Xenobiotic Conjugation Chemistry - American Chemical Society

4 Xenobiotic Conjugation in Higher Plants G. L. Lamoureux and D. G. Rusness

Downloaded by NORTH CAROLINA STATE UNIV on October 2, 2013 | http://pubs.acs.org Publication Date: January 24, 1986 | doi: 10.1021/bk-1986-0299.ch004

Metabolism and Radiation Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Fargo, ND 58105 Xenobiotic conjugation in plants is extensively reviewed. Where appropriate, the following data are tabulated: xenobiotic, class of conjugate, species, and methods of isolation and identification. The following conjugates are discussed: simple and complex glucose conjugates formed from HOOC-, HO-, H N-, HON-, and HS-functional groups; glutathione or homoglutathione conjugates formed from various electrophiles; amino acid conjugates of carboxylic acids; malonic acid conjugates of amines; several lipophilic conjugates; and bound residues. The relationship between metabolism and herbicide selectivity in various plant species is also discussed. 2

The metabolism of xenobiotics in plants has been an area of intense research for approximately 20 years. This interest has been motivated by concern over the widespread use of pesticides in our environment and by the desire to produce pesticides that are more bio-degradable and more selective. As a result, most of the available information concerning xenobiotic metabolism in plants pertains to pesticides or pesticide analogs. Numerous reviews have been written on plant metabolism of pesticides (1-7) and herbicides (8-11). In addition, more specific reviews have dealt with glycoside conjugation (]2), amino acid conjugation (_13.), glutathione conjugation 04), catabolism of glutathione conjugates in plants (15, 16), bound residues (17), in vitro methods for studying xenobiotic metab&lism in plants 7l8), metabolism in cell culture (19*20), plant enzymes involved in xenobiotic metabolism (21_,22), and oxidative enzymes in plants (23). The mechanisms utilized by plants and mammals in the metabolism of xenobiotics are remarkably similar. Similar classes of compounds or functional groups are frequently metabolized by comparable mechanisms. Oxidation, reduction, hydrolysis, and conjugation reactions occur with similar frequency in both. In most instances, however, This chapter not subject to U.S. copyright. Published 1986, American Chemical Society

In Xenobiotic Conjugation Chemistry; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.


4.

LAMOUREUX AND

RUSNESS

Xenobiotic

Conjugation

in Higher

Plants

63

considerably more i s known about the enzymes and mechanisms involved i n xenobiotic transformations i n mammals than i n plants. This i s p a r t i c u l a r l y true i n regards to the oxidative reactions and amino acid conjugation. Although i t i s commonly stated that xenobiotic metabolism occurs more slowly i n plants than i n mammals, many xenob i o t i c transformations i n plants are completed within 6- to 24-hr following exposure to the chemical. Hydrolysis, glucose conjugation, and glutathione conjugation can occur very r a p i d l y i n plants. The t i t e r of glutathione S-transferase enzyme i n corn appears to be comparable to that i n rat l i v e r (24). Several fundamental differences between plants and mammals may be responsible for some of the differences observed i n xenobiotic metabolism. Plants lack a well-developed excretory system, and as a r e s u l t , xenobiotics may be subjected to a greater array of metabol i c transformations over a longer period of time than would occur i n mammals. The autotrophic nature of higher plants may also r e s u l t i n some differences i n metabolism, e.g., reincorporation of CO2 produced from the metabolism of xenobiotics. Plant c e l l s are characterized by a c e l l wall that can be highly l i g n i f i e d . Xenobiotics may be incorporated into the c e l l wall i n nonselective free r a d i c a l reactions u t i l i z e d i n the synthesis of l i g n i n . Xenobiotics may also be incorporated into hemicellulose or other carbohydrate components of the c e l l wall i n more selective reactions. As a r e s u l t , bound residues tend to be much more common i n xenobiotic metabolism i n plants than i n mammals. Plant c e l l s are also characterized by the presence of large c e l l vacuoles. Xenobiotics may be metabolized i n such a manner that they become sequestered from further metabolic processes by storage i n this organelle. Glycoside conjugation appears to be the most common xenobiotic conjugation reaction i n both plants and mammals; however, plants form glucose rather than glucuronic acid conjugates and glucoside conjugation tends to be far more complex i n plants. Plants r a r e l y form sulfate ester conjugates. Glutathione conjugation occurs i n both plants and mammals, probably with s i m i l a r frequency and with a s i m i l a r range of compounds. Glutathione conjugates undergo further catabolism i n both plants and mammals. Although some differences have been observed, the o v e r a l l process of glutathione conjugate catabolism i s very s i m i l a r i n both. Amino acid conjugation i s not common i n plants, but occurs as a f a i r l y general reaction with a r e s t r i c t e d class of compounds. Compounds that form amino acid conjugates i n both plants and mammals have s t r u c t u r a l l y s i m i l a r charact e r i s t i c s . Xenobiotics are rarely acetylated i n common plant species, but the formation of malonyl conjugates i s r e l a t i v e l y common. This appears to be a s t r i k i n g difference between plants and mammals. Several recent reports suggest that plants occasionally form l i p o p h i l i c conjugates, as has also been reported i n mammals* In some plant species, these conjugates may be highly unusual i n structure. In this review, conjugation reactions u t i l i z e d i n xenobiotic metabolism i n plants w i l l be discussed i n reference to functional groups, phase I reactions necessary to produce a functional group suitable for conjugation, r e l a t i v e rates of reactions, competing metabolic pathways, frequency of occurence, plant species, s t a b i l i t y of conjugates, and the relationship between metabolism and herbicide s e l e c t i v i t y . Pesticides discussed herein are l i s t e d i n Table I.


XENOBIOTIC CONJUGATION CHEMISTRY

64


TABLE I .

Nomenclature o f X e n o b i o t i c s Mentioned

i n Tables/Text

Abscisic Acid

5-(1-Hydroxy-2,6,6-trimethyl-4-oxo-2-cyclohexen-1-yl)-3-methyl-2,4-pentadienoic acid

Acifluorfen

5-(2-Chloro-4-trifluoromethylphenoxy)-2nitrobenzoate

Alachlor

2-Chloro-2•, 6'-diethyl-N-(methoxymethyl)acetanilide

Atrazine

2- C h l o r o - 4 - ( e t h y l a m i n o ) - 6 - ( i s o p r o p y l a m i n o ) triazine

Bar ban

4-Chloro-2-butynyl-3 -chlorocarbanilate

BAY NTN 9306

0- E t h y l 0 - [ 4 - ( m e t h y l t h i o ) p h e n y l ] phosphorodithioate

1

S-propyl

3- I s o p r o p y l - ( 1 H ) - b e n z o - 2 , 1 , 3 - t h i a d i a z i n - 4 - o n e Bentazon

2.2- d i o x i d e Methyl

Bidisin

2-chloro-3-(4-chlorophenyl)propionate

2,6-Dichloro-4-nitroaniline Botran 2-sec-Butylphenyl BPMC

N-methylcarbamate

Butachlor

2- Chloro-2•,6»-diethyl-N-butoxymethylacetanilide

Buthidazole

3[5«(1,1-Dimethylethyl)-1,3,4-thiadiazol-2yl]-4-hydroxy-1-methyl-2-imidazolidinone

Buturon

3-(4 -Chlorophenyl)-1-methyl-1-(1-methylprop2- y n y l ) u r e a

Captan

1,2,3,6-Tetrahydro-N-(trichloromethylthio)phthalimide

Carbar y l

1- N a p h t h y l

Carbofuran

2.3- D i h y d r o - 2 , 2 - d i m e t h y l b e n z o f u r a n - 7 - y l methylcarbamate

Carboxin

5,6-Dihydro-2-methyl-1,4-oxathiin-3carboxanilide

1

CDAA

N-methylcarbamate

N,N-Diallyl-2-chloroacetamide

Chloral

Hydrate

2,2,2-Trichloro-1,1-ethanediol

Chloramben

3- A m i n o - 2 , 5 - d i c h l o r o b e n z o i c

Chlorpropham

Isopropyl-3 -chlorocarbanilate

acid

Chlorsulfuron

2-

1

Chloro-N-[(4-methoxy-6-methyl-1,3,5-triazin-

2- y l ) a m i n o c a r b o n y l ] b e n z e n e s u l f o n a m i d e Chlortoluron Cisanilide Credazine

3-

(3-Chloro-4-methylphenyl)-1,1-dimethylurea

cis-2,5-Dimethyl-1-pyrrolidinecarboxanilide 1

3-(2 -Methylphenoxy)pyridazine


4.

Xenobiotic

LAMOUREUX AND RUSNESS


Table

I.

Conjugation

in Higher

Plants

65

Continued

Cypermethrin

Cyano(3-phenoxyphenyl)methyl 3-( 2,2-dichloroethenyl)-2,2-dimethylcyclopropanecarboxylate

Cyprazine

2-Chloro-4-(cyclopropylamino)-6-(isopropylamino )-s_-tr iazine

Cytrolane

Diethyl (4-methyl-1,3-dithiolan-2-ylidene)phosphoramidate

2,4-D

(2,4-Dichlorophenoxy)acetic

Diamidafos

NjN'-Dimethylphenylphosphorodiamidate

acid

Diazinon

0,0-Diethyl

0-(2-isopropyl-6-methylpyrimidin-

4- yl)phosphorodithioate DIB

2- (2,4-Dichlorophenoxy)isobutyric acid

Dichlobenil Dichlofluanid

2,6-Dichlorobenzonitrile N-Dichlorofluoromethylthio-N , N -dimethyl-Nphenylsulphamide 1

1

,

Diclofop-methyl

Methyl 2-[4-(2*,4 -dichlorophenoxy)phenoxy]propanoate

Dieldrin

1,2,3,4,10,10-Hexachloro-6,7-epoxy-1,4,4a,5,6,7,8,8a-octahydro-1,4-endo-5,8-exodimethanonaphthalene

Dimethametryn

2- (1,2-Dimethylpropylamino)-4-ethylamino-6methylthio-£-triazine

Dinoben

2,5-Dichloro-3-nitrobenzoic acid

Diphenamid

N,N-Dimethyl-2,2-diphenylacetamide

Diuron

3- (3,4-Dichlorophenyl)-1,1-dimethylurea

EPN

O-Ethyl 0-(4'-nitrophenyl) phenylphosphonothioate

EPTC

5- Ethyl

Flamprop-isopropyl

Isopropyl N-benzoyl-N-(3-chloro-4fluorophenyl)-2-aminopropionate

Fluorodifen

2,4 -Dinitro-4-trifluoromethyl diphenylether

Fluvalinate

a-Cyano-3-phenoxybenzyl 2-[2-chloro-4(trifluoromethyl)anilino]-3-methylbutanoate

GS-13529

2- Chloro-4-(ethylamino)-6-(tert-butylamino)-striazine

Hymexazol

3- Hydroxy-5-methylisoxazole

IAA

2- (Indol-3-yl)acetic acid

Isouron

3- (5-tert-Butyl-3-isoxazolyl)-1,1-dimethylurea

Isoxathion

0,O-Diethyl 0-(5-phenyl-3-isoxazolyl)phosphorothionate

Isoxazolinone

Isoxazolin-5-one

Maleic

1,2-Dihydro-3,6-pyridazinedione

Hydrazide

N,N-dipropylthiocarbamate

f

Continued

on next


page


66


Table

I.

Continued

MCPA

(4-Chloro-2-methylphenoxy)acetic

acid

Mephosfolan

Diethyl(4-methyl-1,3-dithiolan-2-ylidene)phosphoramidate

Methazole

2-(3,4-Dichlorophenyl)-4-methyl-1,2,4oxadiazolidine-3,5-dione

Methidathion

S-2,3-Dihydro-5-methoxy-2-oxo-1,3,4-thiadiazol3-ylmethyl 0,0-dimethyl phosphorodithioate

Metolachlor

ct-Chloro-2 • -ethyl-6 -methyl -N-( 1 -methyl-2methoxyethyl)acetanilide

Metribuzin

4-Amino-6-tert-butyl-4,5-dihydro-3-methylthio1,2,4-triazin-5-one

Mobam

4-Benzothiophene N-methylcarbamate

Molinate

S-Ethyl N,N-hexamethylenethiocarbamate

Monolinuron

3-(4-Chlorophenyl)-1-methoxy-1-methylurea

1

NAA

1-Naphthaleneacetic

Nitrofen

2,4-Dichlorophenyl

Oxamyl

N,N-Dimethyl-2-methylcarbamoyloxyimino-2Tmethylthio)acetamide

PCNB

acid 4-nitrophenyl ether

Pentachloronitrobenzene

Perfluidone Permethrin

1,1,1-Trifluoro-N-[2-methyl-4-(phenylsulfonyl)phenyl]methanesulfonamide 3-Phenoxybenzyl (1RS)-cis, trans-3-(2,2-dichlorovinyl-2,2-dimethylcyclopropanecarboxylate

Picloram

4-Amino-3,5,6-trichloropicolinic acid

Prometryn Pronamide

2,4-bis(Isopropylamino)-6-methylthio-s-triazine 3,5-Dichloro-N-(1,1-dimethyl-2-propynyl)benzamide

Propachlor

2-Chloro-N-isopropylacetanilide

Propanil

3 ,4»-Dichloropropionanilide

Propazine

2-Chloro-4,6-bis(isopropylamino)-s-triazine

Propham

Isopropyl carbanilate

Pyrazon

5-Amino-4-chloro-2-phenylpyridazin-3-one

R-25788

f

N,N-Diallyl-2,2-dichloroacetamide

Ro 12-0470

2-Naphthylmethyl

cyclopropanecarboxylate

Simazine

2-Chloro-4,6-bis(ethylamino)-£-triazine

Solan

N-(3-Chloro-4-methylphenyl)-2-methylpentanamide

Sweep

Methyl

2,4,5-T

(2,4,5-Trichlorophenoxy)acetic acid

N-(3,4-dichlorophenyl)carbamate


4. LAMOUREUX AND RUSNESS

Xenobiotic


Table

I.

