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water partition coefficients
Chemosphere,Vol.26,No.5,pp 1015-1028,1993 Printedin GreatBritain
0045-6535/93$6.00+ 0.00 PergamonPressLtd.
CYTOTOXICITY OF 109 C H E M I C A L S TO G O L D F I S H GFS CELLS AND R E L A T I O N S H I P S WITH 1 - O C T A N O L / W A T E R PARTITION COEFFICIENTS
H o t a k a Saito* , Junko K o y a s u , Kikuo Y o s h i d a , T a d a y o s h i Shigeoka and Sakae Koike * Mitsubishi-kasei I n s t i t u t e of T o x i c o l o g i c a l and E n v i r o n m e n t a l 1000, K a m o s h i d a - c h o , M i d o r i - k u , Y o k o h a m a 227, Japan
Sciences
(Received in Japan 11 October 1992; accepted 27 November 1992)
ABSTRACT T h e m i d p o i n t c y t o t o x i c i t y (NR50 v a l u e s ) of 109 c h e m i c a l s i n c l u d i n g a l c o h o l s , a r o m a t i c s , p h e n o l s a n d p e s t i c i d e s to g o l d f i s h s c a l e G F S c e l l s were compared with the fish acute toxicities a n d w i t h t h e 1octanol/water partition coefficient (Pow). The NR50 values were significantly correlated to t h e a v a i l a b l e acute toxicity data for f a t h e a d m i n n o w (n=31, r=0.958, s=0.480, F=325) a n d g u p p y (n=29, r=0.957, s=0.482, F=294). The P o w gave a g o o d c o r r e l a t i o n in s i m p l e l i n e a r r e g r e s s i o n a n a l y s i s f o r a l l c o m p o u n d s e x c l u d i n g 8 c o m p o u n d s t h a t the NR50 v a l u e s c o u l d n o t be o b t a i n e d f o r t h e i r low s o l u b i l i t y in c u l t u r e m e d i u m . Log 1/NR50 (mM) = (0.87-+0.08) L o g P o w - ( 2 . 1 7 - + 0 . 2 4 ) (n=101, r = 0 . 9 1 4 , s=0.587, F=505) F o r 29 n a r c o t i c t y p e of c o m p o u n d s , the r e g r e s s i o n a n a l y s i s r e v e a l e d a h i g h c o r r e l a t i o n b e t w e e n the NR50 v a l u e s a n d Pow, a n d a p p e a r e d to give a baseline cytotoxicity. Log 1/NR50 (mM) =(0.77 __.0.06) L o g P o w - (2.48--+0.12) ( n = 2 9 , r = 0 . 9 8 4 , s=0.252, F=817) A n d a l s o , the r e g r e s s i o n a n a l y s e s b e t w e e n NR50 a n d P o w f o r s o m e t y p e s of c o m p o u n d s were d i s c u s s e d . T h e s e r e s u l t s s u g g e s t t h a t N R a s s a y is u s e f u l f o r p r e d i c t i o n of a c u t e t o x i c i t y to f i s h a n d s t u d i e s of q u a n t i t a t i v e structure-activity relationships (QSARs).
1015
1016 INTRODUCTION In vitro cytotoxicity assays using cultured fish cells have r e c e n t l y been developed for a simple, rapid, r e p r o d u c i b l e and economical generation of t o x i c o l o g i c a l d a t a in a q u a t i c toxicology, and for p r e d i c t i n g t h e a c u t e t o x i c i t y of a q u a t i c p o l l u t a n t s to f i s h [ 1 - 3 ] . A n d t h e s e m e t h o d s a r e c o n s i d e r e d to be u s e f u l f o r d e t e c t i n g p o l l u t i o n in the environment, for evaluating synergetic and antagonistic interactions b e t w e e n c o m b i n a t i o n s of c h e m i c a l s , a n d f o r g e n e r a t i n g c o m p u t e r - d e r i v e d predictions b a s e d on q u a n t i t a t i v e structure-activity relationships (QSARs). The n e u t r a l red a s s a y (NR a s s a y ) has i n i t i a l l y been developed for use w i t h m a m m a l i a n c e l l s [4] a n d a d a p t e d f o r c y t o t o x i c i t y s t u d i e s w i t h fish cells [5-9]. This technique has been successfully a p p l i e d to investigate t h e e f f e c t s of 15 c h l o r o p h e n o l s [2] a n d 45 p e s t i c i d e s [3] on G F S c e l l s , d e r i v e d f r o m the s c a l e of g o l d f i s h , a n d t h e i r NR50 v a l u e s were significantly c o r r e l a t e d to in vivo acute toxicity t o 13 k i n d s of a q u a t i c s p e c i e s . In t h i s s t u d y , the NR50 v a l u e s of 49 c h e m i c a l s except for the previous s t u d y d a t a f o r 15 c h l o r o p h e n o l s a n d 45 p e s t i c i d e s , were d e t e r m i n e d , a n d the NR50 v a l u e s were c o m p a r e d w i t h the d i f f e r e n t s o u r c e s of LC50 v a l u e s f o r f a t h e a d m i n n o w [ 1 0 - 1 2 ] a n d f o r guppy [13-18]. 1-octanol/water p a r t i t i o n c o e f f i c i e n t ( P o w ) a r e o f t e n e m p l o y e d in Q S A R a n a l y s i s a n d a r e s u p p o s e d to t a k e i n t o a c c o u n t t h e e f f e c t s of the transport through the membranes toward the receptor. Our previous r e s u l t s on 15 c h l o r o p h e n o l s indicate that the NR50 values are well correlated w i t h l o g P o w . T h e P o w d a t a e x c e p t f o r 45 p e s t i c i d e s w e r e o b t a i n e d f r o m t h e p u b l i s h e d d a t a . In t h i s s t u d y , P o w f o r 45 p e s t i c i d e s w e r e d e t e r m i n e d b y t h e m e t h o d b a s e d on r e v e r s e p h a s e - h i g h pressure l i q u i d c h r o m a t o g r a p h y ( R P - H P L C ) [ 1 9 ] . A n d all Pow d a t a were c o m p a r e d w i t h a s e r i e s of c y t o t o x i c i t y d a t a (NR50 v a l u e s ) .
MATERIALS Test chemicals
AND
METHODS
and solutions
T h e 109 c h e m i c a l s a r e l i s t e d in T a b l e 1 w i t h t h e i r l o g P o w . T h e c h e m i c a l s e x c e p t f o r t h e c o m p o u n d s d e s c r i b e d in p r e v i o u s s t u d y [ 2 , 3 ] , w e r e p u r c h a s e d f r o m T o k y o K a s e i K o g y o Co., T o k y o , J a p a n a n d w e r e all reagent-grade and used without further purification. Either dimethyl sulfoxide ( D M S O ) o r a c e t o n e w a s u s e d a s t h e v e h i c l e to d i s s o l v e h y d r o p h o b i c c o m p o u n d s at a c o n c e n t r a t i o n of l e s s t h a n 0.5 % ( v / v ) w h i c h e x h i b i t e d no o b s e r v a b l e e f f e c t on t h e c e l l v i a b i l i t y .
1017
Cell l i n e s The e s t a b l i s h e d cell line, GFS ( G F - S c a l e , RCB No.082), was p u r c h a s e d from the Riken Gene Bank, Ibaraki, Japan. The GFS cells have a fibroblast-like morphology a n d w e r e d e r i v e d f r o m s c a l e t i s s u e of goldfish (C. a u r a t u s ) . T h e c e l l s w e r e m a i n t a i n e d as monolayer c u l t u r e s in c u l t u r e f l a s k s a t 25°C as d e s c r i b e d in p r e v i o u s s t u d y [ 3 ] . The m e d i u m w a s L e i b o v i t z ' s L - 1 5 , s u p p l e m e n t e d w i t h 10 % f e t a l b o v i n e s e r u m ( F B S ) , 100 u n i t s / m l p e n i c i l l i n G, 100 /.z g / m l s t r e p t o m y c i n , 1.25 g/ml amphotericin B a n d 10 m M N - 2 - h y d r o x y e t h y l p i p e r a z i n e - N ' - 2 e t h a n e s u l f o n i c a c i d ( H E P E S ) to m a i n t a i n the pH at 7.4. F o r p r o p a g a t i o n , t h e c e l l s w e r e d i s s o c i a t e d w i t h a s o l u t i o n c o n s i s t i n g of 0.05 % t r y p s i n a n d 0.02 % e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d ( E D T A ) . A l l c u l t u r e m e d i a a n d r e a g e n t s w e r e p u r c h a s e d f r o m S i g m a C h e m i c a l C o . , St. L o u i s , MO, U.S.A.
Cytotoxicity The NR a s s a y with GFS cells was p e r f o r m e d a c c o r d i n g to the p r o c e d u r e d e s c r i b e d in p r e v i o u s s t u d y [ 3 ] . R e s u l t s w e r e e x p r e s s e d as p e r c e n t of absorbance (540 n m ) of n e u t r a l r e d e x t r a c t e d f r o m c o n t r o l c u l t u r e s . D a t a w e r e p l o t t e d a s p e r c e n t of c o n t r o l v s . l o g a r i t h m i c chemical concentration in m g / L . T h e m i d p o i n t c y t o t o x i c i t y N R 5 0 v a l u e s (50 % d e c r e a s e of a b s o r b a n c e ) w e r e c a l c u l a t e d by l i n e a r r e g r e s s i o n a n a l y s i s .
1 - o c t a n o l / w a t e r partition coefficient, Pow 1 - o c t a n o l / w a t e r p a r t i t i o n c o e f f i c i e n t ( P o w ) w e r e d e t e r m i n e d by R P H P L C m e t h o d [ 1 9 ] . A l i n e a r c a l i b r a t i o n of t h e l o g a r i t h m of r e t e n t i o n time with the logarithm of t h e p a r t i t i o n coefficent (logPow) was a t t a i n e d by u s i n g a m i x t u r e of b e n z e n e , b r o m o b e n z e n e , b i p h e n y l , b i b e n z y l , p,p'-DDE and 2,4,5,2',5'-pentachlorobiphenyl as r e f e r e n c e s t a n d a r d s of known LogPow.
