standard conditions for atrazine hydroxylation in the presence of DIMBOA. Labeled atrazine (10 ml 5 15 nmol) from an ethanolic stock solution was added to 2.5 ml of a phosphate buffer solution at pH 5.5 (final atrazine concentration: 6 mM) in the presence of 10 mM DIMBOA. After different incubation periods (between 1 and 24 h), aliquots (30 ml) were deposited on silica gel plates and chromatographed in ethyl acetate/acetic acid/formic acid/ water (20/1/1/2, v/v/v/v). Under such conditions, the Rf values were 1 for atrazine and 0.27 for hydroxyatrazine. The quantitative distribution on TLC of the different labeled compounds was obtained with the use of a linear analyzer (Berthold Analyzer LB 213). This procedure of quantification was routinely used after a careful verification consisting of a separation of atrazine and hydroxyatrazine from the aqueous incubation medium by diethylether (extraction of atrazine, hydroxyatrazine remaining in water). The diethylether and water fractions were evaporated to dryness and the radioactivity of each fraction was measured by scintillation counting (Intertechnique Scintillator, Model SL 4000). Identical results were obtained under the two quantification conditions; the linear analyzer technique was easier to use. Chemicals. 2-chloro-4-ethylamino-6-isopropylamino-s-triazine (purity grade 99%) and 2hydroxyatrazine were purchased from Cluzeau

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(Sainte Foy La Grande, France). Atrazine ringUL-14C (radiochemical purity .98%, sp act 0.92 GBq mmol21) was obtained from Sigma. Atrazine was dissolved in ethanol and hydroxyatrazine in a water/acetic acid mixture (19/1, v/v). Following Castelfranco et al. (9), 2-hydroxyatrazine could be obtained from atrazine after a 5 min 2 N HCl treatment at 1008C. Under these conditions, atrazine was completely transformed, to give 2-hydroxyatrazine which was extracted from the water acidic solution by nbutanol. Labelled 2-hydroxyatrazine was obtained under this procedure and, after concentration, was solubilized in acetic acid/water (1/19, v/v). RESULTS

Existence of a Chemical Hydroxylation of Atrazine in the Presence of Benzoxazinones The transformations of organic compounds in living cells are generally biocatalyzed by enzymes. The hydroxylation of atrazine through a pure chemical process is a very particular mechanism of detoxication in plants, especially in corn. It was therefore necessary to verify with much care whether 2-hydroxylation of atrazine was indeed a chemical reaction occurring in corn only in the presence of benzoxazinones, as first established for simazine by Castelfranco et al. (9, 10). For this purpose, a first assay was carried out with 6 mM 14C-atrazine in water buffered at pH 5.5. The mixture was shaken for 24 h at 258C either in the presence of 10.5 mM of a benzoxazinones mixture (crude acetone extract from corn) or in the absence of benzoxazinones. In the presence of the benzoxazinones mixture, a high proportion (91%) of atrazine was transformed into 2-hydroxyatrazine, in marked contrast with the reference assay in which atrazine remained completely stable (Table 1). Influence of the Experimental Conditions on the Chemical Hydroxylation of Atrazine Temperature effects. The kinetic of hydroxylation of atrazine (6 mM) by benzoxazinones (10.5 mM) at pH 5.5 was performed in vitro at

TABLE 1 Percentage of Atrazine Hydroxylated at Different pH after a 24-h Period of Incubation in the Presence or Absence of Benzoxazinones Medium

pH

Hydroxyatrazine

Atrazine

Water Water 1 BZ Water Water 1 BZ Water Water 1 BZ

5.5

0 91 0 54 0 0

100 9 100 46 100 100

7 9

Note. Atrazine, 6 mM; benzoxazinones (BZ), 10.5 mM; temperature, 258C.

