Environmental Fate and Safety Management of Agrochemicals

Downloaded by PENNSYLVANIA STATE UNIV on September 7, 2012 | http://pubs.acs.org Publication Date: March 8, 2005 | doi: 10.1021/bk-2005-0899.ch018

Chapter 18

Identification of Thiolactic Acid Conjugated Metabolites of Fungicide Diethofencarb in Grape (Vitis vinifera L.) and the Mechanism of Their Formation in Plant and Rat 1

2

3

Takuo Fujisawa , Luis O. Ruzo , Yoshitaka Tomigahara, Toshiyuki Katagi , and Yoshiyuki Takimoto 1

1

1

Environmental Health Science Laboratory, Sumitomo Chemical Company, Ltd., Takarazuka, Hyogo 665-8555, Japan PTRL West, Inc., Hercules, CA 94547 Environmental Health Science Laboratory, Sumitomo Chemical Company, Ltd., Osaka 554-8558, Japan 2

3

The carbamate fungicide diethofencarb [isopropyl 3,4diethoxycarbanilate] is very effective for control of various fungal species, especially Botrytis sp., Cercospora sp. and Venturia sp., that are resistant to benzimidazole fungicide. The metabolism study in grape and rat was conducted using 1 4 C — diethofencarb. In grape, unique metabolite such as thiolactic acid attached to the 5-position of phenyl ring of 3-ethoxy-4hydroxy carbanilate, oxidatively O-deethylated form of diethofencarb, via C-S linkage was detected. The thiolactic acid conjugate of 3-ethoxy-4-hydroxy carbanilate was also demonstrated to be involved in rat metabolism.

© 2005 American Chemical Society In Environmental Fate and Safety Management of Agrochemicals; Clark, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2005.

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Introduction Lamoureux et al had firstly characterized thiolactic acid conjugate of pesticide in various plant species. Through the metabolism of PCNB (pentachloronitrobenzene) in peanut (J), thiolactic acid conjugate, 5(pentachlorophenyl)-2-thiolactic acid, was detected from the root in scarce amount (0.5% of the isolated radiolabeled compounds) while it could not be found from peanut cell suspension culture. With EPTC (S-ethyl dipropylthiocarbamate) using corn, cotton, and soybean suspension cell cultures (2), 5-(^^V-dipropylcarbamoyl)-0-malonyl-3-thiolatic acid was characterized as a major metabolite which accounted for up to 33 % of the isolated radiolabeled compounds. Propachlor (2-chloro-JV-isopropylacetanilide) was rapidly degraded to homoglutathione conjugate in the root and foliage of the soybean plant, and then further degraded to thiolactic acid 3-oxide form (J). Fluorodifen herbicide (2,4'-dinitro-4-trifluoromethyldiphenyl ether) was rapidly metabolized by a spruce cell suspension culture and 5-(2-0-glucosyl)-3-thiolactic acid conjugate was presented as major metabolite (4). Although the above studies have shown possible existence of thiolactic acid conjugates as plant metabolites, an ambiguity still remains for the definite determination of their chemical structure because it was conducted only from the results of mass spectrometric analysis. In this study, extensive spectrometric analysis of plant extracts using liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) spectrometry in conjunction with direct chromatographic comparison with the synthetic standards were conducted to definitively identify thiolactic acid conjugate produced as plant metabolites. Also, metabolism study of diethofencarb in rat was conducted in detail and the involvement of the thiolactic acid conjugate in rat metabolism was thoroughly examined using in vivo and in vitro experiments.

l.MetaboIism of diethofencarb in grape Identification of metabolites For the purpose of collecting enough amount of conjugated metabolites subjected to spectrometric analyses (MS, NMR), Pinot Noir grapes under the actual field conditions were used. Diethofencarb (1) labelled at the phenyl ring (8.88 MBq mg" ) and 2-position of the isopropyl group (7.64 MBq mg" ) was prepared in our laboratory (Figure 1). [ C]-1 in the methanol/water solutions was applied to the grape bunches with a hand sprayer at a rate of 1000 g a.i. ha' , three times with an approximately 40 days interval between two subsequent applications. PHI (pre-harvest interval) is set as 14 days. 1

1

14

1

In Environmental Fate and Safety Management of Agrochemicals; Clark, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2005.