Statistical
analysis
The NR50 values were correlated with the published fish acute t o x i c i t y d a t a (LC50) a n d l o g P o w . Both NR50 a n d LC50 d a t a were c o n v e r t e d to i n v e r s e m i l l i m o l a r c o n c e n t r a t i o n a n d g i v e n in t h e l o g a r i t h m i c f o r m f o r r e g r e s s i o n a n a l y s i s . A l l s t a t i s t i c a l a n a l y s e s w e r e p e r f o r m e d on the s t a t i s t i c a l p a c k a g e ( M i c r o s o f t C h a r t V e r . 3 . 1 ) f o r NEC PC9801 c o m p u t e r . RESULTS
AND
DISCUSSION
Relationships between in vivo and in vitro The 24-h N R 5 0
values for the cytotoxicity of the test c o m p o u n d s
to
1018
GFS cells, together
w i t h t h e i r l o g P o w a n d LC50 d a t a f o r f a t h e a d
minnow
a n d g u p p y a r e s u m m a r i z e d in T a b l e 1. T h e l i n e a r r e l a t i o n s h i p s b e t w e e n c y t o t o x i c i t i e s ( 2 4 - h r N R 5 0 ) to G F S c e l l s a n d a c u t e t o x i c i t i e s ( L C 5 0 ) to the fathead m i n n o w a n d g u p p y a r e s h o w n i n F i g u r e 1. I n v i v o d a t a f o r t h e f a t h e a d m i n n o w a n d g u p p y w e r e a v a i l a b l e f o r 31 a n d 29 of t h e 109 c o m p o u n d s , respectively. The regression analyses of t h e s e d a t a p r o v i d e t h e f o l l o w i n g e q u a t i o n 1 an 2 ( t h e c o n f i d e n c e i n t e r v a l s of t h e regression c o e f f i c i e n t s in p a r e n t h e s e s h a v e a 95 % p r o b a b i l i t y ; n is the number of compounds ; r is t h e c o r r e l a t i o n coefficient ; s is s t a n d a r d e r r o r of r e s i d u e s ; F is t e s t of n u l l h y p o t h e s i s ) : Eqn.1 (fathead Log 1/LC50
minnow) ; = (1.10--+0.12) • Log 1/NR50-t- ( 0 . 8 1 - + 0 . 1 8 ) (n=31, r=0.958, s=0.480, F=325)
Eqn.2
;
(guppy)
Log 1/LC50
= (1.03+0.12) • Log 1/NR50+ ( 0 . 9 1 + 0 . 2 0 ) (n=29, r=0.957, s=0.482, F=294)
(,)
4
"./.
.)i.
(b)
2
2 0 -2
-2
-
4
I
-4
Fig.1
and
• ~o
I
4
I
-2 0 2 Logl/NR50, mM
4
-4
-2 0 2 Logl/NR50, mM
T h e r e l a t i o n s h i p s b e t w e e n t he c y t o t o x i c i t i e s ( l o g l / N R 5 0 ) to G F S c e l l s a n d t he a c u t e t o x i c i t i e s ( l o g l / L C 5 0 ) to f a t h e a d m i n n o w (a) and g u p p y ( b ) .
There were good correlations in vivo LC50 values. And as
between in vitro NR50 values the slopes are nearly one, the
1019
t o x i c r a n g e of t h e s e c o m p o u n d s on G F S cells a p p e a r s to be s a m e as t h o s e on f a t h e a d minnow and guppy. Furthermore, the intercepts of t h e regression lines demonstrate t h a t t h e a b s o l u t e v a l u e s of N R 5 0 a r e significantly h i g h e r t h a n L C 5 0 v a l u e s ( t h e N R a s s a y is a b o u t s e v e n t i m e s l e s s s e n s i t i v e t h a n the LC50 t e s t s f o r f a t h e a d m i n n o w a n d g u p p y ) . A s i m i l a r r e s u l t h a s b e e n n o t e d f o r t h e p r e v i o u s s t u d y w i t h r e s p e c t to the linear relationships b e t w e e n N R 5 0 v a l u e s of f i f t e e n c h l o r o p h e n o l s to G F S c e l l s a n d t h e i r a c u t e t o x i c i t i e s to 12 a q u a t i c s p e c i e s [2] a n d b e t w e e n N R 5 0 v a l u e s of 34 p e s t i c i d e s to G F S c e l l s a n d t h e i r a c u t e toxicities to c a r p [ 3 ] . T h e s e r e s u l t s s t r o n g l y s u g g e s t t h a t the NR a s s a y is u s e f u l f o r p r e l i m i n a r y e c o t o x i c o l o g i c a l s c r e e n i n g of v a r i o u s chemicals, recognizing the different sensitivities between in vitro and in vivo. Saito et a/.[3], however, described the different toxic response between in vitro and in vivo for some pesticides, e.g. carbamates. Therefore, by careful analysis f o r the c h e m i c a l s t r u c t u r e s of the o u t l i e r s , the NR a s s a y c a n be a p p l i e d to t h e p r e d i c t i o n of t h e i n v i v o r e s p o n s e .
Relationships
with
1-octanol/water
partition
coefficients
(Pow)
1. A l l c o m p o u n d s and narcotic type of c o m p o u n d s The P o w gave a g o o d c o r r e l a t i o n in s i m p l e l i n e a r r e g r e s s i o n a n a l y s i s f o r all c o m p o u n d s e x c l u d i n g 8 c o m p o u n d s t h a t the NR50 v a l u e s c o u l d not be o b t a i n e d f o r t h e i r l o w s o l u b i l i t y in c u l t u r e m e d i u m ( F i g . 2 ) . T h e r e g r e s s i o n a n a l y s i s p r o v i d e s the f o l l o w i n g e q u a t i o n 3:
Eqn.3;
Log 1/NR50 (mM) = (0.87___0.08) L o g P o w - ( 2 . 1 7 _ + 0 . 2 4 ) (n=101, r = 0 . 9 1 4 , s=0.587, F=505)
It w a s i n t e r e s t i n g t h a t a g o o d c o r r e l a t i o n b e t w e e n NR50 a n d P o w , varying in logPow from - 0 . 7 4 to 6 . 0 2 , w a s o b t a i n e d for various c h e m i c a l s w i t h a w i d e v a r i e t y of c h e m i c a l s t r u c t u r e a n d w i t h a s p e c i f i c m o d e of t o x i c a c t i o n l i k e p e s t i c i d e s . H o w e v e r , f o r 29 n a r c o t i c t y p e of c o m p o u n d s , i n c l u d i n g a l c o h o l s , a r o m a t i c s and some other c o m p o u n d s which were c l a s s i f i e d into Class 1 c o m p o u n d s as d e s c r i b e d by V e r h a a r et al. [ 1 7 ] , the re gre s sion a n a l y s i s r e v e a l e d an e x c e l l e n t c o r r e l a t i o n b e t w e e n NR50 a n d P o w s h o w i n g a n i n c r e a s e in c y t o t o x i c i t y w i t h h i g h e r v a l u e s of l o g P o w ( F i g . 2 a n d Eqn.4).
Eqn.4;
Log 1/NR50 (mM) = (0.77_+0.06) L o g P o w - ( 2 . 4 8 _ + 0 . 1 2 ) ( n = 2 9 , r = 0 . 9 8 4 , s=0.252, F=817)
1020
O
s"
.'"Eqn.3 s
o
o
• o
o
I
I
c~ e" .~:~o . /
~o
~,"
o
o
Eqn.4
1 t~
0
Z
m/'d~
-1 -2 -3 -
4
I
-2
Fig.2
-1
I
I
0
1
2 3 Log Pow
I
I
I
4
5
6
7
The relationship between logl/NR50 to G F S c e l l s a n d f o r all c o m p o u n d s . [ Onarcotic compounds; Nphenols; O pesticides; .aniline; A salicylaldehyde. ]
logPow
A n d e q u a t i o n 4 p r o b a b l y p r e d i c t s the m i n i m a l c y t o t o x i c i t y ( b a s e l i n e t o x i c i t y ) of o r g a n i c c h e m i c a l s to G F S c e l l s . K S n e m a n n [15] d e s c r i b e s t h a t t h e c o e f f i c i e n t f o r l o g P o w in g u p p y ' s b a s e l i n e t o x i c i t y l e v e l a r e 0.87. T h e l o w e r v a l u e 0.77 in e q u a t i o n 4 is p r o b a b l y r e l a t e d to t h e f a c t t h a t the N R a s s a y r e s u l t s in l o w e r s l o p e in the h i g h l o g P o w b e c a u s e of the a d s o r p t i o n of h y d r o p h o b i c c h e m i c a l s to c u l t u r e m e d i u m . As to an h o m o l o g o u s s e r i e s of c o m p o u n d s , the Q S A R e q u a t i o n s f o r 14 alcoho]s and 9 aromatics including trichlorobenzene a r e g i v e n in e q u a t i o n 5 a n d 6, r e s p e c t i v e l y . B o t h e q u a t i o n 5 a n d 6 g a v e g o o d f i t s to the d a t a ( F i g . 3 ) . Eqn.5;
Log 1/NR50 (mM) = (0.65_+0.12) L o g P o w - ( 2 . 4 1 _ + 0 . 1 2 ) (n=14, r=0.962, s=0.185, F=148)
1021
Eqn.6;
Log 1/NR50 (mM) = (0.85_+0.24) L o g P o w - ( 2 . 7 1 _ + 0 . 9 0 ) ( n = 9, r = 0 . 9 5 4 , s = 0 . 2 6 7 , F = 70)
.~"
Z
.6
¢= Eqn.6
.0*6
""¢= Eqn.5
-1
•
O
-2 -3 -
4
-2
Fig.3
I
I
I
-1
0
1
I
I
2 3 Log Pow
I
I
I
4
5
6
The relationship between logl/NR50 to G F S f o r 14 a l c o h o l s ( ( } ) and 9 a r o m a t i c s ( O ) .
cells
7
and
logPow
2. P h e n o l s
The Q S A R f o r 32 p h e n o l s i n c l u d i n g of the P o w with NR50 ( F i g . 4 ) .