4, 25, and 508C. The half-life of atrazine was 67 h at 48C, 90 min at 258C, and 30 min at 508C as shown in Fig. 1. It is known that several benzoxazinones degrade readily when temperature increases (11–13). At 238C degradation in aqueous solution was negligible (14). The following experiments were performed at 258C, a temperature giving a relatively high hydroxylation rate for atrazine without significantly changing the concentration of benzoxazinones in the reaction mixture. Solvents and pH effects. Under the same experimental conditions, the reaction rates decreased when ethanol or acetone was added to the medium. After 73 h, only 9% of atrazine were transformed in the presence of pure ethanol or acetone (solution at pH 5.5); meanwhile, 100% of atrazine was transformed in aqueous solution. All the following reactions were therefore carried out in water. Under these conditions, three different pH were tested (5.5, 7, and 9). These pH were chosen as approximately representative of the pH of the vacuolar solution, the cytosol, and the phloem sap, respectively. At pH 9, no hydroxylation activity was shown. At pH 7, the rate was relatively low, it was maximum at pH 5.5 (Table 1). Influence of the ratio between atrazine and benzoxazinone concentrations. In a first attempt, atrazine concentration was fixed at 6 mM and the benzoxazinones mixture was added in a range of concentrations between 0.01 and 15.8 mM (solubility limit in water). The rate of

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FIG. 1. Rate of atrazine hydroxylation in vitro as a function of temperature. Atrazine: initial concentration, 6 mM; medium pH, 5.5; (n) 508C; (D) 258C; (●) 48C.

atrazine hydroxylation was very low when the benzoxazinones concentration was lower than 1 mM (Fig. 2). The results can be interpreted using first-order kinetics. For concentrations ranging between 1 and 10.5 mM, the half-life time varied between 19 and 3 h. For a chosen benzoxazinones concentration of 10.5 mM, the hydroxylation rates were measured as a function of atrazine concentration between 6 and 100 mM. The ratio between benzoxazinones and atrazine concentrations varied between 1750 and 100. Figure 3 shows that, in that concentration range, no saturation of the hydroxylation process occurred. The equation of the curve representing the initial hydroxylation rates for atrazine concentrations between 6 and 100 mM was y 5 0.434 x 2 0.212. Effect of the Different Types of Corn Benzoxazinones on the Hydroxylation Rate Three benzoxazinones aglycones were found and described in different varieties of corn:

DIMBOA, 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (15), DIBOA, 2,4-dihydroxy-1,4benzoxazin-3-one (16), and DIM2BOA, 2,4dihydroxy-7,8-dimethoxy-1,4-benzoxazin-3one (12). DIMBOA is the major compound widely represented in different varieties. The DIM2BOA is a minor component. These aglycones can be present as glycosylated derivatives (17) and lead to the formation of the free aglycones after enzymatic action (18). In the corn variety Furio studied here, five compounds were isolated on TLC with Rf values reaching 0.88, 0.81, 0.17 (two products), and 0, respectively. DIMBOA was analyzed by NMR, giving the following results: 1H-NMR (DMSd6) ­: 3.72 (3H, s, CH3O); 5.65 (1H, d, J 5 6.4 Hz, C2-H); 6.65 (1H, dd, J 5 2.4 Hz, J 5 8.8 Hz, C6-H); 6.67 (1H, d, J 5 2.4 Hz, C8-H); 7.15 (1H, d, J 5 8.8 Hz, C5-H); 8.14 (1H, d, J 5 6.4 Hz, OH); 10.83 (1H, s, N-OH). 13C-NMR (DMSd6) ­: 55.68 (OCH3), 92.63 (C2), 103.62 (C8), 107.8 (C 6 ), 113.9 (C 5 ), 122.65 (C 4a ), 14.87 (C 8a ), 156.27 (C3), 157.07 (C7). The UV spectra and

CHEMICAL TRANSFORMATION OF ATRAZINE IN CORN

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FIG. 2. Kinetics of hydroxyatrazine formation in the presence of benzoxazinones at different concentrations. (▫) 15.8 mM; (n) 10.5 mM; (C) 5.3 mM; (●) 1.0 mM; (D) 0.1 mM; and (m) 0.01 mM. Atrazine: initial concentration, 6 mM.

FIG. 3. Initial rate of hydroxyatrazine formation as a function of atrazine concentration. Benzoxazinones concentration, 10.5 mM.

