Metabolism and Function of Sterols in Nematodes - ACS Symposium

Chapter 13

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on March 16, 2018 | https://pubs.acs.org Publication Date: December 24, 1987 | doi: 10.1021/bk-1987-0325.ch013

Metabolism and Function of Sterols in Nematodes David J. Chitwood, Ruben Lozano, William R. Lusby, Malcolm J. Thompson, and James A. Svoboda Insect Physiology Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD 20705

Current knowledge of sterol biochemistry and physiology in nematodes is reviewed. Nematodes possess a nutritional requirement for sterol because they lack the capacity for de novo sterol biosynthesis. The free-living nematode Caenorhabditis elegans has recently been used as a model organism for investigation of nematode sterol metabolism. C. elegans is capable of removal of the C-24 alkyT substituent of plant sterols such as sitosterol and also possesses the remarkable ability to attach a methyl group at C-4 on the sterol nucleus. An azasteroid and several long-chain alkyl amines disrupt the phytosterol dealkylation pathway in C. elegans by inhibiting its ∆ -sterol reductase. These compounds inhibit growth and reproduction in certain parasitic nematodes and provide model compounds for development of novel nematode control agents. 24

Nematodes a r e nonsegmented roundworms which i n c l u d e s e v e r a l d i v e r s e groups. F r e e - l i v i n g nematodes i n c l u d e m i c r o s c o p i c s o i l - d w e l l i n g o r a q u a t i c s p e c i e s t h a t feed on microorganisms and dead o r g a n i c m a t t e r . Of g r e a t e r economic importance i n the s o i l a r e p l a n t - p a r a s i t i c nematodes, which cause an e s t i m a t e d annual l o s s o f s i x b i l l i o n d o l l a r s t o American a g r i c u l t u r e ( 1 ) . Because o f t h e i r f r e q u e n t l y l a r g e r s i z e as w e l l as t h e i r t h r e a t t o human h e a l t h , a n i m a l - p a r a s i t i c nematodes are more f a m i l i a r t o the general p u b l i c and i n c l u d e a s c a r i d s , hookworms, pinworms, the dog heartworm, and the c a u s a l agents o f t r i c h i n o s i s , e l e p h a n t i a s i s , and r i v e r blindness. Readers w i t h f u r t h e r c u r i o s i t y about the l i f e h i s t o r y o r b i o l o g y o f nematodes are r e f e r r e d t o r e c e n t monographs by P o i n a r (2) and Maggenti (3). The p r e s e n t d i f f i c u l t y i n r o u t i n e c u l t u r e of p a r a s i t i c nematodes through t h e i r e n t i r e l i f e c y c l e s away from t h e i r p l a n t o r animal hosts has s e v e r e l y h i n d e r e d i n v e s t i g a t i o n s o f t h e i r b i o c h e m i s t r y and p h y s i o l o g y . C o n s e q u e n t l y , most s t u d i e s of s t e r o l n u t r i t i o n and metabolism i n nematodes have n e c e s s a r i l y i n v o l v e d the This chapter not subject to U.S. copyright. Published 1987, American Chemical Society

Fuller and Nes; Ecology and Metabolism of Plant Lipids ACS Symposium Series; American Chemical Society: Washington, DC, 1987.


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use of f r e e - l i v i n g genera such as C a e n o r h a b d i t i s , T u r b a t r i x and Panagrellus. These can be e a s i l y propagated upon b a c t e r i a or i n s t e r i l e l i q u i d media c o n t a i n i n g s e m i d e f i n e d components such as y e a s t e x t r a c t and soy peptone o r , w i t h g r e a t e r d i f f i c u l t y , i n c h e m i c a l l y d e f i n e d l i q u i d media. H i s t o r i c a l l y , p a r a s i t i c nematodes have been d i f f i c u l t to c o n t r o l f o r s e v e r a l r e a s o n s , i n c l u d i n g the r e s i s t a n c e of the nematode c u t i c l e t o p e n e t r a t i o n by p o t e n t i a l n e m a t i c i d e s , the r e s i s t a n c e of the s o i l to the m i g r a t i o n of n e m a t i c i d e s a p p l i e d t o i t , the h i g h mammalian t o x i c i t y of many a n t h e l m i n t i c s , and the general s i m i l a r i t y of the m e t a b o l i c pathways found i n these p a r a s i t e s and t h e i r h o s t s . However, the e x i s t e n c e of key d i f f e r e n c e s i n s t e r o l metabolism between nematodes and t h e i r animal or p l a n t hosts have presented the p o s s i b i l i t y t h a t nematode s t e r o l metabolism c o u l d be s e l e c t i v e l y i n h i b i t e d . Moreover, the l i k e l y f u n c t i o n of nematode s t e r o i d s i n the hormonal r e g u l a t i o n of i m p o r t a n t l i f e processes such as m o l t i n g and r e p r o d u c t i o n t o g e t h e r w i t h the p o t e n t i a l b e n e f i t of s e l e c t i v e l y d i s r u p t i n g these l i f e p r o c e s s e s have p r o v i d e d f u r t h e r impetus f o r the r e c e n t i n t e n s i f i c a t i o n of r e s e a r c h e f f o r t s i n the area of nematode s t e r o i d biochemistry. Nutritional

