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9 The Disposition and Biotransformation of Organochlorine Insecticides in Insecticide-Resistant and -Susceptible

Downloaded by UNIV LAVAL on July 12, 2016 | http://pubs.acs.org Publication Date: May 24, 1979 | doi: 10.1021/bk-1979-0099.ch009

Mosquitofish JAMES D. YARBROUGH and JANICE E. CHAMBERS Department of Biological Sciences, Mississippi State University, Mississippi State, MS 39762 S e l e c t i v e pressures from a g r i c u l t u r a l contamination have r e s u l t e d i n the development o f a r e s i s t a n t (R) population of mosquitofish (Gambusia a f f i n i s ) which demonstrates up to a 500 f o l d greater tolerance of i n s e c t i c i d e s than does a corresponding s u s c e p t i b l e (S) population (Table I). This r e s i s t a n c e was caused by a g r i c u l t u r a l runoff o f p e s t i c i d e s used on cotton and soybean f i e l d s i n t o drainage ditches s u b j e c t i n g the f i s h to chronic exposures to i n s e c t i c i d e s . The r e s i s t a n c e i s g e n e t i c a l l y based, and i s not merely an expression of environmentally-induced tolerances. The major s e l e c t i v e pressure was organochlorine i n s e c t i c i d e s and the highest l e v e l s o f r e s i s t a n c e are to the c h l o r i n a t e d a l i c y c l i c i n s e c t i c i d e s (40 - 500 f o l d d i f f e r e n c e between the 48 hr LC50 values o f S and R p o p u l a t i o n s ) . The S population i s not abnormally s e n s i t i v e to organochlorine i n s e c t i c i d e s , as i n d i c a t e d by a l d r i n acute t o x i c i t i e s in other f i s h species {]_). Table I - Comparative 48-hr LC50 Values ( y g / l ) f o r I n s e c t i c i d e S u s c e p t i b l e (S) and R e s i s t a n t (R) Mosquitofisha

Insecticide

S

R

Fold

difference

DDT

18.9

96.2

5.0

Aldrin

36.2

2735.0

93.5

Dieldrin

8.0

433.6

54.0

Endrin

0.6

314.1

499.0

458.7

388.5

Toxaphene a

11.6 From C u l l e y and Ferguson

(2).

0-8412-0489-6/70/47-099-145$05.00/0 © 1979 American Chemical Society Khan et al.; Pesticide and Xenobiotic Metabolism in Aquatic Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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Work in our laboratory on various parameters i n R and S f i s h has i n v e s t i g a t e d the f a c t o r ( s ) responsible f o r r e s i s t a n c e . The r e s u l t s have i n d i c a t e d t h a t r e s i s t a n c e is m u l t i f a c t o r i a l , i n v o l v i n g a b a r r i e r to i n s e c t i c i d e p e n e t r a t i o n , i n s e c t i c i d e s t o r a g e , i n s e c t i c i d e metabolism, and an apparent " i n s e n s i t i v i t y " at the t a r g e t s i t e to the t o x i c e f f e c t s of the i n s e c t i c i d e . The present report concentrates on two of these f a c t o r s : insecticide d i s p o s i t i o n and metabolism.


Disposition In g e n e r a l , aquatic vertebrates absorb i n s e c t i c i d e s through the g i l l s (3). As such, the i n s e c t i c i d e enters the e f f e r e n t limb of c i r c u l a t i o n and may p o t e n t i a l l y enter the brain d i r e c t l y as one of the f i r s t major organs r e c e i v i n g blood from the g i l l s . The s i t e of a c t i o n o f some organochlorine i n s e c t i c i d e s i s b e l i e v e d to be the central nervous system and a b a r r i e r to i n s e c t i c i d e penetration into the CNS would protect the t a r g e t organ. A comparison of the r a t i o of i n s e c t i c i d e accumulated in the l i v e r to that i n the brain (L/B) of R and S animals f o l l o w i n g in vivo exposure would be an i n d i c a t i o n of the e f f e c t i v e n e s s o f the brain b a r r i e r (Table II). In every c a s e , there i s c o n s i d e r ably more i n s e c t i c i d e in the l i v e r s than in the brains of resistant f i s h . In only one case is t h i s pattern seen in the S f i s h and in the case of endrin there i s 1.7 times as much in the brains as in the l i v e r s . Table II

- Ratios of I n s e c t i c i d e Accumulated in L i v e r s to That Brains in S u s c e p t i b l e (S) and Resistant (R) Fish Exposed to the ' ^ c - l a b e l l e d I n s e c t i c i d e f o r 6 hr

