PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY 16, 136- 140 (1981)

Metabolites of Carbofuran: Effect on Indole-3-acetic Acid Degradation

CESAR FRANCO AND NELSON DURAN

llnstituto de Quimica, Universidade Estadual de Campinas. C.P. 1170, CEP 13100, Campinas, Site Paula. Brazil

Received April 6, 1981: accepted June 17, 1981

Oxygen uptake, product formation, and photon emission in the indole-3-acetic acid/ peroxidase/O, system were inhibited by carbofuran and its metabolites, the 3-ketocarbofuran phenol and carbofuran phenol. These metabolites acted as competitive inhibitors and concomi- tantly were degraded. Photon emission, together with uv spectrophotometry and oxygen mea- surements, shows this to be a rapid and reproducible method to study insecticide interaction with the indole-3-acetic oxidase svstem in \jitro. 3-Ketocarbofuran phenol was metabolized by peroxidase with excited-state production.

Carbofuran insecticides have been found to be degradated rapidly into various me- tabolites in plants, insects. and mammals (l-5); therefore attention was turned to its metabolites. The effects on plant growth of the interaction, between metabolites of carbofuran and indole-3-acetic acid (IAA)’

were confirmed (3, 6, 7). We want to report the interaction of car-

bofuran metabolites (Fig. 1) with the IAAi HRP/O, system (8- 10).

MATERIALS AND METHODS

HRP (Type VI) was obtained from Sigma Chemical Company. IAA was from Merck. Carbofuran phenol and 3-ketocarbofuran phenol were from the U.S. Environmental Protection Agency. Environmental Tox- icology Division, Research Triangle Park.

Absorption spectra were taken on a Zeiss DMR-21 recording spectrophotometer using l-cm cells. The chemiluminescence was measured in a Beckman LS 100~ liquid scintillation counter or in a Hamamatsu TV Photocounter C-767 (11). Oxygen uptake was determined with a Yellow Springs In- struments Model 53 Oxygen Monitor. The

’ Abbreviations used: IAA, indole-3-acetic acid; HRP, horseradish peroxidase; DPAS, 9.10-diphe- nylanthracene-2-sulfonate: DBAS 9,10-dibromo- anthracene-2-sulfonate.

conductivity measurement was carried out in a Beckman RC16 B2 conductivity ap- paratus.

HRP-I was prepared by adding to a sam- ple of HRP exhaustively dialyzed in pen- tadistilled water (resistivity of 1.22 x lo5 R), hydrogen peroxide in a 1.1 to 1.0 pro- portion (12). HRP-II was prepared from HRP-I by the method of Hewson and Dun- ford (13).

RESULTS AND DISCUSSION

Oxygen uptake. Carbofuran phenol de- lays oxygen uptake by the IAAiHRPi O,/Mn*+ system (Fig. 2). 3-Ketocarbofuran phenol inhibited the oxygen consumption at concentrations above 13 pM (Fig. 3). With increasing 3-ketocarbofuran phenol at con- centrations higher than 40 pM an increase of oxygen uptake was observed (results not shown), possibly due to substrate oxida- tion. The 3-ketocarbofuran phenol oxida-

136 0048-3575/81/050136-05$02.00/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction m any form reserved.

METABOLITES OF CABBOFURAN 137

2 4 6 8 MINUTES

FIG. 2. Inhibition of oxygen uptake in the IAAI HRPIMt?+IO, system by different concentrations oj carbofuran phenol: (Cl) 0.0; (m) I PM; (0) 2 PM; (0) 5 PM. Standard conditions: IAA (0.2 mM). HRP (3.0 nM), Mn2+ (0.1 mM), and p-chlorophenol (0.1 mM) in phosphate buffer (25 mM) at pH 5.9 at 25°C.

presence of carbofuran phenol, the enzyme activity was recovered after a lag period with the same rate of product formation as control; the lag time was dependent on the inhibitor concentration. In the case of 3- ketocarbofuran phenol the enzyme activity was not recovered.

Excited-state generation. It is known that the IAA is generated in its electronically excited state by the IAA/HRP/O, system (9, 14, 15). In this system at pH 5.9 and above, correlation between oxygen uptake, prod- uct formation, and photon emission was observed. This means that the IAA degra- dation produces electronically excited state IAI, presumably through a dioxetane in- termediate (16, 17):

IAA

tion catalyzed by peroxidase was confirmed by means of oxygen uptake; photon emis- sion accompanies this oxidation.

Product formation. Product formation from IAA was followed at 262 nm in the presence of carbofuran metabolites; results were the same as observed by Lee and Chapman (4). Carbofuran phenol was more inhibitory to enzymatic degradation of IAA than was 3-ketocarbofuran phenol. In the

60 c .

1 2 4 6 8

MINUTES

FIG. 3. Inhibition of oxygen aptake in the IAAI HRPlMn’+IO, system by different concentrations of 3-ketocarbofuran phenol: (0) 0.0; (W) 13 PM: (0) 26 PM; (a) 40 PM; under standard reaction conditions.