Eqn.7;
naphthol
gave a good correlation
Log 1/NR50 (mM) = (0.81_+ 0.14) L o g P o w - (1.88_+ 0 . 4 2 ) (n=32, r=0.910, s=0.345, F=145)
The i n t e r c e p t v a l u e of e q u a t i o n 7 is h i g h e r t h a n t h a t of e q u a t i o n 4 for narcotic compounds. This result suggests that phenols have a higher toxicity level than narcotic compounds. A similar result has been noted for QSAR studies with fish LC50 data. Veith et al.[20], present
1022
that the fish acute toxicities of m a n y p h e n o l s a n d a n i l i n e s are underestimated by the b a s e l i n e n a r c o s i s QSAR. V e r h a a r et a / . [ 1 7 ] . d e s c r i b e that phenols are c l a s s i f i e d into "less inert c h e m i c a l s " which are not r e a c t i v e when c o n s i d e r i n g o v e r a l l fish acute e f f e c t s , but are s l i g h t l y m o r e t o x i c t h a n b a s e l i n e t o x i c i t y . In a d d i t i o n , 4 - n i t r o p h e n o l w a s m o r e c y t o t o x i c t h a n p r e d i c t i o n f r o m e q u a t i o n 7. This r e s u l t m a y be e x p l a i n e d by a d i f f e r e n c t m o d e of a c t i o n , s u c h as a s t r o n g o x i d a t i v e p h o s p h o r y l a t i o n u n c o u p l i n g model.
Eqn.7
O
ss °S
O
1
o°°
Eqn.4
¢~
s o ~ SSS
z_.
nl
o
•
~
0
o oo° oo,OC52
•
0
-1
-
so S
2
1
I
I
2
3
I
I
4
5
6
Log Pow Fig.4
The r e l a t i o n s h i p b e t w e e n ] o g l / N R 5 0 to G F S c e l l s a n d l o g P o w f o r 32 p h e n o l s . [ O phenols c o n t a i n i n g only C, H and O ; O h a l o g e n a t e d phenols ( O 1 ; 2,6-C1, 02; 2,4,6-C] phenol); Initrophenols (ll; 4-nitrophenol, .2; 2-nitrophenol, .3; 4-methyl-2-nitrophenol). ]
F o r 10 p h e n o l s c o n t a i n i n g only C, H and O, e x c l u d i n g h a l o g e n a t e d and n i t r o p h e n o l s f r o m e q u a t i o n 7, the r e g r e s s i o n a n a l y s i s y i e l d e d b e t t e r c o r r e l a t i o n c o e f f i c i e n t s and predictive c a p a b i l i t i e s (Eqn.8):
1023
Eqn.8;
Log 1/NR50 (mM) = (0.75_+0.13) L o g P o w - ( 1 . 8 2 _ + 0 . 3 2 ) (n=10, r=0.980, s=0.151, F=190)
And the QSAR for h a l o g e n a t e d phenols is given in e q u a t i o n 9.
Eqn.9;
Log 1/NR50 (mM) = (0.83_+0.21) L o g P o w - ( 1 . 9 2 _ + 0 . 7 0 ) (n=19, r=0.901, s=0.335, F= 74)
The o r t h o - s u b s t i t u t e d c h l o r o p h e n o l s such as 2 , 6 - d i c h l o r o - and 2,4,6trichlorophenols w e r e l e a s t t o x i c a m o n g the c y t o t o x i c i t i e s of t h e i s o m e r s h a v i n g the s a m e n u m b e r of c h l o r i n e a t o m s [2]. T h a t is b e c a u s e t h e t o x i c e f f e c t s of the O H - g r o u p on the c e l l s a r e i n h i b i t e d b y the i n t r a m o l e c u l a r b o n d i n g of the O H - h y d r o g e n and the two o r t h o - c h l o r i n e substituents, as w e l l as b y t h e i r s h i e l d i n g of t h e O H - g r o u p [21]. T h e r e f o r e , e x c l u d i n g b o t h 2 , 6 - d i c h l o r o - and 2 , 4 , 6 - t r i c h l o r o p h e n o l s f r o m equation 9, t h e r e g r e s s i o n analysis yielded better correlation c o e f f i c i e n t s and predictive c a p a b i l i t i e s (Eqn.10): Eqn.10;
3.
Log 1/NR50 (mM) =(0.84_+0.13) L o g P o w - (1.85_+0.45) (n=17, r=0.961, s=0.212, F=180)
Pesticides
The equation
regression 11 ( F i g . 5 ) :
Eqn.ll;
analysis
for
pesticides
provides
following
Log 1/NR50 (mM) =(0.52_+ 0.21) L o g P o w - (0.67_+ 0.77) (n=38, r=0.640, s=0.589, F= 25)
The r e g r e s s i o n a n a l y s i s f o r 18 o r g a n o p h o s p h a t e s p r o v i d e s the f o l l o w i n g e q u a t i o n 12:
Eqn.12;
the
of 38 p e s t i c i d e s
Log 1/NR50 (raM) =(0.45__.0.23) L o g P o w (0.46_+0.79) (n=18, r=0.719, s=0.343, F= 17)
Both regression analyses provided the significant correlation c o e f f i c i e n t s b e t w e e n the NR50 v a l u e s and P o w , b u t b o t h r v a l u e s w e r e p o o r e r t h a n t h o s e of e q u a t i o n s 4 t h r o u g h 10. The i n t e r c e p t v a l u e s of e q u a t i o n s 11 a n d 12 a r e h i g h e r t h a n t h o s e of e q u a t i o n s 4 t h r o u g h 10, w h i l e the s l o p e s a r e l o w e r . T h i s r e s u l t s u g g e s t s t h a t m a n y p e s t i c i d e s a r e m u c h m o r e t o x i c t h a n p r e d i c t e d s o l e l y f r o m l o g P o w d u e to t h e i r s p e c i f i c m o d e s of a c t i o n .
1024
0
o o
0
qn.ll
o ; ~
o
..-.
z_.
•
o
~'~0
00
Q O
.-*"~
Eqn.4
"*°**
***
O s ~S Bs~
-2 1
I
I
2
3
l
I
I
4
5
6
7
Log Pow Fig.5
The relationship f o r 38 p e s t i c i d e s .
between logl/NR50 to G F S c e l l s a n d [ O organophosphates, O others. ]
logPow
4. A n i l i n e a n d s a l i c y l a l d e h y d e Both a n i l i n e and s a l i c y l a l d e h y d e were more c y t o t o x i c t h a n p r e d i c t e d f r o m b a s e l i n e c y t o t o x i c i t y of e q u a t i o n 4 ( F i g . 2 ) . T h e s e c o m p o u n d s a r e n o t c l a s s i f i e d i n t o C l a s s 1 t y p e c o m p o u n d s [ 1 7 ] . A n i l i n e s as w e l l as p h e n o l s a r e c l a s s i f i e d i n t o C l a s s 2 t y p e c o m p o u n d s . I n d e e d , t h e NR50 v a l u e f o r a n i l i n e c o u l d be p r e d i c t e d f r o m e q u a t i o n s 7 t h r o u g h 10 f o r p h e n o l s . A l t h o u g h s a l i c y l a l d e h y d e w a s a c o m p o u n d of p h e n o l d e r i v a t i v e s c o n t a i n i n g an a l d e h y d e as a s u b s t i t u e n t , the c y t o t o x i c i t y was h i g h e r t h a n p r e d i c t e d f r o m e q u a t i o n s 7 t h r o u g h 10 f o r p h e n o l s . T h i s r e s u l t s u g g e s t s t h a t s a l i c y l a l d e h y d e is c l a s s i f i e d i n t o C l a s s 3 t y p e c o m p o u n d s w i t h u n s p e c i f i c r e a c t i v i t y b e c a u s e of c o n t a i n i g a n a l d e h y d e [ 1 7 ] . In f u t u r e , the e x p a n s i o n of the c h e m i c a l s e t s to i n c l u d e c o m p o u n d s w i t h the r e a c t i v e f u n c t i o n a l g r o u p s s u c h as a l d e h y d e s a n d e p o x i d e s w o u l d be useful.