Conjugation

in Higher Plants

67

Continued

Tridiphane

2-(3,5-Dichlorophenyl)-2-(2,2,2trichloroethyl)oxirane

Triforine

1,4-bis(2,2,2-Trichloro-1-formamidoethyl)piperazine

Triton

Polyethylene g l y c o l 4-isooctylphenyl ether

Zectran

4-Dimethylamino-N-methyl-3,5-xylylcarbamate

Glycoside conjugates Higher plants have an extremely we11-developed capacity to convert various endogenous and xenobiotic substrates to glucose conjugates (4-6,11,12,21,22). The functional groups most frequently involved i n glucose conjugation i n plants are HO-X, HOOC-X, H2N-X, or HN=X; i n addition, there are several reports o f xenobiotic-glucosides formed from HS-X and HON-X intermediates. Many xenobiotics that do not contain the functional groups described can be metabolized to glucose conjugates after a functional group i s introduced by a phase I reaction. Phase I reactions that lead to glucose conjugation i n plants are described i n Table I I .

TABLE I I . Phase I Reactions that Produce Metabolites Susceptible to Glucose Conjugation i n Higher Plants Phase I Reaction Hydrolysis

Class of Xenobiotic Carbamate Anilide Phosphorothioate Ester N-Hydroxyl Deriv.

Metabolite Susceptible to Glucoside Conjugation Phenols Anilines, Carboxylic Acids Phenols Carboxylic Acids, Alcohols N-Hydroxyls

GSH

Diphenyl

Phenols

Conjugation

ether

Reduction

Nitroaromatic

Anilines

Oxidation

Alkyl Aryl

Alcohols Phenols

Isomer izat ion

Cyclic amides

Alcohols, Amines

O-Glucosldes Phenols and alcohols, or xenobiotics that are metabolized through phenols and alcohols as intermediates, are most commonly metabolized to B-O-D-glucosides (3,4.,H, 12). The a b i l i t y o f d i f f e r e n t plant families to form glucosides from phenols was f i r s t extensively investigated by Pridham (25), who showed that 20 out of 23 species In Xenobiotic Conjugation Chemistry; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.


68


of angiosperms assayed with p- and m-dihydroxybenzene formed high l e v e l s o f g l u c o s i d e s — t h e only angiosperms that did not form glucosides were three aquatic species. I t was also shown that f i v e species of gymnosperms had a high capacity to form glucosides, but generally lower or variable rates of glucosylation were observed i n ferns, mosses and a liverwort. No glucosylation was observed i n ten species o f algae and two species o f fungi. More recent studies with a wide variety of pesticides i n various agronomic and h o r t i c u l t u r a l l y important plant species has c l e a r l y established O-glucosides as the most common class of xenobiotic metabolites i n plants (Table I I I ) . Generally, these O-glucosides have been characterized as B-

Table I I I .

Xenobiotics Metabolized

to O-Glucosides i n Plants 0

Phase I Reaction

Species

GSH

Soybean

C,HPLC,TLC,B-Glcase, Glc-Anal,Acet-Conj-MS

HYD

Cotton

C,TLC,Synth,H+-Hyd, B-Glcase,Aglycone-Co-TLC

Bentazon

OXD

Rice

C,TLC,B-Glcase,H -Hyd, Glc-Anal,Aglycone-MS,NMR

BPMC (Bassa)

OXD

Rice

C,TLC,H+-Hyd,B-Glcase, Aglycone-Co-TLC,MS

Car bo fu ran

OXD

Bean

C,TLC,H+-Hyd,B-Glcase, Aglycone-Co-TLC

Carboxin

HYD OXD

Peanut

Xenobiotic Acifluorfen BAY

9306

NTN

Chloral Hydrate

Methods of I s o l a t i o n and Characterization 14

14

14

+

REF 94 J64 J65

14

166

1U

167

C,CC,HPLC,Synth, Acet-Conj-MS

14

121

Gourd

Synth,Acetylate,MP,Spec. Rotation,Elemental-Anal

168

C,CC,H -Hyd,B-Glcase, Acet-Aglycone-GC,NMR,MS

14

+

161

Chlorpropham

OXD

Soybean

Chlorsulfuron

OXD

Wheat,Wild C, HPLC, H+-Hyd, B-Glcase, Oat,Grasses Aglycone-MS,NMR,IR

Chlortoluron

OXD

Wheat,Wild Oat,Cotton

C,TLC,B-Glcase, Aglycone-Co-TLC

Cisanilide

OXD

Carrot, Cotton

C,CC,TLC,B-Glcase,GlcAnal, Aglycone-IR,MS

Credazine

HYD ISO

Tomato Barley

3H,CC,TLC,Synth,8-Glcase, 170 Acet-Conj-IR,Aglycone-IR

Cotton

C,CC,TLC,B-Glcase, Aglycone-MS

Soybean, Corn

C,TLC,B-Glcase, Aglycone-Co-TLC,PC

Cytrolane

121

OXD 2,4-D

OXD

14

14

31 JH 169

14

171

14

J72


4.



Xenobiotic

Phase I Reaction

a

Xenobiotic

Conjugation

in Higher

69

Plants

Table I I I . Continued Methods o f I s o l a t i o n and C h a r a c t e r i z a t i o n Species

Ref

Diamldafos

HYD

Tobacco

C,TLC,PC,B-Glcase, Aglycone-Co-TLC

Dichlofop-Methyl

HYD OXD

Wheat, Oat

C,TLC,HPLC,H -Hyd,BGlcase,Aglycone-Glc-Anal

Diphenamid

OXD

Tomato, Pepper

C,TLC,H -Hyd,B-Glcase, Glc-Anal,Acet-Conj-MS

OXD

Cotton, Barley, Wheat


Pea

CC,TLC,H -Hyd,B-Glcase, GLC-Anal

213

Peanut

CC,B-Glcase,Acet-Conj MS,NMR

174

Diuron Ethanol

—

Fluorodifen

GSH

Hymexazol

—

,4

14

14

173

+

32 68

+

14

+

n 41

14

175

14

40

14

49

Rice, Cucumber, Tomato

C,CC,TLC,B-Glcase, Glc-Anal,TMS-Conj-MS, IR,NMR

Isouron

OXD

K. Bean Sugar Cane

C,TLC,B-Glcase, Aglycone-Co-TLC,MS

Isoxathion

HYD

Cabbage, Bean

C,Synth,TLC,H+-Hyd,BGlcase,Acet-Conj-IR,NMR

176

Maleic Hydrazide ISO

Tobacco

Monolinuron

OXD

Cress, Potato, Spinach


—

Soybean

C,HPLC,B-Glcase, Acet-Conj-MS

C,CC,B-Glcase,Glc-Anal, YfQ Aglycone-Co-TLC,HPLC,GC,MS

Pentachlorophenol Perfluidone

OXD

Peanut

Permethrin

HYD

Cotton, Bean

14

C,TLC,B-Glcase,GlcAnal ,Acet-Conj-MS

14

1 4

1 4

C,Synth,TLC,B-Glcase, Aglycone-Co-TLC

Angiosperms PC,Diazotization Gymnosperms

m-Phenoxybenzyl Alcohol

Cotton Pea

a

D

36

14

Phenols (m- or pDihydroxybenzene)

Triton X-100

177

179 25

14

30

14

148

C,TLC,B-Glcase, Acet-Conj-MS C,TLC,B-Glcase, Acet-Conj-MS

Phase I reaction proceeding conjugation: OXD=oxidation, HYD= hydrolysis, GSH=glutathione conj, IS0=isomerization. Acet=Acetylated; CC or PC=column or paper chromatography; Conj= Conjugate; Glc=glucose; Glc-Anal=Glucose Analysis; B-Glcase= B-glucosidase; H+-Hyd=Acid Hydrolysis; Synth=Synthesis.



70


g l u c o s i d e s by h y d r o l y s i s w i t h B - g l u c o s i d a s e , s y n t h e s i s and chromat o g r a p h i c comparison (.12), o r i n more r e c e n t s t u d i e s , by NMR analys i s as d e s c r i b e d by F e l l i n C h a p t e r 9. Although p l a n t s are capable o f f o r m i n g g a l a c t o s e , g l u c u r o n i c a c i d , and o t h e r c a r b o h y d r a t e c o n j u g a t e s w i t h endogenous s u b s t r a t e s such as f l a v o n e s (27-29), monosaccharide conjugates o f xenobiotics i n v o l v i n g carbohydrate moieties o t h e r than g l u c o s e have been e x t r e m e l y r a r e . However, as m e t a b o l i s m s t u d i e s a r e conducted w i t h more d i v e r s e c h e m i c a l s and p l a n t s p e c i e s , and w i t h more s o p h i s t i c a t e d methods o f a n a l y s i s , i t seems l i k e l y t h a t o t h e r c a r b o h y d r a t e m o i e t i e s w i l l be found c o n j u g a t e d to xenobiotics . I n p l a n t s , B - O - g l u c o s i d e s o f x e n o b i o t i c s can be formed v e r y rapidly. For example, m-phenoxybenzyl a l c o h o l was m e t a b o l i z e d t o O - g l u c o s i d e s i n e x c i s e d c o t t o n l e a v e s i n c a . 90J y i e l d w i t h i n 8 h r f o l l o w i n g treatment (30). The most common r e a c t i o n ( s ) t h a t appear t o compete w i t h g l u c o s i d e f o r m a t i o n i n the m e t a b o l i s m o f p h e n o l s a r e t h o s e t h a t l e a d to bound r e s i d u e . I f a phase I o x i d a t i o n i s r e q u i r e d t o g e n e r a t e a f r e e h y d r o x y l group p r i o r to g l u c o s i d e f o r m a t i o n , the f r e e p h e n o l or a l c o h o l may not be observed o r the p h e n o l may be observed o n l y a t a v e r y low c o n c e n t r a t i o n (3)* I t appears t h a t uptake or o x i d a t i o n a r e more f r e q u e n t l y the l i m i t i n g s t e p s . C h l o r s u l f u r o n i s m e t a b o l i z e d i n wheat by r i n g - h y d r o x y l a t i o n followed by g l u c o s i d e f o r m a t i o n ( E q u a t i o n 1). In c h l o r s u l f u r o n - t r e a t e d wheat, 60% o f the h e r b i c i d e was i n the form o f the 5 - h y d r o x y g l u c o s i d e 24 h r a f t e r treatment and no f r e e c h l o r s u l f u r o n was o b s e r v e d . The h a l f - l i f e o f c h l o r s u l f u r o n was e s t i m a t e d t o be o n l y 2-3 h r (3D* D i c l o f o p - m e t h y l i s m e t a b o l i z e d i n wheat by h y d r o l y s i s o f an e s t e r group f o l l o w e d by r i n g - h y d r o x y l a t i o n and subsequent f o r m a t i o n o f a B-O-glucoside (Equation 2). The g l u c o s i d e accounted f o r 85J o f the h e r b i c i d e 24 h r a f t e r t r e a t m e n t and the f r e e r i n g - h y d r o x y l a t e d form(s) accounted f o r only 3.U (32). 1,Hydrolysis

2,Glucose

CHLORSULFURON

(1)

3,Glucose

DICLOFOPMETHYL (2)

When more than one h y d r o x y l group i s p r e s e n t , considerable v a r i a t i o n may be observed among p l a n t s p e c i e s r e g a r d i n g the s i t e t h a t w i l l become g l u c o s y l a t e d . When c e l l c u l t u r e s o f e i g h t s p e c i e s were t r e a t e d w i t h o - h y d r o x y b e n z y l a l c o h o l , s i x s p e c i e s formed the g l u c o s i d e a t the b e n z y l i c h y d r o x y l group ( N i c o t i a n a , D a t u r a , D u b o i s i a , C a t h a r a n t h u s , and Bupleurum, 97-100J) w h i l e two s p e c i e s formed p r i m a r i l y the p h e n o l i c g l u c o s i d e ( G a r d e n i a and Llthospermum, 69 and 81J, r e s p e c t i v e l y ) (33). Most B-O-D-glucosides a r e thought t o be formed by g l u c o s y l t r a n s f e r a s e enzymes t h a t r e q u i r e UDPG as the g l u c o s y l donor. However, a t r a n s - g l u c o s y l a t i o n system t h a t can u t i l i z e c e r t a i n g l u c o s i d e s i n the f o r m a t i o n o f new O - g l u c o s i d e s and a system t h a t r e q u i r e s ATP and CoA f o r the s y n t h e s i s o f some g l u c o s e e s t e r c o n j u g a t e s have been r e p o r t e d (J2.,21.). A l t h o u g h g l u c o s y l t r a n s f e r a s e



4.

