bk-1979-0097.ch003

3 Pesticide Metabolism in Higher Plants: In Vitro Enzyme Studies

1

G. L. LAMOUREUX and D. S. FREAR

Downloaded by UNIV OF MINNESOTA on October 2, 2013 | http://pubs.acs.org Publication Date: April 5, 1979 | doi: 10.1021/bk-1979-0097.ch003

Metabolism and Radiation Research Laboratory, Agricultural Research, Science and Education Administration, U. S. Department of Agriculture, Fargo, ND 58105

The study of x e n o b i o t i c metabolism i n p l a n t s has developed p r i m a r i l y as a r e s u l t of the use of p e s t i c i d e s to p r o t e c t p l a n t crops from damage by weeds, i n s e c t s and other pests. Great stimulus f o r research i n t h i s f i e l d was experienced i n the e a r l y I960 s when increased concern was r a i s e d over the p o s s i b l e hazards of p e s t i c i d e residues to man and h i s environment. The complexity of x e n o b i o t i c metabolism s t u d i e s i s a continuum with i n v i v o s t u d i e s conducted i n the n a t u r a l environment at one end, and the use of i s o l a t e d enzymes to study molecular r e a c t i o n s at the other. Each technique i n t h i s continuum has i t s own p a r t i c u l a r l i m i t a t i o n s , but can be used to great advantage under the proper c o n d i t i o n s . In the previous two r e p o r t s , the use of c e l l c u l t u r e s , t i s s u e c u l t u r e s , i s o l a t e d c e l l s and i s o l a t e d p l a n t organs were considered. This report w i l l examine the use of i s o l a t e d p l a n t enzyme systems i n x e n o b i o t i c metabolism s t u d i e s . The primary t o p i c w i l l be a d i s c u s s i o n of the enzymes involved i n the four b a s i c metab o l i c r e a c t i o n s of x e n o b i o t i c s i n p l a n t s : o x i d a t i o n , r e d u c t i o n , h y d r o l y s i s and conjugation. The l i t e r a t u r e regarding key enzymes w i t h i n these c l a s s e s w i l l be reviewed. S p e c i f i c examples of i n v i t r o p l a n t enzyme systems used to study x e n o b i o t i c metabolism w i l l be presented. A d e t a i l e d d i s c u s s i o n of v a r i o u s techniques w i l l not be attempted. 1

OXIDATION REACTIONS: Oxidations are among the most important r e a c t i o n s i n the metabolism of p e s t i c i d e s because they are f r e q u e n t l y the primary 1 Mention of a trademark or p r o p r i e t a r y product does not cons t i t u t e a guarantee or warranty of the product by the U. S. Department of A g r i c u l t u r e and does not imply i t s approval to the e x c l u s i o n of other products that may a l s o be s u i t a b l e .

This chapter not subject to U.S. copyright. Published 1979 American Chemical Society

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.


78

XENOBIOTIC METABOLISM

r e a c t i o n that r e s u l t s i n d e t o x i c a t i o n o r a c t i v a t i o n o f a p e s t i cide. In mammals and i n s e c t s , o x i d a t i o n r e a c t i o n s have been the subject of many i n v i t r o s t u d i e s . I t i s now evident that mixed f u n c t i o n oxidases are r e s p o n s i b l e f o r many o f the o x i d a t i o n r e a c t i o n s i n these organisms. Although p l a n t s have mixed f u n c t i o n oxidase systems, t h e i r r o l e i n p e s t i c i d e metabolism has not been widely demonstrated. I n a d d i t i o n to mixed f u n c t i o n oxidases, peroxidases, laçasses and polyphenol oxidases are found u n i v e r s a l l y i n the p l a n t kingdom and some o f these enzymes may a l s o p l a y a r o l e i n o x i d a t i v e p e s t i c i d e metabolism. At the present, our knowledge o f the enzyme systems i n v o l v e d i n many of the p e s t i c i d e o x i d a t i o n r e a c t i o n s that occur i n p l a n t s i s l i m i t e d and there i s great need f o r more research. Peroxidases : The p l a n t peroxidases c a t a l y z e two general types o f o x i d a t i o n r e a c t i o n s , the c l a s s i c a l p e r o x i d a t i v e r e a c t i o n that r e q u i r e s hydrogen peroxide and the o x i d a t i v e r e a c t i o n that u t i l i z e s molecular oxygen. The h o r s e r a d i s h peroxidase (HRP) p e r o x i d a t i v e r e a c t i o n normally proceeds by the f o l l o w i n g mechanism (1, 2) : HRP + H2O2 HRP-I HRP-I + AH2 + HRP-I I + AHHRP-II + AH 2 -* HRP + AH2 AH-> Products The o x i d a t i v e r e a c t i o n s are not as w e l l understood as the peroxidative reactions. I n Mn++ i n h i b i t e d r e a c t i o n s , the o x i d a t i v e h y d r o x y l a t i o n of s e v e r a l substrates can be c a t a l y z e d by peroxidase i n the presence o f dihydroxyfumaric a c i d (3). Other o x i d a t i v e r e a c t i o n s r e q u i r e an aromatic c o - f a c t o r such as 2,4d i c h l o r o p h e n o l and Mn++ (4·) . I n some o x i d a t i v e r e a c t i o n s , a c a t a l y t i c amount of hydrogen peroxide i s needed as an i n i t i a t o r , but oxygen i s used i n s t o i c h i o m e t r i c amounts ( 5 ) . Plant peroxidases c a t a l y z e the o x i d a t i o n o f a l a r g e and d i v e r s e c l a s s of endogenous and exogenous substrates such as phenols, aromatic amines, e n e d i o l s , ascorbate, ferrocyanide, cytochrome C., i n d o l e - 3 - a c e t i c a c i d , and the leuco form of many dyes. Phenols and aromatic amines are among the most commonly used substrates and the r e a c t i o n c a t a l y z e d i s g e n e r a l l y an o x i d a t i v e condensation o f the substrate (6). I n a d d i t i o n t o o x i d a t i v e condensations, decarboxylations, s u l f u r o x i d a t i o n s , Ndemethylations, r i n g h y d r o x y l a t i o n s , carbon-halogen bond élevages, and o x i d a t i o n o f aromatic methyl groups have a l l been a t t r i b u t e d to peroxidases (3) (Figure 1). Peroxidases are ubiquitous i n the p l a n t kingdom ( 3 ) . They occur throughout the p l a n t c e l l and have been found i n the cytoplasm, c e l l w a l l , membranes, n u c l e i , mitochondria and ribosomes (7). Peroxidase isozymes have been demonstrated i n h o r s e r a d i s h (4, 8-11) and s e v e r a l other species (4, 8-13). S t r i k i n g d i f f e r e n c e s have been


LAMOUREUX


3.

A N D FREAR

Plant

Enzyme

79

Studies

I)

OXIDATIVE CONDENSATIONS Of PHENOLS AND AROMATIC AMINES

2)

DECARBOXYLATION

3)

4)

oca

oca

SULFUR OXIDATIONS

N—DE METHYL AT IONS OH il ;-N-CH

t

Ο H il ι 0-C-N-CH

s

CHÎ^^CH,

CH^^CH,

CH^CHj 5)

CH^%

RING HYDROXYLATIONS COOH

6)

COOH

COOH

ô

û ~ - j ù r

OXIDATIONS OF AROMATIC METHYL GROUPS OH OH

OH

CH, 7Î

5

0

CH OH 2

CARBON —HALOGEN BOND CLEAVAGES F F~ + COMPLEX CONDENSATION PRODUCTS I NH

8)

2

IODIDE OXIDATION I"

Figure 1.

Types of reactions attributed to plant peroxidases ( 3 , 2 2 , 1 8 8 , 1 8 9 , 1 9 0 )



80


noted i n the s u b s t r a t e s p e c i f i c i t y of s e v e r a l HRP isozymes (14). In s p i t e of t h e i r widespread occurrence, the p h y s i o l o g i c a l r o l e of peroxidases i s u n c e r t a i n . T h e i r a b i l i t y to u t i l i z e i n d o l e - 3 - a c e t i c a c i d (15-18) and f l a v o n o i d s (19) as substrates suggests that peroxidases may be important i n the metabolic r e g u l a t i o n of these endogenous s u b s t r a t e s . Peroxidases are a l s o thought to be i n v o l v e d i n the l i g n i f i c a t i o n process (20). The h i g h c o n c e n t r a t i o n of peroxidase a c t i v i t y a s s o c i a t e d w i t h the c e l l w a l l (12) i s c o n s i s t e n t with t h i s theory. Several r e p o r t s have a s s o c i a t e d peroxidase a c t i v i t y w i t h p e s t i c i d e metabolism. The o x i d a t i o n of p a r a t h i o n to paraoxon and the h y d r o l y s i s of both p a r a t h i o n and paraoxon can be c a t a l y z e d by HRP (21) (Figure 2). The r e a c t i o n occurs under both o x i d a t i v e and p e r o x i d a t i v e c o n d i t i o n s . A peroxidase was a l s o i s o l a t e d from bean hypocotyl that was approximately 50% as a c t i v e as HRP i n the o x i d a t i o n and h y d r o l y s i s of parathion (21). T h i s enzyme was equal to HRP i n the o x i d a t i o n of g u i c a c o l . The i n v i t r o r e a c t i o n of ten [l^C-carbonyl]carbamate i n s e c t i c i d e s w i t h hydrogen peroxide i n the presence of HRP has been examined (22). Conversion to e t h e r - s o l u b l e products that could be d i f f e r e n t i a t e d from the r e a c t a n t s by TLC or conversion to waters o l u b l e products were used as the c r i t e r i a f o r a r e a c t i o n . Of the ten substrates t e s t e d (banol, baygon, c a r b a r y l , HRS-1422, i s o l a n , mesurol, UC-10854, m a t a c i l and z e c t r a n ) , only m a t a c i l and z e c t r a n r e a c t e d . The highest percentage of r a d i o a c t i v i t y was i n the form of u n i d e n t i f i e d water-soluble products; however, s e v e r a l e t h e r - s o l u b l e products were i d e n t i f i e d (Figure 3). The e t h e r - s o l u b l e products represented v a r i o u s stages of N-dealkylation. T h i s may suggest a minor r o l e f o r peroxidases i n Nd e a l k y l a t i o n . Mesurol, a carbamate i n s e c t i c i d e that contains an a r y l m e t h y l s u l f i d e , and c y s t e i n e , t h i o g l y c o l i c a c i d , mercaptoethanol, t h i o u r e a and t h i o u r a c i l were not substrates f o r HRP (22, 23); however, chloropromazine, a h e t e r o c y c l i c s u l f i d e , was a s u b s t r a t e ( 3 ) . The p r e c i s e r o l e of peroxidases i n s u l f u r o x i d a t i o n i s thus u n c e r t a i n . A n i l i n e s are known degradation products of phenylcarbamates, phenylureas, and a c y l a n i l i d e h e r b i c i d e s ; t h e r e f o r e , the f a t e of a n i l i n e s i n the environment i s an important c o n s i d e r a t i o n . A number of workers have shown that v a r i o u s a n i l i n e s are converted to azobenzenes and other products by the a c t i o n of HRP and hydrogen peroxide (3). However, i t i s of p a r t i c u l a r note that the c h l o r o a n i l i n e s that formed azobenzenes i n v i t r o as the r e s u l t of HRP and hydrogen peroxide were the same as those that formed azobenzenes i n the s o i l as a r e s u l t of m i c r o b i a l a c t i o n (24) (Figure 4 ) . The HRP system served as a model to p r e d i c t the l i k e l i h o o d of azobenzene formation i n the s o i l . In these s t u d i e s , no e f f o r t was made to i d e n t i f y products other than the azobenzene analogs or to q u a n t i t a t e the r e a c t i o n ; t h e r e f o r e , the f u l l scope of products produced and the y i e l d s are not known.


3.

LAMOUREUX AND FREAR

0-P(OC H ) 2

5

2

Plant Enzyme

BEAN HYPOCOTYL PEROXlDASE(lOuq) pH4.7 DIHYDROXY MALEIC ACID

81

Studies

0-P(OC H5) 2

2

+ HYDROLYSIS PRODUCTS


14%

36% HYDROLYSIS AND 10% OXIDATION OBTAINED WITH 50jjg HRP Figure 2.

