Physiological Effects of Photodynamic Action: Special Reference to

Chapter 8

Physiological Effects of Photodynamic Action: Special Reference to Insects Joseph E. Weaver

Downloaded by CORNELL UNIV on July 18, 2016 | http://pubs.acs.org Publication Date: May 7, 1987 | doi: 10.1021/bk-1987-0339.ch008

Division of Plant and Soil Sciences, West Virginia University, Morgantown, W V 26506

Results of studies involving photodynamic action and i t s effect on the physiology of organisms, especially insects, are summarized and discussed. With regards to insects, few studies on the photooxidative reactions were conducted until the early to mid-seventies. Since that time, there has been a renewed effort to determine how photodynamically active compounds affect insect physiology. This chapter is a review of findings on this phenomenon as it affects the organism at (1) the subcellular level, (2) the cellular level, (3) the systems level, and (4) aspects of photodynamic action as i t affects development and reproduction.

The p h y s i o l o g i c a l e f f e c t s o f photodynamic a c t i o n have been s t u d i e d i n a v a r i e t y o f o r g a n i s m s i n t h e l a s t e i g h t decades. However, o n l y f o u r o r i g i n a l papers f r o m 1900 t o 1970 r e p o r t e d o n i n s e c t s a s t h e e x p e r i m e n t a l e n t i t y . W h i l e some e a r l i e r s t u d i e s (1970-1975) designed t o evaluate the phototoxic e f f e c t s of photodynamically a c t i v e substances i n c l u d e d o b s e r v a t i o n s o n p h y s i o l o g i c a l e f f e c t s , t h i s s u b j e c t r e c e i v e d l i t t l e a t t e n t i o n from researchers u n t i l l a t e r i n t h e decade. B e g i n n i n g i n t h e m i d - s e v e n t i e s , and t o t h e p r e s e n t t i m e , s e v e r a l s t u d i e s have been conducted i n renewed e f f o r t s t o e l u c i d a t e t h e e f f e c t s t h a t a r e a s s o c i a t e d w i t h dyes e n s i t i z e d p h o t o o x i d a t i v e r e a c t i o n s i n i n s e c t s . These s t u d i e s have added g r e a t l y t o our knowledge o f how p h o t o d y n a m i c a l l y a c t i v e s u b s t a n c e s a f f e c t i n s e c t s (and o t h e r o r g a n i s m s ) f r o m t h e c e l l u l a r l e v e l t o the systems l e v e l . The phenomenon o f photodynamic a c t i o n has been t h e s u b j e c t o f s e v e r a l r e v i e w s . Some o f t h e works c i t e d i n those r e v i e w s have a l s o been c i t e d i n t h i s c h a p t e r where r e l e v a n t . These r e v i e w s a r e not a l l i n c l u s i v e and d e a l w i t h a s p e c t s o f photodynamism o t h e r t h a n p h y s i o l o g i c a l r a m i f i c a t i o n s i n l i v i n g organisms. The f o l l o w i n g a r e g e n e r a l r e v i e w s o f photodynamic e f f e c t s o n c e l l s a n d m u l t i c e l l u l a r o r g a n i s m s t h a t may be o f i n t e r e s t t o t h e r e a d e r : 0097-6156/87/0339-0122$06.00/0 © 1987 American Chemical Society

Heitz and Downum; Light-Activated Pesticides ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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Physiological Effects of Photodynamic Action

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Blum Q ) , E r r e r a (2), C l a r e (3), F o w l k s (4), S a n t a m a r i a (5-6). S a n t a m a r i a and P i n t o (J), S p i k e s and G h i r o n (8), Simon (£), S p i k e s and S t r a i g h t (10), Bourdon and S c h n u r i z e r (11), S p i k e s (12), S p i k e s and L i v i n g s t o n (13). P o o l e r and Valenzeno (14), and R o b i n s o n (15). The purpose of t h i s r e v i e w i s t o summarize and d i s c u s s f i n d i n g s on the p h y s i o l o g i c a l e f f e c t s of the photodynamic a c t i o n a t (1) t h e s u b c e l l u l a r l e v e l , ( I I ) the c e l l u l a r l e v e l , ( I I I ) t h e systems l e v e l , and (IV) a s p e c t s of the photodynamic a c t i o n as i t a f f e c t s development and r e p r o d u c t i o n i n i n s e c t s .