LAMOUREUX AND

RUSNESS

Xenobiotic

Conjugation

in Higher

Plants

71

enzymes t h a t u t i l i z e p h e n o l s as s u b s t r a t e s have been i s o l a t e d from s e v e r a l p l a n t s p e c i e s , the c o n v e r s i o n o f x e n o b i o t i c a l c o h o l s and p h e n o l s to B-O-D-glucosides has not been e x t e n s i v e l y s t u d i e d a t the enzymatic l e v e l (t2,2J_). A s o l u b l e U D P G t g l u c o s y l t r a n s f e r a s e from g e r m i n a t i n g mung bean was shown to have a broad s u b s t r a t e s p e c i f i c i t y f o r v a r i o u s p h e n o l s and a l k y l a l c o h o l s . S u b s t r a t e s p e c i f i c i t y was based on m o l e c u l a r s i z e ( 3 4 ) . A s o l u b l e UDPG:glucosyl t r a n s f e r a s e from tomato u t i l i z e d v a r i o u s h y d r o x y - c i n n a m i c a c i d s as s u b s t r a t e s and c a t a l y z e d the f o r m a t i o n o f O - g l u c o s i d e s a t pH 8 and g l u c o s e e s t e r s a t pH 7 ( 3 5 ) . P a r t i c u l a t e UDPG:glucosyl t r a n s f e r a s e s t h a t u t i l i z e s t e r o l s as s u b s t r a t e s have a l s o been r e p o r t e d (21). R e c e n t l y , a s o l u b l e UDPG:glucosyl t r a n s f e r a s e t h a t u t i l i z e d p e n t a c h l o r o p h e n o l as a s u b s t r a t e was i s o l a t e d from wheat and soybean c e l l c u l t u r e s (36). A non-UDPG-dependent enzyme was i s o l a t e d from pea t h a t c a t a l y z e s the t r a n s - g l u c o s y l a t i o n o f p h e n o l s , but the r o l e o f t h i s enzyme system i n x e n o b i o t i c m e t a b o l i s m has not been w e l l explored (37,38). A d i r e c t r e l a t i o n s h i p has been o b s e r v e d between the a b i l i t y o f some p l a n t s p e c i e s t o form O - g l u c o s i d e s o f h e r b i c i d e s and r e s i s t a n c e o f t h o s e s p e c i e s t o the h e r b i c i d e s ; however, i n most c a s e s a phase I r e a c t i o n p r o c e e d s g l u c o s e c o n j u g a t i o n and i t i s not known whether the phase I r e a c t i o n or c o n j u g a t i o n r e s u l t s i n h e r b i c i d e d e t o x i f i c a tion. Chlorpropham i s m e t a b o l i z e d i n p l a n t s by r i n g - h y d r o x y l a t i o n and subsequent c o n j u g a t i o n w i t h g l u c o s e ( E q u a t i o n 3 ) . In v i t r o , the

2,Glucose

2,Glucose

p h e n o l i c i n t e r m e d i a t e was a t l e a s t as t o x i c by s e v e r a l c r i t e r i a as the p a r e n t h e r b i c i d e , but the O - g l u c o s i d e was not b i o l o g i c a l l y a c t i v e (39). In c o n t r a s t , i s o u r o n , a d i m e t h y l u r e a h e r b i c i d e , was r a p i d l y m e t a b o l i z e d i n r e s i s t a n t sugarcane by o x i d a t i o n and subsequent g l u c o s i d e c o n j u g a t i o n ( E q u a t i o n 4). In s u s c e p t i b l e k i d n e y bean, m e t a b o l i s m o c c u r e d more s l o w l y by N - d e m e t h y l a t i o n . O x i d a t i o n o f the t e r t - b u t y l group r e s u l t e d i n l o s s o f a c t i v i t y as a p h o t o s y n t h e t i c i n h i b i t o r i n C h l o r e l l a , but i t was not determined whether C h l o r e l l a formed an O - g l u c o s i d e o f the o x i d i z e d form o f i s o u r o n . Nd e m e t h y l a t i o n r e s u l t e d i n o n l y a p a r t i a l l o s s o f a c t i v i t y (40,211). R e s i s t a n c e o f p l a n t s to c h l o r s u l f u r o n h e r b i c i d e i s r e l a t e d t o metabolism. R e s i s t a n t s p e c i e s r a p i d l y m e t a b o l i z e d c h l o r s u l f u r o n by r i n g - o x i d a t i o n f o l l o w e d by O - g l u c o s i d e f o r m a t i o n w h i l e s u s c e p t i b l e s p e c i e s d i d not m e t a b o l i z e c h l o r s u l f u r o n a t an a p p r e c i a b l e r a t e (3D ( E q u a t i o n 1). I t was not proven whether o x i d a t i o n o r g l u c o s e c o n j u g a t i o n r e s u l t e d i n d e t o x i f i c a t i o n . The p h e n y l u r e a h e r b i c i d e , c h l o r t o l u r o n , was r a p i d l y m e t a b o l i z e d i n r e s i s t a n t s p e c i e s by o x i d a t i o n and g l u c o s i d e c o n j u g a t i o n o f a m e t h y l group s u b s t i t u t e d on the p h e n y l r i n g , but m e t a b o l i s m i n s u s c e p t i b l e s p e c i e s o c c u r e d more s l o w l y by a l t e r n a t e r o u t e s such as N - d e m e t h y l a t i o n (4j0. Diclofopm e t h y l h e r b i c i d e was more r a p i d l y m e t a b o l i z e d by r i n g - h y d r o x y l a t i o n f o l l o w e d by g l u c o s i d e f o r m a t i o n i n a r e s i s t a n t s p e c i e s than by g l u c o s e e s t e r f o r m a t i o n i n s u s c e p t i b l e s p e c i e s (32) ( E q u a t i o n 2).



72


The s t a b i l i t y o f most xenobiotic O-glucosides i n plants has not been studied over the l i f e of the plant. In some cases, they are c l e a r l y stable for moderate periods of time. In other cases, they may be metabolized rapidly to more complex glycosides as discussed l a t t e r . A recent study on the metabolism of the herbicide chlortoluron i n wheat suggested that the O-glucoside of this herbicide was r e l a t i v e l y stable u n t i l the plant reached maturity. At that time, the O-glucoside appeared to be hydrolyzed and the hydroxylated chlortoluron moiety then underwent additional oxidation (41). Studies with m-phenoxybenzyl alcohol i n cotton leaves suggested that the B-O-glucoside of t h i s xenobiotic undergoes rapid turn-over within the c e l l (30). N-Glucosides Aromatic or heterocylic xenobiotics that contain a primary or secondary amino group can be metabolized i n plants to N-glucosides (Table IV). Alternate routes of metabolism of amino groups u t i l i z e d by plants include conjugation with malonic acid, or perhaps more commonly, the formation o f bound residue. N-Glucoside formation i s a common mechanism of xenobiotic metabolism and has been observed i n a variety of plant species. In some cases, resistance or susceptib i l i t y to a herbicide has been correlated to the a b i l i t y of a plant species or c u l t i v a r to detoxify a herbicide by formation of an Nglucoside. Chloramben herbicide appeared to be metabolized almost exclusively to an N-glucoside i n resistant soybean, but i n suscept i b l e barley metabolism was much slower and only 15$ N-glucoside was formed (42). Further studies with tissue sections treated for 7 hr with chloramben v e r i f i e d that N-glucoside formation was a major route of metabolism i n resistant species (morning glory, 76J; squash, 84$; snapbean, 67$; soybean, 62$), but was less signficant i n susceptible species (velvet l e a f , 23$; barley, 19$; giant foxt a i l , 15$) (26). The N-glucoside of chloramben i s a terminal r e s i due i n soybean grown to maturity (44). Dinoben, the nitro-analogue of chloramben, i s metabolized by reduction of the nitro-group followed by N-glucoside formation (45). Metribuzin herbicide i s metabolized to an N-glucoside i n tomato and the l e v e l of resistance to metribuzin among different tomato c u l t i v a r s was correlated to the l e v e l of UDPG:glucosyl transferase i n the young tomato seedlings (46). The N-glucoside of metribuzin i s not a terminal residue, but i s rapidly metabolized to a 6-0-malonylglucose derivative (46). The metabolism of pyrazon herbicide to an N-glucoside i s also thought to be related to plant resistance, but evidence for this i s not as good as with the previous two herbicides (47). The primary route of picloram metabolism i n sunflower i s by formation of the N-glucoside of the primary amino group (15$, 1 day; 55$, 3 days; and 72$, 7 days). Sunflower i s very susceptible to picloram and i t was proposed that metabolism occured too slowly to prevent phytotoxicity (48). The N-glucoside of picloram was also formed i n French bean, 4$; barley, 2.4$; and cucumber, 20$; however, the primary route of picloram metabolism i n most species other than sunflower appears to be by glucose ester formation (48). The N-glucoside of picloram was not phytotoxic and appeared to be resistant to further metabolism i n French bean. Another excellent example of N-glucoside formation i n


4.

Table IV.

Xenobiotic


Xenobiotic


Conjugation

in Higher Plants

73

Xenobiotics Metabolized to N-Glucosides i n P l a n t s Phase I Reaction

Chloramben

—

Dinoben

RED

Halogenated Anilines

Squash,Corn Soybean,etc 0

0

Hymexazol

ISO

Rice Cucumber

Isoxathion

HYD ISO OXD

Cabbage Bean Sweet Pea, Pea

REF

l4

C,CC,TLC,H+-Hydrol, Glc-Anal,Acet-Conj-MS,NMR 14

Soybean Soybean

Isoxazolinone

Methods of Isolation and Characterization

Species

a

+

C,TLC,Synth,H -Hydrol, Aglycone-Co-TLC

43 45 50

+

TLC,H -Hydrol,Color Analysis 14

175

14

49

C,CC,TLC,H+-Hydrol,GlcAnal , TMS-Con j-MS, IR, NMR C,Synth,TLC,H+-Hydrol, Acet-Conj-IR,NMR 14

181

C,TLC,Color Analysis

c

Metribuzin

Tomato

Picloram

46

1 4

C,TLC,HPLC,H+-Hydrol, Glc-Anal,Acet-Conj-MS, NMR,TMS -Conj-NMR d

Propanil

HYD

Pyrazon a

0

c

d

Sunflower Cucumber

14

C,CC,PC,TLC,H+-Hydrol, Glc-Anal,Acet-Conj-IR,MS

48

Rice

14

C,Synth,PC,TLC,H+Hydrol,Glc-Anal,TMSCon j-GC,IR

182

Red Beet

H,CC,TLC,H -Hydrol,GlcAnal ,Aglycone-Glc-GC,IR

3

+

47

Abbreviations are described i n Table I I I . Reduction (RED). Enzymatic synthesis from plant extracts. TMSstrimethyl s i l y l ether.

plants i s the metabolism of the i n s e c t i c i d e , isoxathion i n bean, cabbage, and Chinese cabbage. In bean, N-glucoside formation occured slowly (4.3$, 1 day; 21.6$, 6 days; and 54.1$; 15 days), but i t was the major route of metabolism (49) (Equation 5). Glucose'^

ISOXATHION

n

Glucose^

N-Glucosides can be formed by soluble UDPG:glucosyl transferase enzymes comparable to the transferases that form O-glucosides. A soluble UDPGrglucosyl transferase with a broad substrate s p e c i f i c i t y for arylamines was isolated from soybean (50) and a similar enzyme


74



that catalyzed the formation of an N-glucoside of metribuzin was detected i n tomato (46). A g-configuration would be expected for Nglucosides formed by a UDPG:glucosyl transferase system. NMR e v i dence supporting such a configuration was reported for the Nglucoside of metribuzin ( 4 6 ) . N-Glucosides appear to be resistant to hydrolysis by 8-glucosidase TV2,49) and i n some cases they may be somewhat resistant to acid hydrolysis (X2). Although N-glucosides can undergo additional metabolism i n plants, e.g., to malonylglucosides ( 4 6 ) , they are considered non-toxic and r e l a t i v e l y stable i n plant tissue ( 1 2 ) . Glucose ester conjugates Xenobiotic and endogenous substrates that contain free or potential carboxyl groups are commonly metabolized i n plants to 0-1 glucose ester conjugates. At least 13 pesticides as well as a variety of endogeneous substrates are known to be metabolized to glucose esters i n various plant species (Table V). Amino acid conjugation and f o r mation of bound residue are two metabolic reactions involving the carboxyl group that appear to compete with glucose ester conjugat i o n ; however, reactions not involving the carboxyl group, such as r i n g hydroxylation/glucoside formation or N-glucoside formation may also compete with glucose conjugation (32,53). Glucose ester conjugation i s more common and important i n plants than amino acid conjugation (60. Most xenobiotics known to form glucose ester conjugates i n plants are aromatic or heterocyclic carboxylic acid derivatives. Glucose ester conjugates can be formed very rapidly i n plants: m-phenoxybenzoic acid was metabolized i n 81J y i e l d to a glucose ester conjugate within 18 hr i n excised cotton leaves (51_), Nbenzoyl-N-(3-chloro-4-fluorophenyl)-DL-alanine was metabolized i n 85< y i e l d to simple and complex glucose esters within 24 hr i n oat (52), and diclofop-methyl was metabolized by hydrolysis and glucose ester conjugation i n 54% y i e l d within 24 hr i n oat root ( 3 2 ) . In plants, most xenobiotic glucose ester conjugates are probably formed by a UDPG:glucosyl transferase mechanism ( 3 5 , 5 3 - 5 6 ) , but an ATP/CoA-dependent system for the synthesis of the glucose ester of IAA has also been reported (56). The enzymatic synthesis of a variety of glucose esters, primarily with endogenous substrates, has been accomplished with crude and p a r t i a l l y p u r i f i e d enzyme systems isolated from several plant tissues ( 3 5 , 5 3 - 5 6 ) . A soluble UDPG:glucosyl transferase from tomato f r u i t catalyzed the synthesis of both glucosides (pH 8) and glucose esters (pH 7) ( 3 5 ) . A single enzyme was thought to catalyze both reactions. The enzyme displayed a broad substrate s p e c i f i c i t y for hydroxylated benzoic and cinnamic acid derivatives and had Km values of 0.8 to 10 uM for both the donor and acceptor substrates. A soluble UDPG:glucosyl transferase enzyme from oak leaves that u t i l i z e d a variety of benzoic and c i n namic acid derivatives had much higher Km values for both donor and acceptor substrates ( 5 3 ) . I t did not appear to form simple 0 glucosides. The glucosyl transferases from both tomato f r u i t and oak leaves were unstable. The glucose ester conjugation reaction catalyzed by these enzymes was either highly reversible (53) or the glucose esters were readily hydrolyzed by esterases present i n the enzyme preparations ( 3 5 ) . Chloramben herbicide i s metabolized to a glucose ester i n some plant species and t h i s metabolite i s hydro-


4.