1025
Table
1
Data
of logPow,
NR50
Compound
Alcohols (narcotics) 1. 2 - methoxyethanol 2. methanol 3 ethanol 4 2 - ethoxyethanol 5 2 - propanol 6 1 - propanol 7 tert - butanol 8 2 - butanol 9. iso- butanol 10. 2- buthoxyethanol 11. 1 - butanol 12. tert- amylalcohol 13. 2-pentanol 14. 1-octanol
for
GFS
Log Pow
narcotics 1,4-dioxane acetonitrile acetone pyridine diethyl ether chloroform
and
LC50
for
LOgl/NR50 (retool/L)
fathead
-2.36 __ -2.38 -2.26
13 13
-2.07
13
_-1.68
13
-2.90 -2.70 -2.35 -2.54 -2.71 -2.18 -2.38 -2.13 -1.80 -1.86 -1.81 -2.05 -1.78 -0.26
2 3 3 3 3 3 4 4 5
13 12 15 20 37 76 18 79 15
19 24 2( ~ 2( 19 1( 12 19
-1.24 0.22 -0.23 0.08 0.28 0.91 0.69 1.22 1.51
-0 -0
42 34
~s 13
-0
30 66 88 97
12 26 is 2~
-2.73 -3.02 -2.42 -1.99 -2.43 -1.07
. -1.60 -2.09 . ---
-0.16
0.85
~s
-1.05
Phenols 31. phenol 32. 3,5-dimethoxyphenol 33. 4-nitrophenol 34. 3-methylphenol 35. 2-methylphenol 36. 4- methylphenol 37. 2-chlorophenol 38. 2- nitrophenol 39. 2-bromophenol 40. 3,5-dimethylphenol 41. 2,4-dimethylphenol 42. 4-chlorophenol 43. 3-chlorophenol 44. 4-bromophenol 45. 3 - bromophenol 46. 2,6-dichlorophenol 47. 2-naphthol 48. 4 - methyl - 2- nitrophenol 49. 1-naphthol 50. 2,3-dichlorophenol
1.46 1.64 1.87 1.94 1.95 1.96 2.17 2.24 2.27 2.35 2.36 2.41 2.48 2.59 2.63 2.84 2.84 2.94 2.98 3.15
~7 ~2 2~ 25 25 2~ 27 22 z2 12 19 s~ 2T 22 ss 2~ 12 29 19 ~
-0.90 -0.46 0.53 -0.41 -0.36 -0.07 -0.29 -0.64 0.23 -0.27 0.04 -0.11 0.07 0.31 0.58 -0.21 0.27 0.01 0.47 0.66
0.37 0.40 0.57 -1.32 . . -. . . .
~
2
~ 9
~
~
guppy
--2.96 13 -2.50 19 --2.16 lo -1.88 10 -. . . -1.28 10 --1.37 10 . . . . . . 0.97 19
13 22 ~2 13 12 ~1 22 22 22 13 ~ 12 12 13
Anilines 30. aniline
and
(retool/L) Guppy
74 71 28 21 16 05 36 61 75 85 88 89 34 92
0 0 1
minnow
Logl/LC50 F.minnow
-0 -0 --0 -0 -0 O 0 0 0 0 0 0 1 2
Aromatics (narcotics) benzene 15 16 o- xylene 17 p - xylene 18 m - xylene 19 naphthalene 20 diphenyl 21 1,2,4 - trichlorobenzene 22 bibenzyl 23. di - n - butylphthalate Other 24. 25. 26. 27. 28. 29.
cells
0.42 . 0.55 0.29 0.77 0.58 0.97 -0.06 . . 0.87 -_. . -. . 1.49 .
. _-0.92 _-
__
13 .
.
. .
. .
.
.
1o 10 .
.
1o
1.88
13
-1.60 -2.04
12 13
-1.46 0.07
12 13
-0.13
l(
___ __ _1.06 _. .
15
.
10 11 11 11 10 lg
1~
. .
l0
. .
. .
. .
. .
. .
. .
.
.
lo .
13 13 13 13
0.50 .
. .
0.09 0.48 0.48 0.45 _-
.
10
.
13
. .
1~ 12 12
.
13
_1.18 1.30
12 l~
1.32
ls
__
1026
Table
1
(cont'd)
Compound
51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62.
Log Pow
3.21 3.24 3.24 3.44 3.56 3.75 3.80 4.10 4.42 4.50 4.64 5.04
2T 37 22 27 37 2T 37 27 2~ 22 " ~7
0.84 0.78 1.03 1.20 1.39 0.38 1.68 1.79 1.60 1.82 1.68 2.17
Aldehydes 63. salicylaldehyde
1.81
12
1.09
Organophosphorus pesticides 64. cyanophos 65. dioxabenzofos 66. naled 67. methidathion 68. malathion 69. fenitrothion 70. phenthoate 71. iprofenfos 72. propaphos 73. edifenphos 74. fenthion 75. diazinon 76. isoxathion 77. disulfoton 78. chlorpyrifosmethyl 79. EPN 80. piperophos 81. chlorpyrifos
2.01 2.12 2.19 2.41 2.68 2.96 3.32 3.34 3.44 3.48 3.56 3.70 3.73 3.84 3.92 4.02 4.04 4.73
Carbamate pesticides 82 thiophanate - methyl 83 carbofuran * 84 XMC* 85 carbaryl * 86 isoprocarb * 87 molinate 88 fenobucarb
1.28 1.60 1.94 1.99 2.07 3.13 3.18
<-0.07 <-O <-0 <-0 <-0 -0 -0
2.34 2.35 2.41 2.80 2.88 2.89 2.98 3.08 3.66 3.73 3.86 3.88 3.90
<-0.08 1.66 -0.49 0.33 0.06
Other 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 101.
2,4-dichlorophenol 2,5-dichlorophenol 4-chloro-3,5-dimethylphenol 3,4-dichlorophenol 3,5-dichlorophenol 2,4,6-trichlorophenol 2,3,4-trichlorophenol 2,4,5-trichlorophenol 2,3,4,6-tetrachlorophenol 2,4,6-triiodophenol 3,5-di-tertbutylphenol pentachlorophenol (PCP)
Logl/NR50 (mmol/L)
Pesticides bentazone * captan simetryn propanil isoprothiolane chlorothalonil captafol procymidone * T -BHC benzoximate chlorobenzilate anilazine pyrazolate
*
0.73 0.43 1.01 0.27 0.43 1.32 0.89 0.38 0.84 1.20 1.35 0.97 1.33 0.85 1.42 1.82 1.63 1.77
26 35 003 03 20 17
2.26 1.71 <0.15 1.21 1.23 1.76 1.19 1.44
Logl/LC50 (retool/L) F.minnow Guppy
2 3
1.32 . . . -1.33 . 2.34 __ . . 2.99
3 2 2 3 2 2
2
10 . . .
.
__ . . . __ __ . . . . . 1.50 . . . . . 3.24
3 s 3 a 3 3 3
3 a s 3 3 3
3 3 3 3 3 3
.
. .
13
1.78 1.92
13 13
__ 2.32
18
2.77
13
1.25
17
2.40 2.00
1~ 17
.
11 . .
1.59 . . .
10
. .
10
1.72
3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
. . .
10
. . .
. . .
. . . . .
. . .
. . . . .
. . . . .
. . . . .
. . . . .
11 . . . . . 11
__
__
1027
Table 1 (cont'd) Compound 102. 103 104. 105. 106. 107. 108. 109.
oxadiazon teflubenzuron chlornitrofen trifluralin propargite dicofol quintozene p,p-DDD
Log Pow
*
4.55 4.56 4.71 4.88 5.00 5.02 5.02 6.02
x~
Logl/NR50 (mmol/L) 1.44 <1.58 1.95 1.60 2.96 2.18 2.16 1.79
3 s 3 3 s 3 3
Log1/LC50 (retool/L) F.minnow Guppy __ __ __ 3.46 -__ __ __
xl
* could not be used i n a n analysis of linear relationship between NR50 and logPow because the N R . values could not be obtained because of no observable effect on cell viability at the maximum c o n c e n t r a t i o n a t which the chemicals could be dissolved or dispersed in the culture medium.
CONCLUSIONS T h e p r e s e n t s t u d y s t r o n g l y s u g g e s t s t h a t t he N R a s s a y w i t h g o l d f i s h G F S c e l l s is u s e f u l f o r p r e l i m i n a r y fish acute toxicity screening of various organic chemicals except for some specific chemicals, e.g. carbamate pesticides. Furthermore, t h i s a s s a y is u s e f u l f o r Q S A R generatation because a good relationship b e t w e e n N R 5 0 a n d P o w is o b t a i n e d f r o m the c o m p o u n d s c o m p r i s e d a d i v e r s i t y of c h e m i c a l s t r u c t u r e s Particularly, the cytotoxicities of "unspecific narcotic type of chemicals" a n d " l e s s i n e r t c h e m i c a l s ( e . g . p h e n o l s ) " to G F S c e l l s a r e w e l l p r e d i c t e d f r o m l o g P o w . In f u t u r e , t he N R a s s a y t e c h n i q u e w i t h G F S cells will be applied to the investigation for the potentiation of toxicity by chemicals mixtures and detecting pollution in aquatic environment.
REFERENCES
[ 1] [ 2] [ [ [ [
3] 4] 5] 6]
[ 7]
Babich,H. and Borenfreund,E. Toxic.in Vitro 5, 91-100(1991). Saito,H., Sudo,S., Shigeoka,T. and Yamauehi,F. Environ. Toxicol. Chem. 10, 235-241 (1991). Saito,H., Iwami,S. and Shigeoka,T. Chernosphere 23, 525-537(1991). Borenfreund,E. and Puerner,J.A. Toxicol.Lett. 24, 119-124(1985). Babich,H., Shopsis,C. and Borenfreund,E. Ecotoxicol. Environ. Safety 11,91-99(1986). Babich,H., Puerner,A. and Borenfreund,E. Arch.Environ. Contam. Toxicol. 15, 31-37 (1986). Babich,H. and Borenfreund,E. Toxicol. Lett. 36, 107-116(1987).