E f f e c t s a t the S u b c e l l u l a r L e v e l Many s t u d i e s on v e r t e b r a t e s and i n v e r t e b r a t e s have shown t h a t "almost no c e l l p r o c e s s o r s t r u c t u r e i s immune f r o m ... photosens i t i z e d m o d i f i c a t i o n under the r i g h t c o n d i t i o n s " (14). S e n s i t i z e d m o l e c u l e s are a b l e t o o x i d i z e a w i d e range o f c e l l u l a r components i n c l u d i n g i n t e r m e d i a t e s , p r o t e i n s , and membranes. P o o l e r and Valenzeno (14) c i t e s e v e r a l examples of photoc h e m i c a l damage and m o d i f i c a t i o n s o c c u r r i n g t o i n t r a c e l l u l a r components by p h o t o s e n s i t i z i n g agents. Once p e r m e a b i l i t y of the c e l l membrane has been a l t e r e d , c e l l u l a r f u n c t i o n can be g r e a t l y modified. Cande e t a l . (16) r e p o r t e d t h a t l y s e d c e l l s of kangaroo r a t k i d n e y a r e permeable t o s m a l l m o l e c u l e s such as e r y t h r o s i n B. They observed t h a t h o l e s were p r e s e n t i n the plasma membrane and the m l t o c h r o n d i a were s w o l l e n and d i s t o r t e d ; o t h e r membrane-bound o r g a n e l l e s were not n o t i c e a b l y a l t e r e d . Haga and S p i k e s (17) a l s o reported s w e l l i n g of i s o l a t e d r a t l i v e r mitochondria s e n s i t i z e d w i t h e o s i n Y and methylene b l u e . On the b a s i s of c e r t a i n m e t a b o l i c measurements, they concluded t h a t the observed e f f e c t s o f t h e s e p h o t o t o x i n s s u g g e s t e d t h a t the s w e l l i n g i s caused by i n h i b i t i o n of enzymatic a c t i v i t i e s i n the e l e c t r o n t r a n s p o r t system and by the u n c o u p l i n g o f p h o s p h o r y l a t i o n f r o m r e s p i r a t i o n . H i l f e t a l . (18) have a l s o proposed t h a t m l t o c h r o n d i a may be a c r i t i c a l s u b c e l l u l a r s i t e of p h o t o m o d i f i c a t i o n . L o s s o f c e l l u l a r p o t a s s i u m has been r e p o r t e d and, i n t u r n , p r o t e i n s y n t h e s i s and c e l l membrane p o t e n t i a l s a r e a f f e c t e d . L y s o s o m a l damage by p h o t o s e n s i t i z e r s can r e s u l t i n secondary c e l l u l a r damage by a l t e r i n g the f i n e s t r u c t u r e o f m i t o c h o n d r i a and e n d o p l a s m i c r e t i c u l u m . U n s a t u r a t e d l i p i d s , n u c l e i c a c i d s , DNA, and RNA may be m o d i f i e d photo c h e m i c a l l y . P r o t e i n s and c e r t a i n o f t h e i r amino a c i d s i d e c h a i n s a r e s u s o e p t a b l e t o p h o t o s e n s i t i z e d attack. Palm (19) noted t h a t b a s i c dyes l i k e methylene b l u e and n e u t r a l r e d can produce g r a n u l e s i n the c y t o p l a s m of i n s e c t c e l l s w i t h n e u t r a l red g r a n u l e s o f t e n r e p r e s e n t a t i v e o f v a c u o l e s . Other w o r k e r s (see P o o l e r & Valenzeno (14)) have r e p o r t e d cytoplasmic v a c u o l i z a t i o n or blebs appearing i n photosensitized c e l l s . Only a l i m i t e d number o f s t u d i e s o f photodynamic a c t i o n i n i n s e c t s a t the s u b c e l l u l a r l e v e l have been conducted. C a r p e n t e r e t a l . (20) s t u d i e d the s y n e r g i s t i c e f f e c t o f f l u o r e s c e i n on r o s e bengal i n t h e presence of a p u r i f i e d enzyme. They showed t h a t f l u o r e s c e i n enhanced the photodynamic a c t i v i t y o f r o s e bengal i n


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the i n h i b i t i o n o f g l y c e r a l d e h y d e - 3 - p h o s p h a t e dehydrogenase of m o s q u i t o l a r v a e (Aedes t r i s e r i a t u s (Say)). Fondren and H e i t z (21) o b s e r v e d L T and t i s s u e l e v e l s o f dyes and suggested t h a t the f a c e f l y (Musca autumnal I s DeGeer) i s susceptable t o t o x i c I n t r a c e l l u l a r r e a c t i o n s of c e r t a i n s e n s i t i z e d xanthene dyes. Fondren e t a l . (22) conducted s i m i l a r s t u d i e s on t h e house f l y (Musca d o m e s t i c a L.) where " r e l a t i v e t o x i c i t i e s were d e s c r i b e d by means of r a t e c o n s t a n t s of p h o t o o x i d a t i o n c a l c u l a t e d f o r (6 xanthene dyes) w h i c h i n c l u d e d both t h e L T ^ Q and t h e t i s s u e dye l e v e l . " C a l l a h a m e t a l . (23) r e p o r t e d t h a t d y e - s e n s i t i z e d p h o t o o x i d a t i o n of the a c e t y l c h o l i n e s t e r a s e i n whole-head homogenates o f t h e i m p o r t e d f i r e ant ( S o l e n o p s l s r i c h t e r i ( F o r e l ) ) c o u l d be i n d u c e d i n the presence o f s e v e r a l xanthene dyes. L a c t i c dehydro genase and a c e t y l c h o l i n e s t e r a s e of the b o l l w e e v i l (Anthonomus g r a n d i s g r a n d i s Boheman) were i n a c t i v a t e d by d y e - s e n s i t i z e d p h o t o o x i d a t i o n mediated by s u b s t i t u t e d xanthenes (24). There i s c o n s i d e r a b l e e v i d e n c e t h a t many p h o t o d y n a m i c a l l y a c t i v e compounds a r e mutagenic. I t has been suggested t h a t a l l mutagenic c h e m i c a l s r e a c t w i t h the p r o t e i n p a r t of the gene m o l e c u l e r a t h e r t h a n w i t h the n u c l e i c a c i d (25). C l a r k (25) s t u d i e d the mutagenic a c t i v i t y of s e v e r a l dyes i n D r o s o p h i l a m e l a n o g a s t e r Meigen and suggested t h a t the dye m o l e c u l e may r e a c t w i t h e i t h e r the n u c l e i c a c i d or p r o t e i n m o i e t y of the gene m o l e c u l e . Evidence from the s t u d y suggested t h a t p y r o n i n (a t h i a z i n e dye) produces a g e n e t i c e f f e c t by c o m b i n i n g d i r e c t l y w i t h n u c l e i c a c i d . He a l s o suggested t h a t the mutagenic a c t i v i t y of a dye i s r e l a t e d t o i t s a f f i n i t y f o r u n p o l y m e r i z e d n u c l e i c a c i d . I n t h i s s t u d y , he r e p o r t e d t h a t n e u t r a l red was c a p a b l e of p r o d u c i n g 0.33$ l e t h a i s and t h a t rhodamine appears t o be d e f i n i t e l y a l t h o u g h w e a k l y mutagenic c a u s i n g 0.27$ l e t h a l s . The m u t a g e n i c i t y of a c r i d i n e compounds i n d i f f e r e n t b i o l o g i c a l systems was r e v i e w e d by Nasim and B r y c h c y (26). A c r i f l a v i n e and a c r i d i n e orange were r e p o r t e d t o i n c r e a s e s e x - l i n k e d and second chromosome r e c e s s i v e l e t h a l m u t a t i o n s i n both m a l e s and f e m a l e s o f D. melanagaster. I n the s i l k w o r m , Bombyx m o r l (L.), a c r i d i n e orange produced mutagenic e f f e c t s i n e g g - c o l o r l o c i i n f e m a l e but not male pupae. However, mutagenic e f f e c t s were observed w i t h p a r e n t a l chromosomes i n m i t o t i c cleavage n u c l e i . B l a n c h i e t a l . (27) s t u d i e d t h e e f f e c t s o f methylene blue on f i x e d e u k a r i o t i c chromosomes of the m o s q u i t o C u l i s e t a l o n g i a r e o l a t a under a e r o b i c c o n d i t i o n s w i t h v i s i b l e l i g h t i r r a d i a t i o n . They found t h a t d i l u t e d s o l u t i o n s of the dye d r a m a t i c a l l y a l t e r e d chromosomal s t r u c t u r e . R e s u l t s o f t h i s s t u d y suggested t h a t e l e c t r o n i c a l l y e x c i t e d Op, a s p e c i f i c p r o d u c t of the i n t e r a c t i o n among v i s i b l e l i g h t , methylene b l u e , and 0 , may be r e s p o n s i b l e f o r chromosomal DNA a l t e r a t i o n . S i m i l a r c o n c l u s i o n s were drawn by G rue ne r and Lockwood (28) i n a study on photodynamic m u t a g e n i c i t y o f r o s e bengal i n Chinese h a m s t e r embryo c e l l s . Commercial rhodamine 6G and rhodamine Β have been shown t o induce r e v e r s i o n m u t a t i o n s i n S a l m o n e l l a and s i n g l e - s t r a n d b r e a k s i n Chinese ham s t e r ovary c e l l s (29). However, a n o c h l o r 1254-induced r a t l i v e r homogenate (S9) i s r e q u i r e d f o r p r o d u c t i o n of g e n e t i c a c t i v i t y by these dyes. The photomutagenic e f f e c t s o f c h l o r promazine i n


