Xenobiotic Metabolism - American Chemical Society

2 Xenobiotic Metabolism in Higher Plants: In Vitro Tissue and Cell Culture Techniques

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on October 2, 2013 | http://pubs.acs.org Publication Date: April 5, 1979 | doi: 10.1021/bk-1979-0097.ch002

RALPH O. MUMMA and ROBERT H. HAMILTON Pesticide Research Laboratory and Graduate Study Center and Departments of Entomology and Biology, Pennsylvania State University, University Park, PA 16802 M i l l i o n s of pounds of x e n o b i o t i c s have been a p p l i e d to p l a n t s in our environment f o r the c o n t r o l of pests and p l a n t growth. Some of these chemicals have recognized and w e l l c h a r a c t e r i z e d b i o l o g i c a l e f f e c t s on animals and p l a n t s , while other x e n o b i o t i c s , such as o i l s , adjuvants, e m u l s i f i e r s and i n e r t m a t e r i a l s , are a p p l i e d i n even greater q u a n t i t y , but have been assumed to cause l i t t l e ecological effect. I t i s important to understand what happens to a l l chemicals a p p l i e d t o our environment and to p r o p e r l y i n t e r p r e t the e c o l o g i c a l s i g n i f i c a n c e of these chemicals and of t h e i r degradation products. I f we cannot know t h i s i n d e t a i l , then a general knowledge of p e r s i s t a n c e and metabolic products i s of importance. Since the t a r g e t organism of these x e n o b i o t i c s i s o f t e n p l a n t s , i t i s of the utmost importance to understand the f a t e of these chemicals i n p l a n t s . U l t i m a t e l y , animals and even humans are exposed to these chemicals and/or t h e i r subsequent metabolites and degradation products. Most i n v e s t i g a t i o n s of x e n o b i o t i c metabolism i n p l a n t s have focused on b i o l o g i c a l l y a c t i v e pest c o n t r o l chemicals. Thus, t h i s review w i l l a l s o focus p r i m a r i l y on p l a n t metabolism of p e s t i c i d e s . There are many ways to study the metabolism of x e n o b i o t i c s by p l a n t s , but whatever technique i s employed, i t should p r e d i c t what would a c t u a l l y happen under f i e l d c o n d i t i o n s . Metabolism s t u d i e s have i n v o l v e d whole p l a n t s , excised p l a n t p a r t s (meristems, shoots, stems, l e a v e s , r o o t s , l e a f d i s k s ) , p l a n t c e l l c u l t u r e s , s u b c e l l u l a r p a r t i c l e s , and i s o l a t e d enzymes. Any metabolism study conducted i n the l a b o r a t o r y i s l e s s than i d e a l because i t i s d i f f i c u l t to d u p l i c a t e many f a c t o r s that a f f e c t the degradation of x e n o b i o t i c s under f i e l d c o n d i t i o n s such as weather, l i g h t , microsymbionts, s o i l or method of a p p l i c a t i o n . Metabolism of xenobiot i c s by p l a n t t i s s u e c u l t u r e o f f e r s the obvious advantages of s t e r i l i t y , space, economical use of l a b e l e d chemicals, l e s s p i g ments, e t c . These advantages w i l l be discussed i n d e t a i l l a t e r . The metabolism of x e n o b i o t i c s by p l a n t t i s s u e c u l t u r e (1) and the metabolism of endogenous and exogenous chemicals (2) have been reviewed r e c e n t l y . W i t h i n the l a s t few y e a r s , many new

0-8412-0486-l/79/47-097-035$10.50/0 © 1979 American Chemical Society

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

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i n v e s t i g a t i o n s of x e n o b i o t i c metabolism by p l a n t t i s s u e c u l t u r e s have been reported. This review w i l l focus on these more recent papers with an emphasis on p e s t i c i d e metabolism. We w i l l attempt to evaluate and compare the r e s u l t s obtained w i t h t i s s u e c u l t u r e techniques versus those obtained w i t h whole p l a n t s and to explore the many f a c t o r s ( l i m i t a t i o n s ) that a f f e c t metabolism s t u d i e s with plant tissue culture. H i s t o r y and P r i n c i p l e s of P l a n t T i s s u e Culture Plant t i s s u e c u l t u r e r e f e r s to the growth of r e l a t i v e l y und i f f e r e n t i a t e d p l a n t c e l l s or d i f f e r e n t i a t e d organs on s o l i d or l i q u i d n u t r i e n t medium. The u n d i f f e r e n t i a t e d p l a n t t i s s u e growing on s o l i d i f i e d medium i s u s u a l l y r e f e r r e d to as a c a l l u s c u l t u r e s i n c e they a r e f r e q u e n t l y obtained from cut or wounded surfaces and maintain the appearance o f wound c a l l u s t i s s u e . When such t i s s u e s are placed i n l i q u i d medium w i t h shaking, many small aggregates of c e l l s and even some s i n g l e c e l l s may be obtained. Subcultures of small aggregates or c e l l clumps i n l i q u i d c u l t u r e are u s u a l l y designated as suspension c u l t u r e s . The c u l t u r e of excised d i f f e r e n t i a t e d organs i s , of course, organ c u l t u r e . F r e quently, c a l l u s c u l t u r e s w i l l d i f f e r e n t i a t e w i t h the formation of xylem elements and sometimes buds and/or r o o t s . In many c a l l u s c u l t u r e s with t h i s p o t e n t i a l u s u a l l y a high k i n e t i n / a u x i n concent r a t i o n r a t i o i n the medium favors bud formation while the reverse favors root formation. In a few cases, by proper manipulation of the medium, thousands of pseudoembryos can be induced and grown i n t o normal p l a n t s of the same genotype. Although the c u l t u r e of p l a n t t i s s u e s was attempted i n 1902 (3), success was not achieved u n t i l White c u l t u r e d excised tomato roots i n 1934 and tobacco c a l l u s i n 1939 (4). Gautheret and Nobêcourt a l s o described c u l t u r e of p l a n t c a l l u s t i s s u e at about the same time (5, 6) . White and Gautheret both developed n u t r i e n t media that a r e used widely today (5> 2) · In a d d i t i o n to e s s e n t i a l s a l t s and sucrose, White added thiamine, n i c o t i n i c a c i d , pyridoxine and g l y c i n e while Gautheret added thiamine, pantothenic a c i d , b i o t i n , i n o s i t o l and c y s t e i n e . I t appears that only thiamine i s e s s e n t i a l ; but, i n some cases, b e t t e r growth may be obtained by the a d d i t i o n of other v i t a m i n s . White d i d not add an auxin f o r c u l t u r e of excised root organ c u l t u r e s or tobacco tumor t i s s u e , but Gautheret added naphthaleneacetic a c i d (NAA). Van Overbeek et a l . (8) introduced the use of coconut milk and much l a t e r M i l l e r et a l . (9) found that k i n e t i n was necessary to c u l t u r e tobacco stem p i t h . In g e n e r a l , excised root organ c u l t u r e s , tumor and some c a l l u s c u l t u r e s (so c a l l e d habituated) do not r e q u i r e e i t h e r auxin or k i n e t i n . Some t i s s u e s do not r e q u i r e k i n e t i n e s p e c i a l l y i f 2,4-dichlorophenoxyacetic a c i d (2,4-D) i s used as the auxin (10). The a d d i t i o n of coconut milk i s u s u a l l y not e s s e n t i a l , and a requirement f o r g i b b e r e l l i c a c i d has been reported r a r e l y . I t i s probable that adaptation to the medium