Table V.

Conjugation

Xenobiotics Metabolized

Xenobiotic

Phase I Rxn

Abscisic Acid


Xenobiotic

Methods of Isolation and Characterization

14

121

Peanut C e l l Culture

Chloramben

——

Corn,Barley, C,CC,TLC,H -Hyd,GlcAnal ,Acet-Conj-MS,NMR Barnyardgrass, etc.

Cypermethrin

HYD

Lettuce, Cotton

C,PC,TLC,HPLC,H -Hyd, Glc-Anal,Acet-Conj-MS

Cyclohexanecarbo x y l i c Acid

Bush Bean

C,TLC,Synth,Base-Hyd, Aglycone-Co-TLC

DIB

Tomato

C,TLC,H -Hyd,Glc-Anal, Acet-Conj-MS,NMR,IR

Oat

C,TLC,HPLC,H -Hyd, Synth,Acet-Conj-MS

Soybean, Corn

C,TLC,B-Glcase, Aglycone-Co-TLC,PC

2,4-D

REF 183

H,CC,HPLC,TLC,B-Glcase, Acet-Conj-CC,MS,NMR,UV

HYD

HYD

a

3

Carboxin

Diclofop-Methyl

75

to Glucose Esters i n P l a n t s

Species Xanthium, Spinach

in Higher Plants

C,CC,HPLC, Acet-Conj-GC,MS 14

+

1 4

+

1 4

14

14

+

+

43

11

130 70 32

14

172

1 4

184

14

FlampropIsopropyl

HYD

Barley


Fluvalinate

HYD

Cabbage

C,TLC,HPLC,AcetCon j-MS, NMR

185

Hydroxcinnamic Acids

Duckweed

Co-HPLC

186

IAA

Pine

C,TLC,B-Glcase, Aglycone-Co-TLC,Conj-NMR

14

187

14

179

14

51

Permethrin

HYD

Cotton, Bean

C,Synth,TLC,B-Glcase, Aglycone-Co-TLC

3-Phenoxybenzoic Acid

——

Soybean, Grape, Tomato

C,TLC,HPLC,H+-Hyd, IR,Acet-Conj-NMR,MS

Abbreviations

are described i n Table I I I .

lyzed readily to free chloramben i n v i t r o and i n vivo (43). The formation of glucose and myo-inositol esters of IAA i n young corn seedlings was also reported to be a r e v e r s i b l e process (56). Several herbicides are metabolized to glucose ester conjugates i n species that are suspectible to the herbicides. Diclofop-methyl i s metabolized to a glucose ester i n susceptible oat, but i n r e s i s tant wheat i t i s metabolized more r a p i d l y by ring-hydroxylation followed by glucoside formation (32). Chloramben i s metabolized to a glucose ester and bound residue i n susceptible barley, but i t appears to be metabolized more r a p i d l y to an N-glucoside i n r e s i s -




76

tant morning glory (43). I t i s not clear whether the phytotoxicity i n the two examples above was due to an apparent lower rate of metabolism or the r e v e r s i b i l i t y o f the glucose ester conjugation react i o n . Glucose esters of natural plant hormones and auxin are thought to serve as an inactive chemical reservoir of these compounds and i t has been suggested that glucose esters of herbicides may function i n a similar manner 0 2 ) . As with other xenobiotic glucose conjugates, glucose ester conjugates can be further metabol i z e d by conjugation reactions that involve the glucose moiety. These additional metabolic processes may not s i g n i f i c a n t l y s t a b i l i z e the 0-1 glucose ester bond from non-enzymatic hydrolysis, but they may block enzymatic processes such as B-glucosidase hydrolysis or the reverse reaction i n the synthesis of the 0-glucose ester. Thus, the formation of these more complex metabolites may r e s u l t i n d e t o x i f i c a t i o n of the xenobiotic. Most xenobiotic glucose ester conjugates appear to be B-0-1 esters; however, the glucose ester o f chloramben appeared to be an a-0-1 glucose ester (43). The 0-1 glucose esters can undergo trans e s t e r i f i c a t i o n under mildly basic conditions (NaHCOj) (52); a c y l migration to y i e l d 0-2, 0-4, and 0-6 glucose esters T 5 6 ) ; or they can undergo ammonolysis to y i e l d amide derivatives of the aglycone (43). As a r e s u l t , a r t i f a c t s of glucose esters have occasionaly been reported as metabolites (52). N-0-Glucosldes At least two we11-documented cases o f plant xenobiotic metabolism to N-0-glucosides have been reported (57,58). Oxamyl i n s e c t i c i d e was converted to an N-0-glucoside i n tobacco, young peanut plants, a l f a l f a , and the f r u i t o f orange and tomato (57) (Equation 6 ) . This

(CH3) 2NJLC=N-0-ILNHCH3 SCH3

>

>

(CH3) N«iLc=N-0-Glucose SCH3 2

(6)

OXAMYL glucoside was a major metabolite (35-90% o f the residue) i n orange f r u i t , tobacco, young peanut plants, and a l f a l f a . In some tissues, the mono-N-demethylated form of this glucoside was also observed. These N-0-glucosides were not hydrolyzed by B-glucosidase. In apple f r u i t , potato tubers, and hay from mature peanut plants, more polar metabolites were produced which apparently yielded these glucosides upon hydrolysis with B-glucosidase. These more polar residues were suggested to be polyglucosides o f the simple N-0-glucosides (57). The simple glucosides were i d e n t i f i e d by chromatography, EI/MS, GC/MS, and synthesis. Methazole herbicide was metabolized to an N-0-glucoside (13T i n spinach plants within 48 hr following treatment (58). As with oxamyl, the R-N-0H group was apparently formed by hydrolysis prior to glucoside f o r mation (Equation 7). Both the glucoside and the malonyl ester o f the glucoside were i d e n t i f i e d by a combination o f techniques including H - and 13c -NMR, CI/MS, 3-glucosidase hydrolysis, and synthesis. In contrast to the mono-N-0-glucosides of oxamyl, the 1


4.

LAMOUREUX AND

RUSNESS

Xenobiotic

Conjugation

in Higher Plants

11


N-O-glucoside of methazole was hydrolyzed by &-glucosidase; however, the 6-0-malonyl ester of t h i s glucoside was not hydrolyzed. S-OLUCOSIDES S-Glucoside conjugates have only rarely been reported as xenobiotic metabolites i n higher plants; however, glucosinolates respresent a major class of natural products found i n cruciferous plants (59-61). In Tropaeolum majus L., glucosinolates are synthesized from t h i o hydroxamates by a UDPG:glucosyl transferase that accepts a wide variety of thiohydroxamates as glucose acceptors. The products of these reactions are desulfoglucosinolates (Equation 8) which can then be converted to glucosinolates (Equation 9). The glucosyl transferase involved i n t h i s reaction can also u t i l i z e TDPG as the glucosyl donor, but this substrate i s only 10% as e f f i c i e n t as UDPG. UDP-xylose and UDP-galactose did not serve as glycosyl donors. The enzyme was not tested with a broad range of thio-phenols or t h i o alcohols; therefore, i t i s not known whether t h i s enzyme could function i n the metabolism of a broad range of xenobiotics (62). R/Aryl-C-S-glucose

R/Aryl-C-S-glucose

N-OH

N-OSO3H

DESULFOGLUCOSINOLATE

(8)

GLUCOSINOLATE

(9)

Two xenobiotics that have been reported to form thioglucosides as metabolites i n higher plants are dimethyldithiocarbamate (63) and 4-chloro-4»,6-bis(isopropylamino)-6 ethylamino-di(s-triazinylT^ s u l f i d e (64). Radioactive substrates were not used i n either of these studies, but i n both cases the reported S-glucoside was synthesized and compared to the isolated metabolite. The thioglucoside of dimethyldithiocarbamate was produced i n cucumber, broad bean, and potato (Equation 10). I t was isolated from potato by 1

I

(CH ) N-C-S-Na 3

2

+

>

I

(CH ) N-C-glucose

paper chromatography and was i d e n t i f i e d by parison to the synthetic S-glucoside. The l i t e appears analagous to the formation of mechanism by which a thio-glucoside would t r i a z i n e s u l f i d e i s not clear (64).

3

2

(10)

chromatographic comformation of t h i s metaboglucose esters. The be formed from the


78



Complex G l u c o s e C o n j u g a t e s I n h i g h e r p l a n t s , x e n o b i o t i c g l u c o s e c o n j u g a t e s f r e q u e n t l y undergo a d d i t i o n a l metabolism by c o n j u g a t i o n o f the g l u c o s e m o i e t y w i t h o t h e r endogenous s u b s t r a t e s . This occurs frequently with various x e n o b i o t i c s i n many p l a n t s p e c i e s ; t h e r e f o r e , complex g l u c o s i d e s s h o u l d be c o n s i d e r e d i n any m e t a b o l i s m s t u d y where g l u c o s e c o n j u g a t i o n might be expected ( T a b l e V I ) . B - G e n t i o b i o s i d e s [ B - O — > 6 ) g l u c o s y l B - O — > 0 ) g l u c o s i d e s ] were p r o b a b l y the f i r s t complex g l u c o s i d e s o f x e n o b i o t i c s i d e n t i f i e d i n p l a n t s . Both o - c h l o r o p h e n o l and c h l o r a l h y d r a t e were m e t a b o l i z e d t o g e n t i o b i o s e c o n j u g a t e s i n tomato and i t was s u b s e q u e n t l y shown t h a t c h l o r a l h y d r a t e and c l o s e l y r e l a t e d compounds were a l s o m e t a b o l i z e d i n the same manner i n g l a d i o l u s and h o r s e r a d i s h (65,66). Gentiob i o s e c o n j u g a t e f o r m a t i o n , i l l u s t r a t e d i n E q u a t i o n 11 w i t h o - c h l o r o CI

(11) HO-

OH

OH

p h e n o l , was shown t o be a two-step e n z y m a t i c r e a c t i o n by i n v i t r o methods t h a t u t i l i z e d two d i s t i n c t UDPG:glucosyl t r a n s f e r a s e enzymes i s o l a t e d from wheat germ (67). The UDPG:glucosyl t r a n s f e r a s e t h a t formed t h e s i m p l e B - O - g l u c o s i d e o f the x e n o b i o t i c was s e p a r a t e d from the UDPG:glucosyl t r a n s f e r a s e t h a t formed t h e B-O-O—>6) g l u c o s y l bond ( E q u a t i o n 11). The second g l u c o s y l t r a n s f e r a s e had a broad substrate s p e c i f i c i t y for phenolic glucosides, suggesting that a wide range o f x e n o b i o t i c s c o u l d be m e t a b o l i z e d t o g e n t i o b i o s e c o n jugates. T h i s enzyme d i d not c a t a l y z e t h e f o r m a t i o n o f g e n t i o b i o s e c o n j u g a t e s from f r e e p h e n o l s , n o r d i d i t c a t a l y z e the f o r m a t i o n o f t r i - o r t e t r a - s a c c h a r i d e c o n j u g a t e s . Diphenamid h e r b i c i d e was a l s o shown t o be m e t a b o l i z e d t o a g e n t i o b i o s e c o n j u g a t e i n tomato and t h i s c o n j u g a t e a l s o appeared t o be formed i n a s e q u e n t i a l manner t h r o u g h a s i m p l e B - O - g l u c o s i d e i n t e r m e d i a t e (68,69). A l t h o u g h the g l u c o s y l t r a n s f e r a s e system from wheat germ d i d not form t r i - and t e t r a - s a c c h a r i d e c o n j u g a t e s , r e c e n t r e p o r t s s u g g e s t t h a t i n v i v o systems do form o l i g o s a c c a r i d e c o n j u g a t e s o f xenob i o t i c s (57,70,71). The p l a n t growth r e g u l a t o r , DIB, was metabol i z e d t o a 1—>0 g l u c o s e e s t e r (6$) a s w e l l a s a d i - ( 2 8 % ) , t r i (44%), and p o s s i b l y a t e j t r a - g l u c o s e c o n j u g a t e i n tomato (70l ( E q u a t i o n 12). 2,4-D may be p a r t i a l l y m e t a b o l i z e d i n c e r e a l g r a i n s t o o l i g o - s a c c a r i d e c o n j u g a t e s , a s was observed w i t h DIB i n tomato (71). Oxamyl was r e p o r t e d t o form o l i g o - s a c c a r i d e c o n j u g a t e s i n t h e

(12)


4.