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chemical reactivity of the isolated compounds were typical of the benzoxazinone family. DIMBOA (l EtOH max , 218, 263, and 288 nm) and DIBOA (lEtOHmax, 200, 263, and 291 nm). Rf values and spectra were compared with those obtained with authentic samples. They corresponded, respectively, to the Rf 0.88 and 0.81 (Fig. 4a). After incubation with a b-glucosidase mixture, the spot at Rf 0.17 (lEtOHmax, 204, 224, and 265 nm) gave DIMBOA and DIBOA. The spot at Rf 0 (lEtOHmax 213, 266, and 320 nm) was hydrolyzed into DIMBOA after the same b-glucosidase action. The seedlings of corn (c.v. Furio) therefore contained DIMBOA, DIBOA, a monoglucoside of DIMBOA and a monoglucoside of DIBOA (which were likely to be the 2glucosyl derivatives of these aglycones), and a

more polar glycosylated derivative of DIMBOA. DIMBOA, DIBOA, 2-monoglucosyl-DIMBOA 1 2-monoglucosyl-DIBOA, and di(or tri-)-glycosylated DIMBOA extracted from corn were separated on TLC. The effect on the hydroxylation rate of atrazine was tested under usual conditions for each of these compounds. Figure 5 and Table 2 show that aglycones and monoglucosides were approximately as effective as pure DIMBOA. The di(or tri-)-glycoside derivative was much less effective. The high efficiency of the 2-monoglucosyl derivative allows us to suggest that the chemical reactivity of benzoxazinones toward atrazine involved the 4-N-OH of the nucleus and not the 2-C-OH. As shown by ¨ Virtanen and Wahlroos (19) and Petho (20, 21), DIMBOA and DIBOA are transformed through

FIG. 4. Spectral analysis of different corn benzoxazinones used: (a) UV spectra before and after chelation with AlCl3; (b) Chelation hypothesis.

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FIG. 5. Effects of different purified benzoxazinones on the hydroxylation rate of atrazine. (▫) DIMBOA; (n) DIBOA; (●) Glc-DIMBOA 1 Glc-DIBOA; (C) Glc2-DIMBOA.

heating into BOA (1,3-benzoxazol-2-one) and MBOA (6-methoxy-1,3-benzoxazol-2-one), respectively. The UV spectra of these derivatives differ clearly from those of the parent compounds (14). These derivatives were unable to induce atrazine hydroxylation. In contrast, the 2-monoglucosyl derivatives remained resistant to heat degradation, even for 15 min at 1008C. Their UV spectra remained poorly changed and their hydroxylative activity remained significant. When AlCl3 was added to a solution of DIMBOA or DIBOA, bathochromic shifts (20 nm for DIMBOA and 18 nm for DIBOA) were

spectrophotometrically shown (Fig. 4a) resulting from a chelation process. The chelation of benzoxazinones with metals has been extensively studied (22–24). The spectral changes with Al 3+ were typical of a stable binding between the oxygen of the oxo group and an hydroxyl in the a-position on the nucleus. The spectra of the 2-monoglucosyl derivatives remained unaffected by aluminum, indicating the absence of chelation with Al3+. It is therefore suggested that the chelation of Al 3+ occurs between the 2-hydroxyl and the adjacent cetonic group (as shown in Fig. 4b). As the atrazine

TABLE 2 Rates of Hydroxyatrazine Formation in the Presence of Various Benzoxazinones Extracted from Corn Seedlings DIMBOA Initial rate of atrazine hydroxylation (until 30 min) Average rate of atrazine hydroxylation during the first 8 h of experiment

5.6 10

24

2.8 6 1.5 1024

DIBOA 7.2 10

24

3.4 6 2.1 1024

Glc-DIMBOA 1 Glc-DIBOA 3.2 10

24

2.1 6 0.9 1024

Glc2-DIMBOA 0 0.08 6 0.05 1024

Note. Results are expressed in nmol hydroxyatrazine formed h21 nmol21 benzoxazinones (6 s: 6 replicates).

