Plant Cell Reports

Plant Cell Reports (1982) 1 : 176-179

© Springer-Verlag 1982

Consistency of Metabolism of Aldrin and Dieldrin in Suspension Cultures from Phaseolus vulgaris Root K e i t h R. Brain a n d D a v i d S. Lines Welsh School of Pharmacy, UWIST, Cardiff, CF1 3NU, UK Received April 29, 1982

Abstract

aldrin

R e p r o d u c i b i l i t y of m e t a b o l i s m of a l d r i n and d i e l d r i n in c e l l s u s p e n s i o n c u l t u r e s f r o m P h a s e o l u s v u l g a r i s ( F r e n c h B e a n ) root, a n d effects of variations in hormonal s u p p l e m e n t a t i o n and light, studied. Aldrin a l w a y s c o n v e r t e d to dieldrin, a l d r i n - t r a n s dihydrodiol, and other unidentified more polar metabolites, but not to d i h y d r o c h l o r d e n e - d i c a r b o x y l i c acid. D i e l d r i n a l w a y s c o n v e r t e d to p h o t o d i e l d r i n and m o r e p o l a r m e t a b o l i t e s , but not to a l d r i n - t r a n s dihydrodiol. Metabolism reasonably consistent from one subculture to next, basically unaffected by alterations in m e d i u m tested, or by p r e s e n c e or a b s e n c e of light. Variations observed quantitative rather than q u a l i t a t i v e s u p p o r t i n g v a l i d i t y of use of tissue culture in m e t a b o l i c studies on xenobiotics. P h o t o d i e l d r i n s h o w n to be a direct product of dieldrin m e t a b o l i s m a n d n o t t h e r e s u l t of m i c r o b i a l or e n v i r o n m e n t a l interference.

CICICICICI~~ ,

dieldrin CI

~1

C,c, CI

-I

OH

CICI aldrin-fmns-diol

CI Clio 212 c, v H photodieldrin

F i g u r e 1 I d e n t i f i e d m e t a b o l i t e s of a l d r i n a n d d i e l d r i n in s u s p e n s i o n cultures from P h a s e o l u s v u l g a r i s root

Introduction W e h a v e r e p o r t e d p r e v i o u s l y (Brain and Lines 1977) t h a t s u s p e n s i o n cultures from P h a s e o l u s v u l g a r i s (French Bean) and S o l a n u m tuberosum (Potato) can p r o d u c e the s a m e m e t a b o l i t e s f r o m a l d r i n and d i e l d r i n as h a v e been reported from cell-free preparations (Fig.l). D i e l d r i n w a s the m a j o r m e t a b o l i t e of aldrin, together with low levels of aldrin-trans-dihydrodiol , occasionally photodieldrin, and s o m e u n i d e n t i f i e d p o l a r m a t e r i a l w h i c h w a s not the d i h y d r o c h l o r d e n e d i c a r b o x y l i c acid r e p o r t e d in field trials . Exogenous dieldrin was transformed to photodieldrin but aldrin-trans-dihydrodiol was not produced from added dieldrin, although our studies using trans-stilbene and t r a n s - s t i l b e n e oxide as m o d e l c o m p o u n d s (Ross et al, 1 9 7 8 a , 1978b) showed the presence of both epoxidase and epoxide h y d r a s e e n z y m e s in these cultures.

0721-7714/82/0001/0176/$

01.00

If p l a n t t i s s u e c u l t u r e is to be a u s e f u l t o o l in t h e s t u d y of p e s t i c i d e m e t a b o l i s m t h e r e s u l t s of m e t a b o l i c s t u d i e s m u s t be reproducible. The o b j e c t i v e s of the p r e s e n t w o r k w e r e to e s t a b l i s h that the m e t a b o l i s m of aldrin and dieldrin in suspension cultures from Phaseolus vulgaris root did not vary significantly from one subculture to another under the same cultural conditions, and to determine whether v a r i a t i o n s in the m e d i u m c o m p o s i t i o n , or in the i l l u m i n a t i o n , m a r k e d l y a l t e r e d g r o w t h or metabolism. Results

and D i s c u s s i o n

Reproducibility metabolism

of

growth,

uptake

and

177 It w a s o b v i o u s l y essential to demonstrate first that the behaviour of a culture did not vary significantly from one subculture to the next, under the same conditions. Comparative d a t a for g r o w t h and m e t a b o l i s m in three successive 28 d s u b c u l t u r e s of suspension cultures from Phaseolus vulgaris r o o t i n c u b a t e d w i t h i0 m g ~ ~ ) o f [14C3 a l d r i n or [ 1 4 C ] d i e l d r i n are g i v e n in T a b l e I. Although the experimental conditions w e r e h e l d as c o n s t a n t as p o s s i b l e p r a c t i c a l constraints resulted in some difference in inoculum size between each batch of subcultures. It was immediately clear that the overall pattern of growth and metabolism was consistent but that there were detailed quantitative differences between subcultures. The usual metabolites were p r e s e n t in all cases. W i t h e i t h e r a l d r i n or dieldrin each batch of subcultures took up a b o u t the s a m e t o t a l a m o u n t o f m a t e r i a l f r o m t h e m e d i u m b u t less of this w a s c o n v e r t e d b y the smallest inoculum (90 mg), w h i c h a l s o showed the least growth inhibition. The higher growth inhibition with the larger i n o c u l a s u g g e s t e d t h a t p e r h a p s c o n v e r s i o n to m o r e p h y t o t o x i c p r o d u c t s is a p r e - r e q u i s i t e for suppression of growth. As only relatively minor quantitative differences were observed and our normal experimental design involved inoculation of all flasks with the same number of cells at the same time this degree of reproducibility was considered quite acceptable.