2.

M U M M A

A N D

H A M I L T O N

Plant Tissue and

Cell

Cultures

37

and the s e l e c t i o n of t i s s u e that grows w e l l on a p a r t i c u l a r medium accounts f o r the wide v a r i a t i o n i n n u t r i e n t requirements f o r a t i s s u e such as tobacco c a l l u s . Other media used widely f o r plant t i s s u e c u l t u r e i n c l u d e those of Murashige and Skoog (11), N i t s c h and N i t s c h (12) and Gamborg (13). In a d d i t i o n , some media formul a t i o n s are now a v a i l a b l e from a commercial source (Flow Laborat o r i e s , P. 0. Box 2226, 1710 Chapman Ave., R o c k v i l l e , MD 20852). In theory c a l l u s may be derived from any p l a n t t i s s u e cont a i n i n g parenchyma c e l l s . Some species form c a l l u s r e a d i l y and others do not. Tissue s t e r i l i z a t i o n may be accomplished with 70% a l c o h o l (1-2 min dip) and/or a s i m i l a r treatment with 1 to 5 or 1 to 10 d i l u t e d commercial bleach (0.5-1% sodium hypochlor i t e ) containing 0.1% Tween 20. In e i t h e r case, the t i s s u e i s r i n s e d 2-3 times i n s t e r i l e water. Seeds are germinated i n s t e r i l e p e t r i dishes and b i t s of root or stem t i s s u e are t r a n s f e r r e d to s o l i d i f i e d agar medium c o n t a i n i n g r e l a t i v e l y high auxin l e v e l s (0.5-1.0 mg/1 NAA or 2,4-D). S t e r i l i z e d t i s s u e from bud, leaves or stems may a l s o be used. Formation of enough c a l l u s to s u b c u l ture may vary from 2 weeks to 2 months. Considerable v a r i a t i o n i n appearance and texture between c a l l u s pieces from the same source may be observed. Three to 4 b i t s of c a l l u s (5-7 mm i n dia.) are t r a n s f e r r e d to s o l i d i f i e d agar medium (50 ml i n a 125 ml Erlenmeyer f l a s k ) . D i f f i c u l t t i s s u e s may r e q u i r e the a d d i t i o n of 50 ml of a u t o c l a v e d - f i l t e r e d coconut l i q u i d and/or 2 g of c a s e i n hydrolysate per l i t e r . Growth rates vary, but i t i s convenient to subculture b i t s of c a l l u s onto f r e s h medium every 4^6 weeks. Temperature and l i g h t requirements may not be c r i t i c a l f o r most t i s s u e s . High temperatures (30°C or more) have caused the l o s s of k i n e t i n dependence of tobacco c a l l u s (14) and i n h i b i t e d growth. We have maintained c u l t u r e s under continuous low l e v e l f l u o r e s c e n t l i g h t at 27°C. Simple equipment i s needed f o r p l a n t t i s s u e c u l t u r e s ; a temperature c o n t r o l l e d incubator or c u l t u r e room, an autoclave, and a shaker, i f suspension c u l t u r e s are grown. I t i s a l s o d e s i r a b l e to have a s t e r i l e t r a n s f e r hood. The genetic and p h y s i o l o g i c a l s t a b i l i t y of some plant t i s s u e c u l t u r e s i s of concern. T i s s u e from the same o r i g i n a l c u l t u r e may change i n appearance and growth r a t e over a period of time. It i s commonly observed that the a b i l i t y to regenerate normal p l a n t s by the formation of buds and roots i s l o s t with time. I t may even be necessary to r e i s o l a t e c a l l u s from the same source, i f the c a l l u s t i s s u e appears a t y p i c a l or slow growing. A low growth r a t e i s observed f r e q u e n t l y i n August or September. V a r i ations i n growth r a t e and t i s s u e appearance may be important f a c t o r s i n metabolism studies and should be examined c a r e f u l l y . A m i t o t i c index at two weeks a f t e r t r a n s f e r may give a good i n d i c a t i o n of growth r a t e (15) or f r e s h weights at the end of 4 or 5 weeks may be used. The source of c a l l u s t i s s u e (root, stem, l e a f , cotyledons, etc.) a l s o may i n f l u e n c e metabolism. The appearance of soybean cotyledon, l e a f and root c a l l u s was s i m i l a r and l i m i t e d