Requirement f o r S t e r o l

i n Nematodes

I n t e r e s t i n nematode s t e r o l metabolism was s t i m u l a t e d by the d i s c o v e r y t h a t the DD-136 s t r a i n of Steinernema f e l t i a e , an i n s e c t a s s o c i a t e , would not grow and reproduce upon b a c t e r i a l c u l t u r e s u n l e s s a s t e r o l was p r e s e n t ( 4 ) . Several compounds were c a p a b l e of s a t i s f y i n g t h i s n u t r i t i o n a l requirement, i n c l u d i n g c h o l e s t e r o l , cholestanol, s i t o s t e r o l , stigmastanol, 22-dihydrobrassicasterol, c h o l e s t - 4 - e n - 3 - o n e , 7 - d e h y d r o c h o l e s t e r o l , and l a t h o s t e r o l . Growth and r e p r o d u c t i o n d i d not o c c u r i n c u l t u r e s supplemented w i t h s t i g m a s t e r o l o r e r g o s t e r o l , two s t e r o l s c o n t a i n i n g A22-bonds. Hieb and R o t h s t e i n (5) demonstrated a s i m i l a r requirement i n C a e n o r h a b d i t i s propagated upon the b a c t e r i u m E s c h e r i c h i a c o l i ; a d d i t i o n of c h o l e s t e r o l , 7 - d e h y d r o c h o l e s t e r o l , e r g o s t e r o l , s t i g m a s t e r o l or s i t o s t e r o l r e s u l t e d i n e x c e l l e n t growth and reproduction. T u r b a t r i x a c e t i reproduced s u c c e s s f u l l y upon B a c i l l u s s u b t i l i s supplemented w i t h c h o l e s t e r o l , c h o l e s t a n o l , d e s m o s t e r o l , l a t h o s t e r o l , 7-dehydrocholesterol, 25-norcholesterol, c h o l e s t - 4 - e n - 3 - o n e , cholest-5-en-3-one, campesterol, 2 4 - m e t h y l e n e c h o l e s t e r o l , s t i g m a s t e r o l , s i t o s t e r o l , or f u c o s t e r o l ; two s t e r o l s w i t h a c i s - A / B r i n g c o n f i g u r a t i o n , c o p r o s t a n o l and c o p r o s t - 7 - e n o l , were not u t i l i z e d (6). In o t h e r e x p e r i m e n t s , squalene o r l a n o s t e r o l i n c r e a s e d T. a c e t i and C a e n o r h a b d i t i s p o p u l a t i o n s i n c h e m i c a l l y d e f i n e d media supplemented w i t h casamino a c i d s and myoglobin or cytochrome c (7). S i m i l a r l y , some p o p u l a t i o n i n c r e a s e o c c u r r e d i n c u l t u r e s of the s n a i l a s s o c i a t e R h a b d i t i s maupasi i n c h e m i c a l l y d e f i n e d media c o n t a i n i n g l a n o s t e r o l , a l t h o u g h the r e p r o d u c t i v e r a t e was g r e a t e r i n c h o l e s t e r o l or ergosterol-supplemented cultures (8). D i f f i c u l t i e s i n i n t e r p r e t a t i o n of the r e s u l t s of some of these n u t r i t i o n a l experiments are a consequence of p o s s i b l e s t e r o l contaminants i n media i n g r e d i e n t s , p o s s i b l e i m p u r i t i e s i n the added