207


* indicates label positions Figure l.The chemical structure of diethofencarb and its radiolabeled positions. *H NMR spectra were measured with a Varian Unity 300 FT-NMR spectrometer. Liquid chromatography-atmospheric chemical ionization-mass spectrometry (LC-APCI-MS) in positive and negative ion modes was performed using a Hitachi M-1000 spectrometer. Unknown metabolites were isolated and purified from the grape fruits extracts with solid phase extraction method (Porapak Q) and HPLC. Isolated metabolites were analyzed by free form or derivatized (methylated, acetylated) form for identification. A l l of the nonradiolabeled reference standards 1-6 are synthesized in our laboratory and their chemical structures are shown in Figure 2.

Identification of metabolites 2 and 3 The chemical structure of 2 was easily confirmed by HPLC and TLC cochromatographies of the isolated metabolite with the synthetic standard. In the case of 3, the corresponding HPLC fraction of extracts was collected and divided into two portions. One portion was incubated with cellulase (37 °C, over night) in 10 mM phosphate buffer at pH 5 and a new peak corresponding to 2 appeared via concomitant decrease of the peak due to 3. To further investigate the chemical nature of 3, the remained portion was subjected to LC-APCI-MS. The metabolite 3 was shown to have a molecular weight of 401. These results strongly suggest glucose, or equivalent, as the conjugation moiety. Definitive identification of 3 was achieved by synthesizing the glucose conjugate of 2 and carrying out direct comparison of the isolated 3 with this synthetic standard by HPLC and TLC co-chromatographies.

Identification of metabolites 4 and 5 The metabolite 5 was incubated with cellulase and a new peak corresponding to 4 appeared via concomitant decrease of the peak due to 5. LCAPCI-MS analysis showed that the molecular weight of 5 was 521. With the methylated 5, introduction of the two methyl groups were shown by a MS peak at m/z 548 [M-H]\ When 5 was acetylated, a MS peak at m/z 730 [M-H]" was observed. In order to estimate the functional groups of 5 in detail, the successive derivatization (methylation followed by acetylation) was conducted. LC-APCI-



208

MS showed a MS peak at m/z 716 [M-H]" and hence, the two methyl and four acetyl groups were added to the molecule. Assuming that the aglycone of 5 is 2, one methyl group may be introduced via methylation of the activated phenol at 4-position, while the other one may be introduced from methylation of a carboxylic acid functionally associated with a conjugating moiety. The acetyl groups introduced are probably due to hydroxy functionalities of a monosaccharide which is also consistent with observed molecular weight. The NMR spectrum of the methylated derivative of 5 was investigated. Only two aromatic protons were observed at 6.95 and 7.05 ppm with the coupling constant of 2.0 Hz, indicating the presence of two aromatic protons in the meto-position and hence, some metabolic conversion at 5-position of the phenyl ring was most likely. Furthermore, the isopropyl and one ethoxy groups were found to remain intact. A large number of coupled resonance possibly due to glucopyranosyl protons appeared at 3.10-4.60 ppm, secondary hydroxyl groups, implying that 5 was a sugar conjugate. Taking account of all the spectrometric evidences together with the conjugation form of other pesticides (1-4), the chemical structure of 5 was proposed. Finally, the chemical identity of 5 as 3-{3-ethoxy-2hydroxy-5-[(isopropoxycarbonyl)amino]phenylthio}-2-jS-glucopyranosyloxypropionic acid was definitely confirmed by both HPLC and TLC cochromatographies with the reference standard. Considering from the metabolic pathway, 4 was assumed to exist in grape as an intermediate metabolite of 5. Therefore, the synthetic standard of 4 was used to confirm its existence in the extract of grape by HPLC and TLC cochromatographies. As a result, the metabolite corresponding to 4 was detected in grape fruit.

Identification of metabolite 6 The metabolite 6 was incubated with cellulase, however, no degradation of 6 was observed. LC-APCI-MS spectrum of 6 showed the same molecular weight as 5 (m/z 520 [M-H]"), indicating isomeric structure. LC-MS analysis showed a MS peak of the methylated 6 at m/z 534 ([M-H]) and that of methylated and acetylated derivative of 6 at m/z 744 ([M-H]"). The molecular weight of the latter derivative indicated the addition of five acetyl unites to the methylated molecule. These results together with the data on metabolite 5, implied that 6 was a monosaccharide derivative, but only four acetyl groups was possibly to be introduced. Therefore, the metabolite should have an additional center sensitive to acetylation. Considering all the above spectrometric evidences, and taking chemical structure of 5 into account, 6 was proposed as 3-{3-ethoxy2-j8-glucopyranosyloxy-5-[(isopropoxycarbonyl)amino]phenylthio-2-hydroxy-


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propionic acid}] which was definitely confirmed by both HPLC and TLC cochromatographies with the synthetic standard. Based on these results, the metabolic pathway of 1 in grape plants is proposed in Figure 2.