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XENOBIOTIC

M E T A B O L I S M

work on 2,4-D metabolism i n d i c a t e d no major q u a l i t a t i v e d i f f e r ences (16). Of much more importance was the age or stage of grow th of the c u l t u r e s (15). Most c a l l u s c u l t u r e s e x h i b i t a l a g i n growth f o r 4-7 days a f t e r t r a n s f e r , a l o g growth phase followed by a slower growth phase a f t e r 4-5 weeks. Cultures 5-9 weeks o l d are more a c t i v e i n metabolism of 2,4-D than 3 week o l d c u l t u r e s (15). I t should be noted that prolonged p e s t i c i d e treatment of o l d e r c a l l u s i n f r e s h medium may put the t i s s u e i n t o l o g phase growth again w i t h probable changes i n metabolism. Metabolism of Xenobiotics

by Plant Tissue

Culture

Most i n v e s t i g a t i o n s w i t h p l a n t t i s s u e c u l t u r e s and xenobiot i c s have been concerned w i t h p e s t i c i d e metabolism. Tables I and I I i l l u s t r a t e the v a r i e t y of h e r b i c i d e s , i n s e c t i c i d e s , and p l a n t t i s s u e c u l t u r e s that have been used i n metabolism s t u d i e s . The metabolism of lindane probably represents one of the more extreme cases where fourteen d i f f e r e n t p l a n t t i s s u e s were used. As i s evident, the source of the p l a n t t i s s u e c u l t u r e , the type of c u l ture (suspension or nonsuspension) and the media used a l s o v a r i e d . This v a r i a t i o n i n p l a n t c u l t u r e s and techniques makes i t extremely d i f f i c u l t to c r i t i c a l l y compare metabolism s t u d i e s . As w i l l be pointed out l a t e r , there are a l s o many other f a c t o r s that a f f e c t metabolism s t u d i e s with p l a n t t i s s u e c u l t u r e s . HERBICIDES I t i s apparent that h e r b i c i d e s exert t o x i c or p h y s i o l o g i c a l e f f e c t s on s e n s i t i v e plant species and that some p h y s i o l o g i c a l e f f e c t s can be expected on t i s s u e c u l t u r e s of these species. An exception may be photosynthetic i n h i b i t o r s . Due to the sugar i n the p l a n t t i s s u e c u l t u r e medium, photosynthetic i n h i b i t o r s should not be s t r o n g l y i n h i b i t o r y unless they have secondary s i t e s of action. I f t o l e r a n c e i s due to metabolism, t h i s might be expected to lead to q u a l i t a t i v e and q u a n t i t a t i v e d i f f e r e n c e s i n metabolism by p l a n t t i s s u e c u l t u r e s from s u s c e p t i b l e and r e s i s t a n t v a r i e t i e s or s p e c i e s . Of course, such t i s s u e c u l t u r e s should a l s o show differences i n actual tolerance. The metabolism of the h e r b i c i d e f l u o r o d i f e n (p-nitrophenyl α,α,a-trifluoro-2-nitro-p-tolyl ether) has been i n v e s t i g a t e d (21) with tobacco c e l l s i n suspension c u l t u r e (42). The c e l l s were incubated f o r 15 days w i t h 1-2 ppm of e i t h e r C i - or CF3-labeled f l u o r o d i f e n . A l l of the a p p l i e d f l u o r o d i f e n was metabolized. Recovery of added r a d i o a c t i v i t y v a r i e d between 52 and 76%. The c e l l s contained 60 to 80% of the recovered r a d i o a c t i v i t y and the remainder was found i n the medium and c e l l wash. With ^Ci-label ed f l u o r o d i f e n , 4-nitrophenol (7%) was i s o l a t e d only from the medium. Aqueous-soluble conjugated forms of 4-nitrophenol (93%), p r i m a r i l y the 3-D-glucoside and other a c i d i c conjugates, were pre sent i n both the c e l l s and the medium as summarized i n Figure 1. l l f

1

lf



3

2

ι H

>-N-c-cH cH

Propanil

\=J

CI- • f

0

Diphenimid

CI

^

3

v CH

ÇH3_

Ο

^ N-CCH^ CH ^ ^

Cisanilide

3

ι

0

3

Rice

1

(Oryza s a t i v a L. v a r . Starbonnet)

1

Leaves

Source

Root

Soybean (Glycine max [L.] Merr. Root t i p s Wilkin )

Carrot (Daucus c a r o t a L.) Cotton (Gossypium hirsutum L.)

Species

H, S

B5, S

B5,

Medium and ^ C u l t u r e Type

Plant T i s s u e C u l t u r e

Metabolism of H e r b i c i d e s by P l a n t Tissue C u l t u r e s .

Herbicide

Table I.

20

19

17, 18

Reference.



2

3

Ν — Ν

Cl

0

NH

2,4-D

/Cl

2

OCH2COOH

Metribuzin

3

^-SCH

Fluorodifen

(CH ) C-/

N0

NO2

Table I - page 2

3

( C u l t i v a r Bragg) ( C u l t i v a r Coker 102)

Cotyledon Cotyledon

Soybean (Glycine max L. v a r Root Mandarin) Soybean (Glycine max L. v a r Cotyledon Acme) Jackbean (Canavalia ensiformis)Pod Endosperm Sweet corn (Zea mays) Tobacco (Nicotiana tobacum) Pith Carrot (Daucus c a r o t a v a r Pith Sativa) Sunflower (Helianthus annus) P i t h Root Rice (Oryza s a t i v a v a r Starbonnet) Wheat ( T r i t i c u m monococcum L. ) Stem F i e l d bindweed (Convolvulus a r v e n i s L.)