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s t e r o l s , p o s s i b l e b a c t e r i a l metabolism of the supplemented s t e r o l s , or the p o t e n t i a l a b i l i t y of many s t e r o l s to s u b s t i t u t e f o r c h o l e s t e r o l i n a s t r u c t u r a l r o l e and f r e e small q u a n t i t i e s of endogenous c h o l e s t e r o l i n such c u l t u r a l systems f o r use as a s p e c i f i c p r e c u r s o r of s t e r o i d hormones o r o t h e r m e t a b o l i t e s by a " s p a r i n g " p r o c e s s , as can occur i n many i n s e c t s ( 9 ) . In o r d e r t o m i n i m i z e t h e s e e f f e c t s , we have propagated C a e n o r F a b d i t i s e l e g a n s i n a c h e m i c a l l y d e f i n e d a x e n i c c u l t u r e (CbMM, 10) supplemented w i t h chloroform/methanol ( 2 : 1 , v / v ) - e x t r a c t e d bovine hemoglobin and v a r i o u s h i g h l y p u r i f i e d s t e r o i d s a t 6 \ig/m\ ( u n p u b l i s h e d ) . Nematodes were i n i t i a l l y t r a n s f e r r e d to a s t e r o l - d e f i c i e n t medium, i n c u b a t e d two weeks, and then t r a n s f e r r e d to media supplemented w i t h the a p p r o p r i a t e s t e r o i d . Two s u b c u l t u r e s were performed a t two-week i n t e r v a l s to g r e a t l y minimize the amount of r e s i d u a l s t e r o l . At the c o n c l u s i o n of these e x p e r i m e n t s , t h r i v i n g p o p u l a t i o n s were p r e s e n t i n c u l t u r e s supplemented w i t h c h o l e s t e r o l , l a t h o s t e r o l , d e s m o s t e r o l , 7-dehydrocholesterol, campesterol, ergosterol, s i t o s t e r o l , s t i g m a s t e r o l , s t i g m a s t a n o l , i s o f u c o s t e r o l and c h o l e s t e r y l a c e t a t e . I n t e r e s t i n g l y , 2 9 - f l u o r o s t i g m a s t e r o l , a compound which i s t o x i c to the i n s e c t Manduca s e x t a by v i r t u e of the g e n e r a t i o n of f l u o r o a c e t a t e d u r i n g C - 2 4 d e a l k y l a t i o n (11) s a t i s f i e d the s t e r o l n u t r i t i o n a l requirement i n these e x p e r i m e n t s . ( T o x i c i t y d i d appear a t c o n c e n t r a t i o n s of 50 Mg/ml.) In the same e x p e r i m e n t s , r e p r o d u c t i o n and movement e v e n t u a l l y ceased i n media c o n t a i n i n g coprost-7-enol, 4a-methylcholest-8(14)-enol, lanosterol, 2 2 , 2 3 - d i h y d r o x y s i t o s t e r o l , p r o g e s t e r o n e , ecdysone, or 20-hydroxyecdysone. Although a d d i t i o n a l s t e r o i d s s h o u l d be i n v e s t i g a t e d i n t h i s s t e r i l e , h i g h l y d e f i n e d system b e f o r e c o n c l u s i o n s can be drawn, a p p a r e n t l y , 4 a - m e t h y l - , 4 , 4 - d i m e t h y l - , h e a v i l y h y d r o x y l a t e d , or c i s - A / B s t e r o l s cannot s a t i s f y the s t e r o l n u t r i t i o n a l requirement i r T C . e l e g a n s i n c o n t i n u o u s c u l t u r e . Among p a r a s i t i c nematodes, a s t e r o l requirement has been i n v e s t i g a t e d in only Nippostrongylus b r a s i l i e n s i s , a rat parasite whose eggs w i l l develop i n t o t h i r d - s t a g e , i n f e c t i v e l a r v a e i n a c u l t u r e medium c o n t a i n i n g f o r m a l i n - k i l l e d £ . c o l i and c h o l e s t e r o l , 7-dehydrocholesterol, ergosterol, s i t o s t e r o l , stigmasterol, l a n o s t e r o l , or c h o l e s t a n e , but not c o p r o s t a n o l o r coprostanone ( 1 2 ) . In a d d i t i o n , i n v i t r o development of t h i r d - s t a g e j u v e n i l e s of A s c a r i s t o f o u r t h - s t a g e j u v e n i l e s and the s i z e of the r e s u l t a n t j u v e n i l e s are markedly i n c r e a s e d by the a d d i t i o n of c h o l e s t e r o l (13). Lack o f De Novo S t e r o l B i o s y n t h e s i s i n Nematodes. The d i e t a r y requirement f o r s t e r o l r e s u l t s from the l a c k of de novo s t e r o l b i o s y n t h e s i s i n nematodes. S p e c i e s i n which r a d i o l a b e l e d a c e t a t e or mevalonate are not c o n v e r t e d to r a d i o l a b e l e d s t e r o l i n c l u d e T. a c e t i (14, 1 5 ) , C a e n o r h a b d i t i s (15) and the animal p a r a s i t e s A s c a r i s ( 1 6 ) , D i r o f i T a r i a i m m i t i s ( 1 7 ) , and B r u g i a pahangi ( 1 7 ) . On o c c a s i o n , r a d i o l a b e l e d s t e r o l s T ï ï e n t i f i e d by t h i n - l a y e r chromatography have been d e t e c t e d from nematodes i n c u b a t e d w i t h r a d i o l a b e l e d a c e t a t e ; i n the lone case i n which such compounds were f u r t h e r c h a r a c t e r i z e d by g a s - l i q u i d chromatography, the r a d i o l a b e l e d components possessed r e t e n t i o n times much e a r l i e r than c h o l e s t e r o l ( 1 6 ) . I t i s not known which enzymes i n the t y p i c a l de novo


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b i o s y n t h e t i c pathway are absent i n nematodes. Nutritional i n v e s t i g a t i o n r e v e a l e d t h a t i n T. a c e t i and C a e n o r h a b d i t i s the b l o c k o c c u r s subsequent t o f a r n e s o l [7). P a n a g r e l l u s r e d i v i v u s was r e p o r t e d to possess the i n t e r e s t i n g a b i l i t y to c o n v e r t t r i t i a t e d 2 , 3 - o x i d o s q u a l e n e t o l a n o s t e r o l ; t r i t i a t e d C27 s t e r o l s were not detected (18). Experiments w i t h a d d i t i o n a l r a d i o l a b e l e d p r e c u r s o r s a r e necessary t o f u r t h e r i n v e s t i g a t e t h i s i n t e r e s t i n g s u b j e c t .