1028
[ 8] [ 9] [10] [11] [12]
Babich,H. and Borenfreund,E. Toxic. in Vitro 1, 3-9(1987). Babich,H. and Borenfreund,E. Ecotoxicol.Environ.Safety 14, 78-87(1987). Proti6,M. and Sablji6,A. Aquat. ToxicoL 14, 47-64(1989). Journal WPCF, Literature Review. 49(1977), 54(1982) and 61(1989). Handbook of Environmental Data on Organic Chemicals, Verschueren,K. ed., Von Nostrand Reinhold Company, New York (1983). [13] KSnemann,W.H. Toxicol. 19, 209-221(1981). [14] Hermens,J.L.M., Leeuwangh,P. and Musch,A. Ecotoxicol.Environ.Safety 8, 388-394(1984) [151 KSnemann,W.H. and Musch,A. ToxicoL 19, 223-228(1981). [161 Hermens,J.L.M. and Leeuwangh,P. Ecotoxicol.Environ. Safety 6, 302-310(1982). E17~ Verhaar,H.J.M., van Leeuwen,C.J. and Hermens,J.L.M. Chemosphere 25, 471-491(1992). [18] Saarikoski,J. and Viluksela,M Ecotoxicol. Environ. Safety 6, 501-512(1982). [19] Veith,G.D., Austin,N.M. and Morris,R.T. Water Res. 13, 43-47(1979). [20] Veith,G.D., Call,D.J. and Brooke,L.T. Can.J.Fish.Aquat. Sci. 40, 743-748(1983). [21] Kwasniewska,K. and Kaiser,K.L.E. QSAR in Environmental Toxicology, Kaiser, K.L.E. ed., Reidel Publishing Company, Dordrecht, Holland, pp.223-233(1984). [22] Ahlers,J., Benzing,M., Gies,A., Pauli,W. and RSsiek,E. Chemospkere 17, 1603-1615
(1988). [23] [24] [25] [26] [27] [28] [29]
Hermens,J., Busser,F., Leeuwangh,P. and Musch,A. Ecotoxicol.Environ. Safety 9, 17-25(1985). Yoshida,K., Shigeoka,T. and Yamauchi,F. Ecotoxicol.Environ. Safety 7, 558-565(1983). Yokota,I. Seitai Kagaku 9, 9-18(1988). (in Japanese) Blum,D.J.W. and Speece,R.E. Ecotoxicol.Environ.Safety 22, 198-224(1991). Shigeoka,T., Yamagata,T., Minoda,T. and Yamauchi,F. Jpn.J. Toxicol.Environ.Health 34, 343-349(1988). (in Japanese) Lipnick,R.L., Bicking,C.K., Johnson,D.E. and Eastmond,D.A. Aquat. Toxicol.Hazard Assess. 8th Syrup. 153- 176(1985). Cascorbi,I. and Ahler.J. Toxicol. 58, 197-210(1989).
0045-6535/93$6.00+ 0.00 PergamonPressLtd.
CYTOTOXICITY OF 109 C H E M I C A L S TO G O L D F I S H GFS CELLS AND R E L A T I O N S H I P S WITH 1 - O C T A N O L / W A T E R PARTITION COEFFICIENTS
H o t a k a Saito* , Junko K o y a s u , Kikuo Y o s h i d a , T a d a y o s h i Shigeoka and Sakae Koike * Mitsubishi-kasei I n s t i t u t e of T o x i c o l o g i c a l and E n v i r o n m e n t a l 1000, K a m o s h i d a - c h o , M i d o r i - k u , Y o k o h a m a 227, Japan
Sciences
(Received in Japan 11 October 1992; accepted 27 November 1992)
ABSTRACT T h e m i d p o i n t c y t o t o x i c i t y (NR50 v a l u e s ) of 109 c h e m i c a l s i n c l u d i n g a l c o h o l s , a r o m a t i c s , p h e n o l s a n d p e s t i c i d e s to g o l d f i s h s c a l e G F S c e l l s were compared with the fish acute toxicities a n d w i t h t h e 1octanol/water partition coefficient (Pow). The NR50 values were significantly correlated to t h e a v a i l a b l e acute toxicity data for f a t h e a d m i n n o w (n=31, r=0.958, s=0.480, F=325) a n d g u p p y (n=29, r=0.957, s=0.482, F=294). The P o w gave a g o o d c o r r e l a t i o n in s i m p l e l i n e a r r e g r e s s i o n a n a l y s i s f o r a l l c o m p o u n d s e x c l u d i n g 8 c o m p o u n d s t h a t the NR50 v a l u e s c o u l d n o t be o b t a i n e d f o r t h e i r low s o l u b i l i t y in c u l t u r e m e d i u m . Log 1/NR50 (mM) = (0.87-+0.08) L o g P o w - ( 2 . 1 7 - + 0 . 2 4 ) (n=101, r = 0 . 9 1 4 , s=0.587, F=505) F o r 29 n a r c o t i c t y p e of c o m p o u n d s , the r e g r e s s i o n a n a l y s i s r e v e a l e d a h i g h c o r r e l a t i o n b e t w e e n the NR50 v a l u e s a n d Pow, a n d a p p e a r e d to give a baseline cytotoxicity. Log 1/NR50 (mM) =(0.77 __.0.06) L o g P o w - (2.48--+0.12) ( n = 2 9 , r = 0 . 9 8 4 , s=0.252, F=817) A n d a l s o , the r e g r e s s i o n a n a l y s e s b e t w e e n NR50 a n d P o w f o r s o m e t y p e s of c o m p o u n d s were d i s c u s s e d . T h e s e r e s u l t s s u g g e s t t h a t N R a s s a y is u s e f u l f o r p r e d i c t i o n of a c u t e t o x i c i t y to f i s h a n d s t u d i e s of q u a n t i t a t i v e structure-activity relationships (QSARs).
1015
1016 INTRODUCTION In vitro cytotoxicity assays using cultured fish cells have r e c e n t l y been developed for a simple, rapid, r e p r o d u c i b l e and economical generation of t o x i c o l o g i c a l d a t a in a q u a t i c toxicology, and for p r e d i c t i n g t h e a c u t e t o x i c i t y of a q u a t i c p o l l u t a n t s to f i s h [ 1 - 3 ] . A n d t h e s e m e t h o d s a r e c o n s i d e r e d to be u s e f u l f o r d e t e c t i n g p o l l u t i o n in the environment, for evaluating synergetic and antagonistic interactions b e t w e e n c o m b i n a t i o n s of c h e m i c a l s , a n d f o r g e n e r a t i n g c o m p u t e r - d e r i v e d predictions b a s e d on q u a n t i t a t i v e structure-activity relationships (QSARs). The n e u t r a l red a s s a y (NR a s s a y ) has i n i t i a l l y been developed for use w i t h m a m m a l i a n c e l l s [4] a n d a d a p t e d f o r c y t o t o x i c i t y s t u d i e s w i t h fish cells [5-9]. This technique has been successfully a p p l i e d to investigate t h e e f f e c t s of 15 c h l o r o p h e n o l s [2] a n d 45 p e s t i c i d e s [3] on G F S c e l l s , d e r i v e d f r o m the s c a l e of g o l d f i s h , a n d t h e i r NR50 v a l u e s were significantly c o r r e l a t e d to in vivo acute toxicity t o 13 k i n d s of a q u a t i c s p e c i e s . In t h i s s t u d y , the NR50 v a l u e s of 49 c h e m i c a l s except for the previous s t u d y d a t a f o r 15 c h l o r o p h e n o l s a n d 45 p e s t i c i d e s , were d e t e r m i n e d , a n d the NR50 v a l u e s were c o m p a r e d w i t h the d i f f e r e n t s o u r c e s of LC50 v a l u e s f o r f a t h e a d m i n n o w [ 1 0 - 1 2 ] a n d f o r guppy [13-18]. 1-octanol/water p a r t i t i o n c o e f f i c i e n t ( P o w ) a r e o f t e n e m p l o y e d in Q S A R a n a l y s i s a n d a r e s u p p o s e d to t a k e i n t o a c c o u n t t h e e f f e c t s of the transport through the membranes toward the receptor. Our previous r e s u l t s on 15 c h l o r o p h e n o l s indicate that the NR50 values are well correlated w i t h l o g P o w . T h e P o w d a t a e x c e p t f o r 45 p e s t i c i d e s w e r e o b t a i n e d f r o m t h e p u b l i s h e d d a t a . In t h i s s t u d y , P o w f o r 45 p e s t i c i d e s w e r e d e t e r m i n e d b y t h e m e t h o d b a s e d on r e v e r s e p h a s e - h i g h pressure l i q u i d c h r o m a t o g r a p h y ( R P - H P L C ) [ 1 9 ] . A n d all Pow d a t a were c o m p a r e d w i t h a s e r i e s of c y t o t o x i c i t y d a t a (NR50 v a l u e s ) .
MATERIALS Test chemicals
AND
METHODS
and solutions
T h e 109 c h e m i c a l s a r e l i s t e d in T a b l e 1 w i t h t h e i r l o g P o w . T h e c h e m i c a l s e x c e p t f o r t h e c o m p o u n d s d e s c r i b e d in p r e v i o u s s t u d y [ 2 , 3 ] , w e r e p u r c h a s e d f r o m T o k y o K a s e i K o g y o Co., T o k y o , J a p a n a n d w e r e all reagent-grade and used without further purification. Either dimethyl sulfoxide ( D M S O ) o r a c e t o n e w a s u s e d a s t h e v e h i c l e to d i s s o l v e h y d r o p h o b i c c o m p o u n d s at a c o n c e n t r a t i o n of l e s s t h a n 0.5 % ( v / v ) w h i c h e x h i b i t e d no o b s e r v a b l e e f f e c t on t h e c e l l v i a b i l i t y .
1017
Cell l i n e s The e s t a b l i s h e d cell line, GFS ( G F - S c a l e , RCB No.082), was p u r c h a s e d from the Riken Gene Bank, Ibaraki, Japan. The GFS cells have a fibroblast-like morphology a n d w e r e d e r i v e d f r o m s c a l e t i s s u e of goldfish (C. a u r a t u s ) . T h e c e l l s w e r e m a i n t a i n e d as monolayer c u l t u r e s in c u l t u r e f l a s k s a t 25°C as d e s c r i b e d in p r e v i o u s s t u d y [ 3 ] . The m e d i u m w a s L e i b o v i t z ' s L - 1 5 , s u p p l e m e n t e d w i t h 10 % f e t a l b o v i n e s e r u m ( F B S ) , 100 u n i t s / m l p e n i c i l l i n G, 100 /.z g / m l s t r e p t o m y c i n , 1.25 g/ml amphotericin B a n d 10 m M N - 2 - h y d r o x y e t h y l p i p e r a z i n e - N ' - 2 e t h a n e s u l f o n i c a c i d ( H E P E S ) to m a i n t a i n the pH at 7.4. F o r p r o p a g a t i o n , t h e c e l l s w e r e d i s s o c i a t e d w i t h a s o l u t i o n c o n s i s t i n g of 0.05 % t r y p s i n a n d 0.02 % e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d ( E D T A ) . A l l c u l t u r e m e d i a a n d r e a g e n t s w e r e p u r c h a s e d f r o m S i g m a C h e m i c a l C o . , St. L o u i s , MO, U.S.A.