Soybean Soybean

Tobacco (Nicotiana tobacum L. var Xanthi)

24, 26, 27 27 27 27 28 29 30

C C C C

M, M, M, M,

M, C M, C B5, S X, C

23

22

21

M, C

B5

X, S X, S

M, S


W Ο Ε

"s

ο

i

w

X m ο

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

C

MS,

W,

Bark

C C

W,

Crown-gall W,

Stem

M, S

C

S - Suspension; C - C a l l u s .

f

Geranium (Pelagonium hortorum var N i t t a n y Red) Boston i v y (Parthenocissus tricuspidata) Apple (East M a i l i n g r o o t s t o c k 3430) Shamouti orange ( C i t r u s s i n e n s i s Osb.

Cotyledon

C u l t u r e type:

,CH COOH

L. var

B5 - Gamborg's; H - H e l l e r ' s ; M - M i l l e r s ; MS - Murashige

IAA

2,4,5-T

CI

2

^ - 0 C H COOH

Soybean (Glycine max Acme)

B a s i c media: X - Others.

CI-

Table I - page 3

36

34,

33

32

31

35

1

and Skoog s; W - W h i t e s

f



O-C-NHCH3

Potato (Solanum tuberosum)

Purple cockle (Agrostemma githago L.) Soybean (Glycine max) Bedstraw (Galium verum) Carrot (Daucus carota) Clover (Meliotus alba) Tobacco ( N i c o t i a n a tobacum) Tobacco ( N i c o t i a n a g l u t i n o s a ) Tobacco ( N i c o t i a n a s y l v e s t r i s ) Tobacco ( N i c o t i a n a glauca) Lettuce (Lactuca s a t i v a ) (Beta v u l g a r i s ) P a r s l e y (Pstroselinum hortense)

Source

Culture

M,

S

Medium and ^ Culture Type

Plant Tissue

Cultures.

Tobacco ( N i c o t i a n a tobacum L. var Xanthi)

Species

Metabolism of I n s e c t i c i d e s by Plant Tissue

Insecticide

Table I I .


39

37,

38

Reference


i

f

3

x x

B a s i c media:

-Cl

-Cl

P a r s l e y (Petroselinum hortense, Hoffm.) Soybean (Glycine max L.)

P a r s l e y (Petroselinum hortense, Hoffm.) Soybean (Glycine max L.)

Bean (Phaseolus v u l g a r i s var Roots & Canadian Wonder) Shoots Potato (Solanum tuberosum var Tuber Majestic)

S

41 41 B5, S

41

40

40

B5, S

B5, S

W, S

MS,

S - Suspension; C - C a l l u s .

M - M i l l e r ' s ; W - White's; MS - Murashige and Skoog's; B5 - Gamborg's; X - Other.

Kelthane (Ascaroside)

CC1

OTO

OH

£,£ -DDT

CCI 3

~ 0 ~ K

^ C u l t u r e type:

a

Cl-

c

H

Table I I - page 2 Ql _ CL Cl'


44

XENOBIOTIC

M E T A B O L I S M


NO

roducts

(79%) Journal of Agricultural and Food Chemistry Figure 1.

Metabolism of fluorodifen by tobacco cell in suspension culture

These data from plant t i s s u e c u l t u r e s t u d i e s are c o n s i s t a n t with r e s u l t s obtained w i t h whole p l a n t s where the glucoside of 4n i t r o p h e n o l has been reported as the major product of soybean and maize seedlings (43, 44). Whole p l a n t metabolism s t u d i e s with f l u o r o d i f e n (43, 44) suggested that a small percentage of the a p p l i e d p e s t i c i d e may be reduced to 4-aminophenyl 2-amino-4-(trifluoromethyl) phenyl ether, 4-nitrophenyl 2-amino-4-(trifluoromethyl) phenyl ether or 4-aminophenyl 2 - n i t r o - 4 - ( t r i f l u o r o m e t h y l ) phenyl ether, but none of these compounds, i n c l u d i n g 4-aminophenyl, was detected i n the tobacco t i s s u e c u l t u r e s t u d i e s . 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 ) i s an h e r b i c i d e used to c o n t r o l weeds i n r i c e . The r e s i s t a n c e of r i c e to p r o p a n i l i s a t t r i b u t e d to the high l e v e l s of an a r y l a c y l amidase ( p r o p a n i l amidase) that hydrolyzes p r o p a n i l to 3 , 4 - d i c h l o r o a n i l i n e and prop i o n i c a c i d . The enzymatic l e v e l of p r o p a n i l amidase i n r i c e p l a n t s and i n r i c e root suspension c u l t u r e s has been i n v e s t i g a t e d (20, 45). The a c t i v i t y of the enzyme was found to be two to four times greater i n o l d e r p l a n t s (four leaves) than i n younger p l a n t s ( l e s s than four l e a v e s ) . P r o p a n i l amidase was a l s o demonstrated i n the r i c e suspension c u l t u r e , but i n t e r e s t i n g l y , the enzymatic a c t i v i t y could only be demonstrated a f t e r the t i s s u e c u l t u r e had developed to s t a t i o n a r y phase (5.5 days). This i n v e s t i g a t i o n i s important because i t documents a change i n the b i o s y n t h e t i c capacity of p l a n t t i s s u e c u l t u r e s w i t h the age of the c u l t u r e . The dependency of s e v e r a l enzymes, i n c l u d i n g phenylalanine ammonia l y a s e , upon i l l u m i n a t i o n of p a r s l e y c e l l c u l t u r e s a l s o has been shown (46). The metabolism of diphenamid (N-N-dimethyl-2,2-diphenylacetamide) by soybean r o o t t i p c e l l suspension c u l t u r e s has been


2.