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S a l m o n e l l a and Chinese h a m s t e r ovary c e l l s were s t u d i e d by Ben-Hur e t a l . (30). They found a pH r e l a t e d e f f e c t w h i c h f a c i l i t a t e d b i n d i n g o f t h i s p h o t o t o x i n t o DNA, RNA, and p r o t e i n s w i t h i n t h e c e l l s t h a t enhanced p h o t o t o x i c i t y and m u t a g e n i c i t y . Plant-derived f u r a n o q u i n o l i n e s and c e r t a i n t r y p t o p h a n - d e r i v e d a l k a l o i d s were shown t o i n h i b i t m i t o s i s and t o cause chromosomal a b e r r a t i o n s i n m i c r o o r g a n i s m s ( b a c t e r i a , f u n g i ) and i n Chinese h a m s t e r ovary cells (υ).


E f f e c t s a t the C e l l u l a r L e v e l B e s i d e s some of the s u b t l e t o the more o b v i o u s , d r a m a t i c e f f e c t s p h o t o d y n a m i c a l l y a c t i v e compounds have on c e l l u l a r components, s e v e r a l p h o t o s e n s i t i z e r s can cause c o m p l e t e d e s t r u c t i o n o f c e l l s . D e s t r u c t i o n most l i k e l y b e g i n s w i t h the a l t e r e d p e r m e a b i l i t y of the c e l l membrane w h i c h i n t u r n a l l o w s i n t r a c e l l u l a r pertubations t o occur which u l t i m a t e l y r e s u l t s i n the demise of the e n t i r e c e l l (e.g. r e l e a s e of l y s o s o m a l m a t e r i a l t o the c y t o p l a s m w i t h subsequent c e l l l y s i n g and c o m p l e t e d e s t r u c t i o n ) . A d d i t i o n a l l y , the c e l l u l a r s i t e of damage and/or mode of damage i s a p p a r e n t l y dependent on the s e n s i t i z e r and i t s localization. There a r e a number of r e p o r t s on t h e e f f e c t s o f p h o t o s e n s i t i z e r s on whole c e l l s o f o r g a n i s m s o t h e r t h a n i n s e c t s (see r e v i e w a r t i c l e s ) . Blum (_1), i n h i s r e v i e w of photodynamic a c t i o n , c i t e s f i n d i n g s by v a r i o u s w o r k e r s on e f f e c t s of p h o t o t o x i n s on mammalian blood c e l l s . Under l i g h t c o n d i t i o n s , e r y t h r o c y t e s were hemolyzed and reduced i n number by s e v e r a l xanthene dyes. H e m o l y s i s a l s o was observed i n t h e absence of i r r a d i a t i o n by h i g h c o n c e n t r a t i o n s o f rose bengal. More complex changes have been n o t e d i n t o t a l l e u c o c y t e c o u n t s under i n v i t r o i r r a d i a t i o n ; numbers may g r a d u a l l y i n c r e a s e f r o m a normal l e v e l t o a c o n d i t i o n o f l e u c o c y t o s i s f o l l o w e d by l e u c o p e n i a . The r e v i e w by P o o l e r and Valenzeno (14) d i s c u s s e s photodyna mic i n a c t i v a t i o n of e r y t h r o c y t e s . Membrane p h o t o m o d i f i c a t i o n has been e x t e n s i v e l y s t u d i e d i n t h e s e c e l l s . I n t h e presence of an a p p r o p r i a t e s e n s i t i z e r and l i g h t , t h e r e i s p r o g r e s s i v e c e l l s w e l l i n g e v e n t u a l l y c u l m i n a t i n g i n l y s i s w i t h r e l e a s e of c e l l c o n t e n t s ; the s w e l l i n g and l y s i s a r e s a i d t o be of a c o l l o i d osmotic nature. No s i m i l a r o b s e r v a t i o n s have been made on i n s e c t hemocytes. G i v e n the m o r p h o l o g i c a l and presumed f u n c t i o n a l d i v e r s i t i e s o f hemocytes found f r e e i n the hemolymph and a s s o c i a t e d w i t h h e m o p o i e t i c t i s s u e o r organs, some i n t e r e s t i n g o b s e r v a t i o n s a w a i t r e s e a r c h e r s who undertake s t u d i e s o f the e f f e c t s photodyna mic a c t i o n have on these c e l l s i n i n s e c t s . I n t h e o n l y known s t u d y of the e f f e c t s o f p h o t o s e n s i t i z e r s on i n s e c t hemocytes, Weaver e t a l . (32) showed t h a t e r y t h r o s i n e Β s i g n i f i c a n t l y a f f e c t e d t h e a g g r e g a t e o f hemocytes i n the A m e r i c a n c o c k r o a c h ( P e r i p l a n e t a a m e r i c a n a (L.)). L i g h t - e x p o s e d , dyei n j e c t e d roaches showed d i m i n i s h i n g numbers o f hemocytes a t dosages f r o m 0.068 and 0.244 mg of dye/g of body w e i g h t , r e s u l t i n g i n r e d u c t i o n s o f 11 and 40$, r e s p e c t i v e l y . L i g h t - e x p o s e d , d y e - f e d r o a c h e s a l s o tended t o have f e w e r hemocytes than u n t r e a t e d controls. Roaches h e l d i n d a r k n e s s and e i t h e r d y e - f e d o r dye-