LAMOUREUX AND RUSNESS Table VI.

Xenobiotic

Conjugation

in Higher

Complex C a r b o h y d r a t e C o n j u g a t e s o f X e n o b i o t i c s by P l a n t s Bond

Xenobiotic

a

Plant

79

Plants Produced

REF

Species


Gentiobiose Conjugates ( 8 - D - g l u c o s y l 1-6 B-D-glucose)

66

o-Chlorophenol

1-0

tomato

2,2,2-Trichloroethanol Chloralhydrate Chloralcyanohydrin

1-0 1-0 1-0

tomato, g l a d i o l u s , horseradish

Diphenamid

1-0

tomato,

Caviunin flavone)

1-0

Dalbergia

Phenol p-Methoxypheno1° m-Dihydroxybenzene L-Mandelnitrile° £-Dihydroxybenzene° 2-Hydroxymethylphenol°

1-0 1-0 1-0 1-0 1-0 1-0

wheat (germ)

Maleic

1-0

Malus, S a l i x , N i c o t i a n a , wheat

1-ester

tomato

(natural plant

0

0

hydrazide

DIB

green

pepper

sisso

65

68,69

188

67

189 70

P o l y g l u c o s i d e Conjugates DIB

1-ester

tomato

70

2,4-D

1-ester

cereals

II

Oxamyl

1-0-N

apple, potato, peanut

57

Heterodissacharide Conjugates ( g l u c o s y l a r a b i n o s e and g l u c o s y l x y l o s e ) m-Phenoxybenzoic a c i d

Cypermethrin

0

m-Phenoxybenzyl a l c o h o l

1-ester

grape, c o t t o n , and o t h e r s p e c i e s

51

1-ester

cotton

73

cotton

30

1-0

6-0-Malonyl-3-D-Glucose

Conjugates

174

1-0

peanut

1-ester

wheat

75

1-ester

cotton

Methazole

1-0-N

spinach

21 58

Acifluorfen

1-0

soybean

94

Mo bam

1-0

barley

Fluorodifen Flamprop Cypermethrin

0

190

C o n t i n u e d on next page



80 Table VI. Bond

Xenobiotic

Metribuzin Fluvalinate

0

Pen t ach lor o pheno 1 a

D


c

Continued Plant

3

Species

1-N

soybean

1-ester

tomato

1-0

soybean, wheat

Ref.

46 185 36

Designates the bond between glucose residue and the xenobiotic These xenobiotics were tested only by i n v i t r o enzymatic methods Complex pyrethroid i n s e c t i c i d e

f r u i t of apple, peanut, and potato (Equation 6). I t was also speculated that chlorpropham was metabolized to oligo-saccharide conjugates i n a l f a l f a (72). Arabinosylglucose and xylosylglucose conjugates were reported as metabolites of m-phenoxy benzoic acid, m-phenoxybenzyl alcohol, and a cyclopropane carboxylic acid derivative (30,51^73) (Table VI). In excised cotton leaves, m-phenoxybenzyl alcohol was metabolized to the B-O-glucoside, an arabinosyl-glucoside and a xylosylglucoside (30). The addition of the glucose and pentose residues occurred sequentially. The glucose from these conjugates was exchangeable with endogenous glucose, with a h a l f - l i f e of only several hours. Interconversion of metabolites was demonstrated (Equation 13). I t

m-phenoxybenzyl alcohol
,l6.,89). With several exceptions, the primary routes of metabolism of GSH conjugates i n plants are similar to those observed i n mammals as discussed by Bakke i n Chapter 16. In sorghum and corn, the f i r s t step i n the catabolism o f a xenobiotic


4.


Xenobiotic

Conjugation

^GSH C

QJ

H

85

Plants

GSH—< C l ^ ^ V C l

2

r ^ CH2CCI3

^

TRIDIPHANE


in Higher

(23)

^

PCNB

(24)

GSH conjugate i s the hydrolysis of the glycine residue rather than the glutamyl residue as occurs i n mammals (15). I t i s not known whether this difference also occurs i n other plant species. Most xenobiotic GSH conjugates i n plants are metabolized at least to cysteine conjugates and cysteine conjugates appear to be the pivotal point i n metabolism. Cysteine conjugates may be endproducts of metabolism, as observed i n methidathion metabolism i n tomato and peanut c e l l suspension culture, or a c i f l u o r f e n metabolism i n soybean and peanut c e l l suspension culture (Jj6). Xenobiotic cysteine conjugates are frequently N-acylated with malonic acid as shown i n Equation 25. This was demonstrated with the following NH

HN-C0-CH -C00H

2

2

X-S-CH -CH-C00H 2

>

X-S-CH -CH-COOH 2

(25)

xenobiotics i n a peanut c e l l suspension culture: EPTC (78$), molinate cysteine conjugate (93$), fluorodifen (74$), PCNB (30$), propachlor (42$), metolachlor (24$), and butachlor (5$). Malonylcysteine conjugation was demonstrated i n 8 o f 11 plant species when PCNB was the substrate; therefore, the reaction appears to be general i n higher plants. The s u l f i d e bond of malonylcysteine conjugates can be oxidized, as was observed i n the metabolism of propachlor i n soybean (16). Isomerization of an S-cysteine conjugate to an N-cysteine conjugate v i a the Smiles rearrangement has been observed i n the metabolism o f two t r i a z i n e xenobiotics i n higher plants: atrazine metabolism i n sorghum (103,104) and dimethametryn metabolism i n r i c e (105). This nonenzymatic rearrangement (Equation 26) has not NH 2

COOH

2

X-S-CH -CH-C00H

>

X-NH-CH-CH -SH 2

(26)

been reported i n GSH conjugate metabolism i n mammals. A lanthionine conjugate was produced by further metabolism of the N-cysteine conjugate of atrazine i n sorghum. In r i c e , the N-cysteine conjugate of dimethametryn was degraded to an amino-triazine. The deamination of xenobiotic cysteine conjugates can lead to thio-acetic acid conjugates, as observed with PCNB i n peanut (100), or t h i o - l a c t i c acid conjugates, as observed with EPTC i n corn and cotton (106) or PCNB i n peanut (100). Cysteine conjugates can also undergo B-lyase cleavage to thioalcohols, as observed with PCNB i n peanut (100)» The thioalcohols can be methylated by an S-adenosylmethionine methyltransferase system, as demonstrated with an i n v i t r o enzyme system derived from onion root (101). Pentachloro-



86


phenylmethylsulfide, derived from PCNB, has been detected i n several plant species. The formation of methylsulfides as xenobiotic metab o l i t e s present i n the environment i s of current interest (89). The a b i l i t y of plants to form t h i s class of metabolites from a range of xenobiotics i s uncertain. The methyl transferase enzyme system from onion used a number of different thiophenols as substrates (J5), but the d i s t r i b u t i o n of the necessary 3-lyase and methyl transferase enzymes among other higher plants i s unknown. Several xenobiotics that are metabolized by GSH conjugation i n plants, including atrazine, propachlor, and PCNB, produce s i g n i f i cant levels of bound residue (J5). It appears that the bound r e s i due may be formed from the GSH pathway with either a cysteine conjugate or a t h i o l as a precursor (J5). The chemical nature of these bound residues has not been determined. Glutathione S-transferase enzymes have been detected i n a variety of different plant species (24.78,84,97.101,107-110). However, detailed enzyme studies have been limited to corn (24,78, 109,110) and pea (85,107,110). The f o l i a r tissue of corn contains a GST isozyme that appears rather unique i n i t s a b i l i t y to catalyze the conjugation of various 2-chloro-s-triazines with GSH (78). The molecular weight and other properties of t h i s isozyme are similar to those reported for many mammalian GST isozymes; however, the pH optimum of t h i s isozyme for atrazine i s somewhat lower than that reported for many GST isozymes (24,78,109)* When corn i s treated with R-25788, a second isozyme i s induced (24). The induced isozyme i s very e f f e c t i v e i n catalyzing the GSH conjugation of an a-chloroacetamide herbicide (alachlor), but i t was not assayed with atrazine (24). A GST enzyme from pea i s very e f f e c t i v e i n catalyzing GSH conjugation of the herbicide fluorodifen (85). This enzyme has a pH optimum and other properties that are comparable to mammalian GST enzymes (85). This enzyme a c t i v i t y was observed i n other plant spec i e s , but fluorodifen resistant species appeared to have higher l e v e l s of this enzyme than susceptible species (85). Additional studies with pea indicated the presence of two soluble GST isozymes, one that u t i l i z e d fluorodifen and one that u t i l i z e d ^-cinnamic acid as substrates (107). These isozymes appeared to form aggregates during p u r i f i c a t i o n . In addition, a microsomal GST was detected i n pea that u t i l i z e d both t-cinnamic acid and benzo(a)pyrene as substrates (107). Soybean c e l l suspension cultures metabolized t cinnamic acid i n a 6% y i e l d to a product that corresponded to the GSH conjugate of ^-cinnamic acid by paper chromatography (107). A transferase system capable of u t i l i z i n g either cysteine or GSH to form conjugates of isopropyl-3 -chloro-4 -hydroxy-carbanilate was detected i n etiolated oat seedlings (111,112). At least two enzyme systems capable of catalyzing the above reactions were present (112). Excised oat tissues produced a metabolite corresponding to the i n v i t r o reaction product with cysteine. The product appeared to be a cysteine conjugate, but the mechanism of the reaction and the structure of the product were not determined (111). 1

N-Malonyl and 0-malonyl

1

conjugates

Acetylated xenobiotic conjugates are not commonly formed i n higher plants; however, the d i r e c t or i n d i r e c t conjugation of xenobiotics




Xenobiotic

Conjugation

in Higher

Plants

87

with malonic acid i s a common occurrence. D-Amino acids, xenobiotic cysteine conjugates, and aromatic and heterocyclic compounds containing a primary amino group can be metabolized i n plants, v i a amide bond formation, to malonic acid conjugates. Xenobiotic g l u cose conjugates can be metabolized i n plants to malonic acid conjugates v i a a 1-6 ester bond between malonic acid and the glucose residue. Malonylcysteine and malonylglucose conjugates are secondary or t e r t i a r y metabolites derived from GSH- and glucoseconjugates. These malonyl conjugates were discussed i n previous sections dealing with complex glucosides and glutathione conjugates. D-Amino acids are not usually natural plant constituents and when plants are treated with D-amino acids they are generally converted to N-malonyl conjugates (113-115) (Equation 27). NaturallyR-CH -CH-C00H 2

*IH

—p>

R-CH -CH-C00H 2

\

2

Malonyl CoA

HN-C0-CH -C00H

(

2

7

)

2

CoA

occurring L-amino acids are not metabolized i n t h i s manner and i t i s assumed that N-malonyl conjugation i s a mechanism u t i l i z e d by plants to remove D-amino acids from normal metabolic pathways (113-115). Most D-amino acids are substrates for t h i s pathway, the N-malonyl conjugation reaction occurs i n many plant species, and the N-malonyl conjugates appear to be terminal products of metabolism, i e . , they appear to be stable i n the plant once they are formed (114). NMalonyl conjugation of D-amino acids appears to be similar to Nmalonyl conjugation of xenobiotic cysteine conjugates as discussed previously. The natural ethylene precursor, aminocyclopropane carboxylic acid (ACC), i s also converted to an N-malonyl conjugate i n plants (116,117). Soluble enzyme preparations that catalyze the Nmalonyl conjugation of ACC with malonylcoenzyme A as the natural cosubstrate have been isolated from mung bean (118) and peanut (119). D-Amino acids i n h i b i t the i n v i t r o conjugation of ACC with malonic acid (118) and a malonyltransferase from peanut that u t i l i z e s Dtryptophan as a substrate for N-malonyl conjugation also u t i l i z e s ACC as a substrate (119). These malonyltransferase enzymes have not been assayed with xenobiotic cysteine conjugates to determine i f the same enzymes are involved i n both transformations. Likewise, i t i s not known i f the xenobiotic cysteine conjugates undergo an isomerization from the L-to D-configuration i n order for the malonylation reaction to proceed. Aromatic and heterocyclic amines are usually metabolized i n higher plants by formation of N-glucosides or bound residues. In several cases, however, aromatic and heterocyclic amines have been p a r t i a l l y metabolized to N-malonyl conjugates. I t i s not known whether this type of N-malonyl conjugation i s r e s t r i c t e d to a few selected amines nor i s i t known whether t h i s reaction i s u t i l i z e d by more than a few plant species such as peanut and soybean. Botran was one of the f i r s t xenobiotics reported to be metabol i z e d to an N-malonyl conjugate by t h i s mechanism i n plant tissues (120) (Equation 28). The N-malonyl conjugate of botran was the major metabolite i n both soybean and soybean c a l l u s culture. I t was isolated by chromatographic methods and i d e n t i f i e d by synthesis and by chemical and mass spectral methods.