M U M M A

A N D H A M I L T O N

Plant Tissue and Cell

Cultures

45


i n v e s t i g a t e d (19) a t d i f f e r e n t stages o f growth and compared with whole p l a n t s . The metabolites found i n both p l a n t s and suspension c u l t u r e s were N-hydroxymethyl-N-methyl-2,2-diphenylacetamide (MODA), N-methyl-2,2-diphenylacetamide (MMDA), 2,2-diphenylacetamide (DA) and two p o l a r g l y c o s i d e s . One of the g l u c o s i d e s was a c i d i c and was i d e n t i f i e d as an e s t e r of malonic a c i d (Figure 2).

DA

Figure 2.

MMDA

(Acidic Glycoside)

Metabolism of diphenamid by soybean root cells in suspension culture

About 9-22% o f the diphenamid was metabolized by the c e l l c u l t u r e s at e a r l y l o g (3-7 days) and s t a t i o n a r y phases (14-18 days), r e s p e c t i v e l y . However, diphenamid metabolism per gram was about 2 times more r a p i d by e a r l y l o g phase c e l l s than by stationary c e l l s . Cultures o f a l l ages formed the same metabol i t e s with MODA and the d e a l k y l a t e d products predominating. The r e l a t i v e composition o f the metabolites i s presented i n Tables I I I and IV. The hydroxylated m e t a b o l i t e , MODA, was found almost e x c l u s i v e l y i n the medium 92-99%, and the d e a l k y l a t e d products were found predominantly i n the medium (68-94%). Log phase and s t a t i o n a r y phase c e l l s produced l a r g e r amounts of the d e a l k y l a t e d metabolites per mg dry weight per day than d i d e a r l y l o g phase c e l l s . G l y c o s i d e s c o n s i s t e d of only 6-7% of the t o t a l metabolites i n c e l l c u l t u r e s but were the major metabolites (46-48%) of tomato, pepper and soybean p l a n t s . These c e l l suspension c u l t u r e s demonstrate c l e a r l y the same metabolic degradation pathways as i n t a c t p l a n t s , but s i g n i f i c a n t q u a n t i t a t i v e d i f f e r e n c e s do occur e s p e c i a l l y i n the small amount of g l y c o s i d e formation by the soybean root suspension c e l l s . No q u a l i t a t i v e changes occurred i n the metabolism of diphenamid with age of the c u l t u r e . The metabolism of c i s a n i l i d e ( c i s - 2 , 5 - d i m e t h 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 s e l e c t i v e preemergence h e r b i c i d e , has been i n v e s t i g a t e d i n excised leaves and c e l l suspension c u l t u r e s of



46

XENOBIOTIC

M E T A B O L I S M

Table I I I . Diphenamid Metabolism by Growth Phase of Soybean C e l l Suspension c u l t u r e s (19)

Growth Phase

Diphenamid or M e t a b o l i t e

nmol per Gram C e l l s Medium C e l l Extract

E a r l y Log (3-7 Days)

Diphenamid MODA Dealkylated Glucoside

12.8 0.1 0.6 0.6

444.8 31.6 10.8 1.8

Log (7-14 Days)


41.9 2.2 10.6 1.9

99,4 27.4 27.8 1.1

Stationary (14-18 Days)


42.5 1.2 6.7 0.9

144.3 25.4 14.5 1.9

Table IV. R e l a t i v e Composition of Diphenamid or Metabolites i n C e l l s and Medium i n E a r l y Log Phase (19) Diphenamid or M e t a b o l i t e

% C e l l Extract

Diphenamid MODA Dealkylated Glucoside A c i d i c Glucoside

2,.8 0..3 5.,5 25,,0 100..0

Medium 97.,2 99.,7 94,,5 75.,0 0.,0


2.

M U M M A

A N D


H A M I L T O N

Cultures

47


c a r r o t and cotton (17, 18). A f t e r 6 days, excised c a r r o t leaves metabolized ca. 70% o f the a p p l i e d c i s a n i l i d e , but c a r r o t t i s s u e c u l t u r e s metabolized >95% o f the a p p l i e d h e r b i c i d e i n 3 days. Cotton leaves metabolized c i s a n i l i d e t o a greater extent than cotton suspension c u l t u r e s . The metabolism of c i s a n i l i d e by c a r rot and cotton p l a n t s i s shown i n Figure 3 and the r e l a t i v e comp o s i t i o n of metabolites i n p l a n t s and t i s s u e c u l t u r e i s presented i n Table V.

cri

CH

3

Glycoside I

3

Glycoside H Pesticide Biochemistry and Physiology

Figure 3.

Metabolism of cisanilide by carrot and cotton phnts and cells in culture

In the excised p l a n t s , g l y c o s i d e s I and I I were the major methanol s o l u b l e metabolites and only t r a c e q u a n t i t i e s of the aglycons were detected. In c a r r o t and cotton c e l l suspension c u l t u r e s , aglycon I or g l u c o s i d e I was not detected and only t r a c e amounts of g l y c o s i d e I I were detected. Aglycon I I was a major metabolite i n the medium of the c e l l suspension c u l t u r e s . In the excised p l a n t s , l e s s (20-25%) of the * C - l a b e l was found i n the i n s o l u b l e residue f r a c t i o n compared to the c e l l suspension c u l t u r e s (39-40%). ll

American Chemical Society Library

1155 16th St. N. W. In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; Washington, D. C. 20036 ACS Symposium Series; American Chemical Society: Washington, DC, 1979.