Figure 2. Proposed metabolic pathway of I in the grape plants. F; Fruit treatment, L; Leaf treatment Distribution of metabolites in grape For investigation of metabolic profiles, grape vines (Vitis vinifera L., cv. Kyoho) with fruits setting at maturity stages were used. [ C]-1 in the acetonitrile solutions was topically applied at a rate of 500 g a.i. ha" to grape bunches (fruits) and leaves once with PHI 35 days. Distributions of radioactivity and metabolites in the grape fruit are summarized in Table 1. Within the whole grape, 1 remained as the major residue (52.8-60.9 % of total radioactive residue; TRR). Major metabolites detected were 3 (8.118.1 %TRR, 0.153-0.289 ppm), 5 (6.4-7.3 %TRR, 0.102-0.137 ppm), and 6 (8.7-13.5 %TRR, 0.165-0.216 ppm). Minor metabolites 2 and 4 were both detected below 1.7 %TRR. Meanwhile, the distribution of radioactivity from the treated leaves was different from that of the fruits (Table 2). Within the whole leaf, 1 was major residue (95.6-95.8 %TRR) and the amounts of metabolites 3 14

1


210 and 5 were less than 0.6 %TRR. These results indicated more penetration of 1 followed by metabolic degradation infruitsas compared with leaves.

l4

Table 1 Distribution of radioactivities and metabolites of [ C]-l with fruit treatment. 14

i C-phenyl1-l ppm %TRR 0.844 52.75 0.014 0.90 0.289 18.06 0.024 1.51 0.103 6.39 0.216 13.48 0.101 6.30 0.010 0.61 1}


Fruit

1 2 3 4 5 6 others unextractable

100.00

Total

a)

a)

1.599

14

f C-isopropyll-l %TRR ppm 1.149 60.87 ND ND 0.153 8.11 0.032 1.68 0.137 7.25 0.165 8.71 0.164 8.68 0.088 4.68 )

b)

b)

100.00

1.888

1J

ppm of 1 equivalent.

a)

Consisted of more than 6 metabolites, each metabolite below 3.70 %TRR (0.070 ppm).

b )


Source: Reproduced from réf. 18.

14

Table 2 Distribution ofradioactivitiesand metabolites of [ C]-1 with leaf treatment. 14

[ C-phenyl]-l ppm * %TRR 76.257 95.55 ND ND 0.391 0.49 ND ND 0.458 0.57 ND ND 1.856 2.33 0.847 1.06 1

Leaf

1 2 3 4 5 6 others unextractable

Total

a)

100.00

a)

79.809

14

f C-isopropyll-l ppm ' %TRR 61.646 95.80 ND ND ND ND ND ND 0.273 0.43 ND ND 1.969 3.08 0.444 0.69 b)

b)

100.00

64.332

' ppm of 1 equivalent. a)


b)


Source: Reproduced from ref. 18.


211 2. Metabolism of diethofencarb in rats Identification of metabolites For metabolite identification, 8 male Crj:CD(SD) rats (7 weeks old) were treated orally with [ C-phenyl]-l at 300 mg/kg b.w./day for 2 days. Urine and feces were collected for 3 days after the first dose. Feces were not used following isolation. Urine was lyophilized and fractionated by solvent extractions (hexane, ethyl acetate, etc.). Eight metabolites were isolated from extracts by preparative TLC and preparative HPLC. Their chemical structures were determined by NMR (% H-H COSY, HSQC, HMBC, etc.) and MS (ESI, EI) spectroanalyses. Their chemical structures are shown in Figure 3 (8, 9, 10, 11,13,14,15 and 16).