Oxidation and hydrolysis of parathion by plant peroxidases (21)

MATACIL Figure 3. Ν -Demethyhtion of substituted phenylcarbamate insecticides by horse radish peroxidase (22)




82


3.

L A M O U R E U X AND FREAR

Plant Enzyme

83

Studies

Although azobenzenes do not appear t o be metabolites of c h l o r o a n i l i n e s i n higher p l a n t s , c h l o r o a n i l i n e s may be i n c o r p o r ated i n t o l i g n i n or converted to l i g n i n - l i k e products (25). In an e f f o r t to determine the chemical nature of the c h l o r o a n i l i n e residue i n the l i g n i n f r a c t i o n , a model system was developed that uses HRP to c a t a l y z e the f r e e r a d i c a l formation of a s y n t h e t i c l i g n i n - l i k e m a t e r i a l (26). I n t h i s system, the ^ C - l a b e l e d p e s t i c i d e metabolite, 3 - c h l o r o a n i l i n e or 3 , 4 - d i c h l o r o a n i l i n e , was co-polymerized w i t h c o n i f e r y l a l c o h o l , a b u i l d i n g b l o c k of n a t u r a l l i g n i n (Figure 5). The p o l y m e r i z a t i o n was accomplished by simultaneously pumping s o l u t i o n s of (a) c o n i f e r y l a l c o h o l and HRP i n pH 7.2 b u f f e r , (b) hydrogen peroxide i n pH 7.2 b u f f e r , and (c) the l ^ C - l a b e l e d c h l o r o a n i l i n e i n pH 7.2 b u f f e r i n t o a b u f f e r e d s o l u t i o n of v a n i l l y l a l c o h o l and HRP. A f t e r 10 hr i n the dark, the i n s o l u b l e 14c-labeled polymeric product was removed by c e n t r i f u g a t i o n , washed and subjected to g e l permeation chromatography. Based upon an average molecular weight of 1,000, 1.19 r e s i d u e s of 3 - c h l o r o a n i l i n e or 1.68 residues of 3,4-di c h l o r o a n i l i n e were incorporated per molecule of polymer formed. In v i v o i n c o r p o r a t i o n i n t o r i c e l i g n i n was a l s o greater f o r 3 , 4 - d i c h l o r o a n i l i n e than f o r 3 - c h l o r o a n i l i n e . When a c e t y l a t e d a n i l i n e s were used i n the model HRP system, the i n c o r p o r a t i o n r a t e was reduced by over 50%. T h i s may i n d i c a t e the involvement of the a n i l i n e n i t r o g e n i n the co-polymerization r e a c t i o n .


1

A number o f techniques were used f o r the a n a l y s i s of the synthetic l i g n i n . The most s u c c e s s f u l method was p y r o l y t i c GC/MS. P y r o l y s i s r e l e a s e d over 80% of the r a d i o a c t i v i t y i n a v o l a t i l e form. Over 60% o f the r e l e a s e d l^C was 3 - c h l o r o a n i l i n e or 3,4dichloroaniline. S i m i l a r r e s u l t s were obtained with n a t u r a l l i g n i n i s o l a t e d from c h l o r o a n i l i n e - t r e a t e d r i c e , but w i t h much lower y i e l d s . I t was concluded that a high percentage of the c h l o r o a n i l i n e i n the l i g n i n - l i k e m a t e r i a l was c o v a l e n t l y bonded between the a n i l i n e n i t r o g e n and the α-carbon of the polymer (Figure 5 ) . HRP was a l s o used to study the metabolism of b o t r a n (27). Botran was not a d i r e c t s u b s t r a t e f o r HRP, but i t s r e d u c t i o n product (2,6-dichlorophenylenediamine) was r e a d i l y converted to at l e a s t 8 products upon treatment with HRP (Figure 6). One of the most abundant products was i d e n t i c a l t o a s o i l metabolite of botran. High r e s o l u t i o n mass spectrometry of the s o i l metabolite i n d i c a t e d a molecular formula of Cl2H6N4Cl6- A botran metabolite, N-(4-amino-3,5-dichlorophenyl)malonamic a c i d , was found i n soy bean p l a n t s and i n soybean c a l l u s c u l t u r e s (28). The presumed intermediate of t h i s metabolite (2,6-dichlorophenylenediamine) was not detected; however, p l a n t s c o n t a i n a system that i s capable o f reducing a r y l n i t r o groups (29). The diamine of botran could thus be formed i n the p l a n t and serve as a s u b s t r a t e f o r a malonyl t r a n s f e r r e a c t i o n , a peroxidase-catalyzed o x i d a t i o n or l i g n i n i n c o r p o r a t i o n .



84

XENOBIOTIC M E T A B O L I S M

RAT LIVER MICROSOMES, SOIL BACTERIA* PLANTS

AT LEAST 8 PRODUCTS, INCLUDING C H N CI

HRP/HOg 10 MIN 23°C pH 5.1 2

|2

6

4

4

BOTRAN Figure 6.

Table I.

A possible role for peroxidases in botran metabolism (27,28, 29)

R e l a t i o n s h i p between r e s i s t a n c e capable o f o x i d i z i n g iodide.*

t o i o d i d e and absence o f p e r o x i d a s e

Peroxidase A c t i v i t y

Species

G u a i a c o l donor (units/mg protein)

I o d i d e donor (units/mg protein)

Iodide accumulation (mg/g d r y weight)

Resistance

tomato

2.6

0

70

short

buttercup

2.0

9

13

resistant

cabbage

6.7

26

4

resistant

bean

12.7

28

15

susceptible

mettle

15.3

75

5

susceptible

pea

82

123

8

susceptible

*Adapted from ( 3 2 ) .


term


3.

LAMOUREUX A N D FREAR

Plant Enzyme

Studies

85

The f i n d i n g that i o x y n i l l i b e r a t e d i o d i d e when exposed to u l t r a v i o l e t l i g h t or when subjected t o p l a n t or animal metabolism r e s u l t e d i n an examination of the h e r b i c i d a l p r o p e r t i e s of i o d i d e . Subsequently, i o d i d e was shown t o possess some s e l e c t i v i t y as a h e r b i c i d e (30). The a b i l i t y of p l a n t peroxidases to o x i d i z e i o d i d e to i o d i n e (31) v a r i e d g r e a t l y w i t h the p l a n t s p e c i e s (32) and t h i s a b i l i t y was r e l a t e d t o r e s i s t a n c e t o i o d i d e t o x i c i t y (Table I ) . Iodine was shown to be 10^ times more powerful than i o d i d e as an i n h i b i t o r of the H i l l r e a c t i o n (33). T h i s suggested that i o d i d e t o x i c i t y was due, i n p a r t , to i n t r a c e l l u l a r o x i d a t i o n of i o d i d e to i o d i n e . When 30 iodo-benzoic a c i d s were assayed f o r h e r b i c i d a l a c t i v i t y , only those that l i b e r a t e d i o d i d e i n v i v o were t o x i c to Phaseolus v u l g a r i s seedlings. I t was suggested that l i b e r a t i o n o f i o d i d e might be one f a c t o r i n the h e r b i c i d a l a c t i v i t y of these compounds (30). In a d d i t i o n to i n v i t r o s t u d i e s that suggested an a c t i v e r o l e f o r peroxidases i n p e s t i c i d e metabolism, s e v e r a l s t u d i e s have i n d i c a t e d that c e r t a i n p e s t i c i d e s may i n c r e a s e the l e v e l of peroxidases i n p l a n t s . EPTC was r e p o r t e d to i n c r e a s e peroxidase a c t i v i t y and l i g n i f i c a t i o n i n corn s e e d l i n g s ; these i n c r e a s e s could be a l l e v i a t e d by treatment with a p r o t e c t a n t , N , N - d i a l l y l 2,2-dichloroacetamide (9). S t i m u l a t i o n of peroxidase a c t i v i t y was a l s o observed when Phaseolus r a d i a t u s s e e d l i n g s were t r e a t e d with sodium d i e t h y l d i t h i o c a r b a m a t e (34). A microsomal hydroperoxide-dependent o x i d i z i n g system (hydroperoxidase) was r e c e n t l y i s o l a t e d from pea seeds (35, 36) (Figure 7 ) . T h i s enzyme hydroxylated i n d o l e , phenol, α-naphthol, and a n i l i n e to i n d o x y l , hydroquinone, α-napthylhydroquinone, and N^hydroxyaniline, r e s p e c t i v e l y . Hydrogen peroxide, t e r t - b u t y l hydroperoxide, cumene hydroperoxide, or l i n o l e i c a c i d hydro peroxide served as sources of o x i d i z i n g power. A w e l l - d e f i n e d pH optimum a t pH 7.2 was observed w i t h l i n o l e i c a c i d hydroper oxide, but a broad pH optimum around 8.7 was observed with the other hydroperoxides. An a l l o s t e r i c e f f e c t was noted w i t h l i n o l e i c a c i d hydroperoxide, but not w i t h the other hydroper oxides. I t was speculated that the n a t u r a l hydroperoxides f o r t h i s system were produced by the a c t i o n of lipoxygenases on s u b s t r a t e s such as l i n o l e i c a c i d . Studies with 0^-8-labeled l i n o l e i c a c i d hydroperoxide i n d i c a t e d a d i r e c t oxygen t r a n s f e r from the hydroperoxide to the hydroxylated s u b s t r a t e . No p a r t i c i p a t i o n of molecular oxygen was observed. Pre-treatment w i t h j>-chloromercuriobenzoate r e s u l t e d i n a s l i g h t promotion of the hydroperoxidase r e a c t i o n , i n d i c a t i n g no involvement of P450. Other p r o p e r t i e s of t h i s system a l s o i n d i c a t e d that a c t i v i t y was d i s t i n c t from that of P450. Although none of the i n v i t r o peroxidase s t u d i e s d i s c u s s e d proves that these enzymes p l a y an important r o l e i n p e s t i c i d e metabolism i n p l a n t s , i t i s c l e a r that the spectrum of s u b s t r a t e s u t i l i z e d by these enzymes does encompass many p e s t i c i d e s or p e s t i c i d e m e t a b o l i t e s . The apparent u n i v e r s a l occurrence of



86


Figure 7.

H y droper oxide-dependent enzyme system from pea (35, 36)


3.


Plant Enzyme

Studies

87

peroxidases i n the p l a n t kingdom and throughout the c e l l c o n t r i b u t e s g r e a t l y to the p o s s i b l e involvement of these enzymes i n p e s t i c i d e metabolism. A number of p o s s i b l e r o l e s f o r p e r o x i dases i n the metabolism of s p e c i f i c p e s t i c i d e s or c l a s s e s of p e s t i c i d e s have been pointed out. The u t i l i t y of HRP i n model systems to produce metabolites found i n the s o i l and to a i d i n the study of the p e s t i c i d e residues found i n the p l a n t l i g n i n f r a c t i o n have been c l e a r l y demonstrated.