V. a

a

Methanol Soluble 25 13 (Unknown) I n s o l u b l e Residue 18 39 G l y c o s i d e I was not detected and g l y c o s i d e I I was t e n t a t i v e l y as a minor component.

identified

Excised Cotton Leaves (2 Days) Cotton Suspension Cultures (7 Days) Fraction % % Cells % Medium T o t a l Metabolized >95 >76 Methanol Soluble 55 — 24 (Glycosides I>II) (Aglycon I I )

40

Carrot Suspension C u l t u r e (3 D a y s ) % Cells % Medium >95 — 40 (Aglycon I I ) 20

R e l a t i v e Composition of C i s a n i l i d e M e t a b o l i t e s (18).

Excised Carrot Leaves (6 Days) Fraction % T o t a l Metabolized 70 Methanol Soluble 50 (Glycoside 1=11) Methanol Soluble 10 (Unknown) I n s o l u b l e Residue 20

Table



2.

M U M M A


Plant Tissue and

Cell

Cultures

49

At f i r s t approximation, i t would be easy to conclude that the metabolic degradation pathways were d i f f e r e n t i n the excised p l a n t s versus the c e l l suspensions s i n c e aglycon I and g l u c o s i d e I were not detected i n the t i s s u e or the medium. T h i s d i f f e r e n c e may i n d i c a t e r a p i d i n c o r p o r a t i o n of aglycon I i n t o a methanoli n s o l u b l e f r a c t i o n , because when aglycon I was administered to the c e l l suspension c u l t u r e , i t was r a p i d l y converted to methanol i n s o l u b l e products. Neither t i s s u e i s able to cleave a p p r e c i a b l y the urea type s t r u c t u r e of c i s a n i l i d e . T h i s i s a l s o the case f o r the metabolism of other urea h e r b i c i d e s by p l a n t s (43). Both t i s s u e s possess the a b i l i t y to form the hydroxy 1 d e r i v a t i v e , aglycon I I , but the c e l l c u l t u r e s e v i d e n t l y have a reduced c a p a c i t y to form the g l y c o s i d e conjugate and consequently aglycon I I accumulates i n the media. T h i s hydroxylated metabolite i s apparently not incorporated i n t o the methanol i n s o l u b l e p r o d u c t ( s ) . Thus, the metabolic pathways may be somewhat s i m i l a r except f o r l o s s of aglycons i n t o the medium and the increased formation of an i n s o l u b l e product. The p h y t o t o x i c i t y and metabolism of the h e r b i c i d e m e t r i b u z i n (4-amino-6-t-butyl 3-[methylthio]-as-triazin-5-[4H]-one) has been i n v e s t i g a t e d w i t h dark-grown soybean cotyledon suspension c u l t u r e s from s u s c e p t i b l e ("Coker 102") and r e s i s t a n c e ("Bragg") c u l t i v a r s . Bioassays were based on p o p u l a t i o n changes of v i a b l e c e l l s during i n c u b a t i o n with m e t r i b u z i n . V i a b l e c e l l s were c l a s s i f i e d as c e l l s with s t r u c t u r a l i n t e g r i t y and cytoplasmic streaming. Differential r e s i s t a n c e to m e t r i b u z i n was demonstrated by the c e l l suspensions from r e s i s t a n t and s u s c e p t i b l e c u l t i v a r s . M e t r i b u z i n had been reported to i n h i b i t photosynthesis, but the demonstrated phytot o x i c i t y toward both c u l t i v a r s of dark-grown a c h l o r o p h y l l o u s suspension c u l t u r e s i n d i c a t e d that p h y t o t o x i c i t y was not r e s t r i c t e d to photosynthesis. D e t o x i f i c a t i o n of m e t r i b u z i n by soybeans has been a t t r i b u t e d to formation of an N-glucoside. Enzymatic detoxi f i c a t i o n of m e t r i b u z i n d i d not occur i n the s u s c e p t i b l e c u l t i v a r due to the accumulation of a substance which i n h i b i t e d the enzyme. The r e s i s t a n t c u l t i v a r metabolized the i n h i b i t o r to a n o n i n h i b i tory form. Therefore, m e t r i b u z i n r e s i s t a n c e by the Bragg c u l t i v a r was a t t r i b u t e d to the a b i l i t y of t h i s c u l t i v a r to metabolize a common enzymatic i n h i b i t o r . P l a n t t i s s u e c u l t u r e techniques have been used by numerous i n v e s t i g a t o r s (23 - 29) f o r 2,4-D metabolism s t u d i e s . In 1968, the metabolism of 2,4-D-2-i^C by suspension c u l t u r e s of soybean root grown under continuous l i g h t (2000 lux) was examined (23). Most of the C - l a b e l i n the t i s s u e appeared as two spots on paper chromatography. The f a s t e r moving compound has the same Rf as 2,4-D and was assumed to be f r e e 2,4-D. The slower moving spot was a g l y c o s i d e that y i e l d e d glucose and f r e e 2,4-D when t r e a t e d w i t h emulsin. These i n v e s t i g a t o r s assumed that 2,4-D was metabol i z e d only to the 3-D glucose e s t e r of 2,4-D. Presumably, the amino a c i d conjugates, which have been subsequently i d e n t i f i e d as metabolites of 2,4-D, d i d not separate from 2,4-D i n the 1 4