S t e r o l Composition o f P a r a s i t i c Nematodes Because of the p r e v i o u s l y d e s c r i b e d problems i n c u l t u r e of p a r a s i t i c nematodes, i n v e s t i g a t i o n of s t e r o l metabolism i n these organisms has been l a r g e l y l i m i t e d t o comparison of the s t e r o l c o m p o s i t i o n s of host and p a r a s i t e . For example, l a t h o s t e r o l and c h o l e s t e r o l were the major s t e r o l s of H. carpocapsae DD-136 propagated i n wax moth l a r v a e , organisms t h a t c o n t a i n e d c h o l e s t e r o l as t h e i r p r i n c i p a l sterol. R a d i o l a b e l e d c h o l e s t e r o l i n j e c t e d i n t o h o s t l a r v a e was r e c o v e r e d as r a d i o l a b e l e d l a t h o s t e r o l and c h o l e s t e r o l i n the nematode ( 1 9 ) . Not u n e x p e c t e d l y , c h o l e s t e r o l i s the major s t e r o l of the few v e r t e b r a t e - p a r a s i t i c nematodes examined thus f a r ( 1 6 , 2 0 , 2 1 ) . F l e m i n g and F e t t e r e r (22) have demonstrated r e c e n t l y " v i a o c c l u s i o n of the d i g e s t i v e t r a c t , c o n t i n u o u s p e r f u s i o n of the pseudocoelom and c o l l e c t i o n of p e r i e n t e r i c f l u i d from A s c a r i s i n c u b a t e d i n the presence of t r i t i a t e d c h o l e s t e r o l t h a t t r a n s c u t i c u l a r and t r a n s m u s c u l a r t r a n s p o r t i s the primary means of s h o r t - t e r m cholesterol absorption. The i n t e s t i n e of t h i s animal s e l e c t i v e l y absorbs c h o l e s t e r o l a t about t w i c e the r a t e of s i t o s t e r o l ( 2 3 ) . A b s o r p t i o n of p h y t o s t e r o l s i s undoubtedly r e s p o n s i b l e f o r t f i ? f r e q u e n t o c c u r r e n c e of s u b s t a n t i a l q u a n t i t i e s of these 24-methyl or 2 4 - e t h y l s u b s t i t u t e d compounds i n many p a r a s i t i c nematodes, i n c l u d i n g d i g e s t i v e - t r a c t p a r a s i t e s whose host d i e t s can i n c l u d e s u b s t a n t i a l q u a n t i t i e s of p h y t o s t e r o l s . Because a l l of the p h y t o p a r a s i t i c nematodes examined thus f a r c o n t a i n s u b s t a n t i a l l y g r e a t e r r e l a t i v e p r o p o r t i o n s of c h o l e s t e r o l and/or l a t h o s t e r o l than t h e i r h o s t s (24-27) and because most (but not a l l ) phytophagous i n s e c t s (28) as w e l l as c e r t a i n f r e e - l i v i n g nematodes ( s u b s e q u e n t l y d e s c r i b e d T ~ a r e c a p a b l e of c o n v e r t i n g p h y t o s t e r o l s to c h o l e s t e r o l v i a a C - 2 4 d e a l k y l a t i o n p r o c e s s , i t has been s p e c u l a t e d t h a t p l a n t - p a r a s i t i c nematodes are capable of a s i m i l a r d e a l k y l a t i o n (24, 25, 27). O b v i o u s l y , experiments w i t h r a d i o l a b e l e d 2 4 - a l k y l s t e r o l s are needed to c o n c l u s i v e l y e s t a b l i s h whether the e x i s t e n c e of c h o l e s t e r o l or o t h e r 2 4 - d e s a l k y l s t e r o l s i n p h y t o p a r a s i t i c nematodes i s due to d e a l k y l a t i o n r a t h e r than some s e l e c t i v e uptake mechanism, as i n some i n s e c t s ( 2 9 , 3 0 ) . A s c a r i s a d u l t s d i d not c o n v e r t i n j e c t e d [ C ] s i t o s t e r o l ~ i b any o t h e r s t e r o l ( 1 6 ) ; i t i s unknown i f the C - 2 4 d e a l k y l a t i o n pathway i s s i m i l a r l y not f u n c t i o n a l i n o t h e r stages of t h i s i n t e s t i n a l p a r a s i t e or i n o t h e r a n i m a l - p a r a s i t i c nematodes. 1 4

M e t a b o l i s m of S t e r o l s i n F r e e - l i v i n g

Nematodes

D e f i n i t i v e e v i d e n c e f o r e x i s t e n c e of d e a l k y l a t i o n and o t h e r m e t a b o l i c pathways i n nematodes has been o b t a i n e d through e x t e n s i v e