88

>

HN 2

>

H N2

(28)

C-CH -COOH 2

BOTRAN


C a r b o x i n f u n g i c i d e was m e t a b o l i z e d t o m a l o n a n i l i c a c i d and the glucoside of p-hydroxymalonanilic a c i d i n the f r u i t o f peanut p l a n t s and i n peanut c e l l s u s p e n s i o n c u l t u r e s (121) ( E q u a t i o n 29). Both

0-Glucose

(29) 0

CARBOXIN

m e t a b o l i t e s were i d e n t i f e d by mass s p e c t r a l and c h e m i c a l methods as w e l l as by s y n t h e s i s . Other major m e t a b o l i t e s were c a r b o x i n s u l f o x i d e (30$ o f the r e s i d u e ) and bound r e s i d u e (21$). The 0g l u c o s i d e o f m a l o n a n i l i c a c i d i s an example o f an u n u s u a l d i c o n j u g a t e i n which two p o l a r groups have been c o n j u g a t e d a t d i f f e r e n t s i t e s on the same x e n o b i o t i c m o l e c u l e . M e t r i b u z i n i s m e t a b o l i z e d r a p i d l y i n tomato t o an N - g l u c o s i d e which i s s u b s e q u e n t l y c o n v e r t e d t o a m a l o n y l g l u c o s i d e . In soybean, m e t r i b u z i n i s m e t a b o l i z e d more s l o w l y by c o n j u g a t i o n w i t h homoglut a t h i o n e and by f o r m a t i o n o f bound r e s i d u e ; an N-malonyl c o n j u g a t e i s formed i n soybean as a minor p r o d u c t (95) ( E q u a t i o n 30).

(30)

A s o l u b l e enzyme (100,000g s u p e r n a t a n t ) was o b t a i n e d from peanut c e l l s u s p e n s i o n c u l t u r e s t h a t c a t a l y z e d the f o r m a t i o n o f m a l o n a n i l i c a c i d from a n i l i n e and m a l o n y l CoA i n a 16$ y i e l d (121). T h i s enzyme p r e p a r a t i o n d i d not u t i l i z e ACC as a s u b s t r a t e (122). I n a d e t a i l e d s t u d y o f the m a l o n y l t r a n s f e r a s e s from peanut, f o u r d i s t i n c t m a l o n y l t r a n s f e r a s e enzymes w i t h d i f f e r e n t s u b s t r a t e s p e c i f i c i t i e s were i s o l a t e d from s e e d l i n g h y p o c o t y l s and l e a v e s (119). These f o u r m a l o n y l t r a n s f e r a s e enzymes had v e r y d i s t i n c t s u b s t r a t e s p e c i f i c i t y r e q u i r e m e n t s , and e x c e p t f o r one o f t h e s e enzymes, no o v e r l a p i n s u b s t r a t e s p e c i f i c i t y was o b s e r v e d . The f o u r m a l o n y l t r a n s f e r a s e s were a c t i v e w i t h the f o l l o w i n g s u b s t r a t e s : (a) 3,5d i c h l o r o a n i l i n e , (b) a n t h r a n i l i c a c i d , ( c ) 2-methoxyethanol, and (d) D - t r y p t o p h a n and a m i n o c y c l o p r o p a n e c a r b o x y l i c a c i d . The m a l o n y l t r a n s f e r a s e from peanut l e a v e s and h y p o c o t y l s t h a t u t i l i z e d 3,5-dic h l o r o a n i l i n e as a s u b s t r a t e i s p r o b a b l y s i m i l a r t o the t r a n s f e r a s e from peanut c e l l c u l t u r e t h a t u t i l i z e d a n i l i n e as a s u b s t r a t e . These m a l o n y l t r a n s f e r a s e s s h o u l d be s u b j e c t e d t o d e t a i l e d s u b s t r a t e s p e c i f i c i t y s t u d i e s to a s s e s s t h e i r p o t e n t i a l r o l e i n x e n o b i o t i c metabolism i n p l a n t s .




Xenobiotic

Conjugation

in Higher

Plants

89

Although m a l o n y l c o n j u g a t i o n o f D-amino a c i d s was assumed t o be a d e t o x i f i c i a t i o n p r o c e s s , some m a l o n y l c o n j u g a t e s may be b i o l o g i cally active. A s e r i e s o f 25 m a l o n a n i l i c a c i d s were t e s t e d f o r g r o w t h - r e g u l a t i n g p r o p e r t i e s i n h i g h e r p l a n t s . U n s u b s t i t u t e d malon a n i l i c a c i d was a potent s t i m u l a t o r o f r o o t - g r o w t h i n cucumber ( 1 2 3 ) . I t remains t o be determined whether t h i s a c t i v i t y was due t o m a l o n a n i l i c a c i d o r t o t h e h y d r o l y s i s p r o d u c t , a n i l i n e , which may have been l i b e r a t e d i n t h e r o o t s . I n peanut p l a n t s , amidase enzymes t h a t h y d r o l y z e t h e N-malonyl c o n j u g a t e o f ACC a r e a p p a r e n t l y p r o duced a s the p l a n t ages ( 1 1 9 ) . T h e r e f o r e , c a u t i o n s h o u l d be e x e r c i s e d i n making assumptions about t h e s t a b i l i t y o f x e n o b i o t i c m a l o n y l c o n j u g a t e s based on s h o r t - t e r m e x p e r i m e n t s . I t has been h y p o t h e s i z e d t h a t N-malonyl c o n j u g a t i o n may be a mechanism u t i l i z e d to s t o r e p r o d u c t s i n a b i o l o g i c a l l y i n a c t i v e s t a t e i n c e l l v a c u o l e s

(119). O-Malonylconjugates o f phenols o r a l k y l a l c o h o l s have n o t been commonly r e p o r t e d a s m e t a b o l i t e s o f x e n o b i o t i c s i n p l a n t s ; however, 2-methoxyethanol i s a s u b s t r a t e f o r a m a l o n y l - t r a n s f e r a s e i n peanut, s u g g e s t i n g t h a t t h i s c l a s s o f c o n j u g a t e might o c a s s i o n a l y be p r o duced (119). I t s h o u l d be n o t e d , however, t h a t the Km v a l u e f o r 2-methoxyethanol was v e r y h i g h , g r e a t e r than 100 uM. Amino a c i d

conjugation

X e n o b i o t i c s t h a t c o n t a i n a f r e e o r p o t e n t i a l c a r b o x y l group can be m e t a b o l i z e d by amino a c i d c o n j u g a t i o n i n both p l a n t s and a n i m a l s . T h i s r e a c t i o n i s i l l u s t r a t e d by t h e c o n j u g a t i o n o f 2,4-D w i t h a s p a r t i c a c i d ( E q u a t i o n 3 D . I n h i g h e r p l a n t s , amino a c i d c o n j u g a t i o n i s 9

1

P

CI-/~~\OCH COOH 2

0 H C00H

> ci-^~~\OCH -C-N-AH-CH2-COOH 2

(3D

2,4-D r e l a t i v e l y uncommon and i t o c c u r s u s u a l l y i n c o m p e t i t i o n w i t h o t h e r r e a c t i o n s such a s g l u c o s e e s t e r f o r m a t i o n o r a r o m a t i c r i n g hydroxylation/glucoside formation (20). I t i s g e n e r a l l y r e s t r i c t e d to compounds t h a t have p l a n t growth r e g u l a t o r a c t i v i t y : 2,4-D (11, 124,^25), 2,4,5-T (126,127), MCPA ( U ) , i n d o l e - 3 - a c e t i c a c i d (IAA) (128,129), cyclohexane c a r b o x y l i c a c i d (130), and n a p h t h a l e n e a c e t i c a c i d TT31). 2,4-D was p r o b a b l y t h e f i r s t p e s t i c i d e shown t o be m e t a b o l i z e d to an amino a c i d c o n j u g a t e i n a h i g h e r p l a n t ( a s p a r t i c a c i d c o n j u g a t e i n pea) (124); however, i t had p r e v i o u s l y been shown t h a t IAA, a n a t u r a l p l a n t a u x i n , was m e t a b o l i z e d t o an a s p a r t i c a c i d c o n j u g a t e i n pea ( 1 2 8 ) . S u b s e q u e n t l y , 2,4-D was shown t o be p a r t i a l l y m e t a b o l i z e d t o amino a c i d c o n j u g a t e s i n a v a r i e t y o f p l a n t s p e c i e s and t i s s u e s i n c l u d i n g : wheat; r e d and b l a c k c u r r a n t ; soybean; c o r n ; and c a l l u s t i s s u e c u l t u r e s o f soybean, c o r n , c a r r o t , j a c k b e a n , s u n f l o w e r , and t o b a c c o (11,212). The m e t a b o l i s m o f 2,4-D i n p l a n t s i s h i g h l y s p e c i e s dependent and i n most o f the above s p e c i e s o t h e r r o u t e s o f m e t a b o l i s m appear t o be q u a n t i t a t i v e l y more important than amino a c i d c o n j u g a t i o n . G l u t a m i c and a s p a r t i c a c i d



90


c o n j u g a t e s are u s u a l l y the most abundant amino a c i d c o n j u g a t e s , r e g a r d l e s s o f the p l a n t s p e c i e s o r x e n o b i o t i c (13,20); however, 2,4-D c o n j u g a t e s o f a l a n i n e , v a l i n e , l e u c i n e , p h e n y l a l a n i n e , and t r y p t o p h a n have been i s o l a t e d from soybean c a l l u s (132). Some amino a c i d c o n j u g a t e s appear t o be u n s t a b l e i n p l a n t s . The g l u t a m i c a c i d c o n j u g a t e o f 2,4-D was m e t a b o l i z e d t o 2,4-D, 2,4-D a s p a r t i c a c i d and what appeared t o be g l u c o s e c o n j u g a t e s o f 4hydroxy-2,5-D and 4-hydroxy-3,5-D (132). The r a t e o f metabolism o f 2,4-D g l u t a m i c a c i d appeared t o be g r e a t e r than t h a t o f 2,4-D (132). Amino a c i d c o n j u g a t e s o f 2,4-D have been r e p o r t e d t o be h y d r o l y z e d by almond e m u l s i n ; c o n s e q u e n t l y , some c a u t i o n s h o u l d be e x e r c i s e d i n a t t e m p t i n g t o c l a s s i f y c o n j u g a t e s as g l u c o s i d e s by almond e m u l s i n B - g l u c o s i d a s e h y d r o l y s i s (132). The a s p a r t i c a c i d c o n j u g a t e o f 2,4-D i s a c i d l a b i l e and i n one s t u d y the r e c o v e r y o f t h i s c o n j u g a t e