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i n j e c t e d tended t o have h i g h e r l e v e l s o f hemocytes i n a l l cases e x c e p t a t the h i g h e s t i n j e c t e d dose of 0.244 mg; a t t h i s dose, t h e r e was a s i g n i f i c a n t r e d u c t i o n o f n e a r l y 25$. The reason(s) f o r i n c r e a s e d l e v e l s i n roaches not i r r a d i a t e d a r e not known and s i m i l a r phenomena have not been noted i n mammalian systems. I t was suggested t h a t the dye a l o n e may have a f f e c t e d the a d h e s i o n o f c e l l s (e.g. c y s t o o y t e s ) t o t i s s u e , t h u s d r i v i n g n o r m a l l y none!roul a t i n g hemocytes i n t o c i r c u l a t i o n . T h i s study d i d not d e t e r m i n e i f l y s i s was t h e cause of the decreased number of hemocytes observed. The p o s s i b i l i t y was mentioned t h a t the d y e / l i g h t t r e a t ment may have caused an I n c r e a s e i n t h e number of a d h e r i n g hemoc y t e s r a t h e r than c a u s i n g l y s i s . When c o n s i d e r i n g the a g g r e g a t e o f c e l l s and t h e changes noted, the dye a p p a r e n t l y induced an i n i t i a l c o n d i t i o n o f l e u c o c y t o s i s t h a t progressed t o a s t a t e o f l e u c o p e n i a w i t h i n c r e a s e d dosage upon i r r a d i a t i o n ; s i m i l a r o b s e r v a t i o n s have been noted i n mammalian systems. One of the more common e f f e c t s o f p h o t o t o x i n s on i n s e c t c e l l s t h a t has been observed i s t h a t o f l y s i n g . Yoho (33) presented e v i d e n c e of l y s i n g o f midgut e p i t h e l i a l c e l l s i n t h e house f l y . S c h i l d m a c h e r (34) (as c i t e d by R e s p l c i o and H e i t z ( 3 5 ) ) observed c o n s i d e r a b l e d e s t r u c t i o n o f the midgut w a l l i n m o s q u i t o l a r v a e t r e a t e d w i t h a c r i d i n e r e d . C a r p e n t e r and H e i t z (36). i n t h e i r study on l a t e n t t o x i c i t y of rose bengal on l a r v a e of C u l e x p i p i e n s q u i n q u e f a s c i a t u s Say observed t h a t the gut t r a c t i n t r e a t e d l a r v a e a p p e a r e d destroyed. The c o a g u l a t i o n o f i n s e c t hemolymph appears t o be a f f e c t e d by p h o t o s e n s i t i z e r s . I n t h e i r study on hemocytes, Weaver e t a l . (32) observed t h a t g e l a t i o n o f the plasma was o f t e n a d v e r s e l y a f f e c t e d i n e r y t h r o s i n B - t r e a t e d roaches ( u n p u b l i s h e d r e s u l t s ) . Treated roaches b l e d more f r e e l y w i t h l e s s c o a g u l a t i o n t h a n d i d u n t r e a t e d c o n t r o l s . T h i s c o u l d be a d i r e c t r e s u l t of d i m i n i s h e d numbers o f c y s t o o y t e s i n hemolymph; c y s t o o y t e s have been shown t o be a s s o c i a t e d w i t h c o a g u l a b i l i t y o f the b l o o d i n P e r l p l a n e t eu E f f e c t s a t the Systems L e v e l I n f l u e n c e on Components o f Body F l u i d s . B i o c h e m i c a l changes i n I n s e c t s induced by p h o t o t o x i n s have been s t u d i e d by o n l y a few workers. Broome e t a l . (37) conducted i n v i v o s t u d i e s o f the b i o c h e m i c a l changes a s s o c i a t e d w i t h the dark r e a c t i o n o f d i e t a r y rose bengal i n t h e b o l l w e e v i l . I n c l u s i o n o f rose bengal i n t h e d i e t of newly-emerged b o l l w e e v i l s f o r f o u r days decreased l e v e l s o f t o t a l l i p i d s (90$) and t o t a l p r o t e i n s (41$) when compared t o c o n t r o l s . The t o t a l amino a c i d p o o l i n c r e a s e d 3$; l y s i n e , g l y c i n e , t y r o s i n e , h i s t i d i n e , a r g i n i n e and p r o l i n e i n c r e a s e d , whereas the r e m a i n i n g amino a c i d s e i t h e r decreased o r r e m a i n e d the same. I n a r e l a t e d paper, C a l l ah am e t a l . (38). u s i n g d i e t a r y rose bengal, s t u d i e d s i m i l a r b i o c h e m i c a l parameters i n the a d u l t b o l l w e e v i l through f i v e c o n s e c u t i v e days post-emergence. I n t h e c o n t r o l s , p r o t e i n l e v e l s n e a r l y doubled a t two days post-emergence t h e n remained f a i r l y c o n s t a n t f r o m 2-5 days; t r e a t e d a d u l t s m a i n t a i n e d the same l e v e l t h r o u g h the f i v e day p e r i o d . Amino a c i d p o o l s i z e s a t f i v e days f o r t h e c o n t r o l s and t r e a t e d a d u l t s showed d e c r e a s e s o f 14 and 20$, r e s p e c t i v e l y , when compared t o c o n t r o l s