204

e x p e r i m e n t a t i o n w i t h a x e n i c a l l y propagated f r e e - l i v i n g nematodes. Cole and Krusberg (14) c o n c l u s i v e l y demonstrated C-24 d e a l k y l a t i o n i n T. a c e t i by i t s c o n v e r s i o n of [ - ^ s i t o s t e r o l to t r i t i a t e d c h o T e s t e r o l and 7 - d e h y d r o c h o l e s t e r o l . More r e c e n t l y , our l a b o r a t o r y has undertaken a comprehensive i n v e s t i g a t i o n of the metabolism of e i g h t d i f f e r e n t s t e r o l s by C. e l e g a n s i n s t e r i l e , semi d e f i n e d l i q u i d media c o n t a i n i n g c h l o r o f o r m / m e t h a n o l - e x t r a c t e d i n g r e d i e n t s (31-35). D i e t a r y d e s m o s t e r o l , s i t o s t e r o l and s t i g m a s t e r o l were r a d i o l a b e l e d ; when p o s s i b l e t o measure, a l l s t e r o l s recovered from C. elegans i n such experiments c o n t a i n e d a p p r o x i m a t e l y the same s p e c i f i c a c t i v i t y as the o r i g i n a l supplemented s t e r o l and hence were t r u e m e t a b o l i t e s of the d i e t a r y s t e r o l and not t r a c e media contaminants or the products of de novo s y n t h e s i s . These experiments have demonstrated t h a t £ . e l e g a n s performs s e v e r a l d i f f e r e n t s t e r o l m e t a b o l i c p r o c e s s e s , i n c l u d i n g C-7 d e h y d r o g e n a t i o n , A - r e d u c t i o n , 4a-methylation, Δ - s t e r o l to A ( * 4 ) - s t e r o l i s o m e r i z a t i o n , C-24 d e a l k y l a t i o n , and A - r e d u c t i o n . I n i t i a l experiments w i t h supplemented c h o l e s t e r o l demonstrated t h a t C. e l e g a n s can i n t r o d u c e a double bond a t C-7 a n d , to a l e s s e r e x t e n T , reduce the A - b o n d o f the r e s u l t i n g A5,7_diene, as 7 - d e h y d r o c h o l e s t e r o l and l a t h o s t e r o l were major and minor m e t a b o l i t e s (Table I ) . U n e x p e c t e d l y , we d e t e c t e d s u b s t a n t i a l q u a n t i t i e s of two d i f f e r e n t 4 a - m e t h y l s t e r o l s i n C. e l e g a n s f e d c h o l e s t e r o l , and t h e s t e r y l e s t e r f r a c t i o n was e s p e c i a l l y r i c h i n these compounds. Because 4 a - m e t h y l s t e r o l s are g e n e r a l l y regarded as i n t e r m e d i a t e s between l a n o s t e r o l (or c y c l o a r t e n o l ) and c h o l e s t e r o l i n organisms w i t h de novo s t e r o l b i o s y n t h e t i c c a p a b i l i t y , our i n i t i a l r e a c t i o n was "Eiïârf t h e s e compounds were endogenous media c o n t a m i n a n t s . However, attempts to i s o l a t e them from i n c u b a t e d , nematode-free media f a i l e d - Subsequent experiments w i t h [ C ] d e s m o s t e r o l and [ 1 4 c ] s i t o s t e r o l r e v e a l e d t h a t the 4 a - m e t h y l c h o l e s t - 8 ( 1 4 ) - e n o l and 4 a - m e t h y l c h o l e s t - 7 - e n o l contained a p p r o x i m a t e l y the same s p e c i f i c a c t i v i t y as the o r i g i n a l d i e t a r y s t e r o l and were produced by a d i r e c t n u c l e a r m e t h y l a t i o n pathway. The b i o s y n t h e s i s of 4 - m e t h y l s t e r o l s from m e t h y l a t i o n of a 4 - d e s m e t h y l s t e r o l p r e c u r s o r has not been suggested to occur i n any other organism. The n u c l e a r m e t h y l a t i o n pathway i s not unique t o elegans. We have r e c e n t l y d i s c o v e r e d s i m i l a r but not i d e n t i c a l pathways i n T. a c e t i and P_. r e d i y i v u s (Chitwood e t a l . , u n p u b l i s h e d ) . Cysts of Heterodera zeae d i d not c o n t a i n 4 - m e t h y l s t e r o l s (27h but p o s s i b l y o t h e r l i f e stages of H. zeae or o t h e r p a r a s i t i c nematodes may contain 4-methylsteroTs. Experiments w i t h s i t o s t e r o l - s u p p l e m e n t e d media demonstrated the C-24 d e a l k y l a t i o n o f a 24a-ethy1 s t e r o l by C. e l e g a n s (Table I ) . The a b i l i t y of t h i s nematode t o produce 2 4 - d e s a l k y l s t e r o l m e t a b o l i t e s from c a m p e s t e r o l , 2 2 - d i h y d r o b r a s s i c a s t e r o l , 24-methylenecholesterol, s t i g m a s t e r o l and s t i g m a s t a n o l (Table I) i n d i c a t e s t h a t 24a-methyl, 243-methyl, and 24-methylene s u b s t i t u e n t s a r e e f f e c t i v e l y removed and t h a t C-24 α - e t h y l group removal i s not dependent upon l a c k of a A - b o n d o r presence o f a A - b o n d . However, the f a c t t h a t s u b s t a n t i a l l y l a r g e r q u a n t i t i e s of campesterol remained unmetabolized i n C. e l e g a n s , as compared to the o t h e r f i v e p h y t o s t e r o l s , i n d i c a t e s e i t h e r t h a t s u b s t r a t e s p e c i f i c i t y f o r the 5