Cytotoxicity The NR a s s a y with GFS cells was p e r f o r m e d a c c o r d i n g to the p r o c e d u r e d e s c r i b e d in p r e v i o u s s t u d y [ 3 ] . R e s u l t s w e r e e x p r e s s e d as p e r c e n t of absorbance (540 n m ) of n e u t r a l r e d e x t r a c t e d f r o m c o n t r o l c u l t u r e s . D a t a w e r e p l o t t e d a s p e r c e n t of c o n t r o l v s . l o g a r i t h m i c chemical concentration in m g / L . T h e m i d p o i n t c y t o t o x i c i t y N R 5 0 v a l u e s (50 % d e c r e a s e of a b s o r b a n c e ) w e r e c a l c u l a t e d by l i n e a r r e g r e s s i o n a n a l y s i s .
1 - o c t a n o l / w a t e r partition coefficient, Pow 1 - o c t a n o l / w a t e r p a r t i t i o n c o e f f i c i e n t ( P o w ) w e r e d e t e r m i n e d by R P H P L C m e t h o d [ 1 9 ] . A l i n e a r c a l i b r a t i o n of t h e l o g a r i t h m of r e t e n t i o n time with the logarithm of t h e p a r t i t i o n coefficent (logPow) was a t t a i n e d by u s i n g a m i x t u r e of b e n z e n e , b r o m o b e n z e n e , b i p h e n y l , b i b e n z y l , p,p'-DDE and 2,4,5,2',5'-pentachlorobiphenyl as r e f e r e n c e s t a n d a r d s of known LogPow.
Statistical
analysis
The NR50 values were correlated with the published fish acute t o x i c i t y d a t a (LC50) a n d l o g P o w . Both NR50 a n d LC50 d a t a were c o n v e r t e d to i n v e r s e m i l l i m o l a r c o n c e n t r a t i o n a n d g i v e n in t h e l o g a r i t h m i c f o r m f o r r e g r e s s i o n a n a l y s i s . A l l s t a t i s t i c a l a n a l y s e s w e r e p e r f o r m e d on the s t a t i s t i c a l p a c k a g e ( M i c r o s o f t C h a r t V e r . 3 . 1 ) f o r NEC PC9801 c o m p u t e r . RESULTS
AND
DISCUSSION
Relationships between in vivo and in vitro The 24-h N R 5 0
values for the cytotoxicity of the test c o m p o u n d s
to
1018
GFS cells, together
w i t h t h e i r l o g P o w a n d LC50 d a t a f o r f a t h e a d
minnow
a n d g u p p y a r e s u m m a r i z e d in T a b l e 1. T h e l i n e a r r e l a t i o n s h i p s b e t w e e n c y t o t o x i c i t i e s ( 2 4 - h r N R 5 0 ) to G F S c e l l s a n d a c u t e t o x i c i t i e s ( L C 5 0 ) to the fathead m i n n o w a n d g u p p y a r e s h o w n i n F i g u r e 1. I n v i v o d a t a f o r t h e f a t h e a d m i n n o w a n d g u p p y w e r e a v a i l a b l e f o r 31 a n d 29 of t h e 109 c o m p o u n d s , respectively. The regression analyses of t h e s e d a t a p r o v i d e t h e f o l l o w i n g e q u a t i o n 1 an 2 ( t h e c o n f i d e n c e i n t e r v a l s of t h e regression c o e f f i c i e n t s in p a r e n t h e s e s h a v e a 95 % p r o b a b i l i t y ; n is the number of compounds ; r is t h e c o r r e l a t i o n coefficient ; s is s t a n d a r d e r r o r of r e s i d u e s ; F is t e s t of n u l l h y p o t h e s i s ) : Eqn.1 (fathead Log 1/LC50
minnow) ; = (1.10--+0.12) • Log 1/NR50-t- ( 0 . 8 1 - + 0 . 1 8 ) (n=31, r=0.958, s=0.480, F=325)
Eqn.2
;
(guppy)
Log 1/LC50
= (1.03+0.12) • Log 1/NR50+ ( 0 . 9 1 + 0 . 2 0 ) (n=29, r=0.957, s=0.482, F=294)
(,)
4
"./.
.)i.
(b)
2
2 0 -2
-2
-
4
I
-4
Fig.1
and
• ~o
I
4
I
-2 0 2 Logl/NR50, mM
4
-4
-2 0 2 Logl/NR50, mM
T h e r e l a t i o n s h i p s b e t w e e n t he c y t o t o x i c i t i e s ( l o g l / N R 5 0 ) to G F S c e l l s a n d t he a c u t e t o x i c i t i e s ( l o g l / L C 5 0 ) to f a t h e a d m i n n o w (a) and g u p p y ( b ) .
There were good correlations in vivo LC50 values. And as
between in vitro NR50 values the slopes are nearly one, the
1019
t o x i c r a n g e of t h e s e c o m p o u n d s on G F S cells a p p e a r s to be s a m e as t h o s e on f a t h e a d minnow and guppy. Furthermore, the intercepts of t h e regression lines demonstrate t h a t t h e a b s o l u t e v a l u e s of N R 5 0 a r e significantly h i g h e r t h a n L C 5 0 v a l u e s ( t h e N R a s s a y is a b o u t s e v e n t i m e s l e s s s e n s i t i v e t h a n the LC50 t e s t s f o r f a t h e a d m i n n o w a n d g u p p y ) . A s i m i l a r r e s u l t h a s b e e n n o t e d f o r t h e p r e v i o u s s t u d y w i t h r e s p e c t to the linear relationships b e t w e e n N R 5 0 v a l u e s of f i f t e e n c h l o r o p h e n o l s to G F S c e l l s a n d t h e i r a c u t e t o x i c i t i e s to 12 a q u a t i c s p e c i e s [2] a n d b e t w e e n N R 5 0 v a l u e s of 34 p e s t i c i d e s to G F S c e l l s a n d t h e i r a c u t e toxicities to c a r p [ 3 ] . T h e s e r e s u l t s s t r o n g l y s u g g e s t t h a t the NR a s s a y is u s e f u l f o r p r e l i m i n a r y e c o t o x i c o l o g i c a l s c r e e n i n g of v a r i o u s chemicals, recognizing the different sensitivities between in vitro and in vivo. Saito et a/.[3], however, described the different toxic response between in vitro and in vivo for some pesticides, e.g. carbamates. Therefore, by careful analysis f o r the c h e m i c a l s t r u c t u r e s of the o u t l i e r s , the NR a s s a y c a n be a p p l i e d to t h e p r e d i c t i o n of t h e i n v i v o r e s p o n s e .
Relationships
with
1-octanol/water
partition
coefficients
(Pow)
1. A l l c o m p o u n d s and narcotic type of c o m p o u n d s The P o w gave a g o o d c o r r e l a t i o n in s i m p l e l i n e a r r e g r e s s i o n a n a l y s i s f o r all c o m p o u n d s e x c l u d i n g 8 c o m p o u n d s t h a t the NR50 v a l u e s c o u l d not be o b t a i n e d f o r t h e i r l o w s o l u b i l i t y in c u l t u r e m e d i u m ( F i g . 2 ) . T h e r e g r e s s i o n a n a l y s i s p r o v i d e s the f o l l o w i n g e q u a t i o n 3:
Eqn.3;
Log 1/NR50 (mM) = (0.87___0.08) L o g P o w - ( 2 . 1 7 _ + 0 . 2 4 ) (n=101, r = 0 . 9 1 4 , s=0.587, F=505)
It w a s i n t e r e s t i n g t h a t a g o o d c o r r e l a t i o n b e t w e e n NR50 a n d P o w , varying in logPow from - 0 . 7 4 to 6 . 0 2 , w a s o b t a i n e d for various c h e m i c a l s w i t h a w i d e v a r i e t y of c h e m i c a l s t r u c t u r e a n d w i t h a s p e c i f i c m o d e of t o x i c a c t i o n l i k e p e s t i c i d e s . H o w e v e r , f o r 29 n a r c o t i c t y p e of c o m p o u n d s , i n c l u d i n g a l c o h o l s , a r o m a t i c s and some other c o m p o u n d s which were c l a s s i f i e d into Class 1 c o m p o u n d s as d e s c r i b e d by V e r h a a r et al. [ 1 7 ] , the re gre s sion a n a l y s i s r e v e a l e d an e x c e l l e n t c o r r e l a t i o n b e t w e e n NR50 a n d P o w s h o w i n g a n i n c r e a s e in c y t o t o x i c i t y w i t h h i g h e r v a l u e s of l o g P o w ( F i g . 2 a n d Eqn.4).