8.

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a t day 0. Enzyme a c t i v i t y of l a c t i c dehydrogenase and a c e t y l c h o l i n e s t e r a s e a l s o showed a decrease i n t r e a t e d w e e v i l s . I n b o t h s t u d i e s , the a u t h o r s suggest t h a t r o s e bengal can cause a l e t h a l energy s t r e s s on the organism. Hemolymph p r o t e i n s of e r y t h r o s i n B - t r e a t e d a d u l t A m e r i c a n c o c k r o a c h e s have been s t u d i e d by p o l y a c r y l a m i d e d i s c e l e c t r o p h o r e s i s (Weaver, J . E., West V i r g i n i a U n i v e r s i t y a t Morgantown, u n p u b l i s h e d data). P r o t e i n p a t t e r n s i n d y e - s e n s i t i z e d roaches were a l t e r e d i n p o s i t i o n and numbers and the mean c o n c e n t r a t i o n of p r o t e i n w i t h i n s p e c i f i c bands was s i g n i f i c a n t l y d i f f e r e n t f r o m u n t r e a t e d roaches. There were d i s t i n c t d i f f e r e n c e s between sexes and i r r a d i a t e d v e r s u s d a r k - t r e a t e d roaches. I n f l u e n c e on Body F l u i d s . The xanthene dyes, rose bengal and e r y t h r o s i n Β have been shown t o cause v o l u m e t r i c changes i n the hemolymph and c r o p c o n t e n t s o f d y e - s e n s i t i z e d A m e r i c a n and o r i e n t a l cockroaches ( B l a t t a o r i e n t a l i s L.) (39). D i e t a r y o r i n j e c t e d dye were both e f f e c t i v e i n p r o d u c i n g s i g n i f i c a n t changes i n hemolymph and c r o p volumes but changes were more d r a m a t i c , e s p e c i a l l y i n c r o p volumes, i n i n j e c t e d roaches. I n i r r a d i a t e d roaches, hemolymph volumes p r o g r e s s i v e l y decreased and crop volumes i n c r e a s e d o v e r t i m e up t o 63 minutes. I t was suggested i n t h i s study t h a t c e l l membrane p e r m e a b i l i t y may have been a f f e c t e d thereby c r e a t i n g a d i f f e r e n t i a l i n osmotic pressure which allowed hemocoel f l u i d s t o pass i n t o the a l i m e n t a r y c a n a l . Changes i n t h e s p e c i f i c g r a v i t y of hemolymph i n the A m e r i c a n c o c k r o a c h have been observed a f t e r p h o t o s e n s i t i z a t i o n w i t h e r y t h r o s l n B. I r r a d i a t e d , s e n s i t i z e d roaches showed i n c r e a s e s i n s p e c i f i c g r a v i t y o f 0.44 and 0.81$ a f t e r 30 and 60 m i n u t e s of l i g h t response, r e s p e c t i v e l y (Amrine, J. W. J r . , West V i r g i n i a U n i v e r s i t y a t Morgantown, u n p u b l i s h e d data). These changes c o u l d be r e l a t e d t o a l o s s o f w a t e r through the M a l p i g h i a n t u b u l e s i n t o the a l i m e n t a r y c a n a l w i t h a c o n c u r r e n t i m p a i r m e n t ( f a i l u r e ) of the w a t e r r e t r i e v a l system i n the l o w e r M a l p i g h i a n t u b u l e s and r e c t a l membrane p r e c i p i t a t e d by the photodynamic a c t i o n . I n f l u e n c e on the Nervous System. S t u d i e s on a number o f o r g a n i s m s have shown t h a t e x c i t a b l e c e l l s a r e s u s c e p t a b l e t o photomodification. Normal i m p u l s e p r o p a g a t i o n and subsequent e v e n t s t r i g g e r e d by the i m p u l s e may become b l o c k e d or the i m p u l s e d i s t o r t e d i n complex ways I n s e n s i t i z e d c e l l s depending on t h e l i g h t dose. P o o l e r and Valenzeno (14) p r o v i d e a good r e v i e w o f photodynamic i n a c t i v a t i o n o f e x c i t a b l e c e l l s i n nerve axons, s k e l e t a l m u s c l e , c a r d i a c and smooth muscle i n v a r i o u s o r g a n i s m s o t h e r than i n s e c t s (see a l s o p r o c e e d i n g c h a p t e r ) . Kondo and K a s a i (40) s t u d i e d the p h o t o i n a c t i v a t i o n o f s a r c o p l a s m i c r e t i c u l u m v e s i c l e membranes o f r a b b i t by s e v e r a l xanthene dyes ( e r y t h r o s l n B, e o s i n Y, rhodamine B, methylene b l u e , r o s e bengal). They observed t h a t some r e g u l a r r e l a t i o n s h i p s e x i s t between t h e m o l e c u l a r s t r u c t u r e s of xanthene dyes and the i n a c t i v a t i o n o f these e x c i t a b l e c e l l s . Food, drug and c o s m e t i c dyes o f the xanthene t y p e have been shown under dark c o n d i t i o n s t o a c t i n a dose-dependent manner when a p p l i e d t o i s o l a t e d m o i l us can g a n g l i a ; t h e s e dyes a l t e r the p o t a s s i u m p e r m e a b i l i t y of the membrane