8

2 2

5

14

2 2

5


13. CHITWOOD ET AL.

205

Sterob in Nematodes

C-24 d e a l k y l a t i o n enzyme system o c c u r s o r t h a t d i f f e r e n t enzymes are u t i l i z e d f o r d i f f e r e n t substrates (34). S e v e r a l o t h e r i n t e r e s t i n g o b s e r v a t i o n s were noted d u r i n g our comparative i n v e s t i g a t i o n s ( T a b l e I ) . For example, s t i g m a s t a n o l - f e d C. e l e g a n s d i d not c o n t a i n any Δ - or A 5 , 7 _ t e r o l s ; t h e r e f o r e , i t i s l i k e l y t h a t t h i s nematode l a c k s a Δ -dehydrogenase. Although n u c l e a r m o d i f i c a t i o n of d i e t a r y 2 4 - e t h y l s t e r o l s d i d not o c c u r p r i o r to d e a l k y l a t i o n , the nucleus o f d i e t a r y 2 4 - m e t h y l s t e r o l s was d i r e c t l y m e t a b o l i z e d to a s u r p r i s i n g l y l a r g e degree, r e s u l t i n g i n p r o d u c t i o n of s i g n i f i c a n t q u a n t i t i e s o f 2 4 - m e t h y l c h o l e s t a - 5 , 7 - d i e n o l , 2 4 - m e t h y l c h o l e s t - 7 - e n o l , and 4a,24-dimethylcholest-8(14)-enol. A p p a r e n t l y , the n u c l e a r m o d i f i c a t i o n enzymes have l i t t l e a f f i n i t y f o r 2 4 - e t h y l s t e r o l s but might indeed b i n d to s t e r o l s w i t h a l e s s b u l k y 24-methyl group. A l t e r n a t i v e l y , s e p a r a t e enzymes f o r n u c l e a r metabolism of 2 4 - m e t h y l s t e r o l s and 2 4 - d e s m e t h y l s t e r o l s c o u l d e x i s t c o n c u r r e n t l y . R o t h s t e i n (15) o r i g i n a l l y demonstrated the e s t e r i f i c a t i o n of [ l ^ C ] c h o l e s t e r o l ~ B y C a e n o r h a b d i t i s and T. a c e t i . In our i n v e s t i g a t i o n s , e s t e r i f i e d s t e r o l s comprised from 7 . 3 % t o 2 1 . 3 % of the t o t a l s t e r o l from C. e l e g a n s and were r a d i o l a b e l e d when r a d i o l a b e l e d d i e t a r y s t e r o l s were employed ( 3 2 , 3 4 ) . Steryl ester f r a c t i o n s c o n s i s t e n t l y c o n t a i n e d l a r g e r p r o p o r t i o n s of 4a-methyl s t e r o l s than f r e e s t e r o l f r a c t i o n s . Speculative e x p l a n a t i o n s f o r the abundance of 4 a - m e t h y l s t e r y l e s t e r s i n c l u d e an e s t e r i f i c a t i o n requirement f o r 4 a - m e t h y l s t e r o l s y n t h e s i s or t r a n s p o r t or a s p e c i f i c hormonal, pheromonal or o t h e r p h y s i o l o g i c r o l e f o r a 4 a - m e t h y l s t e r y l e s t e r or m e t a b o l i t e .


5

s

I n h i b i t i o n o f S t e r o l M e t a b o l i s m i n Nematodes L i k e nematodes, i n s e c t s n u t r i t i o n a l l y r e q u i r e s t e r o l because they l a c k the c a p a c i t y f o r de novo s t e r o l b i o s y n t h e s i s (9). Many a z a s t e r o i d s and n o n s t e r o i d a l a l k y l a m i n e s and amides i n t e r f e r e w i t h s t e r o i d m e t a b o l i s m , growth and development i n i n s e c t s and have p r o v i d e d model compounds f o r development of novel agents f o r i n s e c t control (36). C o n s e q u e n t l y , s e v e r a l i n v e s t i g a t o r s have e v a l u a t e d these as w e l l as r e l a t e d compounds f o r t o x i c i t y or g r o w t h - i n h i b i t o r y a c t i v i t y towards v a r i o u s nematodes. C o l e and Krusberg (14) demonstrated the a c c u m u l a t i o n of [ H ] - d e s m o s t e r o l i n T. a c e t i s t e r i l e l y propagated i n media c o n t a i n i n g [ H ] - s i t o s t e r o l and t r i p a r a n o l s u c c i n a t e , a v e r t e b r a t e h y p o c h o l e s t e r o l e m i c agent by v i r t u e of i t s i n h i b i t i o n of A ^ - s t e r o l r e d u c t a s e , an enzyme t h a t c o n v e r t s desmosterol to c h o l e s t e r o l . Feldmesser e t a l . (37) demonstrated the t o x i c i t y o f many d i f f e r e n t N - s u b s t i t u t e d l o n g ^ c h a i n (Cn t o C15) a l k y l amines and amides to ? . r e d i y i y u s and the r o o t - k n o t nematode Meloidogyne i n c o g n i t a ; 100% l e t h a l i t y o c c u r r e d a t c o n c e n t r a t i o n s of 5-40 Mg/ml. Other r e l a t e d amines possessed i n v i t r o t o x i c i t y a g a i n s t the pinewood nematode, Bursaphelenchus x y l o p h i l u s ( 3 8 ) . Douvres e t a l . (39) found t o x i c i t y of many of these a l k y l a m i n e s and amides to be as low as 1 . 0 t o 2 . 5 pg/ml i n v i t r o a g a i n s t the c a t t l e stomach worm O s t e r t a g i a ostertagi. Less i s known about the e f f e c t s of a z a s t e r o i d s upon nematodes because o f t h e i r u n d e s i r a b l e resemblance to human s t e r o i d s as w e l l 3

3

2


206


Table I. sterols


R e l a t i v e percentages

of s t e r o l s

i n free sterol

Cholesterol Recovered s t e r o l Cholesterol 7-Dehydrocholesterol Lathosterol Cholesta-5,7,9(ll)-trienol Cholest-8(14)-enol Cholestanol Desmosterol Cholesta-5,7,24-trienol Campesterol Dihydrobrassicasterol 24-Methylenechol e s t e r o l 24-Methylcholesta-5,7-dienol 24-Methyl chol e s t - 7 - e n o l 2 4 - M e t h y l e n e c h o l e s t a - 5 , 7 - d i enol 24-Methylchol e s t a - 5 , 7 , 9 ( 1 1 ) - t r i enol Sitosterol Stigmasterol Stigmastanol Fucosterol 4a-Methylcholest-8(14)-enol 4a-Methylchol e s t - 7 - e n o l 4a,24-Dimethylcholest-8(14)-enol 4a,24-Dimethylchol estanol Contained