Exposure Concentrât!on,yg/l

in

L/B R

S

Reference

DDT

20

5.6

1.1

4

Aldrin

80

3.1

2.1

5

Dieldrin

30

2.9

1.2

5

Endrin

10

1.8

0.6

6

This b a r r i e r can be f u r t h e r i l l u s t r a t e d by comparing t i s s u e i n s e c t i c i d e r a t i o s between the S and R p o p u l a t i o n s . Radioactivity accumulated in major organs f o l l o w i n g exposure to 10 y g / l 14cendrin i s greater i n S f i s h than i n R f i s h f o r a l l organs studied except kidney (Table III). S i m i l a r l y , S f i s h accumulate more a l d r i n , d i e l d r i n and DDT i n t h e i r brains than do R f i s h .

Khan et al.; Pesticide and Xenobiotic Metabolism in Aquatic Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

9.

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Organochlorine Insecticides

A N D CHAMBERS

147

With the exception o f DDT, there was a t l e a s t as much o r more r a d i o a c t i v i t y accumulated i n l i v e r s from S f i s h than i n l i v e r s from R f i s h (Table I V ) . Table III

- R a d i o a c t i v i t y Accumulated i n Tissues o f S u s c e p t i b l e (S) and Resistant (R) F i s h Exposed to 10 y g / l 1*Cendrin f o r 6 hr ng endrin equivalents/mg wet weight


Tissue

S/R

R

S

3

Brain

192.2

+ 19.2

6.9 + 0.5

27.8**

Liver

119.9

+ 23.6

12.5 + 0.6

9.6**

78.0 + 23.4

6.5 + 0.8

12.0**

7.1

20.8 + 0.7

6.8**

+ 7.3

12.8 + 0.3

2.4**

+ 13.0

24.1

Gill Gall

Bladder

140.4+

Intestine

30.1

Spleen

288.1

Kidney

20.2 + 0.5

+ 0.8

12.0**

66.8 + 1.8

0.3**

a

Values are expressed as mean + SEM, 4-6 r e p l i c a t i o n s , 3 f i s h per r e p l i c a t i o n . From Hunsinger (6_). * * S i g n i f i c a n t d i f f e r e n c e s between the means o f the two populations (£_ < 0.01) as determined by the t - t e s t . Table IV - Ratios ( S u s c e p t i b l e / R e s i s t a n t ; S/R) o f I n s e c t i c i d e Accumulated i n Brains and Livers Following 6 hr o f R e l a b e l l e d I n s e c t i c i d e Exposure Exposure Concentration, y g / l

S/R Brain

Li ver

Reference

DDT

20

2.2

0.5

4

Aldrin

80

5.2

3.6

5

Dieldrin

30

2.1

0.9

5

American Chemical Society Library 1155 16th St. N. % Washington, D. C. 20035 Khan et al.; Pesticide and Xenobiotic Metabolism in Aquatic Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