was o n l y 19% (J33). Amino a c i d c o n j u g a t i o n o f 2,4-D appears t o be more important i n c a l l u s c u l t u r e than i n whole p l a n t s (20), but a number o f f a c t o r s can e f f e c t l e v e l s o f amino a c i d c o n j u g a t e s : the c o n c e n t r a t i o n o f 2,4-D used, age o f t i s s u e (134), l e n g t h o f time between treatment and h a r v e s t , form o f 2,4-D used ( f r e e a c i d , amine s a l t , o r e s t e r ) (133), and the s p e c i f i c s o u r c e o f t i s s u e (135). Four days a f t e r t r e a t m e n t o f soybean p l a n t s w i t h 2,4-D propyleneglycol butyl ester, l e v e l s o f 2,4-D g l u t a m i c and a s p a r t i c a c i d c o n j u g a t e s (75 and 57 ppm, r e s p e c t i v e l y ) were much h i g h e r than the l e v e l o f 2,4-D a c i d (43 ppm). I n comparison, l e v e l s o f g l u t a m i c and a s p a r t i c a c i d c o n j u g a t e s were l e s s than 4 ppm w h i l e f r e e 2,4-D was a t 47 ppm i n soybean p l a n t s t r e a t e d w i t h 2,4-D dimethylamine s a l t (133). L e v e l s o f 2,4-D amino a c i d c o n j u g a t e s v a r i e d d r a m a t i c a l l y i n soybean c a l l u s as a f u n c t i o n o f time f o l l o w i n g t r e a t m e n t : from 1.3 ppm g l u t a m a t e and 0.3 ppm a s p a r t a t e a t 2 days t o o n l y 0.16 ppm g l u t a m a t e and 0.03 ppm a s p a r t a t e a t 10 days (133). L e v e l s o f 2,4-D amino a c i d c o n j u g a t e s a l s o d e c l i n e d s i g n f i c a n t l y i n whole soybean p l a n t s between 4 and 10 days f o l l o w i n g t r e a t m e n t . The metabolism o f 2,4-D i n plants i s e x t r e m e l y c o m p l i c a t e d and i t appears t h a t 2,4-D can be m e t a b o l i z e d t o amino a c i d c o n j u g a t e s o f the g l y c o s i d e s o f 4-hydroxy-2,4-D and t o o t h e r h i g h l y p o l a r u n i d e n t i f i e d p r o d u c t s (180). The metabolism o f o t h e r x e n o b i o t i c s t o amino a c i d c o n j u g a t e s i n p l a n t s appears to be comparable t o 2,4-D m e t a b o l i s m . Naphthalene a c e t i c a c i d (NAA) i s a p l a n t growth r e g u l a t o r used f o r a v a r i e t y o f purposes on h o r t i c u l t u r a l c r o p s . I t i s m e t a b o l i z e d t o an a s p a r t i c a c i d c o n j u g a t e i n cowpea, f r u i t o f the mandarin orange, and i n t o b a c c o m e s o p h y l l p r o t o p l a s t s (131,136). Tobacco m e s o p h y l l p r o t o p l a s t s t h a t were induced t o d i v i d e w i t h NAA m e t a b o l i z e d n e a r l y h a l f o f the NAA i n the medium t o the a s p a r t i c a c i d c o n j u g a t e . The c o n j u g a t e was i d e n t i f i e d by s y n t h e s i s and n e g a t i v e i o n CI/MS (NpO/CHuH131). Cyclohexane c a r b o x y l i c a c i d was m e t a b o l i z e d t o a g l u c o s e e s t e r and an a s p a r t i c a c i d c o n j u g a t e i n bush bean l e a f discs. M e t a b o l i t e s were c h a r a c t e r i z e d by TLC comparison t o s y n t h e t i c s t a n d a r d s (130). S e v e r a l endogenous compounds have been o bserved i n p l a n t s as p e p t i d e c o n j u g a t e s . F o r m y l - and m e t h y l t e t r a h y d r o f o l a t e form p o l y g l u t a m a t e c o n j u g a t e s i n c a r r o t , p o t a t o , t u r n i p and beet s t o r a g e t i s s u e s . Mono- and d i - g l u t a m a t e c o n j u g a t e s were a l s o o b s e r v e d , but a t lower l e v e l s (137). A g l y c y l p h e n y l a l a n i n e d i p e p t i d e c o n j u g a t e o f f e r r u l i c a c i d was i s o l a t e d from b a r l e y g l o b u l i n s by p a r t i a l h y d r o l y s i s (138).



4.

LAMOUREUX AND

Xenobiotic

RUSNESS

Conjugation

in Higher

91

Plants

Some amino acid and glucose ester conjugates of 2,4-D, 2,4,5-T, and IAA are b i o l o g i c a l l y a c t i v e , and i n some cases an amino acid conjugate has been reported to be more active than the parent compound (127,139-142). Some amino acid conjugates are unstable i n plant tissues and t h e i r b i o l o g i c a l a c t i v i t y appears to be correlated to t h e i r ease of h y d r o l y s i s . I t may be that amino acid conjugates are not b i o l o g i c a l l y a c t i v e , but y i e l d the active free acid upon hydrolysis as postulated with glucose esters of some herbicides. The b i o l o g i c a l a c t i v i t y of the a s p a r t i c acid and alanine conjugates of IAA was d i r e c t l y related to the ease of hydrolysis of the amino acid conjugates i n a bean stem assay. The a s p a r t i c acid conjugate was more r e s i s t a n t to hydrolysis and r e l a t i v e l y i n a c t i v e compared to the alanine conjugate (139). The a s p a r t i c acid conjugate of NAA was not b i o l o g i c a l l y active and i t was speculated that the amide bond was not hydrolyzed i n vivo (131). The i n v i t r o synthesis of amino acid conjugates has not been demonstrated with enzyme systems i s o l a t e d from plants i n s p i t e of the great importance of these enzymes to our understanding of the mode of action and metabolism of IAA, NAA, and 2,4-D (56). However, i t appears that these enzymes can be induced by exogenous 2,4-D, NAA, or IAA (56). In v i t r o studies with enzymes from mammals suggest that xenobiotic carboxylic acids are activated by a reaction that requires ATP and CoA. The xenobiotic acyl-CoA d e r i v a t i v e i s then released from the enzyme surface. The i n i t i a l a c t i v a t i o n reactions are catalyzed by d i f f e r e n t acyl-CoA synthetase enzymes with d i f f e r e n t substrate s p e c i f i c i t i e s . Benzoic and phenylacetic a c i d derivatives are activated by what appears to be a butyrl-CoA synthetase present i n the mitochondrial matrix. The f i n a l reaction i s catalyzed by an acyl-CoA:amino acid N-acyltransferase. Two c l o s e l y related forms have been p u r i f i e d from bovine l i v e r mitochondria. One i s s p e c i f i c for benzoyl-CoA, salicyl-CoA and short chain f a t t y acids while the other s p e c i f i c a l l y u t i l i z e s phenylacetyl-CoA or indol-3-ylacetyl-CoA (143). The substrates u t i l i z e d by these mammalian enzymes are remarkably s i m i l a r to the substrates metabolized to amino acid conjugates i n plants. L i p o p h i l i c Conjugates Most xenobiotic conjugation reactions i n plants and animals lead i n i t i a l l y to the formation of polar products such as glycoside or glutathione conjugates, but several reports indicate that plants (144-148) and animals (149) may also form l i p o p h i l i c conjugates. An early i n d i c a t i o n that a widely used a g r i c u l t u r a l chemical might be metabolized to a nonpolar conjugate i n plants came from an in v i t r o enzyme study with ^C-labeled surfactants of the T r i t o n family. A crude p a r t i c u l a t e enzyme preparation from corn shoots catalyzed the formation of f a t t y a c i d ester conjugates from the two ^C-labeled polyethoxylated surfactants indicated below (Equation 32). The ester conjugates were formed p r i m a r i l y from p a l m i t i c and l i n o l e i c acids (>85$). They were i d e n t i f i e d by mass spectrometry and by GLC analysis of hydrolysis products (148). In v i v o , r i c e and 1

1

C H - ^ ~ ^ - ( 0 C H C H ) - 0 H , n=6 or 9 8

17

2

2

n

TRITON


(32)


92 1

barley tissue formed small amounts of ^C-labeled l i p o p h i l i c metabol i t e s that were thought to be f a t t y acid esters of the two R e labeled surfactants; however, deethoxylation and glucoside conjugation were the major routes of metabolism (38). A soluble enzyme preparation from pea catalyzed the formation of the B-glucoside conjugates o f these surfactants (148). An experimental acaricide (Ro 12-0470) was reported to be converted to f a t t y acid esters i n apple f r u i t (145). One day after treatment, 22J o f the applied dose was recovered as f a t t y acid methyl esters, primarily saturated f a t t y acids (C-j6, C-jg* 20 C22) (Equation 33). The f a t t y acid esters were present both on the surface o f the apple and i n extracts of washed apples.


c

^y^CH o-c^ 2

Ro 12-0470

^^j^^^

3 1 1 ( 1

(33) n=l4,16,18,20

These metabolites were i d e n t i f i e d by synthesis and GC/MS. The parent compound (Ro 12-0470) and the f a t t y acid ester metabolites were e a s i l y hydrolyzed (145). Additional studies should be conducted to determine i f these metabolites are produced enzymatically and/or i f they might be formed as a r t i f a c t s during i s o l a t i o n and i d e n t i f i c a t i o n . Fatty acid esters of Ro 12-0470 were not detected i n the f o l i a r tissues of apple. Carbofuran i s metabolized to a conjugate of angelic acid i n carrot, (146,147) (Equation 34). This metabolite was i d e n t i f i e d by

(34) -CO-NH-CH3

O-CO-NH-CH3

CARBOFURAN EI/MS, !H FT-NMR, FT-IR and synthesis. I t was the major residue of carbofuran i n carrot 15 days following treatment (approx. 40$), but i t was not detected i n potato or radish (146,147). In potato and radish, carbofuran was metabolized slowly to water-soluble conjugates and bound residue (146,147). The mechanism by which xenobiotic alcohols or esters are converted to fatty acid esters has not been studied. They could be formed by the action o f lyase enzymes i n the presence of fatty acid g l y c e r y l esters, as i n the conversion of farnesol to farnesol fatty acid esters (150). Some l i p o l y t i c acyl hydrolase enzymes from plants readily catalyze the transfer of lipid-bound f a t t y acids to low MW alcohol acceptors (150,151) and enzymes of t h i s class could be responsible for the occasional formation o f f a t t y acid conjugates of xenobiotic alcohols. Mechanisms involving f a t t y acid a c y l CoA, phospholipids, or direct e s t e r i f i c a t i o n with f a t t y acids might also be involved (152). Recently, a very different class of l i p o p h i l i c conjugates of picloram and 2,4-D were isolated from radish and mustard plants



Xenobiotic Conjugation in Higher Plants

93

(144). The conjugates were formed with p-hydroxy-styryl-mustard, vinyl-mustard, and allyl-mustard (Equation 35). In radish, picloram

-CHsChW •C-O-C-NH-R, R= -CH=CH

2

•OH =

^

(35)

-CH -CH=CH


2

2

and 2,4-D were metabolized to mustard o i l conjugates i n y i e l d s of 30J and 12J, respectively, i n 72 hr. Mustard o i l conjugates were also formed i n mustard plants, but the y i e l d was lower. Mustard o i l conjugates appeared to be the only metabolites of 2,4-D i n these species (144). These conjugates were isolated by chromatographic methods and i d e n t i f i e d by UV, IR, MS, and H NMR (360 M Hz). The metabolites were base l a b i l e , but appeared stable when introduced into sunflower. At present, conjugation with mustard o i l s appears to be a highly unusual route of metabolism, possibly r e s t r i c t e d to plants of the Cruciferae family (144). In other species such as sunflower (48) and leafy spurge (153), picloram i s metabolized to Nglycoside and glucose ester conjugates and 2,4-D i s metabolized to a glucose ester, O-glucosides, and to amino acid conjugates (20). The metabolism of other xenobiotic carboxylic acids should be studied i n radish and mustard to determine i f t h i s i s a general pathway of metabolism i n the Cruciferae family. Improved methods and instrumentation for metabolite i s o l a t i o n and i d e n t i f i c a t i o n , such as c a p i l l a r y GC, GC/MS, HPLC, high f i e l d NMR, FAB/MS, CI/MS, FT-IR and HPLC/MS have made the i d e n t i f i c a t i o n of new or unusual metabolites more p r a c t i c a l . As these advanced techniques are employed to study xenobiotic metabolism i n more diverse species of plants, additional classes of xenobiotics w i l l no doubt be discovered. 1

Bound residues Xenobiotics are frequently metabolized i n plants by mechanisms that lead to the incorporation or inclusion of the xenobiotic into b i o l o g i c a l polymers or tissue residues that are not soluble i n commonly used nonreactive solvents. These residues are frequently refered to as bound, insoluble, or nonextractable residues (j_7). Bound r e s i dues i n plants have most commonly been detected i n plant tissues treated with radioactively-labeled pesticides. These residues were an important topic of a symposium held i n V a i l , Colo, i n 1975 (17); they have been discussed i n many more recent papers (11,154-157J~and they were discussed at a symposium at the 188th ACS National Meeting, 1984: Non-extractable Pesticide Residues: Characteristics, B i o a v a i l a b i l i t y and Toxicological S i g n i f i c a n c e . Occasionally, xenobiotics may be extensively metabolized i n plants to C0 or other low MW endogenously occuring products which can produce bound residues by reincorporation into b i o l o g i c a l polymers. Residues of this type are generally of l i t t l e concern to toxicologists and residue chemists because these residues do not represent an unusual hazard to the biosphere. A recently proposed n

n

2



94

d e f i n i t i o n of a bound plant residue s p e c i f i c a l l y excluded residues formed by reincorporation of such endogenously oecuring compounds (158); however, some d i f f i c u l t y can be encountered i n determining i f recycling of this nature has occured. A general protocol to assess the b i o l o g i c a l signficance of bound plant residues which addresses t h i s problem has also been suggested (158). A p a r t i a l l i s t of xenobiotics that form bound residues i n plant tissues i s presented on Table VIII. Many heterocyclic and aromatic


Table VIII.