FS 52.3 40.5 3.6 1.4

-

-

-

-

2.1

SE 41.2 26.7 5.7 1.5

-

-

-

0.1

23.4 1.3

-

-

(FS) and s t e r y l

Desmosterol FS 26.9 31.2 1.7 1.4

SE 18.7 39.0 1.4 1.4

32.3

-

2.4

-

-

-

-

3.8 0.3

-

-

22.8 4.5

-

-

-

10.6 1.6

-

1.5% c a m p e s t e r o l .


ester

Campesterol FS 3.9 29.4 3.7 1.9

-

-

-

35.8

-

3.1 14.1 0.6 1.3 1.0

-

3.6 0.2 0.7 0.4

SE 3.7 10.9 1.8

53.3 3.7 12.9 1.1

-

0.3

-

9.7 0.2 1.4 0.8

207


13. CHITWOOD ET AL.

(SE) f r a c t i o n s from C a e n o r h a b d i t i s e l e g a n s propagated w i t h d i f f e r e n t


Dihydrobrassicasterol FS 5.1 45.0 3.5 6.5

-

SE 8.5 21.5 1.6 3.4

-

Supplemented s t e r o l 24-Methyl e n e cholesterol Sitosterol FS 8.6 49.6 5.0 2.1

-

_

-

24.2 1.3 5.4 0.2 0.3 1.1

31.7 8.8 7.1

6.7

14.7

0.3 0.3

0.6 0.4

-

1.3

-

-

31.0

SE 12.2 43.8 2.5 2.5

-

-

6.3

-

-

-

-

16.0

-

1.1 0.1

14.9 0.7

0.1 4.2 0.7

-

-

-

FS

SE

_

_

68.3

-

-

28.6

-

6.4 5.6

_

_

_

_

_

_ _ _ _

_ _

-

2.5

Stigmastanol

3.7 3.8

_

16.5

SE 11.3 26.5 9.4 2.4

-

1.3

_ _ _ _ _ _

FS 8.6 55.6 3.9 5.8

-

_

-

Stigmasterol

-

0.7

_

-

-

SE 9.3 30.5 3.6 0.3

0.6

_ _

-

FS 6.7 66.5 4.4 0.8

5

dietary

_ _ _ _ _ _

_ _ _ _

_ _ _ _ _

30.3

-

0.1 23.3 1.4 _

20.5

-

5.4 0.2 _

21.9

-_ 27.5 1.0

_

_ _ _

_

_

-

14.2

. 9.3 0.7 _


-

30.1 _

27.0 2.3 _


208


as t h e i r complex s t r u c t u r e and r e s u l t a n t expensive chemical synthesis. The a z a s t e r o i d 2 5 - a z a c o p r o s t a n e s t r o n g l y i n h i b i t e d r e p r o d u c t i o n i n C. e l e g a n s (32) and i η v i t r o development of f i r s t - s t a g e t o t ï ï i r d - s t a g e j u v e n i l e s o f N. b r a s i l i e n s i s and the mouse p a r a s i t e N e m a t o s p i r o i d e s dubius ( 4 0 ) . Specific biological e f f e c t s i n nematodes of many a l k y l a m i n e s and a l k y l a m i des were d e s c r i b e d i n d e t a i l i n 0 . o s t e r t a g i , where e f f e c t s i n c l u d e d reduced s u r v i v a l , decreased m o t i l i t y or induced p a r a l y s i s , delayed development, lowered y i e l d s o f advanced s t a g e s , d e l a y e d or b l o c k e d or i n c o m p l e t e d t h i r d or f o u r t h m o l t , and decreased or n o n e x i s t e n t egg p r o d u c t i o n ( 3 9 ) . A s i m i l a r p a r a l y s i s and i n h i b i t i o n of m o t i l i t y and r e p r o d u c t i o n o c c u r r e d i n £ . e l e g a n s t r e a t e d w i t h v a r i o u s a l k y l a m i n e s (33) or 2 5 - a z a c o p r o s t a n e [3Z). The l a t t e r compound or 2 5 - a z a c h o l e s t a n e induced H. b r a s i l i e n s i s j u v e n i l e s to develop m o r p h o l o g i c a l a b n o r m a l i t i e s observed i n j u v e n i l e s c u l t u r e d i n s t e r o l - d e f i c i e n t media, i n c l u d i n g d e g e n e r a t i o n of i n t e s t i n a l c e l l s , abnormal d i s p e r s i o n of l i p i d g l o b u l e s , and small l e n g t h ( 4 0 ) . E f f e c t s o f i n h i b i t o r s on s t e r o l m e t a b o l i c pathways i n C. e l e g a n s . The most s p e c i f i c e f f e c t s on nematodes of the a z a s t e r o i d s , amines and amides have been o b t a i n e d through our i n v e s t i g a t i o n s of C_. e l e g a n s propagated i n media supplemented w i t h one of s e v e r a l d i f f e r e n t i n h i b i t o r s ( 3 1 , 3 2 , 3 3 , 3 5 ) . Our r e s u l t s have demonstrated t h a t these i n f i T b i t o r s can a c t a t s e v e r a l d i f f e r e n t m e t a b o l i c s i t e s ; moreover, the a c c u m u l a t i o n of many p r e v i o u s l y u n d e t e c t e d s t e r o l s has l e d to the d i s c o v e r y of s e v e r a l key i n t e r m e d i a t e s i n the s t e r o l m e t a b o l i c pathways of t h i s organism (Figure 1). I n i t i a l l y , Z. e l e g a n s was propagated i n media supplemented w i t h 5 . 