PESTICIDE AND XENOBIOTIC M E T A B O L I S M IN AQUATIC ORGANISMS


148

From the data presented, there i s o b v i o u s l y a more e f f e c t i v e b a r r i e r to i n s e c t i c i d e penetration i n R f i s h than i n S f i s h . F u r t h e r , t h i s b a r r i e r apparently operates over a wide range o f exposure l e v e l s . For example, when the t i s s u e concentration i n brains from R f i s h exposed to 10 y g / l are compared to t i s s u e concentrations i n brains o f R f i s h exposed to 314 y g / l e n d r i n , there i s a 10-fold increase i n endrin concentration i n brain t i s s u e (from 6.91 to 73.8 ng endrin equivalents/mg wet weight o f t i s s u e ) , although t h i s represents a 3 0 - f o l d increase i n the i n s e c t i c i d e exposure l e v e l . However, i n S f i s h , the l e v e l o f i n s e c t i c i d e i n brain t i s s u e increased 355-fold (from 0.5 to 192.2 ng endrin equivalents/mg wet weight o f t i s s u e ) when the i n s e c t i c i d e exposure l e v e l was r a i s e d 1 7 - f o l d (from 0.6 to 10 y g / l ) (submitted f o r p u b l i c a t i o n ) . It should be pointed out that the 314 y g / l exposure l e v e l s i n the R f i s h represents the 48-hr LC50 value while the 0.6 y g / l exposure l e v e l i s the 48-hr LC50 value f o r S f i s h . Therefore these data represent comparisons o f the S and R populations at both e q u i t o x i c and equal exposure l e v e l s o f e n d r i n . In most s t u d i e s i n s e c t i c i d e uptake i s measured a t a s p e c i f i c time o f exposure and does not take i n t o account the d i f f e r e n c e s in t o l e r a n c e to the i n s e c t i c i d e w i t h i n a p o p u l a t i o n . In such s t u d i e s , the more t o l e r a n t i n d i v i d u a l s o f a population are a c t u a l l y s e l e c t e d . I f comparisons are made w i t h i n and between the R and S populations based on t o x i c e f f e c t s as expressed by the appearance o f symptoms o f p o i s o n i n g , a c l e a r e r understanding of the r e l a t i o n s h i p between u p t a k e / d i s p o s i t i o n and t o x i c i t y i s possible. The i n s e c t i c i d e concentration i n the brain expressed as a r a t i o o f symptomatic (s) to asymptomatic (a) would give a measure o f the e f f e c t i v e n e s s o f the membrane b a r r i e r and some i n d i c a t i o n o f other f a c t o r s which might be i m p l i c a t e d i n t o x i c i t y . A symptomatic/asymptomatic r a t i o greater than 1 would be expected i f i n s e c t i c i d e concentration i n the t a r g e t organ was the only determining f a c t o r i n t o x i c i t y . L i k e w i s e , i f varying t a r g e t s i t e s e n s i t i v i t y i s a f a c t o r , a r a t i o o f 1 o r l e s s would be expected. Such comparison o f endrin accumulation within the r e s i s t a n t population (R /Ra) demonstrated a r a t i o s i g n i f i c a n t l y greater than 1 f o r the various b r a i n segments (Table V) (7_)· This i s i n d i c a t i v e o f more e f f e c t i v e membrane b a r r i e r s t o i n s e c t i c i d e penetration i n the more t o l e r a n t R f i s h , which leads to varying degrees o f i n s e c t i c i d e t o l e r a n c e w i t h i n the R p o p u l a t i o n . When a s i m i l a r comparison i s made w i t h i n the S population between symptomatic and asymptomatic f i s h ( S / S ) the r a t i o s are l e s s than 1 (Table V ) . Since these comparisons are between l e s s t o l e r a n t i n d i v i d u a l s to more t o l e r a n t i n d i v i d u a l s w i t h i n the S p o p u l a t i o n , the S / S a r a t i o s i n d i c a t e a range i n t a r g e t s i t e sensitivity. Such a range shows a b a s i c f l e x i b i l i t y w i t h i n the unselected population upon which s e l e c t i v e pressures could have acted to produce the R p o p u l a t i o n . s

s

a

s


9.

YARBROUGH AND CHAMBERS

Organochlorine

149

Insecticides

Table V - Ratios of R a d i o a c t i v i t y Accumulated in Brains of S u s c e p t i b l e (S) and Resistant (R) F i s h , Some of Which Were E x h i b i t i n g Symptoms (s) and Others not E x h i b i t i n g Symptoms (a) of I n s e c t i c i d e P o i s o n i n g . Treatment Consisted of a 6 hr Exposure to 1 4 c - e n d r i n . a


10 y g / l

1500 yg/1

Endrin

Endrin

Ss/Sa

Ss/Rab

Sa/Rab

Ss/Rsb

Rs/Rab

Ss/Rab

Forebrain

0.6

3.1**

5.1**

0.5**

3.7**

1.7*

Midbrain

0.7

2.9**

4.4**

0.5**

4.8**

2.2*

Hindbrain

0.6

3.1**

4.9**

0.4**

4.4**

2.0*

a

From Scales {7). b A l l s t a t i s t i c a l comparisons are between components of each ratio. S i g n i f i c a n t d i f f e r e n c e s were determined by the J > t e s t , Ρ < 0.05 (*), Ρ < 0.01 (**). When comparisons are made between p o p u l a t i o n s , both the e f f e c t i v e n e s s of the membrane b a r r i e r in R f i s h and the s e n s i t i v i t y of the t a r g e t s i t e can be demonstrated (Table V ) . When endrin S / R r a t i o s are compared, the r a t i o i s l e s s than 1; t h i s suggests that more i n s e c t i c i d e i s required to e l i c i t symptoms in the R than in the S f i s h . s

s

This implicated target s i t e i n s e n s i t i v i t y i s more e f f e c t i v e l y demonstrated when actual amounts of endrin present in brain t i s s u e of Ss f i s h are compared to R f i s h (7). In S f i s h the endrin concentration in the f o r e b r a i n i s 24 ng endrin equiv alents/mg protein while in the same brain f r a c t i o n of R f i s h there was 1150 ng endrin equivalents/mg p r o t e i n . F u r t h e r , when the amount of endrin in various brain f r a c t i o n s from R f i s h exposed to 1500 y g / l ^ C - e n d r i n i s monitored with time, up to 4560 ng endrin equivalents/mg protein appears in the brain f r a c t i o n s of R f i s h a f t e r 24 hr endrin exposure (Table V I ) . a