Eqn.4;
Log 1/NR50 (mM) = (0.77_+0.06) L o g P o w - ( 2 . 4 8 _ + 0 . 1 2 ) ( n = 2 9 , r = 0 . 9 8 4 , s=0.252, F=817)
1020
O
s"
.'"Eqn.3 s
o
o
• o
o
I
I
c~ e" .~:~o . /
~o
~,"
o
o
Eqn.4
1 t~
0
Z
m/'d~
-1 -2 -3 -
4
I
-2
Fig.2
-1
I
I
0
1
2 3 Log Pow
I
I
I
4
5
6
7
The relationship between logl/NR50 to G F S c e l l s a n d f o r all c o m p o u n d s . [ Onarcotic compounds; Nphenols; O pesticides; .aniline; A salicylaldehyde. ]
logPow
A n d e q u a t i o n 4 p r o b a b l y p r e d i c t s the m i n i m a l c y t o t o x i c i t y ( b a s e l i n e t o x i c i t y ) of o r g a n i c c h e m i c a l s to G F S c e l l s . K S n e m a n n [15] d e s c r i b e s t h a t t h e c o e f f i c i e n t f o r l o g P o w in g u p p y ' s b a s e l i n e t o x i c i t y l e v e l a r e 0.87. T h e l o w e r v a l u e 0.77 in e q u a t i o n 4 is p r o b a b l y r e l a t e d to t h e f a c t t h a t the N R a s s a y r e s u l t s in l o w e r s l o p e in the h i g h l o g P o w b e c a u s e of the a d s o r p t i o n of h y d r o p h o b i c c h e m i c a l s to c u l t u r e m e d i u m . As to an h o m o l o g o u s s e r i e s of c o m p o u n d s , the Q S A R e q u a t i o n s f o r 14 alcoho]s and 9 aromatics including trichlorobenzene a r e g i v e n in e q u a t i o n 5 a n d 6, r e s p e c t i v e l y . B o t h e q u a t i o n 5 a n d 6 g a v e g o o d f i t s to the d a t a ( F i g . 3 ) . Eqn.5;
Log 1/NR50 (mM) = (0.65_+0.12) L o g P o w - ( 2 . 4 1 _ + 0 . 1 2 ) (n=14, r=0.962, s=0.185, F=148)
1021
Eqn.6;
Log 1/NR50 (mM) = (0.85_+0.24) L o g P o w - ( 2 . 7 1 _ + 0 . 9 0 ) ( n = 9, r = 0 . 9 5 4 , s = 0 . 2 6 7 , F = 70)
.~"
Z
.6
¢= Eqn.6
.0*6
""¢= Eqn.5
-1
•
O
-2 -3 -
4
-2
Fig.3
I
I
I
-1
0
1
I
I
2 3 Log Pow
I
I
I
4
5
6
The relationship between logl/NR50 to G F S f o r 14 a l c o h o l s ( ( } ) and 9 a r o m a t i c s ( O ) .
cells
7
and
logPow
2. P h e n o l s
The Q S A R f o r 32 p h e n o l s i n c l u d i n g of the P o w with NR50 ( F i g . 4 ) .
Eqn.7;
naphthol
gave a good correlation
Log 1/NR50 (mM) = (0.81_+ 0.14) L o g P o w - (1.88_+ 0 . 4 2 ) (n=32, r=0.910, s=0.345, F=145)
The i n t e r c e p t v a l u e of e q u a t i o n 7 is h i g h e r t h a n t h a t of e q u a t i o n 4 for narcotic compounds. This result suggests that phenols have a higher toxicity level than narcotic compounds. A similar result has been noted for QSAR studies with fish LC50 data. Veith et al.[20], present
1022
that the fish acute toxicities of m a n y p h e n o l s a n d a n i l i n e s are underestimated by the b a s e l i n e n a r c o s i s QSAR. V e r h a a r et a / . [ 1 7 ] . d e s c r i b e that phenols are c l a s s i f i e d into "less inert c h e m i c a l s " which are not r e a c t i v e when c o n s i d e r i n g o v e r a l l fish acute e f f e c t s , but are s l i g h t l y m o r e t o x i c t h a n b a s e l i n e t o x i c i t y . In a d d i t i o n , 4 - n i t r o p h e n o l w a s m o r e c y t o t o x i c t h a n p r e d i c t i o n f r o m e q u a t i o n 7. This r e s u l t m a y be e x p l a i n e d by a d i f f e r e n c t m o d e of a c t i o n , s u c h as a s t r o n g o x i d a t i v e p h o s p h o r y l a t i o n u n c o u p l i n g model.
Eqn.7
O
ss °S
O
1
o°°
Eqn.4
¢~
s o ~ SSS
z_.
nl
o
•
~
0
o oo° oo,OC52
•
0
-1
-
so S
2
1
I
I
2
3
I
I
4
5
6
Log Pow Fig.4
The r e l a t i o n s h i p b e t w e e n ] o g l / N R 5 0 to G F S c e l l s a n d l o g P o w f o r 32 p h e n o l s . [ O phenols c o n t a i n i n g only C, H and O ; O h a l o g e n a t e d phenols ( O 1 ; 2,6-C1, 02; 2,4,6-C] phenol); Initrophenols (ll; 4-nitrophenol, .2; 2-nitrophenol, .3; 4-methyl-2-nitrophenol). ]
F o r 10 p h e n o l s c o n t a i n i n g only C, H and O, e x c l u d i n g h a l o g e n a t e d and n i t r o p h e n o l s f r o m e q u a t i o n 7, the r e g r e s s i o n a n a l y s i s y i e l d e d b e t t e r c o r r e l a t i o n c o e f f i c i e n t s and predictive c a p a b i l i t i e s (Eqn.8):
1023
Eqn.8;
Log 1/NR50 (mM) = (0.75_+0.13) L o g P o w - ( 1 . 8 2 _ + 0 . 3 2 ) (n=10, r=0.980, s=0.151, F=190)
And the QSAR for h a l o g e n a t e d phenols is given in e q u a t i o n 9.
Eqn.9;
Log 1/NR50 (mM) = (0.83_+0.21) L o g P o w - ( 1 . 9 2 _ + 0 . 7 0 ) (n=19, r=0.901, s=0.335, F= 74)
The o r t h o - s u b s t i t u t e d c h l o r o p h e n o l s such as 2 , 6 - d i c h l o r o - and 2,4,6trichlorophenols w e r e l e a s t t o x i c a m o n g the c y t o t o x i c i t i e s of t h e i s o m e r s h a v i n g the s a m e n u m b e r of c h l o r i n e a t o m s [2]. T h a t is b e c a u s e t h e t o x i c e f f e c t s of the O H - g r o u p on the c e l l s a r e i n h i b i t e d b y the i n t r a m o l e c u l a r b o n d i n g of the O H - h y d r o g e n and the two o r t h o - c h l o r i n e substituents, as w e l l as b y t h e i r s h i e l d i n g of t h e O H - g r o u p [21]. T h e r e f o r e , e x c l u d i n g b o t h 2 , 6 - d i c h l o r o - and 2 , 4 , 6 - t r i c h l o r o p h e n o l s f r o m equation 9, t h e r e g r e s s i o n analysis yielded better correlation c o e f f i c i e n t s and predictive c a p a b i l i t i e s (Eqn.10): Eqn.10;
3.
Log 1/NR50 (mM) =(0.84_+0.13) L o g P o w - (1.85_+0.45) (n=17, r=0.961, s=0.212, F=180)
Pesticides
The equation
regression 11 ( F i g . 5 ) :
Eqn.ll;
analysis
for
pesticides
provides
following
Log 1/NR50 (mM) =(0.52_+ 0.21) L o g P o w - (0.67_+ 0.77) (n=38, r=0.640, s=0.589, F= 25)
The r e g r e s s i o n a n a l y s i s f o r 18 o r g a n o p h o s p h a t e s p r o v i d e s the f o l l o w i n g e q u a t i o n 12:
Eqn.12;
the
of 38 p e s t i c i d e s
Log 1/NR50 (raM) =(0.45__.0.23) L o g P o w (0.46_+0.79) (n=18, r=0.719, s=0.343, F= 17)
Both regression analyses provided the significant correlation c o e f f i c i e n t s b e t w e e n the NR50 v a l u e s and P o w , b u t b o t h r v a l u e s w e r e p o o r e r t h a n t h o s e of e q u a t i o n s 4 t h r o u g h 10. The i n t e r c e p t v a l u e s of e q u a t i o n s 11 a n d 12 a r e h i g h e r t h a n t h o s e of e q u a t i o n s 4 t h r o u g h 10, w h i l e the s l o p e s a r e l o w e r . T h i s r e s u l t s u g g e s t s t h a t m a n y p e s t i c i d e s a r e m u c h m o r e t o x i c t h a n p r e d i c t e d s o l e l y f r o m l o g P o w d u e to t h e i r s p e c i f i c m o d e s of a c t i o n .
1024
0
o o
0
qn.ll
o ; ~
o
..-.
z_.
•
o
~'~0
00
Q O
.-*"~
Eqn.4
"*°**
***
O s ~S Bs~
-2 1
I
I
2
3
l
I
I
4
5
6
7
Log Pow Fig.5
The relationship f o r 38 p e s t i c i d e s .
between logl/NR50 to G F S c e l l s a n d [ O organophosphates, O others. ]
logPow
4. A n i l i n e a n d s a l i c y l a l d e h y d e Both a n i l i n e and s a l i c y l a l d e h y d e were more c y t o t o x i c t h a n p r e d i c t e d f r o m b a s e l i n e c y t o t o x i c i t y of e q u a t i o n 4 ( F i g . 2 ) . T h e s e c o m p o u n d s a r e n o t c l a s s i f i e d i n t o C l a s s 1 t y p e c o m p o u n d s [ 1 7 ] . A n i l i n e s as w e l l as p h e n o l s a r e c l a s s i f i e d i n t o C l a s s 2 t y p e c o m p o u n d s . I n d e e d , t h e NR50 v a l u e f o r a n i l i n e c o u l d be p r e d i c t e d f r o m e q u a t i o n s 7 t h r o u g h 10 f o r p h e n o l s . A l t h o u g h s a l i c y l a l d e h y d e w a s a c o m p o u n d of p h e n o l d e r i v a t i v e s c o n t a i n i n g an a l d e h y d e as a s u b s t i t u e n t , the c y t o t o x i c i t y was h i g h e r t h a n p r e d i c t e d f r o m e q u a t i o n s 7 t h r o u g h 10 f o r p h e n o l s . T h i s r e s u l t s u g g e s t s t h a t s a l i c y l a l d e h y d e is c l a s s i f i e d i n t o C l a s s 3 t y p e c o m p o u n d s w i t h u n s p e c i f i c r e a c t i v i t y b e c a u s e of c o n t a i n i g a n a l d e h y d e [ 1 7 ] . In f u t u r e , the e x p a n s i o n of the c h e m i c a l s e t s to i n c l u d e c o m p o u n d s w i t h the r e a c t i v e f u n c t i o n a l g r o u p s s u c h as a l d e h y d e s a n d e p o x i d e s w o u l d be useful.