Table 1 Uptake and metabolism of [14C] aldrin and dieldrin in successive subcultures of suspension cultures from Phaseolus vulgaris root

subculture inoculum

size

(mg)

I

II

III

mean

108

90

123

107

ALDRIN ADDED growth inhibition a aldrin: medium b cells total dieldrin: medium cells total aldrin-trans-diol c polar metabolites d total conversion

i0 34 ii 45 ii 26 37 4 3 43

3 34 24 58 ii 15 26 4 4 34

19 38 ii 49 12 21 33 4 5 42

Ii 36 15 52 ii 22 34 4 4 40

DIELDRIN ADDED growth inhibition dieldrin:medium cells total photodieldrin c polar metabolites total conversion

42 46 36 82 6 6 12

19 43 40 83 2 4 6

47 45 40 85 1 8 9

36 45 39 83 3 6 9

a b c d

% decrease relative % of added dose f o u n d o n l y in c e l l s % undetected by GLC

to u n t r e a t e d

control

Effect

of v a r i a t i o n

in h o r m o n a l

levels

We previously noted some differences in growth and in m e t a b o l i s m of aldrin and dieldrin when a single culture was grown on three diverse media (Brain and Lines 1977). However these media differed not only in their basic mineral and nutrient composition b u t a l s o i n c l u d e d the u n c o n t r o l l e d variable coconut water. Fig. 2 s h o w s t h e r e s u l t s o f a comparative s t u d y w h e r e i0 m g (14.8 k B q ) [14C]aldrin or [14C]dieldrin was added to cultures on seven closely related welldefined media based on a commercial base formulation (Murashige & Skoog Medium, Flow Labs, Irvine, UK) w h i c h was supplemented w i t h v a r y i n g q u a n t i t i e s a n d r a t i o s of 2 , 4 - D and kinetin in t h e r a n g e 0.5 m g 1 -I t o 2.0 m g 1 -I. C e l l s were grown routinely on a medium containing 0.5 m g 1 -I 2 , 4 - D a n d 0.5 m g 1 -I k i n e t i n and transferred to media containing the other levels of these h o r m o n e s a n d c o n d i t i o n e d for t w o s u b c u l t u r e periods (8 w e e k s ) before addition of s u b s t r a t e s a n d u s e in t h i s e x p e r i m e n t .

Both aldrin and dieldrin were taken up and converted to the usual products ,although there were quantitative differences in the rates of growth, uptake and metabolism° With aldrin there was some correlation b e t w e e n u p t a k e of a l d r i n i n t o the c e l l s a n d accumulation of dieldrin but little correlation between these and growth inhibition. Within the overall conversion there was significant variation from medium to medium in the distribution between dieldrin, aldrin-trans-dihydro-diol and polar material. T h e h i g h e s t r a t e s of u p t a k e and conversion were obtained on the medium used for routine culture, suggesting that the standard medium was near optimal for t h i s p a r t i c u l a r s e r i e s of r e a c t i o n s , or t h a t perhaps the cultures were not completely c o n d i t i o n e d to the n e w m e d i a . W i t h d i e l d r i n there was a similar correlation between uptake and conversion to photodieldrin and p o l a r m e t a b o l i t e s b u t no c o r r e l a t i o n b e t w e e n the concentrations of these and growth inhibition. Distribution between photodieldrin and polar metabolites varied to some extent. The highest rates of uptake and conversion were again found with the standard medium.

The overall conclusion was that variations in h o r m o n a l s u p p l e m e n t a t i o n , o v e r the r a n g e tested, had quantitative rather than qualitative effects on the uptake and metabolism of b o t h a l d r i n and d i e l d r i n . The m a x i m u m e x t e n t of t h e s e v a r i a t i o n s w a s less than four-fold. Minor variations in m e d i u m c o m p o s i t i o n c a n t h e r e f o r e be e x c l u d e d as an i m p o r t a n t s o u r c e of v a r i a b i l i t y of r e s p o n s e of cultures although it is s t i l l p o s s i b l e that radical changes of hormone type can p r o d u c e q u a l i t a t i v e l y s i g n i f i c a n t effects.

178

ALDRIN ADDED

DIELDRIN ADDED

50

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