s

a

a

This varying s e n s i t i v i t y of the t a r g e t s i t e might e x p l a i n why uptake data does not seem to r e l a t e d i r e c t l y to t o x i c i t y (LC50 v a l u e s ) . From our data i t i s p o s s i b l e to suggest that more t o l e r a n t i n d i v i d u a l s within the R population would probably possess a high i n s e n s i t i v i t y to i n s e c t i c i d e s at the target s i t e and an e f f e c t i v e b a r r i e r to i n s e c t i c i d e p e n e t r a t i o n . The l e s s t o l e r a n t might possess only one of these f a c t o r s , or varying degrees of f u n c t i o n a l e f f e c t i v e n e s s of one or both f a c t o r s .


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PESTICIDE A N D XENOBIOTIC M E T A B O L I S M I N AQUATIC

ORGANISMS

I n d i v i d u a l s in the S population would probably not contain both f a c t o r s and the f a c t o r ( s ) , i f p r e s e n t , would not be as f u n c t i o n a l l y e f f e c t i v e as i n i n d i v i d u a l s in the R p o p u l a t i o n . Table VI - R a d i o a c t i v i t y in Brain F r a c t i o n s of Resistant F i s h Exposed to 1500 y g / l 14C-endrin not E x h i b i t i n g Symptoms of P o i s o n i n g 3


Brain F r a c t i o n ^ Exposure Time

Forebrain

Midbrain

Hindbrain

3

970 ± n o

640 +

40

950 +

6

1150 ± 140

940 +

80

1100 ± 130

9

1280 ± 170

890 +

80

1200 ±

12

2370 ± 370

1910 + 170

2240 ± 140

24

4220 ± 230

4350 + 210

4560 ± 270

90

80

a

From Scales (7_). b Each value represents a mean of 5 treatments of 3 f i s h each expressed as ng endrin equivalents/mg p r o t e i n ± SEM. Biotransformation The biotransformation systems involved in i n s e c t i c i d e metabolism have been studied in the R and S populations to determine any d i f f e r e n c e s which might be p o t e n t i a l c o n t r i b u t o r y f a c t o r s to or r e s u l t s of i n s e c t i c i d e r e s i s t a n c e . In a d d i t i o n , the p o s s i b i l i t y of mixed-function oxidase induction has been investigated. S p e c i f i c a l l y , the studies have encompassed a seasonal study of microsomal mixed-function oxidase (mfo) components, and studies of a l d r i n , d i e l d r i n and DDT metabolism. Seasonal Study of Mixed Function O x i d a s e s . — A seasonal study of hepatic microsomal mfo components has been conducted i n female R and S f i s h (submitted f o r p u b l i c a t i o n ) . Components studied were cytochromes P-450 and Jb5, NADPH-cytochrome c reductase, NADPH-dichlorophenolindophenol r e d u c t a s e , NADHcytochrome c^ reductase and NADH-cytochrome J55 reductase. All were monitored at 30°C by standard spectrophotometry methods f o l l o w i n g o p t i m i z a t i o n procedures (8, 9 1J0, 21, 12). Microsomal and t o t a l hepatic p r o t e i n (]3j and l i v e r weight to body weight r a t i o s were a l s o monitored. 9



9.