Mixed Function

Oxidases:

Extensive research over the l a s t 28 years has shown that mixed f u n c t i o n oxidases are of great importance i n x e n o b i o t i c metabolism i n i n s e c t s and animals (37). The presence of mixed f u n c t i o n oxidases i n higher p l a n t s has been e s t a b l i s h e d i n the l a s t 10 years by experiments conducted w i t h both endogenous substrates (38-42) and x e n o b i o t i c s (43-48). Based upon l i g h t r e v e r s i b l e CO i n h i b i t i o n (39, 44) and s p e c t r a l p r o p e r t i e s (49), s e v e r a l mixed f u n c t i o n oxidase (mfo) systems i n p l a n t s appear to resemble the cytochrome P450 mfo systems found i n animals and insects. Other p l a n t mfo systems, however, appear to be q u i t e d i f f e r e n t (38, 40). Based upon our knowledge of i n s e c t and mammalian mfo systems and our knowledge of metabolites produced i n p l a n t s , a number of r e a c t i o n s i n p l a n t s should be considered as p o s s i b l e mixed f u n c t i o n oxidase-catalyzed r e a c t i o n s . Some of these are N-dea l k y l a t i o n , C)-dealkylation, aromatic h y d r o x y l a t i o n , a l k y l o x i d a t i o n , epoxidation, d e s u l f u r a t i o n , s u l f u r o x i d a t i o n , e s t e r h y d r o l y s i s , and n i t r o g e n o x i d a t i o n (Figure 8). The demonstrated mfo r e a c t i o n s i n p l a n t s are more l i m i t e d . There are s e v e r a l examples of mfo-catalyzed N - d e a l k y l a t i o n s i n p l a n t s . A mfo system from cotton that c a t a l y z e s the N-demethylation of phenylurea h e r b i c i d e s (47, 48, 50) and mfo systems from c a s t o r beans (51) and avocado pear (46) that c a t a l y z e the N-demethylation of _rj-chloro-N-methylaniline have been reported. The O-demethylation of £-nitroanisole has been detected with an ±n v i t r o system from avocado pear (46). A s i m i l a r mfo-catalyzed d e a l k y l a t i o n might e x p l a i n the presence of the phenol of 2,4-D that has been reported as a metabolite i n c e r t a i n p l a n t systems. The formation of the phenol of 2,4-D does not seem to i n v o l v e an i n i t i a l d e c a r b o x y l a t i o n (52). The reported i n v i v o a l k y l o x i d a t i o n of the p l a n t growth r e g u l a t o r f l u r e n o l b u t y l e s t e r might a l s o be explained by a mfo (53). Several mixed f u n c t i o n oxidase systems that c a t a l y z e aromatic r i n g h y d r o x y l a t i o n s have been s t u d i e d jln v i t r o w i t h both x e n o b i o t i c (44-46) and endogenous substrates (38-41, 45, 54-57). Because of the l a r g e number of p o t e n t i a l s u b s t r a t e s , t h i s r e a c t i o n i s of p a r t i c u l a r i n t e r e s t . The epoxidation a l d r i n has been demonstrated by jLn v i t r o systems i s o l a t e d from pea and bean (43, 58-62). The a l d r i n epoxidase systems have not been c l e a r l y




88



3.


Plant Enzyme

Studies

89

demonstrated to be mfo systems. The conversion of organophosphorothioates to phosphates i n p l a n t s has been w e l l e s t a b l i s h e d . T h i s conversion i n v o l v e s mixed f u n c t i o n oxidases i n animals and i n s e c t s (63). Mixed f u n c t i o n oxidases may a l s o c a t a l y z e t h i s r e a c t i o n i n p l a n t s , but the p o s s i b i l i t y that other enzyme systems may be i n v o l v e d should be considered (21). The o x i d a t i o n of a number of t h i o - e t h e r s to s u l f o x i d e s and sulfones has been reported i n p l a n t metabolism s t u d i e s ; again, the enzymes r e s p o n s i b l e f o r these r e a c t i o n s have not been w e l l c h a r a c t e r i z e d . An enzyme that c a t a l y z e s the s u l f o x i d a t i o n of phorate has been i s o l a t e d from soybean root (64). A d d i t i o n a l s t u d i e s are needed to c h a r a c t e r i z e t h i s system. In animals, organophosphate e s t e r h y d r o l y s i s can be c a t a l y z e d by both mixed f u n c t i o n oxidases and g l u t a t h i o n e S-transferases (63). In plants, t h i s r e a c t i o n may be c a t a l y z e d by esterases or peroxidases (21, 65, 66), but the r e a c t i o n mechanism needs f u r t h e r study at the i n v i t r o l e v e l . In p l a n t s , N-oxide formation does not appear to be a common r e a c t i o n i n p e s t i c i d e metabolism. However, an enzyme that c a t a l y z e s the formation of the N-oxide of morphine has been detected i n Papaver somniferum l a t e x (67). Since peroxidases and polyphenol oxidases are found i n high concentrations i n p l a n t l a t e x , the p o s s i b i l i t y that t h i s r e a c t i o n may be c a t a l y z e d by one of these enzymes should a l s o be considered. The N-demethylase from cotton (47) and the cinnamic a c i d hydroxylase from cucumber (44) are two mfo systems that have been shown to u t i l i z e p e s t i c i d e s as s u b s t r a t e s . The N-demethylase system appears to have l i m i t e d s u b s t r a t e s p e c i f i c i t y , only N-methyl s u b s t i t u t e d phenylureas were found to be a c t i v e substrates. The N-demethylation r e a c t i o n was s t r o n g l y and competi t i v e l y i n h i b i t e d by carbamate i n s e c t i c i d e s that had high e l e c t r o n d e n s i t y at the p o s i t i o n ortho to the carbamate group (50, 68). I n h i b i t i o n by s u b s t i t u t e d phenylureas was a l s o demonstrated. T h i s i n h i b i t i o n was dependent upon the presence of a proton on the a n i l i n e n i t r o g e n atom (50). F i e l d observations (69, 70) and an i n v i t r o study (71) i n d i c a t e d an a n t a g o n i s t i c i n t e r a c t i o n of carbamates w i t h dimethylphenylurea h e r b i c i d e s . A p o s i t i v e c o r r e l a t i o n between r e s i s t a n c e to phenylurea h e r b i c i d e s and the presence of an a c t i v e N-demethylase system was supported by i n v i t r o determinations of N-demethylase a c t i v i t y i n 12 p l a n t s p e c i e s . In c o t t o n , the s p e c i f i c a c t i v i t y of the mfo system v a r i e d over a 1 0 - f o l d range, depending upon the age of the p l a n t and the s p e c i f i c t i s s u e . A recent study has shown that 2,4-D i s hydroxylated by an i n v i t r o mfo system from cucumber leaves (44). T h i s appears to be the f i r s t reported r i n g - h y d r o x y l a t i o n of a p e s t i c i d e c a t a l y z e d by an i s o l a t e d p l a n t mfo system. Cinnamic a c i d was a l s o hydroxylated by t h i s system. Hydroxylase a c t i v i t y was increased 2- to 3 - f o l d by spraying the cucumber leaves with 2,4-D b e f o r e enzyme i s o l a t i o n . Other s t u d i e s have shown that c e r t a i n p l a n t mfo systems were induced or stimulated by l i g h t



90


(39, 72). The NIH s h i f t was observed i n the h y d r o x y l a t i o n of 2,4-D. The primary product was 4-hydroxy-2,5-dichlorophenoxya c e t i c a c i d . The NIH s h i f t was a l s o demonstrated i n the 4h y d r o x y l a t i o n of cinnamic a c i d by a mfo system from pea (73). These s t u d i e s suggest that a d d i t i o n a l i n v e s t i g a t i o n s on the i n d u c t i o n of p l a n t mfo systems may be warranted. They a l s o suggest the need to conduct k i n e t i c s t u d i e s on the cinnamic a c i d 4-hydroxylase r e a c t i o n w i t h 2,4-D and r e l a t e d p e s t i c i d e s as possible inhibitors. Since microsomal mixed f u n c t i o n oxidases p l a y an important r o l e i n the metabolic pathway of some endogenous substrates (39, 41), the i n d u c t i o n or i n h i b i t i o n of these r e a c t i o n s by p e s t i c i d e s could have important i m p l i c a t i o n s . In animals, the microsomal f r a c t i o n has t y p i c a l l y d i s p l a y e d a broad s p e c i f i c i t y range (37) that can be explained by the presence of a s e r i e s of P450 cytochromes (74). In c o n t r a s t , mixed f u n c t i o n oxidases from p l a n t s appear to have a f a i r l y narrow s u b s t r a t e s p e c i f i c i t y range with some t i s s u e s p e c i f i c i t y . The mfo system from c a s t o r bean (51) d i s p l a y e d 4-hydroxylase a c t i v i t y f o r cinnamic a c i d and N-demethylase a c t i v i t y f o r j>chloro-N-methylaniline, N-methylaniline, and Ν,N-dimethylaniline, but d i s p l a y e d no a c t i v i t y f o r 15 other compounds that contained N-, 0-, or S-methyl groups. The system from avocado pear may have one of the broadest s p e c i f i c i t y ranges of the i s o l a t e d p l a n t systems. I t apparently has hydroxylase a c t i v i t y f o r b i p h e n y l and a n i l i n e , 0-demethylase a c t i v i t y f o r j > - n i t r o a n i s o l e , and N-demethylase a c t i v i t y f o r p_-chloro-N-methylaniline (46). Mixed f u n c t i o n oxidase systems i s o l a t e d from p l a n t s t y p i c a l l y d i s p l a y very low l e v e l s of a c t i v i t y . Specific a c t i v i t i e s are f r e q u e n t l y i n the range of 1- to 10-nmol product/mg protein/hr. T h i s low l e v e l of a c t i v i t y can be explained by the r e p o r t that P450 concentrations i n p l a n t s range from 0.007 to 0.02 nmol P450/mg microsomal p r o t e i n as compared to 0.9 to 1.2 nmol P450/mg microsomal p r o t e i n i n r a t l i v e r (49). Because of the low l e v e l s of mfo a c t i v i t y i n p l a n t t i s s u e s , the use of r a d i o a c t i v e s u b s t r a t e s i n assays i s o f t e n necessary. The p r o p e r t i e s of i s o l a t e d p l a n t mfo systems are h i g h l y v a r i a b l e (Table I I ) . Some mfo systems are microsomal, undergo l i g h t r e v e r s i b l e C O - i n h i b i t i o n , r e q u i r e NADPH as a c o - f a c t o r and behave much l i k e mammalian P450 mfo systems (39). Others r e q u i r e i l l u m i n a t e d c h l o r o p l a s t s as a c o - f a c t o r and may be s o l u b l e (38). Some systems r e q u i r e only one c o - f a c t o r (47), but other systems r e q u i r e s e v e r a l (40). A s c o r b i c a c i d , NADH, NADPH, d i t h i o n i t e , i l l u m i n a t e d c h l o r o p l a s t s , and an u n i d e n t i f i e d n a t u r a l product (probably a p t e r i d i n e ) have a l l been u t i l i z e d i n v a r i o u s i n v i t r o p l a n t mfo s t u d i e s (38, 39, 40, 47). The response of these systems to v a r i o u s i n h i b i t o r s i s a l s o q u i t e v a r i a b l e . Mercaptoethanol was an i n h i b i t o r of phenylalanine hydroxylase (40) and g i b b e r e l l i n Αχ hydroxylase (41), but i t was necessary to demonstrate 4-hydroxylase a c t i v i t y w i t h cinnamic a c i d (39). C h e l a t i n g agents i n h i b i t e d an N-demethylase (47) and a


In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979. 44

CO-light

weak w i t h CO, SKF 525A, CU++, NADH.

mercaptoethanol

s a f r o l e and 3,4-benzopyrene a r e s t i m u l a t o r y t o 2hydroxylation.

39

51

CO-light reversible, azide.

CO l i p a s e s , menadione, 1,4-nap thoquinone, riboflavin.

mercaptoethanol

EDTA

illuminated chloro p l a s t s , or d i t h i o nite

NADPH o r NADH, and an u n i d e n t i f i e d p r o d u c t o r THFA

NADPH, a s c o r b a t e Fe-H-

NADPH

NADPH ( o r NADH w i t h 2nd substrate)

pyroloxygenase

phenylalanine hydroxylase

g i b b e r e l l i n Αχ hydroxylase

cinnamic a c i d hydroxylase

cinnamic a c i d , h y d r o x y l a s e and N-demethylase

ascorbate

NADPH

b i p h e n y l hydroxylase (microsomal)

40

41

aminopteridine, s u l f h y d r y l c o n t a i n i n g compounds; i . e . , mercaptoethanol. mercaptoethanol,

EDTA

38

c h e l a t i n g agents, d i t h i o t h r e i t o l and mercapto ethanol.

45,46

NADPH

2,4-D h y d r o x y l a s e and c i n n a m i c a c i d hydroxylase

reversible.

43

e l e c t r o n a c c e p t o r s , CN, phenols, a n i l i n e .

c h e l a t i n g agents and detergents

NADPH ( a c t i v i t y p r e s e n t a t reduced l e v e l without)

aldrin

epoxidase

47

Reference

CO, i o n i c d e t e r g e n t s s u l f h y d r y l reagents, c h e l a t i n g agents and electron acceptors.

Inhibitors

NaCN, i s o a s c o r b a t e , p o l y c l a r AT

A c t i v a t o r s o r f a c t o r s used t o demonstrate a c t i v i t y

NADH o r NADPH

Co-factors

P r o p e r t i e s o f p l a n t enzymes w i t h mfo t y p e a c t i v i t y .

N-demethylase

Enzyme

Table I I .