Bound Residues of Xenobiotics i n Plants

Xenobiotic Atrazine Bentazon Benzopyrene Buthidazole Buturon Carbaryl Carboxin Chloramben Chloroaniline Chlortoluron Cisanilide 2,4-D DIB Dichlobenil Dieldrin EPN Flamprop Fluorodifen Isoxathion MCPA Mephosfolan Metribuzin Nitrofen Oxamyl PCNB PCP PCTP Perfluidone Prometryn Pronamide Propachlor Pro pan i l Propham Solan Sweep Tr ich 1 or o pheno 1 Triforine Zectran

Plant Species Corn Rice, Soybean Soybean c s c Corn, A l f a l f a Wheat Tobacco Peanut 10 species Rice Wheat Carrot esc Several esc White clover Bean Radish Cotton Wheat Peanut Bean Wheat Rice Soybean Rice, Wheat Peanut Peanut Soybean, Wheat Peanut esc Peanut Oat Alfalfa Soybean Rice Alfalfa 8 species 8 species Tomato Barley Broccoli, Bean b

C

d

e

% Bound Residue 38 25-90 15-25 19 50 40 21 4-39 30 50 40

3

REF 192 m 155 193

i"9"T 195,196 121

11 197 19o"

3-19

T5§ 155

34 20 10 24 42 12-26 17

"P* U9

T62 199 159 200 155"

21 21 20-40 45

155" 201 95 202 57 102 155 15 176" 203 2u¥

38

TfS

30 20 10-20 30 60 75 18,28

205 205" 207 207 205" 209 210

33 41-55 30 50 40 13

11,37

a

Percent of radioactive residue i n plant not extracted by methods used i n that study. csc=Cell suspension culture. PCNB=Pentachloronitrobenzene. PCP=Pentachlorphenol. PCTP=Pentachlorothiophenol. b

c

d

e



4.



95

compounds that contain or can be metabolized to y i e l d functional groups such as H0-, HOOC-, H2N-, and HS- frequently form bound r e s i dues; but chlorinated hydrocarbons generally do not (159)* Bound residues o f xenobiotics are found incoporated or associated with most of the b i o l o g i c a l polymers of plants, including l i g n i n , various carbohydrate polymers, and proteins (JJ_). Lignin has been implicated as the major form of bound pesticide residue i n the greatest number of cases where the residue has been studied (11, 155,158)* Lignin i s a highly insoluble plant c e l l - w a l l polymer of heterogenous nature. I t i s probably formed by polymerization o f cinnamic acid alcohol derivatives and other endogneous substrates i n f r e e - r a d i c a l reactions catalyzed by peroxidase and laccase enzymes (160). Bound residues of l i g n i n are d i f f i c u l t to study because o f the highly insoluble nature of the material. Several methods are frequently used to i s o l a t e l i g n i n (158), but these methods may a l t e r the xenobiotic-lignin bond. There have been no t r u l y s a t i s f a c t o r y methods for the s t r u c t u r a l analysis of xenobiotic-lignin residues. In some cases, such as with 3,4-dichloroaniline, xenobiotics appear to be incorporated into l i g n i n by covalent bonding (155,157), but i n other examples, such as swep, buturon, and carboxin, the xenobiotic may simply become entrapped i n the cage-like matrix o f the l i g n i n polymer (155). A model system for the synthesis of l i g n i n - l i k e polymers showed that enols would react with the quinone-methide intermediates i n t h i s system by a 1-6 addition (160). U t i l i z i n g t h i s model system, chloroanilines were copolymerized with c o n i f e r y l alcohol i n the presence o f horseradish peroxidase Type I I enzyme, hydrogen peroxide, v a n i l l y l alcohol i n i t i a t o r and pH 7.2 buffer (157). The mechanism of this copolymerization reaction i s shown i n Equation 36. The

(36)

copolymers of chloroaniline and c o n i f e r y l alcohol had average MVTs of 1,000 to 1,300 and the molar r a t i o of chloroaniline incorporated into the polymer was 1.19 to 1.68. When N-acetylated chloroanilines were used i n place of chloroanilines, incorporation was much lower. This suggested the involvement of the amino group i n the copolymerization reaction. The bound residue i n l i g n i n from r i c e treated with 3,4-dichloroaniline was s i m i l a r i n some respects to the synthetic l i g n i n . These residues were studied by H NMR and pyrolysis/MS. The incorporation o f 3-chloroaniline and 3,4-dichloroaniline into copolymers with c o n i f e r y l alcohol i n a s i m i l a r model system was confirmed i n a separate laboratory where the research was expanded to include benzo(a)pyrene quinones (155). The copolymers were studied by H-NMR, 3c_NMR, and other techniques. The copolymers o f coni1

1

1



96


f e r y l alcohol and chloroanilines appeared to contain benzylamine bonds, but evidence was obtained i n both studies that suggest that bonds involving the aromatic rings were also formed. I t would be expected that nucleophiles such as -SH, -NH2, -OH, and -COOH would react i n this manner to form l i g n i n - l i k e copolymers (155)* Carbohydrate conjugation o f xenobiotics i s often accompanied by the formation of bound residues. However, i t i s not clear whether carbohydrate conjugation i s i n competition with or i s an intermediate process i n the formation o f some bound residues. Some xenob i o t i c s are metabolized to dissacharide and oligosaccharide conjugates i n certain tissues and i t has been speculated that these xenobiotics may be incorporated into polymeric carbohydrate f r a c t i o n s . Diphenamid was metabolized to a gentiobiose conjugate and bound residue i n tomato and i t was speculated that the bound r e s i due might be a polysaccharide conjugate (69). Chlorpropham was metabolized i n soybean to a glucoside, bound residue, and what appeared to be polysaccharide conjugates. I t was postulated that the bound residue might be a more complex polysaccharide conjugate (161). Oxamyl was metabolized to simple and complex polysaccharide conjugates i n peanut and i t was speculated that the bound residue might be carbohydrate polymers (57). In white clover, DIB was metabolized to simple, d i - , and tri-glucose conjugates and a bound r e s i due that was speculated to be a carbohydrate polymer (162). MCPA and flamprop were metabolized i n spring wheat v i a processes that involved glucose conjugation and the formation o f bound residue (156). In wheat straw, 33% and 42$ of the residues o f MCPA and flamprop, respectively, were bound. Characterization o f these bound residues by various enzymes including a-amylase, protease, pectinase, s n a i l digestive enzymes, hemicellulase, and c e l l u l a s e was not successful; however, fractionation o f the bound residue by s o l u b i l i t y and hydrolysis of the r e s u l t i n g fractions indicated that MCPA may have been incorporated into the polysaccharide and hemicellulose f r a c t i o n s . The products were separated into several d i f f e r e n t molecular weight ranges by gel permeation chromatography. Lignin accounted for only 2% o f the residue from MCPA and Flamprop i n these tissues. I t appears that very similar xenobiotics can be metabolized to d i f f e r e n t types o f bound residue and that considerable quantitative variation i n bound residue can occur as a function of plant species or s p e c i f i c source o f tissue. In wheat c e l l suspension culture, 2,4-D, which i s s t r u c t u r a l l y s i m i l a r to MCPA, appeared to be incoporated into l i g n i n (155). This was i n sharp contrast to MCPA metabolism i n wheat. Carboxin (aniline-14C) metabolism i n peanut c e l l suspension culture and the f r u i t of whole peanut plants i s an example o f tissue v a r i a t i o n . In peanut c e l l suspension culture, only 2.7$ o f the carboxin was incorporated into bound residue, but i n the f r u i t of whole plants, 21$ incorporation into bound residue was observed (121). The metabolism o f metribuzin i n tomato and soybean i s an excellent example of species v a r i a t i o n . In tomato, metribuzin was rapidly metabolized to N-glucosides and only 2% was incorporated into bound residue, but i n soybean, metribuzin was metabolized slowly by homoglutathione conjugation and 20-30$ of the metribuzin was incorporated into bound residue (46,95). Bound residues are normally considered end-products of metabolism; however, some carbohydrate components of c e l l walls may be


4.

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RUSNESS


97

r e u t i l i z e d d u r i n g c e r t a i n s t a g e s o f p l a n t development (163)* Therefore, the i n c o r p o r a t i o n o f a x e n o b i o t i c r e s i d u e i n t o h e m i c e l l u l o s e or o t h e r c a r b o h y d r a t e f r a c t i o n s can not be r e g a r d e d as p r o o f t h a t a r e s i d u e i s no l o n g e r s u b j e c t t o f u r t h e r m e t a b o l i c t r a n s f o r mations .


Conclusions The m e t a b o l i s m o f x e n o b i o t i c s i n h i g h e r p l a n t s has been s t u d i e d e x t e n s i v e l y over the l a s t 20 y e a r s . In common p l a n t s p e c i e s such as c o r n , i t i s f r e q u e n t l y p o s s i b l e t o p r e d i c t the c o n j u g a t i o n r e a c t i o n s t h a t may be u t i l i z e d i n the i n i t i a l phases o f m e t a b o l i s m o f a new xenobiotic. In l e s s commonly s t u d i e d s p e c i e s , p r e d i c t i o n s a r e more u n c e r t a i n and e x o t i c m e t a b o l i t e s a r e o c c a s i o n a l y formed. In t h o s e c a s e s where phase I o x i d a t i v e r e a c t i o n s a r e l i k e l y , i t i s d i f f i c u l t t o p r e d i c t the c o u r s e o f m e t a b o l i s m because phase I o x i d a t i o n r e a c t i o n s i n p l a n t s are f r e q u e n t l y v e r y s u b s t r a t e and s p e c i e s s p e c i f i c . Phase I o x i d a t i v e r e a c t i o n s have a p r o f o u n d e f f e c t on e n s u i n g c o n jugation reactions. The p r e s e n c e o f m u l t i p l e f u n c t i o n a l groups on a x e n o b i o t i c a l s o i n c r e a s e s the u n c e r t a i n t y o f the r o u t e o f m e t a b o l i s m l i k e l y t o be f o l l o w e d i n a p a r t i c u l a r s p e c i e s . A l t h o u g h t h e s e u n c e r t a i n t i e s e x i s t i n our a b i l i t y t o p r e d i c t i n i t i a l c o n j u g a t i o n r e a c t i o n ( s ) , some g e n e r a l i z a t i o n s can be made. Most p h e n o l s , a n i l i n e s , and c a r b o x y l i c a c i d s a r e i n i t i a l l y metabol i z e d to g l u c o s e c o n j u g a t e s and/or bound r e s i d u e s . Occasionally, c a r b o x y l i c a c i d s a r e m e t a b o l i z e d t o amino a c i d c o n j u g a t e s and a n i l i nes t o m a l o n i c a c i d c o n j u g a t e s . Xenobiotics with e l e c t r o p h i l i c s i t e s are f r e q u e n t l y m e t a b o l i z e d t o g l u t a t h i o n e or h o m o g l u t a t h i o n e c o n j u g a t e s , depending upon the s p e c i e s . L i p o p h i l i c conjugates of x e n o b i o t i c p h e n o l s and c a r b o x y l i c a c i d s have been r e p o r t e d , but few g e n e r a l i z i a t i o n s can be made w i t h t h i s c l a s s o f c o n j u g a t e . Most bound r e s i d u e s appear t o be a s s o c i a t e d w i t h l i g n i n o r c a r b o h y d r a t e polymers. I n h i g h e r p l a n t s , x e n o b i o t i c c o n j u g a t e s f r e q u e n t l y undergo a d d i t i o n a l metabolic transformations. I t i s d i f f i c u l t to p r e d i c t the p r e c i s e n a t u r e o f t h e s e s e c o n d a r y or t e r t i a r y transformations. Simple g l u c o s e c o n j u g a t e s may be s t a b l e o r t h e y may be m e t a b o l i z e d to malonylglucose-, g e n t i o b i o s e - , o l i g o s a c c h a r i d e - , or heterodissacharide-conjugates, or t h e y may be i n c o r p o r a t e d i n t o bound residue. At p r e s e n t , we can not p r e d i c t which o f the above r o u t e s o f metabolism w i l l be u t i l i z e d i n a s p e c i f i c c a s e . G l u t a t h i o n e o r h o m o g l u t a t h i o n e c o n j u g a t e s n e a r l y always undergo a d d i t i o n a l metabol i c t r a n s f o r m a t i o n s to c y s t e i n e - , m a l o n y l c y s t e i n e - , malonylcysteine s u l f o x i d e - , t h i o l a c t i c a c i d - c o n j u g a t e s , e t c . , o r t o bound r e s i d u e , but the r o u t e t h a t w i l l be u t i l i z e d i n a s p e c i f i c case can not be predicted. X e n o b i o t i c amino a c i d c o n j u g a t e s and g l u c o s e e s t e r c o n j u g a t e s may be t r a n s i t o r y and f u r t h e r m e t a b o l i z e d i n a manner s i m i l a r t o the p a r e n t compound. Most o f the enzymes t h a t c a t a l y z e the f o r m a t i o n o f x e n o b i o t i c c o n j u g a t e s i n p l a n t s have not been w e l l - s t u d l e d . S i n c e some c o n j u g a t i o n r e a c t i o n s are i n v o l v e d i n h e r b i c i d e s e l e c t i v i t y , i t i s l i k e l y t h a t r e s e a r c h r e l a t i n g t o t h e s e enzymes w i l l i n t e n s i f y as a r e s u l t o f e f f o r t s to develop h e r b i c i d e r e s i s t a n t crops through bi©engineering. Enzymes t h a t may be u s e f u l i n b i o e n g i n e e r i n g f o r h e r b i c i d e r e s i s t a n c e a r e the GST enzymes, N - g l u c o s y l t r a n s f e r a s e s ,



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and perhaps malonyl transferases. Oxidative enzymes are extremely important in regards to herbicide selectivity, but they may present some unusually difficult problems in bioengineering studies. Intensive research on the enzymes responsible for xenobiotic conjugation in plants will greatly increase our knowledge of the behavior and fate of xenobiotics in plants.


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