50

XENOBIOTIC

M E T A B O L I S M

chromatographic s o l v e n t s used. The a d d i t i o n o f glutamine to the media increased the uptake of 2,4-D, but d i d not change the apparent metabolic products when analyzed by t h e i r chromatographic s o l vent systems. Some 2,4-D was a l s o a s s o c i a t e d with p r o t e i n . Two clones of f i e l d bindweed (Convolvulus a r r e n s i s L.) that d i f f e r e d i n t h e i r s u s c e p t i b i l i t y to 2,4-D under f i e l d and greenhouse c o n d i t i o n s a l s o e x h i b i t e d s i m i l a r d i f f e r e n c e s when stem c e l l s were c u l t u r e d i n l i q u i d and agar media (30). When amino a c i d s were added to the c u l t u r e media, the response to 2,4-D was a l t e r e d . The a b s o r p t i o n o f 2,4-D was increased w i t h glutamine and decreased with glutamic a c i d . Glutamic a c i d increased the t o l e r ance of the s u s c e p t i b l e clone, but reduced the t o l e r a n c e of the r e s i s t a n t clone. Glutamine i n c r e a s e d the s u s c e p t i b i l i t y of the s u s c e p t i b l e clone to a much g r e a t e r degree than i t d i d the r e s i s tant clone. There was a c o r r e l a t i o n between 2,4-D s u s c e p t i b i l i t y and n i t r a t e reductase a c t i v i t y . When soybean (Glycine max L.) cotyledon c a l l u s was incubated for 32 days w i t h 2,4-D-l- ^C,metabolites changed q u a l i t a t i v e l y and q u a n t a t i v e l y w i t h time (24). The water s o l u b l e f r a c t i o n from the t i s s u e increased i n r a d i o a c t i v i t y . When i t was t r e a t e d with 3 glucosidase, at l e a s t e i g h t aglycons that changed with time were r e l e a s e d ( F i g . 4 ) . 4-Hydroxy-2,5-dichlorophenoxyacetic a c i d (4OH-2,5-D) was the most abundant aglycon and 4-hydroxy-2,3-dichlorophenoxyacetic a c i d (4-OH-2,3-D) was i d e n t i f i e d as a minor component. Free 2,4-D a l s o was l i b e r a t e d f o l l o w i n g enzymatic treatment. The presumed presence o f 2,4-dichlorophenoxyacetyl-3-0-D glucose, a metabolite reported p r e v i o u s l y (23), was suggested. A

The ether s o l u b l e f r a c t i o n reached a maximum a f t e r 2 days and c o n s i s t e d of seven d i f f e r e n t regions of components on paper chromatography ( E t i - E t 7 ) that v a r i e d with time ( F i g . 5 ) . The major component (Eti*) was i d e n t i f i e d as the glutamic a c i d conjugate of 2,4-D and i t s r e l a t i v e composition was maximal a f t e r one day. The a s p a r t i c a c i d conjugate o f 2,4-D ( E t 2 ) increased g r a d u a l l y i n r e l a t i v e composition. S u r p r i s i n g l y , f r e e 2,4-D ( E t 7 ) d i d not reach i t s maximum u n t i l eight days. These data imply that contrary to some previous s t u d i e s , the metabolism of 2,4-D by plant t i s s u e i s q u i t e complex. Subsequently, f i v e a d d i t i o n a l amino a c i d conjugates of 2,4-D have been i d e n t i f i e d from soybean (27). These i n clude the a l a n i n e , v a l i n e , l e u c i n e , phenylalanine and tryptophan conjugates. A t y p i c a l d i s t r i b u t i o n of 2,4-D metabolites i s o l a t e d from 4-week-old c a l l u s t i s s u e i s presented i n Table VI.


2.

M U M M A



Cultures

ΙΟΟ r


80

u. 60 I UJ

i

$ 40 I

2CLU

4

8

16

INCUBATION TIME (DAYS) Plant Physiology Figure 4. The relative amounts of the aglycons obtained from the water-soluble fractions of soybean callus tissue incubated with 2,4-D-l- C: (Agi), primarily 4-OH-2,5-D and 4-011-2,3-D; (Ag ), 2,4-D. 14

7

Table V I .

R e l a t i v e Percentage of 2,4-D M e t a b o l i t e s i n Soybean C a l l u s T i s s u e (27). a

Ether-Soluble Metabolite Unk

Metabolites % In T i s s u e 1.2

2,4-D-Asp 2,4-D-Gly Unk 2,4-D-Ala,-Val) 2,4-D 2,4-D-Leu,-Phe -Try) TOTAL a

3.7 12.9 1.4 5.3 33.7 4.0 62.2%

Four-week-old c a l l u s t i s s u e with 2,4-D.

(emulsin) Aglycons % In Tissue Metabolite 26.3 (4-OH-2,5-D, 4-OH-2,3-D) 2.5 Unk 1.1 Unk 1.0 Unk 0.8 Unk 0.9 Unk 0.8 2,4-D 0.4 Unk 33.8% (10g) incubated

f o r 8 days

Journal of Agricultural and Food Chemistry



52

XENOBIOTIC

I

2

4

8

INCUBATION TIME

16

24

M E T A B O L I S M

32

(DAYS)

Plant Physiology Figure 5. Relative amounts of ether solubles isolated from soybean callus tissues incubated with 2,4-D-l- C: (Et ), aspartic acid conjugate; (Et ), glutamic acid conjugate; and (Et ), free 2,4-D. 14

2

k

7

A d d i t i o n a l minor aglycons a l s o have been i d e n t i f i e d t e n t a t i v e l y from corn endosperm c a l l u s as 3-hydroxy-2,4-dichlorophenoxyacetic a c i d (3-OH-2,4-D), 4-hydroxy-2-chlorophenoxyacetic a c i d (4-OH-2-C1) (27), and from wheat suspension c u l t u r e s as 6-hydroxy2,4-dichlorophenoxyacetic a c i d (6-OH-2,4-D) and 2-hydroxy-4chlorophenoxyacetic a c i d (2-OH-4-C1) (29). The e t h y l ester o f 2,4-D has been i s o l a t e d from the g l y c o s i d e f r a c t i o n of r i c e c a l l u s t i s s u e c u l t u r e f o l l o w i n g (3-glucosidase treatment (28). The e t h y l ester was presumed to be an a r t i f a c t of the i s o l a t i o n procedure probably being derived from the glucose e s t e r that e x i s t s i n high concentration i n t h i s t i s s u e . The glutamic a c i d conjugate of 2,4-D i s not an end product of 2,4-D metabolism (25) . When C-2,4-D-glutamic a c i d was i n c u bated with soybean c a l l u s t i s s u e , f r e e 2,4-D, the a s p a r t i c a c i d conjugate, and other products were found. These data suggest that amino a c i d conjugates may represent a r e s e r v o i r of bound 2,4-D 1l +



2.