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t h e r e b y i n c r e a s i n g t h e r e s t i n g membrane p o t e n t i a l and conductance of the neurons (41). A l a t e r study by A u g u s t i n e and L e v i t o n (42) showed t h a t l i g h t i n t e n s i t y was a l s o a f a c t o r i n t h e degree of a c t i v i t y observed w i t h e r y t h r o s l n Β on t h e p r e s y n a p t i c e f f e c t o f t h i s xanthene dye a t the f r o g n e u r o m u s c u l a r j u n c t i o n . I n a study on the s y n a p t i c c o n n e c t i v i t y between a u d i t o r y i n t e r n e u r o n s of the c r i c k e t , G r y l l u s b l m a c u l a t u s DeGeer, S e l v e r s t o n e t a l . (43) used the i n t r a c e l l u l a r dye, l u c i f e r y e l l o w , t o s e l e c t i v e l y p h o t o i n a c t i v a t e s i n g l e neurons i n p r o t h o r a c i c g a n g l i o n . Various b e h a v i o r i a l responses observed i n i n s e c t s subjected t o p h o t o r e a c t i v e s u b s t a n c e s s t r o n g l y suggest t h a t xanthene dyes, e s p e c i a l l y , can s e r i o u s l y a f f e c t the nervous system. Yoho e t a l . (44) o b s e r v e d t h a t s e n s i t i z e d house f l i e s a f t e r a dark p e r i o d were i n i t i a l l y v e r y a c t i v e when f i r s t exposed t o l i g h t . Periods of h y p e r a c t i v i t y , c h a r a c t e r i z e d by s p o r a d i c b u r s t s o f f l y i n g and p r o l o n g e d a n t e n n a l and w i n g - c l e a n i n g movements, were f o l l o w e d by p e r i o d s o f quiescence. P r o l o n g e d movement of the l a b e l l u m was a s s o c i a t e d w i t h r e g u r g i t a t i o n and d e f e c a t i o n . Locomotory movements became u n c o o r d i n a t e d as f l i e s l o s t f u n c t i o n a l c o n t r o l of l e g s ; s i m u l t a n e o u s l y , o v i p o s i t o r s of f e m a l e s were o f t e n o s b e r v e d i n an extended p o s i t i o n . F l i e s became p r o g r e s s i v e l y u n c o o r d i n a t e d , o f t e n f a l l i n g o n t o t h e i r s i d e or dorsum. Many f l i e s d i e d w i t h l e g s f o l d e d v e n t r a i l y o v e r the t h o r a x ; o t h e r s d i e d i n a n o r m a l u p r i g h t p o s i t i o n . Broome e t a l . (45) noted t h a t i m p o r t e d f i r e a n t s s e n s i t i z e d w i t h rose bengal e x h i b i t e d i n c r e a s e d i r r i t a b i l i t y , i n c r e a s e d a n t e n n a l grooming, l o s s o f l o c o m o t o r y c o o r d i n a t i o n , f o l l o w e d by a t e t a n i c p a r a l y s i s p r i o r t o death. The a n t s q u i t e o f t e n assumed "a c o n t o r t e d p o s i t i o n a t death c h a r a c t e r i z e d by p o s i t i o n i n g t h e abdomen under the t h o r a x w i t h t h e c e p h a l i c r e g i o n drawn down." Other E f f e c t s D e v e l o p m e n t a l Aspects. I n a r e v i e w by Barbosa and P e t e r s (46), s e v e r a l r e p o r t s ( p r i o r t o 1971) a r e n o t e d w h i c h i n c l u d e d o b s e r v a t i o n s on adverse e f f e c t s o f p h o t o a c t i v e s u b s t a n c e s on i n s e c t development. G r o w t h - r a t e r e t a r d a t i o n appears t o be one o f t h e more commonly observed e f f e c t s . P r o l o n g e d p e r i o d s o f l a r v a l development and u n d e r s i z e d pupae have been r e p o r t e d . Some w o r k e r s s p e c u l a t e d i n t h e s e e a r l y s t u d i e s , and more r e c e n t l y (37-38), t h a t p h o t o t o x i n s i n h i b i t maximum u t i l i z a t i o n of energy s o u r c e s or cause l e t h a l energy s t r e s s e s i n t h e organism. I n more r e c e n t s t u d i e s , decreased body w e i g h t s of I n s e c t s s e n s i t i z e d w i t h s u b s t i t u t e d xanthene dyes have been documented. C o r r e l a t i o n s between the decrease i n body w e i g h t i n the b o l l w e e v i l and e f f i c i e n c y of dyes ( i n c r e a s i n g h a l o g e n a t i o n ) were r e p o r t e d by C a l l a h a m e t a l . (47) as rose b e n g a l > p h l o x i n Β > e r y t h r o s l n Β > e o s i n Y. F u r t h e r d o c u m e n t a t i o n o f rose bengal a f f e c t i n g body w e i g h t i n the b o l l w e e v i l was made by Callaham e t a l . (38); u n t r e a t e d i n s e c t s showed w e i g h t g a i n s o f 30$ w h i l e w e i g h t s of t r e a t e d w e e v i l s r e m a i n e d e s s e n t i a l l y the same. G r o w t h i n h i b i t i o n i n t h e house f l y t r e a t e d w i t h rose bengal and e r y t h r o s l n Β has been i n v e s t i g a t e d (48); l a r v a e r e a r e d on t r e a t e d medium showed i n h i b i t i o n o f p u p a t i o n and decreased p u p a l w e i g h t s .