SU

«s:

co


92


pyroloxygenase (38), but s l i g h t l y stimulated an a l d r i n epoxidase (43). S a f r o l e and 3,4-benzopyrene stimulated a b i p h e n y l 2hydroxylase from avocado, but other mfo a c t i v i t i e s were u n a f f e c t ed (46). Castor bean N-demethylase was s t r o n g l y i n h i b i t e d by K p i b u f f e r and was assayed w i t h t r i c i n e b u f f e r (51). In sharp c o n t r a s t , N-demethylase from cotton was i n h i b i t e d by t r i c i n e , but not by K p i (47). Based on d i f f e r e n c e s i n c o - f a c t o r requirements, responses to d i f f e r e n t i n h i b i t o r s and responses to d i f f e r e n t b u f f e r s , i t would appear that a v a r i e t y of d i f f e r e n t p l a n t mfo systems e x i s t . Although most mfo systems i n p l a n t s are a s s o c i a t e d w i t h p a r t i c u l a t e (microsomal) f r a c t i o n s , a g i b b e r e l l i n Ai hydroxylase (41) and a b i p h e n y l hydroxylase (45, 46) are s o l u b l e systems. The nature of s e v e r a l other mfo systems (38, 40) i s u n c e r t a i n . The s o l u b i l i z a t i o n of Swede root cinnamic a c i d 4-hydroxylase w i t h T r i t o n X-100 has been reported (75) and both s o l u b l e and p a r t i c u l a t e a l d r i n epoxidase (60, 61) and b i p h e n y l hydroxylase (45, 46) have been i s o l a t e d from the same t i s s u e s . I t appears that i s o l a t e d a c t i v e f r a c t i o n s may not always be a true i n d i c a t i o n of the nature and l o c a t i o n of the enzyme i n the n a t i v e s t a t e , but may a l s o be a f u n c t i o n of the combination of techniques and t i s s u e used. A number of d i f f e r e n t procedures and s p e c i a l precautions were used to i s o l a t e mfo a c t i v i t y from p l a n t sources (Table I I I ) . In the i s o l a t i o n of N-demethylase a c t i v i t y from c o t t o n , the t i s s u e was ground under l i q u i d n i t r o g e n (47, 48) and i n the i s o l a t i o n of a l d r i n epoxidase from pea (43), t o t a l l y anaerobic c o n d i t i o n s were used. Most e x t r a c t i o n and i s o l a t i o n procedures u t i l i z e d pH 7.5 b u f f e r s that contained e i t h e r p o l y v i n y l p y r r o l i d i n e or p o l y c l a r AT. The b u f f e r used to e x t r a c t N-demethylase a c t i v i t y from c o t t o n contained i s o a s c o r b a t e , p o l y c l a r AT and sodium cyanide. Mercaptoethanol, c h e l a t i n g agents, sucrose, and bovine serum albumin have a l s o been used w i t h v a r y i n g frequency to help p r o t e c t mfo systems during i s o l a t i o n . Differential centrifugation has been the most commonly used f r a c t i o n a t i o n method, but DEAE chromatography, sephadex chromatography, ammonium s u l f a t e f r a c t i o n a t i o n and d e n s i t y gradient c e n t r i f u g a t i o n have a l s o been employed. Based on t h i s b r i e f survey, i t i s apparent that the number of x e n o b i o t i c s shown to be metabolized by i n v i t r o p l a n t mixed f u n c t i o n oxidases i s very l i m i t e d . Some of these o x i d a t i v e systems have not been w e l l d e f i n e d . Considerable e f f o r t i s needed to i s o l a t e and c h a r a c t e r i z e these systems. The methods used f o r enzyme i s o l a t i o n have been h i g h l y v a r i a b l e . Because of the extremely low l e v e l s of enzyme a c t i v i t y a s s o c i a t e d w i t h many of the mfo systems, they are f r e q u e n t l y d i f f i c u l t to study and s p e c i a l methods are o f t e n needed to measure r e a c t i o n r a t e s . This i s f u r t h e r complicated by the presence of endogenous i n h i b i t o r s and the i n s t a b i l i t y of many of these systems.


In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979. diff

diff

75

76

EDTA, sucrose, mercaptoethanol

mercaptoethanol, EDTA, serum albumin, p o l y c l a r AT, mannitol.

homogenize i n pH 7.5 b u f f e r , f i l t e r , d i f f e r e n t i a l centrifug.

homogenize i n pH 7.5 b u f f e r , f i l t e r , d i f f e r e n t i a l c e n t r i f u g . and d e n s i t y gradient c e n t r i f u g .

aged Swede r o o t

wounded Jerusalem a r t i c h o k e tuber

cinnamic a c i d hydroxylase (microsomal)


*Also i s o l a t e d from other sources.

51

EDTA, sucrose

p u l v e r i z e i n pH 7.5 b u f f e r , f i l t e r , d i f f e r e n t i a l c e n t r i f u g . or d e n s i t y gradient c e n t r i f u g .

c a s t o r bean

41


PVP

sucrose,

homogenize decoated imbibed seeds (pH 6.5), d i f f e r e n t i a l c e n t r i f u g .

snap bean

40

38

45,46

43

47

g i b b e r e l l i n Αχ hydroxylase

none apparent

remove high inhibitor

m.w.

isolation, AT

none apparent

PVP

anaerobic polyclar

AT,

Reference

p u l v e r i z e f r o z e n t i s s u e , watere x t r a c t , f i l t e r , acetone f r a c t i o n a t i o n , DEAE-cellulose and calcium phosphate g e l a d s o r p t i o n .

water-extract, (NH4)2SO4 f r a c t i o n a t i o n , DEAE and Sephadex G-100

e x t r a c t (pH 7.4), f i l t e r , erential centrifug.

e x t r a c t (pH 7.5), f i l t e r , erential centrifug.

e x t r a c t (pH 7.5), f i l t e r , Sephadex G-100 and d i f f e r e n t i a l c e n t r i f u g .

NaCN, p o l y c l a r isoascorbate

S p e c i a l Precautions

spinach leaves

*wheat germ

avocado pear

*cucumber leayes

pea root

Procedure

p u l v e r i z e d i n l i q u i d N2» e x t r a c t (pH 7.5), f i l t e r , d i f f e r e n t i a l and d e n s i t y g r a d i e n t c e n t r i f u g .

General I s o l a t i o n

phenylalanine hydroxylase

pyroloxygenase

b i p h e n y l hydroxy l a s e (microsomal and s o l u b l e )

2,4-D and cinnamic A. hydroxylase (microsomal)

a l d r i n epoxidase (microsomal)

* c o t t o n leaves

T i s s u e Source

Methods used to I s o l a t e mixed f u n c t i o n o x i d a s e - l i k e a c t i v i t y from higher p l a n t s .

N-demethylase (microsomal)

Enzyme

Table I I I .


94



REDUCTION REACTIONS: Although r e d u c t i v e r e a c t i o n s have not been demonstrated t o p l a y a major r o l e i n x e n o b i o t i c metabolism i n higher p l a n t s , s e v e r a l i n v e s t i g a t o r s have reported the r e d u c t i o n of aromatic n i t r o groups i n p l a n t s (77-82) and c a l l u s c u l t u r e (82). A r e d u c t i v e dehalogenation (83) and the r e d u c t i o n o f a s u l f o x i d e to a s u l f i d e (84) have a l s o been reported. The reported a r y l n i t r o reductions of pentachloronitrobenzene (PCNB) (77) and f l u o r o d i f e n (j>-nitrophenyl-a,a,a-trifluoro-2-nitrophenyl-£-tolyl ether) (7£, 80, 85, 86) were i n competition w i t h g l u t a t h i o n e conjugation. In studies with f l u o r o d i f e n , a r y l n i t r o reduction was a minor metabolic pathway. However, i n s t u d i e s w i t h pentachloronitrobenzene, about 28% of the p e s t i c i d e was converted to pentachloroaniline. The only r e d u c t i v e r e a c t i o n s i n v o l v i n g p e s t i c i d e s that appear to have been studied i n d e t a i l i n p l a n t s a r e those c a t a l y z e d by a r y l n i t r o r e d u c t a s e s . A r y l nitroreductase a c t i v i t y has been i s o l a t e d from peanut (77) and pea (78). The s o l u b l e a r y l n i t r o r e d u c t a s e system from peanut was i s o l a t e d i n c o n j u n c t i o n with s t u d i e s on GSH S-transferase a c t i v i t y . Magnesium c h l o r i d e was used i n the i s o l a t i o n procedure to p r e c i p i t a t e the microsomal f r a c t i o n (87) and to avoid h i g h speed c e n t r i f u g a t i o n . With PCNB as the s u b s t r a t e , reductase a c t i v i t y was detected w i t h enzyme p r e p a r a t i o n s from r o o t s and hypocotyls o f 7-day-old e t i o l a t e d peanut seedings. A r y l n i t r o r e d u c t a s e a c t i v i t y was detected only when the r e a c t i o n was run under a n i t r o g e n atmosphere i n the presence of both FAD and NADPH. The a r y l n i t r o r e d u c t a s e i s o l a t e d from soybean s e e d l i n g r o o t s c a t a l y z e d the r e d u c t i o n of dinoben ( 2 , 5 - d i c h l o r o - 3 - n i t r o b e n z o i c acid) when the system was incubated under n i t r o g e n a t pH 8.2 w i t h e i t h e r NADPH or NADH (78). When t h i s enzyme system was p u r i f i e d f u r t h e r , FAD or FMN was r e q u i r e d i n a d d i t i o n t o NADH or NADPH. The f a c t that i n v i t r o r e d u c t i o n could be demonstrated only under a n i t r o g e n atmosphere suggests that these r e a c t i o n s may become more important i n v i v o under c o n d i t i o n s o f low oxygen t e n s i o n . In many cases i n which r e d u c t i o n r e a c t i o n s have been reported, p l a n t s have been grown and t r e a t e d i n such a manner that m i c r o b i a l a c t i o n could have accounted f o r the r e a c t i o n . This p o s s i b i l i t y should be considered i n any study i n which r e d u c t i v e r e a c t i o n s a r e reported i n x e n o b i o t i c metabolism i n p l a n t s . The r e d u c t i o n of the a r y l n i t r o groups of PCNB and dinoben with i n v i t r o enzyme systems i s o l a t e d from p l a n t s and the apparent r e d u c t i o n of the a r y l n i t r o group of 2 , 6 - d i c h l o r o - 4 - n i t r o a n i l i n e i n soybean c a l l u s c u l t u r e suggests, however, that a r y l n i t r o reductions may r e s u l t from p l a n t metabolism. HYDROLYTIC REACTIONS: A r y l Acylamidases:


3.