1025
Table
1
Data
of logPow,
NR50
Compound
Alcohols (narcotics) 1. 2 - methoxyethanol 2. methanol 3 ethanol 4 2 - ethoxyethanol 5 2 - propanol 6 1 - propanol 7 tert - butanol 8 2 - butanol 9. iso- butanol 10. 2- buthoxyethanol 11. 1 - butanol 12. tert- amylalcohol 13. 2-pentanol 14. 1-octanol
for
GFS
Log Pow
narcotics 1,4-dioxane acetonitrile acetone pyridine diethyl ether chloroform
and
LC50
for
LOgl/NR50 (retool/L)
fathead
-2.36 __ -2.38 -2.26
13 13
-2.07
13
_-1.68
13
-2.90 -2.70 -2.35 -2.54 -2.71 -2.18 -2.38 -2.13 -1.80 -1.86 -1.81 -2.05 -1.78 -0.26
2 3 3 3 3 3 4 4 5
13 12 15 20 37 76 18 79 15
19 24 2( ~ 2( 19 1( 12 19
-1.24 0.22 -0.23 0.08 0.28 0.91 0.69 1.22 1.51
-0 -0
42 34
~s 13
-0
30 66 88 97
12 26 is 2~
-2.73 -3.02 -2.42 -1.99 -2.43 -1.07
. -1.60 -2.09 . ---
-0.16
0.85
~s
-1.05
Phenols 31. phenol 32. 3,5-dimethoxyphenol 33. 4-nitrophenol 34. 3-methylphenol 35. 2-methylphenol 36. 4- methylphenol 37. 2-chlorophenol 38. 2- nitrophenol 39. 2-bromophenol 40. 3,5-dimethylphenol 41. 2,4-dimethylphenol 42. 4-chlorophenol 43. 3-chlorophenol 44. 4-bromophenol 45. 3 - bromophenol 46. 2,6-dichlorophenol 47. 2-naphthol 48. 4 - methyl - 2- nitrophenol 49. 1-naphthol 50. 2,3-dichlorophenol
1.46 1.64 1.87 1.94 1.95 1.96 2.17 2.24 2.27 2.35 2.36 2.41 2.48 2.59 2.63 2.84 2.84 2.94 2.98 3.15
~7 ~2 2~ 25 25 2~ 27 22 z2 12 19 s~ 2T 22 ss 2~ 12 29 19 ~
-0.90 -0.46 0.53 -0.41 -0.36 -0.07 -0.29 -0.64 0.23 -0.27 0.04 -0.11 0.07 0.31 0.58 -0.21 0.27 0.01 0.47 0.66
0.37 0.40 0.57 -1.32 . . -. . . .
~
2
~ 9
~
~
guppy
--2.96 13 -2.50 19 --2.16 lo -1.88 10 -. . . -1.28 10 --1.37 10 . . . . . . 0.97 19
13 22 ~2 13 12 ~1 22 22 22 13 ~ 12 12 13
Anilines 30. aniline
and
(retool/L) Guppy
74 71 28 21 16 05 36 61 75 85 88 89 34 92
0 0 1
minnow
Logl/LC50 F.minnow
-0 -0 --0 -0 -0 O 0 0 0 0 0 0 1 2
Aromatics (narcotics) benzene 15 16 o- xylene 17 p - xylene 18 m - xylene 19 naphthalene 20 diphenyl 21 1,2,4 - trichlorobenzene 22 bibenzyl 23. di - n - butylphthalate Other 24. 25. 26. 27. 28. 29.
cells
0.42 . 0.55 0.29 0.77 0.58 0.97 -0.06 . . 0.87 -_. . -. . 1.49 .
. _-0.92 _-
__
13 .
.
. .
. .
.
.
1o 10 .
.
1o
1.88
13
-1.60 -2.04
12 13
-1.46 0.07
12 13
-0.13
l(
___ __ _1.06 _. .
15
.
10 11 11 11 10 lg
1~
. .
l0
. .
. .
. .
. .
. .
. .
.
.
lo .
13 13 13 13
0.50 .
. .
0.09 0.48 0.48 0.45 _-
.
10
.
13
. .
1~ 12 12
.
13
_1.18 1.30
12 l~
1.32
ls
__
1026
Table
1
(cont'd)
Compound
51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62.
Log Pow
3.21 3.24 3.24 3.44 3.56 3.75 3.80 4.10 4.42 4.50 4.64 5.04
2T 37 22 27 37 2T 37 27 2~ 22 " ~7
0.84 0.78 1.03 1.20 1.39 0.38 1.68 1.79 1.60 1.82 1.68 2.17
Aldehydes 63. salicylaldehyde
1.81
12
1.09
Organophosphorus pesticides 64. cyanophos 65. dioxabenzofos 66. naled 67. methidathion 68. malathion 69. fenitrothion 70. phenthoate 71. iprofenfos 72. propaphos 73. edifenphos 74. fenthion 75. diazinon 76. isoxathion 77. disulfoton 78. chlorpyrifosmethyl 79. EPN 80. piperophos 81. chlorpyrifos
2.01 2.12 2.19 2.41 2.68 2.96 3.32 3.34 3.44 3.48 3.56 3.70 3.73 3.84 3.92 4.02 4.04 4.73
Carbamate pesticides 82 thiophanate - methyl 83 carbofuran * 84 XMC* 85 carbaryl * 86 isoprocarb * 87 molinate 88 fenobucarb
1.28 1.60 1.94 1.99 2.07 3.13 3.18
<-0.07 <-O <-0 <-0 <-0 -0 -0
2.34 2.35 2.41 2.80 2.88 2.89 2.98 3.08 3.66 3.73 3.86 3.88 3.90
<-0.08 1.66 -0.49 0.33 0.06
Other 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 101.
2,4-dichlorophenol 2,5-dichlorophenol 4-chloro-3,5-dimethylphenol 3,4-dichlorophenol 3,5-dichlorophenol 2,4,6-trichlorophenol 2,3,4-trichlorophenol 2,4,5-trichlorophenol 2,3,4,6-tetrachlorophenol 2,4,6-triiodophenol 3,5-di-tertbutylphenol pentachlorophenol (PCP)
Logl/NR50 (mmol/L)
Pesticides bentazone * captan simetryn propanil isoprothiolane chlorothalonil captafol procymidone * T -BHC benzoximate chlorobenzilate anilazine pyrazolate
*
0.73 0.43 1.01 0.27 0.43 1.32 0.89 0.38 0.84 1.20 1.35 0.97 1.33 0.85 1.42 1.82 1.63 1.77
26 35 003 03 20 17
2.26 1.71 <0.15 1.21 1.23 1.76 1.19 1.44
Logl/LC50 (retool/L) F.minnow Guppy
2 3
1.32 . . . -1.33 . 2.34 __ . . 2.99
3 2 2 3 2 2
2
10 . . .
.
__ . . . __ __ . . . . . 1.50 . . . . . 3.24
3 s 3 a 3 3 3
3 a s 3 3 3
3 3 3 3 3 3
.
. .
13
1.78 1.92
13 13
__ 2.32
18
2.77
13
1.25
17
2.40 2.00
1~ 17
.
11 . .
1.59 . . .
10
. .
10
1.72
3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
. . .
10
. . .
. . .
. . . . .
. . .
. . . . .
. . . . .
. . . . .
. . . . .
11 . . . . . 11
__
__
1027
Table 1 (cont'd) Compound 102. 103 104. 105. 106. 107. 108. 109.
oxadiazon teflubenzuron chlornitrofen trifluralin propargite dicofol quintozene p,p-DDD
Log Pow
*
4.55 4.56 4.71 4.88 5.00 5.02 5.02 6.02
x~
Logl/NR50 (mmol/L) 1.44 <1.58 1.95 1.60 2.96 2.18 2.16 1.79
3 s 3 3 s 3 3
Log1/LC50 (retool/L) F.minnow Guppy __ __ __ 3.46 -__ __ __
xl
* could not be used i n a n analysis of linear relationship between NR50 and logPow because the N R . values could not be obtained because of no observable effect on cell viability at the maximum c o n c e n t r a t i o n a t which the chemicals could be dissolved or dispersed in the culture medium.
CONCLUSIONS T h e p r e s e n t s t u d y s t r o n g l y s u g g e s t s t h a t t he N R a s s a y w i t h g o l d f i s h G F S c e l l s is u s e f u l f o r p r e l i m i n a r y fish acute toxicity screening of various organic chemicals except for some specific chemicals, e.g. carbamate pesticides. Furthermore, t h i s a s s a y is u s e f u l f o r Q S A R generatation because a good relationship b e t w e e n N R 5 0 a n d P o w is o b t a i n e d f r o m the c o m p o u n d s c o m p r i s e d a d i v e r s i t y of c h e m i c a l s t r u c t u r e s Particularly, the cytotoxicities of "unspecific narcotic type of chemicals" a n d " l e s s i n e r t c h e m i c a l s ( e . g . p h e n o l s ) " to G F S c e l l s a r e w e l l p r e d i c t e d f r o m l o g P o w . In f u t u r e , t he N R a s s a y t e c h n i q u e w i t h G F S cells will be applied to the investigation for the potentiation of toxicity by chemicals mixtures and detecting pollution in aquatic environment.
REFERENCES
[ 1] [ 2] [ [ [ [
3] 4] 5] 6]
[ 7]
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