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Clement e t a l . (49) examined t h e e f f e c t o f rose b e n g a l on development o f l a r v a e o f t h e b l a c k cutworm ( A g r o t i s I p s i l o n (Hufnagel)). U s i n g t h e number o f f e c a l p e l l e t s produced, they found t h a t t r e a t e d l a r v a e produced s i g n i f i c a n t l y f e w e r p e l l e t s t h a n c o n t r o l s . Retarded l a r v a l g r o w t h observed i n t h i s study may have been t h e r e s u l t o f t e m p o r a r y i n h i b i t i o n o f i n g e s t i o n . I n s e c t g r o w t h r e g u l a t o r s (IGRs) a r e c u r r e n t l y a new t e c h n o l o g y b e i n g developed f o r i n s e c t c o n t r o l . There i s c o n s i d e r a b l e evidence t h a t some p h o t o t o x i n s a f f e c t i n s e c t s s i m i l a r l y . Barbosa and P e t e r s (46) i n t h e i r r e v i e w m e n t i o n d e s c r i p t i o n s o f developmental a b n o r m a l i t i e s of s e n s i t i z e d i n s e c t s r e s e m b l i n g those t h a t more r e c e n t l y have been n o t e d i n I G R - t r e a t e d i n s e c t s . N e u t r a l red was r e p o r t e d t o cause 80$ m o r t a l i t y i n e l a t e r i d l a r v a e a s t h e m o u l t i n g phase began (50). Morphological a b b e r r a t i o n s were noted i n l a r v a e o f t h e b u t t e r f l y C o l l a s eurytheme (Bols.) when f e d d i e t s o f n e u t r a l r e d . In a related s p e c i e s (Ç. p h i l o d i c e (L.)), n e u t r a l r e d caused p u p a l deformations. B r i d g e s e t a l . (51) t e s t e d a new f l u o r e s c e n t compound a g a i n s t l a r v a e o f Aedes a e g y p t i (L.) and found i t produced morphogenetic e f f e c t s s i m i l a r t o methoprene. L a r v a e f a i l e d t o c o m p l e t e t h e m o u l t i n g p r o c e s s and l a r v a l - p u p a l i n t e r m e d i a t e s were formed. Specimens t h a t formed a p p a r e n t l y normal pupae o f t e n d i e d b e f o r e t h e a d u l t emerged o r d i e d o n l y p a r t i a l l y emerged. A d u l t males t h a t emerged n o r m a l l y d i d n o t complete g e n i t a l i a r o t a t i o n . 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 i n l a r v a e and pupae f r o m rose b e n g a l t r e a t e d m o s q u i t o l a r v a e have been s u g g e s t e d t o r e s u l t f r o m i m p r o p e r f o r m a t i o n o f c h i t i n (52) o r f r o m a dye-induced energy s t r e s s on t h e i n s e c t (36). The IGR e f f e c t has been observed i n pupae o f t h e f a c e f l y when l a r v a e were t r e a t e d w i t h rose bengal and e r y t h r o s l n Β (53). Most f l i e s d i e d i n t h e p u p a l s t a g e as t h e a d u l t a t t e m p t e d t o emerge f r o m t h e puparium; some m o r p h o l o g i c a l l y normal f l i e s emerging f r o m e r y t h r o s l n B - t r e a t e d manure had s h o r t e r l i f e spans t h a n t h e c o n t r o l s . Downum e t a l . (54), i n a study on t h e p h o t o t o x i c e f f e c t s o f a l p h a - t e r t h i e n y l on t h e t o b a c c o hornworm (Manduca s e x t a (L.)), noted d r a s t i c d e v e l o p m e n t a l a l t e r a t i o n s i n t r e a t e d l a r v a e . Dietary alpha-terthienyl, with i r r a d i a t i o n of larvae a f t e r i n g e s t i o n , r e s u l t e d i n d e l a y e d and abnormal pupal f o r m a t i o n w i t h no subsequent a d u l t emergence; a d d i t i o n a l l y , l a r v a l g r o w t h was d e l a y e d f o r f o u r days a f t e r t r e a t m e n t when l a r v a e r e f u s e d d i e t . Pronounced t i s s u e n e c r o s i s was observed a t a p p l i c a t i o n s i t e s o f t o p i c a l l y t r e a t e d , i r r a d i a t e d l a r v a e ; a t p u p a t i o n , normal s c l e r o t i z a t i o n and i n d e n i z a t i o n were a f f e c t e d i n v a r i o u s a r e a s o f t h e p u p a l case. R e p r o d u c t i v e Aspects. There a r e a p p a r e n t l y o n l y a few r e p o r t s of the adverse e f f e c t s o f photodynamically a c t i v e s u b s t a n c e s on t h e r e p r o d u c t i v e p o t e n t i a l of i n s e c t s . I n an e a r l y r e p o r t , David (55) s p e c u l a t e d t h a t methylene b l u e was "somehow a f f e c t i n g gametogenesis" o f D r o s o p h l l a i n a study o f t h e e f f e c t s o f t h i s dye on s u c c e s s i v e g e n e r a t i o n s . I n a l a t e r study, David (56) observed t h a t methylene b l u e caused a marked decrease by a f a c t o r o f f o u r i n the f e c u n d i t y o f t r e a t e d Drosophlla females. More r e c e n t l y , P i m p r i k a r e t a l . (57) r e p o r t e d t h a t f e c u n d i t y