14

Excretion of diethofencarb and metabolites in rats 14

Four male Crj:CD(SD) rats (7 weeks old) was treated orally with [ Cphenyl]-l at 10 mg/kg b.w. Urine and feces were collected for seven days. C Residue in residual carcass was also analyzed on the 7th day after administration. Identification of the metabolite was carried out by TLC co-chromatography with radiolabeled metabolites mentioned above and unlabelled authentic standards (1, 2, and 12). The radiocarbon was rapidly eliminated from the body, and the Cresidue was low on 7 days after administration ( C-Residue in residual carcass on the 7th day after administration was 0.1 % of the dose). Total C-recoveries within 7 days after administration were 98.6 % of the dose (urine; 89.6 %; feces; 9.0 %). Pooled (0-2 day) urine and feces were analyzed for quantification of metabolites. 1 was detected only in feces (0.3 % of the dose). More than 16 metabolites were detected and quantified. The conjugates (sulfate and/or glucuronide) of 2 and a mixture of conjugates (sulfate and/or glucuronide) of 11, 12, and 13 were the major metabolites, accounting for 55.1 % and 18.9 % of the dose, respectively. S-containing metabolites such as free and conjugates (sulfate and/or glucuronide) of 8,14,15, and 16 were detected, accounting for 0.9 % or less of the dose. Other metabolites accounted for 2.8 % or less of the dose. The major metabolic reactions were 1) 0-deethylation of 4-ethoxy group; 2) cleavage of the carbamate linkage, 3) acetylation of the amino group; 4) conjugation of the resultant phenols with sulfuric acid and/or glucuronic acid. The minor reactions were 1) oxidation of the isopropyl group; 2) cyclization of the oxidized isopropyl carbamate group; 3) oxidation of the 4-ethoxy group; 4) conjugation of the phenols with glutathione; 5) γ-glutamyltranspeptidation and depeptidation of the glutathione to form cysteine conjugate; 6) iV-acetylation of the cysteine; 7) cleavage of C-S linkage of the cysteine followed by 5methylation; 8) oxidation of the 5-methyl group. Proposed metabolic pathways of 1 in rat are shown in Figure 3. 14

14

14

14



212


213


In vitro metabolism of 7 Three Crj:CD(SD) male rats were sacrificed by bleeding from the abdominal artery under ansesthesia with diethyl ether. Liver was taken from these three rats. The liver was homogenized at 4 °C with 10 mM potassium phosphate buffer (pH 7.5) containing 1.15 % KC1, 10 μΜ (4-amidinophenyl)methanesulphonyl fluoride and 50 μΜ pyridoxal 5'-phosphate, and then the homogenates was centrifuged at 9000xg for 20 min. The supernatant was subsequently centrifuged at 105000xg for 60 min to separate cytosol and microsomal fractions. The reaction mixtures (n=3) contained 100 mM Trisacetate buffer (pH7.0), 2 mg of rat liver cytosol protein (final conc.;4 mg/mL), 5 mM α-ketoglutaric acid, 1 mM NADH, 1 mM NADPH, 1 % (v/v) dimethylsulfoxide and 1 μτηοΐ of [ C-phenyl]-7 (final cone; 2 mM), in a volume of 0.5 mL. Control experiment (n=3) were performed by excluding rat liver cytosol from the reaction mixtures. The reaction mixtures were incubated at 37 °C for 16 hours. The metabolites were analyzed by TLC and quantified by the scraping-LSC method. 4 was only detected in the reaction mixture containing rat liver cytosol fraction. In 16 hours of reaction, 5.3 % of 7 was biotransformed to 4. The formation rate of 4 in rat liver cytosol was 1.67 nmol/hr/mg protein. The metabolic pathway of 7 to 4 in rats was proposed. The metabolic reactions were 1) transamination of amino group of cysteine; 2) reduction of ketone group of pyruvic acid. Above-mentioned step 1) and 2) were catalyzed by cysteine conjugate transaminase and 3-mercaptopyruvic acid 5-conjugate reductase, respectively. Proposed metabolic pathway of 7 to 4 in rat (in vitro) are shown in Figure 4. 14

7

pyruvic acid conjugate

4

Figure 4. Proposed pathways of 7 to 4 in rats.

3. Discussion The mechanism on formation of the thiolactic acid conjugate was postulated from the various works conducted with acetaminophen (/V-acetyl-/?aminophenol). Acetaminophen is a widely used analgesic medicine which has p-