0 pg/ml 2 5 - a z a c o p r o s t a n e h y d r o c h l o r i d e , a c o n c e n t r a t i o n p r e v i o u s l y shown t o decrease r e p r o d u c t i v e r a t e i n £ . e l e g a n s by about 50%. E x c l u d i n g d i e t a r y s i t o s t e r o l , n e a r l y 96% of the s t e r o l s from such organisms were A 2 4 . t e r o l s n o r m a l l y p r e s e n t i n no more than trace q u a n t i t i e s : c h o l e s t a - 5 , 7 , 2 4 - t r i e n o l , desmosterol, c h o l e s t a - 7 , 2 4 - d i e n o l , and f u c o s t e r o l (Table I I ) . The abundance of these compounds i n d i c a t e d t h a t the a z a s t e r o i d s i g n i f i c a n t l y i n h i b i t e d Δ ^ - s t e r o l r e d u c t a s e i n £ . e l e g a n s and t h a t A - s t e r o l s a r e major i n t e r m e d i a t e s i n the £ . e l e g a n s pathway for sitosterol dealkylation. In a d d i t i o n , the predominance o f A - 4 a - m e t h y l s t e r o l s r e v e a l e d t h a t the a z a s t e r o i d i n h i b i t s the isomerase t h a t c o n v e r t s Δ - t o Δ ( ) - 4 a - m e t h y l s t e r o l s . Four n o n s t e r o i d a l d i m e t h y l amines s i m i l a r l y i n h i b i t e d the A 2 4 - s t e r o l r e d u c t a s e i n s i t o s t e r o l - f e d C_. e l e g a n s , b u t to a l e s s e r extent (Table I I ) . Among these compounds, maximal i n h i b i t i o n o c c u r r e d upon a d d i t i o n of N , N , 3 , 7 , l l - p e n t a m e t h y l d o d e c a n a m i n e , f o l l o w e d by Ν,Ν-dimethyldodecanamine, N,N-dimethyltetradecanamine, and Ν,Ν-dimethylhexadecanamine. A l t h o u g h i n h i b i t o r y to growth and r e p r o d u c t i o n i n C. e l e g a n s , the c o r r e s p o n d i n g C12 d i e t h y l a m i d e possessed l i t t l e e f f e c t on s i t o s t e r o l ( T a b l e I I ) o r s t i g m a s t e r o l ( T a b l e I I I ) m e t a b o l i s m ; perhaps s u b s t i t u t i o n of an amide group f o r the amine group d e s t r o y e d the a b i l i t y of the i n h i b i t o r t o c o m p e t i t i v e l y b i n d to the £ . e l e g a n s A 4 - s t e r o l r e d u c t a s e . The b r a n c h e d - c h a i n a l k y l a m i n e r e s u l t e d i n an a c c u m u l a t i o n of u n m e t a b o l i z e d d i e t a r y s i t o s t e r o l , p o s s i b l y because i t s g r e a t e r s

2 4

7

8

1 4

2


Fuller and Nes; Ecology and Metabolism of Plant Lipids ACS Symposium Series; American Chemical Society: Washington, DC, 1987. 4a -METHYLCH0LEST-8( 14)-EN0L

i

4a -METHYLCHOLEST-7-ENOL

LATHOSTEROL

CH0LESTA-7.24-DIEN0L

TRIENOL

CHOLESTA-5,7,24-

DESMOSTEROL

FUCOSTEROL

SITOSTEROL

24-METHYLCHOLESTA-5.7-DIENOL

4a ,24-DIMETHYLCHOLEST-8(14)-ENOL

CHOLESTANOL

4a,24-DIMETHYL-

24-METHYLCHOLEST-7-ENOL

CHOLESTA-5.7-DIENOL

24-METHYLENE-

24-METHYLCHOLESTEROL

F i g u r e 1. Major pathways o f s t e r o l metabolism i n C a e n o r h a b d i t i s elegans. Dotted l i n e s r e p r e s e n t p o s s i b l e but unproven s t e p s .

TETRAENOL

CHOLESTA-5.7,22,24-

TRIENOL

CHOLESTA-5.22,24- -

TRIENOL

STIGMASTA-5,22.24(28)-

STIGMASTEROL



210

Table I I . R e l a t i v e percentages of t o t a l s t e r o l s from C a e n o r h a b d i t i s metabolic i n h i b i t o r s . D i e t a r y s i t o s t e r o l c o n t a i n e d 1.5% c a m p e s t e r o l .


Recovered

sterol

None

Cholesterol 8.1 7-Dehydrochol e s t e r o l 56.4 5.5 Lathosterol 2.0 C h o l e s t a - 5 , 7 , 9 ( 1 1 ) - t r i enol Desmosterol Chol e s t a - 5 , 7 , 2 4 - t r i e n o l Cholesta-7,24-dienol Cholesta-5,7,9(ll),24-tetraenol Cholesta-8,24-dienol Campesterol 0.6 Fucosterol 0.1 Sitosterol 18.2 4

Metabolism and Function of Sterols in Nematodes - ACS Symposium

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