LAMOUREUX AND FREAR

Studies

95

A r y l acylamidases that hydrolyze the h e r b i c i d e p r o p a n i l ( 3 , 4 - d i c h l o r o p r o p i o n a n i l i d e ) have been i s o l a t e d from b a c t e r i a (88), f u n g i (89), b i r d s (90), mammals (91, 92) and p l a n t s (93110) (Figure 9). Of the h y d r o l y t i c enzymes that p l a y important r o l e s i n the metabolism of p e s t i c i d e s i n p l a n t s , the a r y l a c y l amidases appear to be the most thoroughly s t u d i e d . Studies w i t h homogenates of r i c e p l a n t s (101, 105, 106) e s t a b l i s h e d the enzymatic nature of p r o p a n i l h y d r o l y s i s . H y d r o l y t i c a c t i v i t y was a s s o c i a t e d w i t h a p a r t i c u l a t e f r a c t i o n (93, 94). The enzyme was s t a b l e , r e q u i r e d no c o - f a c t o r s , and had a pH optimum of 7.5-7.9. A comparison of the enzyme a c t i v i t y i n r i c e and barnyard grass showed a 60-fold higher l e v e l of enzyme a c t i v i t y i n r i c e (94). T h i s suggested that p r o p a n i l r e s i s t a n c e was based on the presence of the a r y l acylamidase i n s u f f i c i e n t c o n c e n t r a t i o n to d e t o x i f y the h e r b i c i d e before s e n s i t i v e s i t e s could be attacked. Enzyme a c t i v i t y i n r i c e s e e d l i n g s was a f u n c t i o n of the developmental stage of the p l a n t and maximum a c t i v i t y was reached at the f o u r t h l e a f stage. In f i e l d s t u d i e s , however, seedlings i n the t h i r d and f o u r t h l e a f stages were more s e n s i t i v e to p r o p a n i l than were younger seedlings (98). A r y l acylamidase a c t i v i t y was a l s o reported i n r i c e r o o t c a l l u s suspension c u l t u r e s (98), but i n v i t r o a c t i v i t y was demonstrated only i n c u l t u r e s that were at l e a s t 120 hr o l d . T h i s d i d not correspond w i t h the much e a r l i e r appearance of 3 , 4 - d i c h l o r o a n i l i n e i n p r o p a n i l - t r e a t e d c u l t u r e s . f


Plant Enzyme

f

I n h i b i t o r s t u d i e s showed that carbamate i n s e c t i c i d e s were powerful competitive i n h i b i t o r s of the a r y l acylamidase from r i c e (94). The phosphorothioate i n s e c t i c i d e s , p a r a t h i o n and sumithion were much weaker i n h i b i t o r s of the enzyme, but t h e i r o x i d i z e d analogs, paraoxon and sumioxon, were r e s p e c t i v e l y 100X and 200X more e f f e c t i v e as i n h i b i t o r s (95). Phosphorothioate i n s e c t i c i d e s are p a r t i a l l y converted to t h e i r oxygen analogs i n p l a n t s (103). The s t r e n g t h of paraoxon as an i n h i b i t o r of the h y d r o l y s i s r e a c t i o n i n v i t r o was c o r r e l a t e d w i t h i t s s y n e r g i s t i c e f f e c t i n reducing the f r e s h weight of p r o p a n i l - t r e a t e d r i c e seedlings (95). The s y n e r g i s t i c e f f e c t between p r o p a n i l and the organophosphorthioate and carbamate i n s e c t i c i d e s (101, 107) can thus be explained on the b a s i s of competitive i n h i b i t i o n of the a r y l acylamidase enzyme necessary f o r the d e t o x i c a t i o n of propanil. A p a r t i c u l a t e a r y l acylamidase enzyme has a l s o been i s o l a t e d from red r i c e (108) and s o l u b l e a r y l acylamidases have been i s o l a t e d from t u l i p (99) and dandelion (109). Substrate s p e c i f i c i t i e s of these enzymes are shown i n Tables IV and V. Significant and p o t e n t i a l l y u s e f u l d i f f e r e n c e s e x i s t i n the s u b s t r a t e preferences of these enzymes f o r d i f f e r e n t halogenated propionanilides. 2, 3 D i c h l o r o p r o p i o n a n i l i d e was the p r e f e r r e d s u b s t r a t e f o r the amidase from c u l t i v a t e d r i c e , but i t was the poorest s u b s t r a t e f o r red r i c e . In c o n t r a s t , p r o p a n i l was the best s u b s t r a t e f o r red r i c e , but had a r e l a t i v e r a t e of metabolism of only 42% i n c u l t i v a t e d r i c e . Substrate preferences f o r f

L


96


*

,

0

CI - ^ J V N - C - C H

Cl'

-CH

»»

3

PRORANIL

Figure 9.


ARYL ACYLAMIDASE 2

. . C.,vO/~ Cl'

mz

Hydrolysis of 3' ,4'-dichloropropionanilide amidase

"**

M

0

0

~

C

C

H

2~

C

H

3

(propanil) by an aryl acyl-

d i f f e r e n t a l k y l s i d e chains were s i m i l a r f o r these enzymes, each had a marked preference f o r the p r o p i o n i c s i d e c h a i n . These a r y l acylamidases were not capable o f h y d r o l y z i n g carbamate o r urea herbicides. Some d i f f e r e n c e s were found i n the response o f these enzymes to i n h i b i t o r s (Table V I ) . These d i f f e r e n c e s between the a r y l acylamidases suggest that s u b s t r a t e s p e c i f i c i t y t e s t s might be used t o develop more s e l e c t i v e compounds o r compounds with more d e s i r a b l e b i o l o g i c a l s t a b i l i t y . Differences i n response to s u l f h y d r y l i n h i b i t o r s suggests that i n h i b i t o r s t u d i e s w i t h i s o l a t e d enzyme systems could provide an e f f e c t i v e means f o r the development of s e l e c t i v e p e s t i c i d e s y n e r g i s t s .

Table IV.

Substrate s p e c i f i c i t y of a r y l acylamidase from red r i c e (108), r i c e (94), t u l i p (99) and dandelion (109): the e f f e c t of c h l o r i n e r i n g s u b s t i t u t i o n . R e l a t i v e A c t i v i t y (%)

Substrate

1

1

1

1

2 ,3 -dichloropropionanilide 2 ,4 -dichloropropionanilide 2 -chloropropionanilide 3 -chloropropionanilide 3 ^'-dichloropropionanilide 3 ,5 -dichloropropionanilide 2 ,5 -dichloropropionanilide 4 -chloropropionanilide 2 ,6 -dichloropropionanilide propionanilide 1

1

1

1

1

1

1

1

1

1

Red Rice

29 47 29 73 100 33 27 38

—

58

Rice

Tulip

Dandelion

100 84 60 42 42 30 27 21 1

41 100 66 27 100

14 49 37 51 100

— —

— —

—

—

—

99 2


54 0


3.

Table V.

Fiant Enzyme

97

Studies

Substrate s p e c i f i c i t y of a r y l acylamidases from red r i c e (108), r i c e (94), t u l i p (99) and dandelion (109): the e f f e c t of v a r i o u s 3, 4 d i c h l o r o a n i l i d e a l k y l analogs. f

L

Relative A c t i v i t y Substrate 1

1

3 ,4 -dichloroacetanilide 3 ,4'-dichloropropionanilide 3 ,4 -dichlorobutyranilide 3 ,4 -dichlorovaleranilide 3 ,4 -dichloro-2-methyl propionanilide 3 ,4 -dichloro-2-methyl acrylanilide 3 ,4 -dichloro-3-methyl butyranilide


f

f

T

1

1

f

1

1

1

1

1

Red

Rice

82 100 18 40

— 0

—

(%)

Rice

Tulip

Dandelion

59 100 32 39 2

49 100 18 3

49 100 37 12

—

—

0

2

0

—

8

—

T h i r t y - e i g h t d i f f e r e n t h o r t i c u l t u r a l and agronomic crop species r e p r e s e n t i n g 10 d i f f e r e n t p l a n t f a m i l i e s were assayed f o r a r y l acylamidase a c t i v i t y with p r o p a n i l as the substrate (96). Enzyme a c t i v i t y was reported i n over h a l f of the s p e c i e s . Only one f a m i l y , leguminosae, was devoid of a c t i v i t y . A s i m i l a r study was conducted with 19 genera of weeds. Enzyme a c t i v i t y was assayed w i t h p r o p a n i l , 1,l-dimethyl-3-phenylurea (fenuron), and i s o p r o p y l c a r b a n i l a t e (propham) (97). P r o p a n i l was hydrolyzed at widely v a r y i n g r a t e s by approximately 70% of the s p e c i e s . Fenuron and propham, however, were hydrolyzed by the enzyme p r e p a r a t i o n from only one s p e c i e s , w i l d cucumber. The d i s t r i b u t i o n of a r y l acylamidases i n other members of the p l a n t kingdom has a l s o been reported (100, 110). Extensive s t u d i e s w i t h a r y l acylamidases have shown that these enzymes are widely d i s t r i b u t e d i n the p l a n t kingdom. A c t i v i t y v a r i e s widely w i t h i n d i f f e r e n t p l a n t t i s s u e s and among d i f f e r e n t plant species. Resistance to p r o p a n i l i s dependent upon the presence of these enzymes. D e t a i l e d substrate s p e c i f i c i t y s t u d i e s w i t h four a r y l acylamidases revealed s u b t l e d i f f e r e n c e s i n s u b s t r a t e s p e c i f i c i t y and response to i n h i b i t o r s . I n h i b i t o r s t u d i e s with carbamate and organophosphate i n s e c t i c i d e s have c l e a r l y shown that these compounds are strong competitive i n h i b i t o r s of the a r y l acylamidases from r i c e . Interactions observed i n the f i e l d between p r o p a n i l and these i n s e c t i c i d e s can be a t t r i b u t e d to the i n h i b i t i o n of a r y l acylamidases.


In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979. 29

— —

0.2mM

0.2mM

0.2mM

— —

iodoacetate

2,4-dichlorophenoxyacetic

pyrocatecol

3

6

35

**Catechol used instead of pyrocatechol.

FeCl

*CuCl2 used instead of CUSO4

2

0.2mM

N-ethylmaleimide

C0CI2

6

0.2mM

j>-benzoquinone

4

0.2mM

j>-chloromercuribenzoate

60

0.2mM

2

— — —

CUSO4

HgCl

Na2As02

— — —

Red Rice Cone. % Inhib.

74

O.lmM

—

— — — — —

18

44

47

50

0.5mM

0.5mM

0.5mM

0.5mM

59

74

l.OmM

0.2mM

74

Rice % Inhib.

1 0

0.50mM

— —

2

0.50mM

— —

0.50mM

9

— —

— — 0.50mM

16

16 6

l.OmM

73 l.OmM

0.5mM**

—

2

l.OmM

—

23

50

61

l.OmM

0.50mM

0.25mM

86

89

0.5mM

100

0.25mM

0.25mM 0.5mM*

—

—

—

—

5

0.25mM

27

Dandelion Cone. % Inhib

0.25mM

Tulip Cone. % Inhib.

Source of Enzyme

(108), r i c e (94), t u l i p (99) and dandelion (109).

0.5mM

Cone.

I n h i b i t i o n of a r y l acylamidases from red r i c e

_o-iodosobenzoate

Inhibitor

Table VI.


3.

L A M O U R E U X A N D FREAR

Plant Enzyme

Studies

99


Esterases: Many c a r b o x y l i c a c i d e s t e r p e s t i c i d e s are r e a d i l y hydrolyzed to f r e e a c i d s i n v i t r o . In most cases, the f r e e a c i d form of the p e s t i c i d e i s presumed or known to be the a c t i v e agent. Esterases that hydrolyze c a r b o x y l i c a c i d e s t e r p e s t i c i d e s are important, not only because they p l a y a r o l e i n the degradation of the p e s t i c i d e , but a l s o because they may be i n v o l v e d i n a c t i v a t i o n , s e l e c t i v i t y or d e t o x i c a t i o n . Some esterases may be i n d u c i b l e (111). Common i n h i b i t o r s of these enzymes i n c l u d e a number of the organophosphate and carbamate i n s e c t i c i d e s (112-114). The p o s s i b i l i t y of p e s t i c i d e i n t e r a c t i o n between c a r b o x y l i c a c i d e s t e r h e r b i c i d e s and c e r t a i n i n s e c t i c i d e s e x i s t s . Research on the i n d u c t i o n , a c t i v a t i o n , i n h i b i t i o n , and s u b s t r a t e s p e c i f i c i t y of esterases should have important and d i r e c t a p p l i c a t i o n to the improvement and b e t t e r use of p e s t i c i d e s . Esterases are probably ubiquitous and have been i s o l a t e d from many p l a n t s p e c i e s . The s t a b i l i t y of p l a n t esterase pre p a r a t i o n s v a r i e s with the source and may be r e l a t e d to the presence of phenol oxidases and polyphenols. Gel e l e c t r o p h o r e s i s has been a v a l u a b l e t o o l i n studying p l a n t e s t e r a s e s and has shown that these enzymes are a complex f a m i l y of isozymes with d i f f e r e n c e s i n s u b s t r a t e preference and s u s c e p t i b i l i t y to i n h i b i t o r s (115-118). P o l y a c r y l a m i d e - g e l e l e c t r o p h o r e s i s separated 7 esterases from pea and 14 esterases from green bean (114). Separated isozymes responded d i f f e r e n t l y to the assay s u b s t r a t e s , a-naphtylacetate, α-naphthylbutyrate, and a-naphthylpropionate. D i f f e r e n t i a l response to i n h i b i t o r s such as p a r a t h i o n , paraoxon and d i i s o p r o p y l p h o s p h o r o f l u o r i d a t e was a l s o observed. Gel e l e c t r o p h o r e s i s of enzyme p r e p a r a t i o n s from cabbage i n d i c a t e d the presence of 6 esterases (113). With 2naphthylacetate as the s u b s t r a t e , i n h i b i t i o n was demonstrated w i t h carbofuran, e s e r i n e and s e v e r a l other compounds. The p a r t i a l l y p u r i f i e d e s t e r a s e from cabbage had a pH optimum of approximately 7, and an estimated molecular weight of 69,000. T h i s enzyme l o s t 50% of the o r i g i n a l a c t i v i t y when i t was s t o r e d f o r 22 days a t -22°. A few s t u d i e s have reported the h y d r o l y s i s of p e s t i c i d e s by i n v i t r o esterase systems (Figure 10). Recent s t u d i e s showed that apple and cucumber leaves c o n t a i n esterases that hydrolyze f u n g i c i d a l n i t r o p h e n y l e s t e r s (115). Esterase a c t i v i t y was detected w i t h 18 d i f f e r e n t s u b s t r a t e s . Hydrolysis rates varied 100-fold, depending upon the s u b s t r a t e . Selective i n h i b i t i o n with paraoxon and s e v e r a l other compounds was a l s o demonstrated. E s e r i n e and EDTA were not i n h i b i t o r y . D i f f e r e n c e s i n isozyme patterns were observed between the two s p e c i e s . The c a r b o x y l i c a c i d e s t e r h e r b i c i d e chlorfenprop-methyl (methyl 2-chloro-3-(4-chlorophenyl)propionate) i s hydrolyzed i n v i v o and i n v i t r o to an a c t i v e h e r b i c i d e (119). A c t i v e esterases were i s o l a t e d from 2 c u l t i v a r s of oat, w i l d oat, wheat and beet


100


0

O-c'-R

NOo

OH

(FUNGICIDE)

NOo

(ACTIVE)

Ο Downloaded by UNIV OF MINNESOTA on October 2, 2013 | http://pubs.acs.org Publication Date: April 5, 1979 | doi: 10.1021/bk-1979-0097.ch003

OH NO,

N0

O

v.