214 aminophenol as a fundamental chemical structure. The p-aminophenol moiety is considered to undergo two-electron oxidation and it is easily transformed to a highly reactive benzoquinoneimine. The in vitro oxidation of acetaminophen has been extensively investigated using various enzymes such as cytochrome P-450, prostaglandin H synthase [PHS], and peroxidase (5-11) and the three possible routes to generate benzoquinoneimine from /j-aminophenol by enzymatic oxidation was postulated. Firstly, acetaminophen is oxidized with peroxidase or PHS via one-electron oxidation to produce N--acetyl-p-benzosemiquinoneimine and the resulting semiquinoneimine disintegrates to give acetaminophen and pbenzoquinoneimine. Secondly, acetaminophen is oxidized with P-450 and PHS directly via two-electron oxidation to form benzoquinoneimine. Thirdly, acetaminophen is oxidized with P-450 to form hydroxylated intermediate (Nacetyl-JV-hydroxy-p-aminophenol), subsequently followed by dehydration to form benzoquinoneimine. The main metabolite of 1 at the early stage of metabolism is most likely to be 2, possessing the common p-aminophenol structure to acetaminophen. Therefore, 2 would be similarly oxidized to form the corresponding benzoquinoneimine as an intermediate. Additionally, Finley et al. (12) had discussed the specificity of reacting positions on benzene ring of N,N'-quinonediimine against a nucleophilic substitution. It was proposed that the 1-, 3- and 5-positions of N,N'quinonediimine ring was highly susceptible to nucleophilic substitution. Actually, it has been reported in metabolism of acetaminophen (13-16) that the 3- and 5positions of quinoneimine is highly reactive to the nucleophilic attack by glutathione (GSH). In summary, 1 primary underwent O-deethylation at 4-position of the phenyl ring to form 2. Successively, 2 was likely to be converted via twoelectron oxidation to the corresponding benzoquinoneimine whose 5-position of the ring was attached by cysteine via C-S linkage. Cysteine is the most probable nucleophile, since it is known to be formed by catabolism of the corresponding conjugates of GSH, a major ingredient in grape fruit (17). Actually, acetyl cysteine conjugate of 2 was detected as rat metabolites to support our assumption. Then, the amino group of cysteine was considered to be transformed to the pyruvate structure as an intermediate by cysteine conjugate transaminase and finally the pyruvate form to the thiolactic acid by 3-mercaptopyruvic acid Sconjugate reductase (Fig 4).

References 1. 2.

Lamoureux, G. L.; Gouot, J. M.; Davis, D. G.; Rusness, D. G. J. Agric. Food Chem. 1981, 29, 996-1002. Lamoureux, G. L.; Rusness, D. G. J. Agric. Food Chem. 1987, 35, 1-7.


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7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

Lamoureux, G. L.; Rusness, D. G. Pestic. Biochem. Physiol. 1989, 34, 187204. Lamoureux, G. L.; Rusness, D. G.; Schröder, P.; Rennenberg, H. Pestic. Biochem. Physiol. 1991, 36, 291-301. Hinson, J. Α.; Monks, T. J.; Hong, M.; Highet, R. J.; Pohl, L. R. Drug Metab. Dispos. 1982, 10, 47-50. Morgan, E. T.; Koop, D. R.; Coon, M. J. Biochem. Biophys. Res. Commun. 1983, 112, 8-13. Albano, E.; Rundgren, M.; Harvison, P. J.; Nelson, S. D.; Moldéus, P. Mol. Pharmacol. 1985, 28, 306-311. Potter, D. W.; Miller, D. W.; Hinson, J. A. Mol. Pharmacol. 1985, 29, 155162. Potter, D. W.; Hinson, J. A. Mol. Pharmacol. 1986, 30, 33-41. Potter, D. W.; Hinson, J. A. J. Biol. Chem. 1987, 262, 966-973. Potter, D. W.; Hinson, J. A. J. Biol. Chem. 1987, 262, 974-980. Finley, T. K.; Tong J. L. K.; Patai, S., Eds.; Interscience publishers, London, 1970; pp 663-729. Gemborys, M. W.; Gribble, G. W.; Mudge G. H. J. Med. Chem. 1978, 21, 649-652. Miner, D. J.; Kissinger, P. T. Biochem. Pharmacol. 1979, 28, 3285-3290. Buckpitt, A. R.; Rollins D. E.; Nelson, S. D.; Franklin, R. B.; Mitchell, J. R. Anal. Biochem. 1977, 83, 168-177. Pascoe, G. Α.; Calleman, C. J.; Baille, T. A. Chem.-Biol. Intractions 1988, 68, 85-98. Park, S. K.; Boulton, R. B.; Noble, A. C. Food Chem. 2000, 68, 475-480. Fujisawa, T.; Ichise-Shibuya, K.; Katagi, T.; Ruzo, L. O.; Takimoto, Y. Metabolism of Fungicide Diethofencarb in Grape (Vitis vinifera L.): Definitive Identification of Thiolactic Acid Conjugated Metabolites. J. Agric. Food Chem. 2003, 51, 5329-5336.


Environmental Fate and Safety Management of Agrochemicals

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