130


i n t h e house f l y was d i r e c t l y r e l a t e d t o t h e d i e t a r y c o n c e n t r a t i o n o f rose bengal and t h e frequency of dye f e e d i n g ; up t o 10% reduc t i o n i n f e c u n d i t y was observed i n f e m a l e s m a i n t a i n e d on a c o n t i n u o u s d i e t f o r 15 days. As noted i n t h e p r e v i o u s s e c t i o n , s e v e r a l s t u d i e s have shown t h a t pupal and a d u l t w e i g h t s o f some i n s e c t s are decreased by t r e a t m e n t s w i t h xanthene dyes. However, no o b s e r v a t i o n s were made i n these s t u d i e s on how these developmental a b n o r m a l i t i e s might a f f e c t reproduction. Since i t i s known f o r some I n s e c t s p e c i e s t h a t a c o r r e l a t i o n e x i s t s between p u p a l w e i g h t and number of eggs produced by f e m a l e s , i t seems r e a s o n a b l e t o assume t h a t s e n s i t i z e d i n s e c t s p r o d u c i n g a b n o r m a l l y s m a l l e r pupae o r a d u l t s w h i c h f a i l t o show normal w e i g h t g a i n s may not be capable of p r o d u c i n g a normal complement of eggs. O v i c i d a l p r o p e r t i e s of p h o t o t o x i n s have not been s t u d i e d e x t e n s i v e l y . P i m p r i k a r e t a l . (57) i n v e s t i g a t e d the o v i c i d a l a c t i v i t y of s i x xanthene d e r i v a t i v e s a g a i n s t the house f l y . Rose bengal and e r y t h r o s l n Β were most a c t i v e c a u s i n g n e a r l y 30$ inhi b i t i o n of egg h a t c h w h i l e rhodamine Β and e o s i n Y were the l e a s t a c t i v e c a u s i n g about 15$ i n h i b i t i o n each. T y p i c a l t o x i c symptoms observed by the a u t h o r s were: some eggs f a i l e d t o h a t c h (presumably due t o embryo death p r i o r t o hatch); some l a r v a e e c l o s e d f r o m a l o n g i t u d i n a l l i n e o f weakness a t the me s a l dorsum of the c h o r i o n ; some l a r v a e f r e e d t h e head c a p s u l e , but were u n a b l e t o f r e e the c a u d a l end from the c h o r i o n . Kagan and Chan (58) s t u d i e d t h e p h o t o o v i c i d a l a c t i v i t y of t h r e e n a t u r a l l y o c c u r r i n g m o l e c u l e s a g a i n s t D. melanogaster. P h e n y l h e p t a t r i y n e , a l p h a - t e r t h i e n y l , and 8-methoxypsoralan a l l prevented egg h a t c h . The f i r s t two were t o x i c t o eggs i n the dark, but upon i r r a d i a t i o n , e f f e c t i v e n e s s was I n c r e a s e d 37 and 4,333-fold, r e s p e c t i v e l y . Summary and Remarks I n g e n e r a l , we know f a r l e s s about t h e p h y s i o l o g i c a l e f f e c t s o f photodynamic a c t i o n on i n s e c t s t h a n we do about t h i s phenomenon as i t a f f e c t s t h e p h y s i o l o g y o f o t h e r i n v e r t e b r a t e s and mammals. T h i s i s e s p e c i a l l y t r u e a t the s u b c e l l u l a r and c e l l u l a r l e v e l s . At t h e systems l e v e l , we have gained c o n s i d e r a b l e knowledge d u r i n g the l a s t decade of how p h o t o t o x i n s can m o d i f y the b i o c h e m i c a l processes and p h y s i o l o g y of i n s e c t s . But s t i l l , o n l y a l i m i t e d number of s t u d i e s have been done a t t h i s l e v e l , p a r t i c u l a r l y w i t h t h e nervous system; a t t h i s p o i n t i n t i m e we can o n l y s p e c u l a t e from abnormal b e h a v i o r a l p a t t e r n s observed i n s e n s i t i z e d i n s e c t s t h a t p e r t u b a t i o n s a r e o c c u r r i n g i n t h e nervous system. There i s s t r o n g e v i d e n c e f r o m r e c e n t s t u d i e s t h a t some pho t o a c t i v e s u b s t a n c e s can m o d i f y the p h y s i o l o g i c a l processes i n i n s e c t s i n much the same manner as IGRs. There appears t o be a need f o r f u r t h e r study on how the p h o t o t o x i n s a f f e c t t h e r e p r o d u c t i v e p o t e n t i a l of i n s e c t s ; many IGRs have been shown t o reduce f e c u n d i t y and induce s t e r i l i t y , but o n l y a c o u p l e o f r e c e n t s t u d i e s have d e a l t w i t h t h i s a s p e c t i n any d e t a i l u s i n g the more e f f e c t i v e p h o t o t o x i n s . I n s t u d i e s where pupal and a d u l t w e i g h t s


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were observed t o be a l t e r e d I n s e n s i t i z e d i n s e c t s , t h e r e was no f o l l o w u p a s t o what e f f e c t these a b n o r m a l i t i e s may have on r e p r o ductive ability. I t has been suggested t h a t the photodynamic a c t i o n mechanism may not be a v i a b l e o p t i o n f o r i n s e c t c o n t r o l (15). Recognizing t h a t a l l the easy c h e m i s t r y on i n s e c t i c i d e s has been done, i s n ' t i t time t o explore phototoxins as an a l t e r n a t i v e t o , o r p o s s i b l e use i n a n a d j u n c t i v e r o l e , t o the more c o n v e n t i o n a l i n s e c t i c i d e s ? Whatever some may t h i n k about i n v e s t i g a t i n g " i n s i d i o u s and l i t t l e understood mechanisms (of p h o t o t o x i n s ) t o rescue ... a b o r d e r l i n e t e c h n o l o g y " (15). i t s h o u l d r e m a i n a c h a l l e n g e t o r e s e a r c h e r s t o d e v e l o p t h a t t e c h n o l o g y t o combat I n s e c t p e s t s . The more we understand about the modes o f a c t i o n o f p h o t o d y n a m i c a l l y a c t i v e s u b s t a n c e s , t h e more i n t e l l i g e n t l y we w i l l be a b l e t o use them t o our b e n e f i t .

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RECEIVED November 20, 1986


Physiological Effects of Photodynamic Action: Special Reference to

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