M U M M A AND HAMILTON


Cultures

53

that may be i n v o l v e d i n the r e g u l a t i o n of 2,4-D l e v e l s i n the tissue. Comparative metabolism s t u d i e s o f 2,4-D i n c a r r o t , jackbean, sunflower, tobacco, corn and r i c e c a l l u s t i s s u e c u l t u r e s (27, 28) and a wheat suspension c u l t u r e (29) a r e shown i n Tables V I I and V I I I . A l l t i s s u e s examined, except r i c e , formed amino a c i d conjugates and a l l formed g l y c o s i d e s (phenolic g l y c o s i d e s as w e l l as glucose e s t e r s ) . The d i c o t s formed a higher r e l a t i v e percentage of amino a c i d conjugates w h i l e the monocots produced a higher percentage o f g l y c o s i d e s . The metabolism o f 2,4-D i n soybean and corn p l a n t s has been compared w i t h 2,4-D metabolism i n soybean c a l l u s t i s s u e (26). These data are presented i n Tables IX and X. In t h i s experiment, the 2,4-D-l- *C was d i r e c t l y i n j e c t e d i n t o the c a l l u s t i s s u e growing on agar r a t h e r than i n t o f r e s h l i q u i d medium w i t h suspended c a l l u s . The c a l l u s , t h e r e f o r e d i d not r e v e r t i n t o l o g phase growth and i t s n u t r i e n t s t a t u s was not changed. The metabolites found i n the p l a n t s a r e a l s o present i n the c a l l u s t i s s u e , but differ quantitatively. Of p a r t i c u l a r note i s the f a c t that f r e e hydroxylated 2,4-D e x i s t s i n both c a l l u s and i n p l a n t s i n c o n t r a s t to e a r l i e r t i s s u e c u l t u r e experiments. Free hydroxylated 2,4-D a l s o has been reported i n bean p l a n t s (48). These experiments a l s o suggest that the unknown compounds, Unki and U n k 2 , a r e amino a c i d conjugates o f hydroxylated 2,4-D metabolites ( p r i m a r i l y the glutamic a c i d conjugate of 4-OH-2,5-D) and are common to a l l t i s sue examined. A summary of the metabolism of 2,4-D i n p l a n t s i s presented i n F i g u r e 6. Recently, the metabolism of 2,4-D has been reported i n s i x i n t a c t p l a n t s : wheat, timothy, green bean, soybean, sunflower and strawberry (47). The r e l a t i v e percentage of amino a c i d conjugates i s low compared to the p r e v i o u s l y c i t e d work w i t h t i s s u e c u l t u r e experiments (Table X I ) . Of p a r t i c u l a r n o t i c e i s the unusually h i g h percentage of the d i c h l o r o p h e n o l g l y c o s i d e i n strawberry. In c o n t r a s t to use of t r u e suspension c u l t u r e s by most other l a b o r a t o r i e s , Feung e t a l . (24-28) u s u a l l y incubated a f a i r l y l a r g e amount (~10 gms) of small p i e c e s of 4-5 week o l d c a l l u s taken from s o l i d medium i n 25-40 ml l i q u i d medium w i t h shaking during treatment w i t h 2,4-D-l- ^C. T h i s technique r e s u l t s i n n e a r l y t o t a l uptake o f the a p p l i e d 2,4-D w i t h i n 48 hours, the accumulation o f s i g n i f i c a n t amounts of g l y c o s i d e metabolites i n the t i s s u e (phenolic g l y c o s i d e s and glucose e s t e r ) , i n s i g n i f i c a n t amounts of primary hydroxylated products and no accumulation of products i n the medium. In c e l l suspension c u l t u r e s , a b s o r p t i o n a l s o i s r a p i d but metabolites o f t e n accumulate i n the medium. In the study (26) where c a l l u s t i s s u e was i n j e c t e d d i r e c t l y w i t h 2,4-D-l- C., a d d i t i o n a l metabolites were found. Thus, the method of p e s t i c i d e a d m i n i s t r a t i o n may be important. Data w i t h i n t a c t p l a n t s (Tables IX, X and XI) a r e c o n s i s t a n t w i t h metabolism data obtained w i t h p l a n t t i s s u e c u l t u r e s , but s i g n i f i c a n t q u a n t i t a t i v e d i f f e r e n c e s do occur. The monocots ll

1



ll+

a

4.1 0.8 0.5 0.8 0.6 3.2

2.6 0.4 0.3 0.3 1.1 1.2

12.8

3.2 0.7 0.4 0.1

0.9 1.0

13.2

42.2

2.6 7.7

8.3 2.0 1.9 2.3

17.4

64.6

10.2

1.6

19.4 0.5 1.2

31.7

Corn

A l l were c a l l u s t i s s u e c u l t u r e s (27, 28) except wheat (29).

18.3

8.3

6.9

6.9

(4-OH-2,3-D, 4-OH-2,5-D) Unk Unk Unk Unk Unk Unk 2,4-D Ethyl-2,4-D Others TOTAL

% Total i n Tissue Sunflower Tobacco

Carrot

Jackbean

14.9 12.9 1.5 29.7

0.4

Rice

23.9

Ν/

10. 2

\

Wheat

R e l a t i v e Percentage of Water-Soluble 2,4-D-l- C M e t a b o l i t e s I s o l a t e d from Seven Species of Plant T i s s u e C u l t u r e s as the Aglycons.

Metabolites

Table V I I .



M U M M A AND H A M I L T O N

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Xenobiotic Metabolism - American Chemical Society

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