(FUNGICIDE)

(ACTIVE) R^l R X

Ο CH -ÇH-C-OCH I Cl CHLORFENPROP-METHYL (HERBICIDE) 2

CH.-CH-COOH I Cl (ACTIVE)

3

Figure 10. Hydrolysis of fungicides and herbicides by plant esterases (115, 119)

S (CH 0) P 3

(CH OÎ P-OH

2

3

2

S

+ CH3O-P-OH + CH3O-P-OH

S i

ÇH

S I

CH

2

I C-N-CH3 ' 1

S S I CH I -OH II 0 2

2

I _ _ II H 0 2

C

H

0

N

z

C

H

C

DIMETHOATE

S 0 Il II (CH 0) P-S-CH-C-OC H 3

2

2

5

CH^-C-OC^H. II 0 2

2

S II (CH 0) P-OH 3

2

S + (CH 0) P-SH 3

2

5

MALATHION Figure 11.

Hydrolysis of organophosphorus insecticides by plant esterases (127, 128)



3.


Vlant Enzyme

Studies

101

(119). Estimates of i n v i v o r a t e s of h y d r o l y s i s based upon i n v i t r o data i n d i c a t e d that h e r b i c i d e s e l e c t i v i t y was not r e l a t e d to esterase a c t i v i t y . Two r e l a t e d h e r b i c i d e s , flamprop-isopropyl [(±)-N-benzoyl-N-(3-chloro-4-fluorophenyl)alanine i s o p r o p y l e s t e r ] and benzoylprop-ethyl [(±)-N-benzoyl-N-(3,4-dichloropheny1) a l a n i n e e t h y l e s t e r J are a l s o hydrolyzed to a c t i v e h e r b i c i d e s , presumably by p l a n t esterases (120, 121). Very low l e v e l s of a c t i v i t y have been reported f o r the i n v i t r o h y d r o l y s i s of b i f e n o x [methyl 5-(2,4-dichlorophenoxy)-2-nitrobenzoate] i n homogenates of v e l v e t g r a s s (122). These i n v i t r o and i n v i v o s t u d i e s have shown that p l a n t esterases can c a t a l y z e the h y d r o l y s i s of a number of p e s t i c i d e s . These s t u d i e s do not, however, answer important questions regarding the l o c a l i z a t i o n of the enzymes w i t h i n the c e l l or the d i s t r i b u t i o n of the enzymes i n v a r i o u s t i s s u e s of the p l a n t . In the case of h e r b i c i d e s , these f a c t o r s may p l a y an important r o l e in selectivity. A number of organophosphorous compounds are metabolized i n higher p l a n t s to products that are c o n s i s t e n t w i t h the i n v o l v e ment of h y d r o l y t i c enzymes (123-129). In v i v o and i n v i t r o s t u d i e s showed that wheat and sorghum g r a i n s r a p i d l y degrade dimethoate [(),O-dimethyl-S-(N-methylcarbamoylmethyl)phosphorot h i o l o t h i o n a t e ] to a number of products i n c l u d i n g j),0-dimethylphosphoro thionate, mono-C^-me thy 1 JS-N-me t h y l - c a r b amo y lme t hy l p ho s p h o r o t h i o l o t h i o n a t e and mono-O-methyl-S-carboxymethyl-phosphorot h i o l o t h i o n a t e (127) (Figure 11). The apparent h y d r o l y s i s of malathion (J3,0-dimethyl-S-bis(carboethoxy)ethyl phosphorodit h i o a t e ) to dimethylphosphorothionate and dimethylphosphorothiolt h i o n a t e with crude e x t r a c t s from wheat germ was a l s o demonstrated (128). Unfortunately, these s t u d i e s do not e s t a b l i s h the s i g n i f i c a n c e of esterases or phosphohydrolyases i n the h y d r o l y s i s of organophosphorous p e s t i c i d e s . In animals and i n s e c t s , g l u t a t h i o n e S-transferases and mixed f u n c t i o n oxidases p l a y important r o l e s i n the metabolism of organophosphorus compounds (126). These enzymes may form some of the same products produced by e s t e r a s e s . Unless experiments are conducted p r o p e r l y , i t may not be p o s s i b l e to d i s c e r n whether GSH S - t r a n s f e r a s e , esterase or mixed f u n c t i o n o x i d a s e - r e l a t e d r e a c t i o n s are r e s p o n s i b l e f o r e s t e r hydrolysis. In a d d i t i o n , p l a n t peroxidases may a l s o c a t a l y z e the cleavage of organophosphorous compounds (21). In v i v o and jLn v i t r o s t u d i e s have shown that chloramben (3amino-2-5-dichlorobenzoate) i s r a p i d l y metabolized to a s t a b l e N-glucoside i n r e s i s t a n t p l a n t species (130). In a d d i t i o n to the N-glucoside, s u s c e p t i b l e species a l s o form the chloramben glucose e s t e r (130, 131). The glucose e s t e r was not s t a b l e i n v i v o and appeared to a c t as a chloramben r e s e r v o i r (Figure 12). Crude homogenates from cucumber and b a r l e y t i s s u e s hydrolyzed the chloramben glucose e s t e r q u i t e r a p i d l y . Since other benzoic a c i d or phenoxy h e r b i c i d e s may form glucose e s t e r conjugates, these h y d r o l y t i c enzymes could p l a y an important r o l e i n r e g u l a t i n g


102



CHLORAMBEN (ACTIVE)

TRANSIENT PRODUCT Figure 12.

Chloramben conjugation and hydrolysis (ISO, 131)

α — CHLOROACETAMIDES (2 EXAMPLES) Figure 13.

OIPHENYL ETHERS (I EXAMPLE)

4—HYDROXYANILINOS (2 EXAMPLES)

Agricultural chemicals known to be enzymatically conjugated to GSH in phnts


3.


Phnt

Enzyme

103

Studies

the l e v e l s of'these h e r b i c i d e s i n v i v o . CONJUGATION: Glutathione

S-Transferase:

In animals, g l u t a t h i o n e (GSH) conjugation i s an important r e a c t i o n i n the metabolism o f a wide range o f x e n o b i o t i c s and has been the subject of a number o f reviews (132-136).


HN-CH2-C00H

HN-CH -C00H 2

C=0 R-X

+

I CHo-CH

I

C=0 GSH S - t r a n s f e r a s e | ^ CH2-CH

I

HX

I I

SH HN-C=0

COOH

S

CH2-CH -CH

R

2

NH GSH

HN-C=0

COOH

I I I

I I Substrate

+

CH -CH -CH 2

2

NH

2

2

GSH Conjugate

Recent f i n d i n g s (137-146) have e s t a b l i s h e d that GSH conjugation i s a l s o important i n p l a n t s . Although the range of x e n o b i o t i c compounds that have been demonstrated to undergo GSH conjugation i n p l a n t s i s l i m i t e d (Figure 13), i t can be assumed that GSH conjugation o f a d d i t i o n a l c l a s s e s of compounds w i l l be shown as more s t u d i e s a r e conducted. Glutathione conjugation should be considered as a p o s s i b l e metabolic r e a c t i o n with any x e n o b i o t i c that possesses an e l e c t r o p h i l i c center and an a p p r o p r i a t e l e a v i n g group, o r with x e n o b i o t i c s that can be a c t i v a t e d by o x i d a t i o n or by some other means to a f f o r d an a c t i v e s u b s t r a t e . Glutathione conjugation i s of p a r t i c u l a r importance i n p l a n t s (a) because of the wide range of p o t e n t i a l s u b s t r a t e s , (b) because i t may determine the nature of the t e r m i n a l r e s i d u e s i n the p l a n t , and (c) because i t may be a major f a c t o r i n p e s t i c i d e d e t o x i c a t i o n or h e r b i c i d e s e l e c t i v i t y . Glutathione S^-transferases have been found i n 21 p l a n t s p e c i e s (Table VII) and appear to be widespread i n the p l a n t kingdom. G l u t a t h i o n e S-transferases from animals (133) and p l a n t s (140, 142, 143, 145, 146) appear to be s o l u b l e enzymes. G l u t a t h i o n e ^ - t r a n s f e r a s e a c t i v i t y may not be detected i n crude enzyme p r e p a r a t i o n s u n t i l a f t e r g e l chromatography (140) or may have l i m i t e d s t a b i l i t y when p a r t i a l l y p u r i f i e d (142).


104


Table V I I .

Species which have been examined i n v i t r o f o r GSH a c t i v i t y and c y s t e i n e S - t r a n s f e r a s e a c t i v i t y .

S-transferase

Substrate Used to Assay f o r A c t i v i t y


Plant

Atrazine (143)

barley

N. D.

corn

H

Fluorodifen (142)

PCNB (146)

EPTC (144)

4-hydroxy chlorpropham

C191)

cotton crabgrass foxtail lambsquarter johnsongrass

H

oat

Ν. D.

okra pea peanut pigweed soybean sorghum sudangrass sugarcane squash tomato wheat cucumber rice



3.