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hydroxylating activity of DIMBOA or DIBOA remained unchanged in the presence of AlCl3, the reactive group involved in the hydroxylation process was probably the 4-N-OH (the average rate of hydroxylation between 0 and 8 h of the experiment was 2.7 6 1.2 1024 nmol hydroxyatrazine h21 nmol21 benzoxazinones (6 s with five replicates). In the presence of AlCl3 this rate of hydroxylation remained unchanged: DIMBOA 1 AlCl3 5 2.2 6 1.5 1024, GlcDIMBOA 1 Glc-DIBOA 1 AlCl3 5 2.7 6 1.3 1024, 6 s: five replicates). DISCUSSION

The chemistry of biologically active benzoxazinones has been recently reviewed (25), showing that the chemical hydroxylation of atrazine by benzoxazinones is not yet clearly established. Our results definitively discard the possibility of an enzyme-catalyzed hydroxylation with a relatively lipophilic enzyme which might have partly remained in extracts. Our results show that this hydroxylation process is a first-order reaction, excluding, therefore, the hypothesis of a catalytic role of DIMBOA as previously suggested (26). Figure 6 summarizes

our hypothetical pathway leading to atrazine hydroxylation in the presence of benzoxazinones. This hypothetic scheme remains to be carefully demonstrated, particularly concerning the presence of a chlorinated derivative of DIMBOA. Our first attempts in this way were not successful. Is the reaction between atrazine and benzoxazinones acting as well in vivo as in vitro? In vitro, a high concentration of benzoxazinones was required for the reaction to occur. An optimal rate was obtained for an average concentration of 10 mM. In the studied seedlings, the benzoxazinones content was measured and reached a concentration of 14 6 7 mmol g21 fresh weight in the roots and 17 6 6 mmol g21 fresh weight in the aerial green parts. These results are in good agreement with those of Argandona and Corcuera (27) and indicate that the average concentration of the benzoxazinones mixture in corn plantlets is close to 10–20 mM. So, the in vitro experimental ratio atrazine/ benzoxazinones used in this work seems close to the ratio obtained in vivo in treated seedlings. In fact, after freezing the corn material in liquid nitrogen and reequilibrating it at 148C, the aqueous fraction obtained after centrifugation (and

FIG. 6. Atrazine hydroxylation: hypothetical chemical mechanism.

CHEMICAL TRANSFORMATION OF ATRAZINE IN CORN

representing mostly the vacuolar solution) contained close to 80% of the benzoxazinone pool, with the aglycones as well as the glycosides being present in this fraction. This suggests that benzoxazinones were accumulated in the vacuoles, as is the case for numerous compounds representative of secondary metabolisms (28). Inside the vacuole of corn cells, the conditions allowing the chemical atrazine hydroxylation seem to be fulfilled: the concentration of benzoxazinones is appropriate and the slightly acidic pH of the vacuolar sap is a favorable factor. Atrazine metabolization in the seedlings of corn of the studied variety (70 to 80% of hydroxyderivatives in roots and aerial parts) seems to depend greatly, under field conditions and after a preemergence treatment, on the root chemical hydroxylation (29). The conjugation with glutathione only occurs in the aerial parts. Moreover, it has to be underlined that hydroxyatrazine is not a substrate for glutathione conjugation. Therefore, under such conditions, only small amounts of atrazine reach leaves, leading to small amounts of glutathione conjugates. After a post-emergence treatment, the relative importance of glutathione conjugates would probably be much greater. However, the common agronomic conditions for corn culture in France show that atrazine preemergence treatment is mostly used and, at the legally authorized amount of 1.5 kg a.i./ha, most of atrazine is absorbed by corn roots at the seedling stage, as shown by Tasli et al. (30). Afterward, the concentrations in soil water are very low, and the formation of conjugates in the leaves of well-developed plants cannot therefore represent an intense process. ACKNOWLEDGMENTS The authors thank Professor Niemeyer (Universidad de Chile, Santiago, Chile) for the generous gift of an authentic sample of DIMBOA.

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