Figures 4 and 5 show the inhibitory effect of carbofuran phenol on the rate of emis- sion. The presence of an excited species other than IA1 at 200 @4 3-ketocarbofuran phenol is evident (Fig. 6). In fact the 3- ketocarbofuran phenol/HRP/O, system was analyzed and a strong emission was ob- served initially during the reaction.

MINUTES

FIG. 4. Inhibition of photon emission of the IAAI HRPIMn”l0, system by different concentrations of carbofaran phenol under standard reactions condi- tions: (0) 0.0: (m) I PM; (0) 2 @I: (0) 5 PM.

138 FRANC0 AND DURAN

FIG. 5. Changes in lag period of IAA degradation by delayed addition of carbofuran phenol (2 pM) in the IAAIHRPIMnZ+I02 system under standard reaction conditions: (0) control. Carbofuran phenol added after: (W) 60 set; (0) 30 set; and (0) 10 sec.

Table 1 shows the enhanced emission when 3-ketocarbofuran phenol was de- graded by peroxidase in the presence of DPAS and DBAS which are singlet and trip- let excited-state counters, respectively (11).

Peroxidase alferations. HRP-I or HRP-II, the intermediate forms of HRP, were trans- formed almost instantaneously to native peroxidase. in the presence of carbofuran phenol and 3-ketocarbofuran phenol at 10 @f concentration. In contrast, the thiocarbamate herbicides transformed HRP-I into HRP-II (8) and had no effect upon the latter.

The formation of HRP-II was followed at 420 nm and that of native peroxidase at 400 nm; the results agreed with those of Lee (6).

Our results on oxygen consumption and photon emission together with the product formation in the inhibitor IAA/HRP/O, system showed that carbofuran phenol in- hibited the enzymatic degradation of IAA but this was not persistent, indicating that the inhibitor was unstable. Similar results were published with phenolic inhibitors

v 1 ;

-A-- 1~--- 5 6

MINUTES

FIG. 6. Photon emission of IAAIHRPIMnZ+I02 system in the presence of diffeerent concentratiotts of 34etocarbofuran phenol under standard reaction conditions: (0) control; (0) 40 PM: (0) 200 PM; (H) 800 ~.LM. Photon emission by the jr-ketocarbofuran phenol (200 pM)IHRPI02 system (- - - ).

such as scopolotin and ferulic acid (18, 19). Due to the partially competitive nature of the inhibition by carbofuran phenol the rate of IAA degradation reached the same level as that of the control without the inhibitor after the lag time. Probably, this was due to breakdown of the metabolite.

By following spectrophotometrically the reaction, Lee and Chapman (4) concluded that 3-ketocarbofuran phenol was stable under the experimental conditions used. However, our results on oxygen uptake and photon emission showed that this com- pound consumes oxygen and produces an electronically excited product. Anthracenic acceptors enhanced the emission which lies between 400 and 500 nm.

3-Ketocarbofuran phenol competes with IAA for an HRP active site as was shown by the fact that the emission from IAA oxi- dation disappears and another one appears in the first minutes of reaction (Fig. 6). This emission in the region 400-500 nm indi- cates probably a generation of carbonyl ex- cited species via a dioxetane intermediate:

METABOLITES OF CARBOFURAN 139

TABLE 1 Enhanced Emission from Ihe 3-Ketocarbofiran

PhenollHRPIO, System”

Integrated Singlet emission at triplet

60 secC energy Acceptor” (counts) (kcal/mol)

Control 20.000 - - Anthracene-2-sulfonate 118.000 68 42.5 9, IO-Diphenylanthra-

cene-2-sulfonate 94.000 65 40.6 9, IO-Dibromoanthra-

cene-2-sulfonate 95.000 66 40.2

o 3-Ketocarbofuran phenol (200 PM); HRP (3 nM), IAA (0.2 mM), Mn2+ (0.1 mM) in phosphate buffer (25 mA4) pH 5.9 at 25°C.

* Acceptor concentration. 5 PM. r Measured in a Hamamatsu TV Photocounter C-767

(10).

This is similar to cell-free extract from a Pseudomonas sp. growing on (+)-catechin, oxidizing dehydrogossypetin by cleaving the A-ring to form oxalacetic and 5-(3,4- dihydroxyphenyl)-4-hydroxy-3-oxovalero- Slactone (20). For these processes the for- mation and cleaving of the dioxetane ring was proposed (21) (Eq. [3]):

This carbonyl compound in its excited state presumably has (zT,~)* as (n.r)* character, giving enhanced emission with singlet and triplet counter. It is known that the (rr,rr)* character gives more singlet ex- cited carbonyl compounds (22). The prod- ucts and chemiluminescence of this system are under study.

ACKNOWLEDGMENTS

The financial support of CNPq (Brasilia), FINEP (Rio de Janeiro), FAPESP (Sao Paulo). CAPES (Brasilia), and UNESCO is appreciated. The authors wish to express their gratitude to Quality Assurance Section, Environmental Toxicology Division, Re- search Triangle Park, North Carolina, for the gift of pesticidal chemicals and to Dr. G. Cilento for a critical reading of the manuscript as well as for several en- lightening discussions.

?” 0 r 0” 9 ?

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