Plant Enzyme

Studies

105

The f i r s t demonstrated GSH conjugation of a p e s t i c i d e i n p l a n t s was reported i n sorghum (137). A t r a z i n e (2-chloro-4ethylamino-6-isopropylamino-j3-triazine) was i s o l a t e d as a GSH conjugate. The conjugation r e a c t i o n was enzymatic (143) and a c t i v e enzyme systems were subsequently i s o l a t e d from a t r a z i n e r e s i s t a n t corn, sorghum, johnsongrass, sudangrass and sugarcane. A t r a z i n e - s u s c e p t i b l e species (pea, oats, wheat, b a r l e y and pigweed) contained no d e t e c t a b l e enzyme a c t i v i t y . The f i n d i n g that GSH S - t r a n s f e r a s e a c t i v i t y was e a s i l y i s o l a t e d from r e s i s t a n t s p e c i e s , but not from s u s c e p t i b l e s p e c i e s , i n d i c a t e d that r e s i s t a n c e to a t r a z i n e was based on the presence of t h i s enzyme. Subsequent s t u d i e s w i t h an inbred corn l i n e showed that GSH JS-transferase a c t i v i t y was present i n high concentrations i n the r e s i s t a n t l i n e , but not i n the s u s c e p t i b l e l i n e . T h i s was c o r r e l a t e d to the production of the atrazine-GSH conjugate and l a c k of photosynthetic i n h i b i t i o n i n the r e s i s t a n t l i n e (147) (Table V I I I ) . I t was f u r t h e r shown that a number of r e s i s t a n t corn l i n e s contained h i g h concentrations of GSH S - t r a n s f e r a s e activity. These s t u d i e s confirmed the importance of GSH St r a n s f e r a s e a c t i v i t y i n the s e l e c t i v i t y of a t r a z i n e . In the t o l e r a n t s p e c i e s , corn and sorghum, GSH JS-transferase a c t i v i t y was concentrated i n the f o l i a r t i s s u e . Partially p u r i f i e d enzyme p r e p a r a t i o n s from corn leaves were s t a b l e and could be s t o r e d w i t h l i t t l e l o s s i n a c t i v i t y . Unfortunately, attempts to f u r t h e r p u r i f y the enzyme were not s u c c e s s f u l (148). Substrate s p e c i f i c i t y s t u d i e s with s e v e r a l 2 - c h l o r o - s — t r i a z i n e , 2-methoxy-s-triazine, and 2-methylmercapto-_s-1 r i a z ine h e r b i c i d e s showed that the 2 - c h l o r o - s - t r i a z i n e s were the only e f f e c t i v e s u b s t r a t e s . A l a t e r study showed that the s u l f o x i d e of a 2methylmercapto-j5-triazine was a good s u b s t r a t e f o r GSH conjugation (149). The i s o l a t e d corn enzyme was s p e c i f i c f o r GSH. D i t h i o t h r e i t o l , mercaptoethanol, 2,3-dimercaptopropanol or Lc y s t e i n e d i d not f u n c t i o n as s u l f h y d r y l s u b s t r a t e s . R e s u l t s from 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 w i t h the corn enzyme were comparable to those obtained w i t h e x c i s e d sorghum leaves (Table I X ) . Although i n v i t r o s t u d i e s i n d i c a t e d that 2-chloro-4amino-6-isopropyl-amino-s-triazine was a poor s u b s t r a t e f o r GSH jS-transferase i n corn, i n v i v o s t u d i e s with sorghum (150) showed that t h i s s u b s t r a t e was conjugated with GSH, T h i s d i f f e r e n c e may be a t t r i b u t e d to d i f f e r e n c e s i n the s u b s t r a t e s p e c i f i c i t y of the GSH ^ - t r a n s f e r a s e s from sorghum and corn. The GSH jS-transferase mediated cleavage of f l u o r o d i f e n (p_n i t r o p h e n y l α,α,α-trifluoro-2-nitro-p-tolyl ether) to S - ( 4 - t r i f l u o r o - 2 - n i t r o p h e n y l ) g l u t a t h i o n e and 4-nitrophenol appears to be the f i r s t demonstrated cleavage of a diphenylether by a GSH t r a n s f e r a s e system (142). When j v - ( 4 - t r i f l u o r o m e t h y l - 2 - n i t r o p h e n y l ) g l u t a t h i o n e was f i r s t detected i n v i v o , the nature of t h i s metabolite was not understood. A crude enzyme that produced the same product i n the presence of GSH was i s o l a t e d from pea epicotyl tissue. Subsequent l a r g e - s c a l e enzyme i n c u b a t i o n s



106


Table VIII.

Relationship between GSH-transferase a c t i v i t y , formation of GSH conjugates, and i n h i b i t i o n of photosynthesis i n atrazine resistant and susceptible corn.

GSH transferase* activity in vitro

% GSH conjugate i n leaf disc after 5 hours

% Inhibition of photosynthesis i n leaf discs after 2 hours

Corn l i n e

Response to atrazine

GT112RfRf

resistant

1.63

43.1

8.6

GT112

susceptible

0.03

0.4

64.9

6 other resistant

resistant

2.62 ±

1.0

*nmoles/mg protein/hour. **adapted from (147).


3.

LAMOUREUX AND FREAR

Plant Enzyme

107

Studies

produced S - ( 4 - t r i f l u o r o m e t h y l - 2 - n i t r o p h e n y l ) g l u t a t h i o n e i n good y i e l d and g r e a t l y f a c i l i t a t e d the development of a n a l y t i c a l techniques that were used i n the i s o l a t i o n and i d e n t i f i c a t i o n of the i n v i v o metabolite (141, 142).


Table IX.

Comparison of the r e l a t i v e s u b s t r a t e s p e c i f i c i t y of excised sorghum leaves (138, 150) w i t h the s u b s t r a t e s p e c i f i c i t y of the g l u t a t h i o n e S-transferase from corn (143).

Substrate

atrazine GS-13529 cyprazine propazine simazine 2-chloro-4-amino-6i s ο ρ ropylamino-s_triazine 2-hydroxy-4-ethylamino 6-isopropylamino-striazine

Relative activity in vitro (corn)

0.68 1.00 0.52 0.42 0.05 0.01

Relative a c t i v i t y i n vivo (sorghum)

1.00 0.93 0.92 0.90 0.62 active*

0.01

* G l u t a t h i o n e - r e l a t e d conjugates were the primary products produced, but experimental c o n d i t i o n s were d i f f e r e n t from those used w i t h the other j>-triazines. The i s o l a t e d GSH jS-transferase from pea was s t a b l e , but crude enzyme preparations from corn, peanut and c o t t o n underwent i r r e v e r s i b l e i n h i b i t i o n when s t o r e d f o r s e v e r a l hours a t 4 ° . Enzyme a c t i v i t y was detected i n higher concentrations i n r e s i s t a n t species ( c o t t o n , corn, peanut, pea, soybean and okra) than i n s u s c e p t i b l e s p e c i e s (cucumber, tomato and squash). F l u o r o d i f e n s e l e c t i v i t y appeared to be based on enzyme d i s t r i b u t i o n and c o n c e n t r a t i o n . A broad s u b s t r a t e s p e c i f i c i t y study was not conducted w i t h t h i s enzyme, but two other diphenylether h e r b i c i d e s were t e s t e d and found to be i n a c t i v e . I t was hypo t h e s i z e d that only diphenylethers that were h i g h l y a c t i v a t e d a t the C - l p o s i t i o n would a c t as s u b s t r a t e s . Mercaptoethanol, 2,3-dimercaptopropanol, d i t h i o t h r e i t o l and c y s t e i n e would not f u n c t i o n as the s u l f h d r y l s u b s t r a t e with the enzyme from pea. Recent s t u d i e s have shown that the f u n g i c i d e PCNB (pentachloronitrobenzene) i s a l s o converted to GSH conjugates w i t h an enzyme system i s o l a t e d from pea (145). A d d i t i o n a l s t u d i e s w i l l be needed to determine i f the same enzyme i s i n v o l v e d i n the metabolism of both PCNB and f l u o r o d i f e n . A unique f e a t u r e of the




108

PCNB GSH S - t r a n s f e r a s e assay system was the i n c l u s i o n of t e r t ^ butanol to i n c r e a s e PCNB s o l u b i l i t y and enzymatic a c t i v i t y (Figure 14). The use of t e r t - b u t a n o l i n enzyme systems has been p r e v i o u s l y reported (151). T h i s system has not been completely s t u d i e d w i t h r e s p e c t to the PCNB-GSH conjugation r e a c t i o n . D e t a i l e d i n h i b i t o r s t u d i e s were conducted w i t h the GSH S~ t r a n s f e r a s e s i s o l a t e d from both corn and pea (Table X ) . Both enzymes gave s i m i l a r responses to s u l f h y d r y l compounds and to the c l a s s i c a l i n h i b i t o r s of mammalian GSH S - t r a n s f e r a s e a c t i v i t y , sulfobromophthalein and 1,2-dichloronitrobenzene. I n h i b i t i o n by sulfobromophthalein was competitive i n mammalian systems and a l s o appeared to be competitive i n both p l a n t systems, Propachlor ( 2 - c h l o r o - N - i s o p r o p y l a c e t a n i l i d e ) and barban (4-chloro~2-butynyl*m-chlorocarbanilate) were i n h i b i t o r s of the enzymatic r e a c t i o n s with a t r a z i n e and f l u o r o d i f e n . I n h i b i t i o n by a l k y l a t i n g agents was a l s o observed. The f a c t that sulfobromophthalein was a competitive i n h i b i t o r of both enzymes suggested some commonality of the a c t i v e s i t e ( s ) . On the other hand, the f a c t that a t r a z i n e was n e i t h e r a s u b s t r a t e nor an i n h i b i t o r of the pea enzyme suggested important d i f f e r e n c e s . I n h i b i t i o n of the pea enzyme by other diphenylether compounds, phenylureas and acetamide h e r b i c i d e s suggested the p o s s i b i l i t y of p e s t i c i d e i n t e r a c t i o n s w i t h these compounds. A number of s - t r i a z i n e s were t e s t e d as i n h i b i t o r s of the corn enzyme. These s t u d i e s suggested that the bis(alkylamino)-methoxy-s-1riaz ines and the methylmercapto analogs were probably competitive i n h i b i t o r s capable of b i n d i n g at the a c t i v e s i t e , but i n c a p a b l e of undergoing r e a c t i o n , Hydrox y t r i a z i n e s and the d e a l k y l a t e d t r i a z i n e s were not e f f e c t i v e inhibitors. I t i s of p a r t i c u l a r i n t e r e s t to note that a known s y n e r g i s t o f a t r a z i n e , 2 , 3 , 6 - t r i c h l o r o p h e n y l a c e t i c a c i d , was an i n h i b i t o r of the GSH S - t r a n s f e r a s e from corn. EPTC ( S - e t h y l dipropylthiocarbamate) does not appear to be a GSH S - t r a n s f e r a s e s u b s t r a t e . However, a f t e r o x i d a t i o n to the s u l f o x i d e , i t r e a d i l y undergoes conjugation i n the presence of GSH S - t r a n s f e r a s e s i s o l a t e d from r a t l i v e r (152) or corn r o o t (144). 0 tt

0 0 f C2H5-S-C-NCC3H7)2 M

C H5-S-C-N(C3H )2 2

7

EPTC

EPTC S u l f o x i d e

0 It GSH-transferase EPTC S u l f o x i d e

;-cS-Carbamyl^-GSH


3.


Plant Enzyme

109

Studies

14 -,


12 -I

ίο 1

φ ε

6 J

4 J

2Η

ImM GSH 9.2 μΜ PCNB 120 mM Pi 0.32 mg enzyme

TO

Τ 15

—ι 20

% (v/v) tert-butanol in system Figure 14.

Effect of tert-butanol on GSH conjugation of PCNB catalyzed by an enzyme system isolated from pea (192)


In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979. 72 0

29 29 34 41 54 —

86 0 3 —

—

28 60 88 79 55 57 68

1 1 0.1 0.06

0.1 0.1 0.1 0.1 1 0.03 0,1 0.1 1.0

2,3-dimercaptopropanol

cysteine

atrazine

2,4-bis(isopropylamino-6-methylmercapto-s-triazine*

2,3,6-trichlorophenyl acetic acid

propachlor

barban

sulfobromophthalein

1,2-dichloro-4-nitrobenzene

nitrofen*

diuron*

propanil

l-chloro-3-tosylamldo-7-amino-L-2 2-heptane«HCl* ( a l k y l a t i n g agents)

*Related compounds were t e s t e d w i t h s i m i l a r r e s u l t s .

58

11

6

1

S-methyl g l u t a t h i o n e

61

N.A.

40

14

1

dithiothreitol

Inhibitor

% I n h i b i t i o n of GSH-transferase (corn)

% I n h i b i t i o n of GSH-transferase (pea)

I n h i b i t o r s t u d i e s w i t h the g l u t a t h i o n e S-transferases i s o l a t e d from corn (143) and pea (142). Inhibitor concentration (mM)

Table X.



3.


Phnt

Enzyme

Studies

111

T h i s i s comparable to a previous observation that a methylm e r c a p t o - s - t r i a z i n e would not undergo conjugation u n t i l a f t e r o x i d a t i o n t o the s u l f o x i d e (149). Recent s t u d i e s have a l s o shown that chlorpropham ( i s o p r o p y l m-chlorocarbanilate) i s not a s u b s t r a t e f o r GSH S-transferase a c t i v i t y i n oat, but i t s o x i d a t i o n product, 4-hydroxychlorpropham, r e a d i l y undergoes an enzymatic r e a c t i o n w i t h c y s t e i n e or GSH (139, 140). These observations i l l u s t r a t e the need to consider the p o s s i b l e r o l e of a c t i v a t i n g r e a c t i o n s i n GSH conjugation. When corn i s t r e a t e d w i t h Ν,N-diallyl-2,2-dichloroacetamide (R-25788), h e r b i c i d a l i n j u r y due to EPTC i s g r e a t l y reduced (153). Glutathione J3-transferase a c t i v i t y and the c o n c e n t r a t i o n o f GSH were increased 2- to 3 - f o l d by treatment w i t h 0.3 to 30 ppm of R-25788. I t was concluded that decreased h e r b i c i d a l i n j u r y was due to an increased r a t e of GSH conjugation brought about by the elevated l e v e l s of GSH and GSH S-transferase a c t i v i t y (152). I n EPTC-susceptible o a t s e e d l i n g s , the l e v e l s o f GSH and GSH j>t r a n s f e r a s e d i d not i n c r e a s e i n response t o R-25788. The a c t i o n of R-25788 appears t o be s e l e c t i v e . No i n c r e a s e i n a c t i v i t y was noted when the i s o l a t e d corn system was t r e a t e d w i t h R-25788; t h e r e f o r e , R-25788 does not appear to be a simple a c t i v a t o r . Increased l e v e l s o f GSH ^ - t r a n s f e r a s e a c t i v i t y were observed i n both crude and p a r t i a l l y p u r i f i e d enzyme preparations a f t e r treatment w i t h R-25788. Although i t was not proven, the r e s u l t s suggest that enzyme i n d u c t i o n o r p o s s i b l e removal o f endogenous i n h i b i t o r s may be r e s p o n s i b l e f o r the observed increases i n enzyme a c t i v i t y . Twenty-eight compounds were compared to R-25788 f o r t h e i r e f f e c t i v e n e s s i n i n c r e a s i n g GSH S-transferase a c t i v i t y and GSH content i n corn s e e d l i n g r o o t s . Although s i g n i f i c a n t exceptions were noted, the e f f e c t i v e n e s s of these compounds as a n t i d o t e s g e n e r a l l y c o r r e l a t e d w i t h increased GSH and GSH Sr-transferase l e v e l s . Chlorpropham ( i s o p r o p y l m-chlorocarbanilate) and c i s a n i l i d e ( c i s - 2 , N - p h e n y l - l - p y r r o l i d i n e c a r b o x a n i l i d e ) a r e metabolized to hydroxylated d e r i v a t i v e s i n c e r t a i n p l a n t species (139, 154). Recent evidence i n d i c a t e s that the 4-hydroxylated d e r i v a t i v e s of chlorpropham and c i s a n i l i d e a r e converted to GSH and c y s t e i n e conjugates i n oat shoot s e c t i o n s (139, 140) (Figure 15). The s o l u b l e enzyme complex that c a t a l y z e s conjugate formation was i s o l a t e d from oat. When c y s t e i n e and 4-hydroxychlorpropham were incubated with the enzyme, a p o l a r metabolite was formed. When GSH was s u b s t i t u t e d f o r c y s t e i n e , a more p o l a r product was formed. The i n v i t r o enzyme system was used to produce s u f f i c i e n t metabolite from the r e a c t i o n w i t h c y s t e i n e and 4-hydroxychlor propham to allow i s o l a t i o n and p a r t i a l c h a r a c t e r i z a t i o n of the product (139). Because o f the low y i e l d o f t h i s product i n the i n v i v o system and d i f f i c u l t i e s encountered i n i t s i s o l a t i o n , the use of an i n v i t r o system f o r product formation g r e a t l y f a c i l i t a t e d the c h a r a c t e r i z a t i o n of t h i s product. In the c h a r a c t e r i z a t i o n o f the c y s t e i n e conjugate, c y s t e i n e C-S_ l y a s e


XENOBIOTIC


112

METABOLISM

SG

Figure 15.

Reactions thought to he catalyzed by a GSH/cysteine from oat (139,140)

S-transferase

R-S-CH -C-C00H 2

NH

2

-[c-s (ORGANIC)

LYASE]

(H 0) 2

0 0

RSH CH

II II -C-C-OH ^ - N A D H

MASS

f [LACTIC \~~[ DEHYDROGENASE |

SPECTROMETER

^^NAD OH I CH,-C-COOH I H 3

Δ Α 340nm Figure 16.

Cysteine C-S lyase cleavage of cysteine conjugates (139,150)



3.


Plant Enzyme

Studies

113

was used to cleave the c y s t e i n e moiety from the a r y l group to y i e l d p y r u v i c a c i d and a thiophenol (Figure 16). The r e a c t i o n was q u a n t i t a t i v e and the l i b e r a t e d p y r u v i c a c i d was measured by c o u p l i n g the c y s t e i n e £-S l y a s e r e a c t i o n to a l a c t i c a c i d dehydrogenase r e a c t i o n . These coupled r e a c t i o n s were p r e v i o u s l y used i n the c h a r a c t e r i z a t i o n of a GSH-related conjugate of a t r a z i n e (150). Glutathione jS-transferase a c t i v i t y w i t h GSH and 4-hydroxychlorpropham was not demonstrated u n t i l the crude enzyme was p a r t i a l l y p u r i f i e d by Sephadex g e l chromatography. This behavior suggested the presence of endogenous i n h i b i t o r s . D e t a i l e d i n h i b i t o r s t u d i e s showed that s e v e r a l n a t u r a l l y o c c u r r i n g aromatic compounds and 3-chloro-4-hydroxyaniline were powerful i n h i b i t o r s of the c y s t e i n e S-transferase a c t i v i t y (155). I t was suggested that these enzyme systems may use n a t u r a l l y o c c u r r i n g hydroxylated aromatic compounds or a r y l hydroxylated x e n o b i o t i c s as s u b s t r a t e s . Two t r a n s f e r a s e enzyme systems were apparently present i n oat shoots. One e x h i b i t e d n e a r l y comparable a c t i v i t y with e i t h e r c y s t e i n e or GSH and the other d i s p l a y e d much greater a c t i v i t y w i t h c y s t e i n e . The l a t t e r enzyme a l s o functioned as a GSH S-transferase when the e t h y l e s t e r of c y s t e i n e was added to the r e a c t i o n mixture (140). The nature and the s i g n i f i c a n c e of t h i s a c t i v a t i o n i s not understood. These are the f i r s t s t u d i e s to suggest that c y s t e i n e may be u t i l i z e d as a s u b s t r a t e much l i k e GSH i n a t r a n s f e r a s e r e a c t i o n (139, 140, 155). T h i s system should be studied i n greater d e t a i l to b e t t e r evaluate i t s s i g n i f i c a n c e to x e n o b i o t i c metabolism. Glutathione j>-transferase a c t i v i t y was r e c e n t l y i s o l a t e d from 10 a g r i c u l t u r a l l y important p l a n t species and screened f o r a c t i v i t y with 8 d i f f e r e n t p e s t i c i d e substrates (146). Of the substrates examined, GSH S-transferase a c t i v i t y was demonstrated i n a l l species w i t h PCNB, propachlor and CDAA ( N , N - d i a l l y l chloroacetamide). The r e s u l t s suggested that c e r t a i n types of GSH j>-transferase a c t i v i t y may be widely d i s t r i b u t e d i n higher plants. These l i m i t e d s t u d i e s have c l e a r l y shown that GSH S-transf e r a s e s p l a y an important 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 . Some GSH S-transferases appear to be widely d i s t r i b u t e d i n the p l a n t kingdom, but others appear to be more l i m i t e d i n t h e i r distribution. Glutathione ^ - t r a n s f e r a s e enzymes p l a y an important r o l e i n the s e l e c t i v i t y of c e r t a i n h e r b i c i d e s , such as the 2 - c h l o r o - s - t r i a z i n e s , f l u o r o d i f e n and EPTC s u l f o x i d e , but t h e i r r o l e i n the s e l e c t i v i t y of h e r b i c i d e s such as the a - c h l o r o acetamides i s u n c e r t a i n . The p o s s i b i l i t y that h e r b i c i d a l s e l e c t i v i t y may be increased by s e l e c t i v i t y s t i m u l a t i n g or inducing GSH ^ - t r a n s f e r a s e l e v e l s has been r a i s e d . A d d i t i o n a l s t u d i e s are needed to determine the d i s t r i b u t i o n of GSH St r a n s f e r a s e s i n higher p l a n t s and to b e t t e r determine the p r o p e r t i e s of the i n d i v i d u a l t r a n s f e r a s e s .


XENOBIOTIC

114


Glucose

METABOLISM

Conjugation:

The r e p o r t s that ethylenechlorohydrin was converted to a 3-0-D-glucoside and a g e n t i o b i o s i d e i n wheat (156) and tomato (157T were among the f i r s t i n d i c a t i o n s that p l a n t s had the a b i l i t y to convert c e r t a i n x e n o b i o t i c s to g l u c o s i d e s . I t was l a t e r shown that most higher p l a n t s (158, 159) had the a b i l i t y to convert exogenous phenols to $-j)-D-glueosides. T h i s a b i l i t y was apparently l a c k i n g i n algae, fungT and c e r t a i n aquatic p l a n t s (158). The formation of 0-, N-, and ^ - g l u c o s i d e s , a c y l a t e d g l u c o s i d e s , g e n t i o b i o s i d e s , and glucose e s t e r s have a l l been demonstrated e i t h e r i n v i t r o or i n v i v o with x e n o b i o t i c s or n a t u r a l substrates (160). The formation of glucosides i s extremely important i n p e s t i c i d e biochemistry f o r the f o l l o w i n g reasons: there i s a wide range of p o t e n t i a l substrates f o r conjugation, g l u c o s i d e formation may a f f e c t the nature of terminal r e s i d u e , and g l u c o s y l a t i o n may play a r o l e i n p e s t i c i d e s e l e c t i v i t y or d e t o x i c a t i o n . The most common types of g l y c o s i d e r e a c t i o n s encountered i n p e s t i c i d e metabolism appear to i n v o l v e an i n i t i a l UDPG-dependent g l u c o s y l t r a n s f e r r e a c t i o n (Figure 17), The formation of simple 0-glucosides from polyhydroxylated phenols was demonstrated w i t h an i n v i t r o system from wheat germ (161). Substrate s p e c i f i c i t y t e s t s showed that the wheat germ g l u c o s y l t r a n s f e r a s e could use a number of polyhydroxy phenols as s u b s t r a t e s , but was not a c t i v e with simple phenols. A f t e r p u r i f i c a t i o n of t h i s enzyme, a c t i v i t y f o r c e r t a i n substrates was l o s t ; thus, the presence of more than one t r a n s f e r a s e was i n d i c a t e d . The i n v i t r o synthesis of 14 phenolic glucosides by crude enzymes from wheat germ and bean was compared with the i n v i v o s y n t h e s i s i n bean (162). The only products detected i n v i t r o were g e n e r a l l y the primary products formed i n v i v o . The enzyme systems from wheat germ and bean could not u t i l i z e simple mono-hydroxylated phenols as substrates; i t i s , t h e r e f o r e , questionable whether these enzymes are i n v o l v e d i n the formation of 3-0-D-glucosides from p e s t i c i d e s or p e s t i c i d e m e t a b o l i t e s . A number of UDPG:sterol g l u c o s y l t r a n s f e r a s e s have been i s o l a t e d from v a r i o u s p l a n t sources (163-166). These enzymes are u s u a l l y a s s o c i a t e d with the p a r t i c u l a t e f r a c t i o n (164). For some phenolic x e n o b i o t i c s , the p o s s i b i l i t y should be considered that UDPG:glucosyltransferase a c t i v i t y may be membrane bound. A UDPG-dependent enzyme that c a t a l y z e s the formation of β-0-D-glucosides with a v a r i e t y of phenols, a l k y l a l c o h o l s and other substrates has been i s o l a t e d from germinating mung bean (167). Attempts to demonstrate the presence of t h i s enzyme i n seedlings were not s u c c e s s f u l . The ammonium s u l f a t e f r a c t i o n a t e d enzyme from germinating mung beans could be stored i n l i q u i d n i t r o g e n with l i t t l e l o s s i n a c t i v i t y , but the more h i g h l y p u r i f i e d enzyme l o s t a l l a c t i v i t y upon f r e e z i n g . T h i s enzyme u t i l i z e d UDPG as the g l u c o s y l donor, had an estimated M.W. of 62,000 and had a pH optimum of approximately 10, The pH optimum



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115

Studies

3) 1-0 GLUCOSE ESTERS

4) Ν-GLUCOSIDES

bk-1979-0097.ch003

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