Organochlorine

Insecticides

151

The r e s u l t s i n d i c a t e d that microsomal mfo a c t i v i t i e s followed a d e f i n i t e seasonal p a t t e r n , with highest a c t i v i t i e s and l e v e l s o c c u r r i n g in the c o l d weather months. A l l parameters measured, except protein c o n c e n t r a t i o n , followed the same t r e n d s . Microsomal p r o t e i n concentration was r e l a t i v e l y constant throughout the study. Cytochrome P-450 and NADPH-cytochrome c reductase are presented as r e p r e s e n t a t i v e of the parameters i n v e s t i g a t e d ( F i g s . 1 and 2 ) . Both the seasonal patterns and the o v e r a l l range of a c t i v i t i e s were s i m i l a r in both p o p u l a t i o n s . The c y c l i c nature of the parameters i n v e s t i g a t e d may be the r e s u l t of the r e l a t i v e magnitude of microsomal hydroxylations during the year in r e l a t i o n s h i p to other microsomal processes such as b i o synthesis. Although the ranges of enzyme s p e c i f i c a c t i v i t i e s and cytochrome l e v e l s were about the same in both p o p u l a t i o n s , the c o n s i s t e n t l y greater r e l a t i v e l i v e r s i z e in the R than the S f i s h suggests a greater p o t e n t i a l f o r x e n o b i o t i c o x i d a t i o n ( F i g . 3 ) . Lower r e l a t i v e l i v e r s i z e in the summer in both populations r e f l e c t s the greater proportion of eggs in the t o t a l body mass. There has a l s o been a report of seasonal trends in mfo a c t i v i t i e s in the f e r a l roach (Leuciscus r u t i l u s ) with highest a c t i v i t i e s in the summer and of induction of mfo a c t i v i t y by environmental contaminants (14). A l d r i n and D i e l d r i n Metabolism.— The j_n vivo metabolism of the c h l o r i n a t e d a l i c y c l i c i n s e c t i c i d e s , a l d r i n and d i e l d r i n , has been measured. Fish were exposed to l ^ C - l a b e l l e d a l d r i n or d i e l d r i n f o r 6 hours. The metabolism of each compound was monitored by t h i n l a y e r chromatography of hexane and chloroformmethanol e x t r a c t s of l i v e r homogenates, followed by l i q u i d s c i n t i l l a t i o n counting of the spots ( 5 , 1 5 , 1 6 ) . When f i s h of both populations were exposed to 80 y g / l "I^Ca l d r i n , d i e l d r i n was the only detectable metabolite in the organic e x t r a c t s of the l i v e r . The percentage of r a d i o a c t i v i t y in the w a t e r - s o l u b l e f r a c t i o n in both populations was s m a l l . Although previous work had i n d i c a t e d a greater production of w a t e r - s o l u b l e m e t a b o l i t e ( s ) in the R population (1_5), more recent work i n d i c a t e d that the r e l a t i v e proportion of r a d i o a c t i v i t y in the w a t e r - s o l u b l e f r a c t i o n was s i m i l a r in both populations (Table VII). The r e l a t i v e conversion of a l d r i n to d i e l d r i n a l s o v a r i e d with the season, with the greatest conversion o c c u r r i n g in the winter (Table V I I I ) . This seasonal phenomenon c o r r e l a t e s with the above mentioned seasonal data i n d i c a t i n g greater mixedf u n c t i o n oxidase component a c t i v i t y in winter in both populations ( F i g . 1 and 2 ) .



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PESTICIDE AND XENOBIOTIC M E T A B O L I S M I N AQUATIC

Figure

1.

ORGANISMS

Cytochrome P-450 contents in liver microsomes from insecticideresistant and -susceptible mosquitofish

Figure 2. Specific activity of ΝADPH-Cytochrome c reductase in liver micro somes from insecticide-resistant and -susceptible mosquitofish. A unit (U) of enzyme activity is defined as 1 μτηοΐ product formed/min.


Organochlorine

Insecticides




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PESTICIDE AND XENOBIOTIC M E T A B O L I S M I N AQUATIC ORGANISMS

Table VII

- R a d i o a c t i v i t y i n Water-soluble F r a c t i o n s of L i v e r s of S u s c e p t i b l e (S) and Resistant (R) F i s h Exposed to 80 y g / l 1 4 C - a l d r i n f o r 6 h r a

Population


a

ng a l d r i n e q u i v a l e n t s / mg protein

Water s o l u b l e r a d i o a c t i v i t y χ 100/ t o t a l hepatic r a d i o a c t i v i t y

S

9 ± 3(11)

1.7 ± 0.3(11)

R

10 ± 2(24)

1.8 ± 0.2(20)

Values are expressed as mean ± SEM (N).

Table VIII -

Proportion o f D i e l d r i n Formed (percent o f t o t a l hepatic r a d i o a c t i v i t y ) in L i v e r s of S u s c e p t i b l e (S) and Resistant (R) Fish Exposed to 80 y g / l Caldrin for 6 hr 1

4

a

Season

s

R

Dec-Feb

47.5 ± 7.2(4)

52.8 ± 2.6(10)

Mar-May

29.1 ± 4.3(2)

13.7 ± 0.0(2)

Sep-Nov

25.0 ± 3.9(4)

28.1 ± 8.7(4)

a


No metabolites o f d i e l d r i n were observed i n orqanic e x t r a c t s of l i v e r s of e i t h e r S or R f i s h exposed to 30 y g / l ' ^ C - d i e l d r i n f o r 6 hr (5) (Table IX). A small percentage o f the r a d i o a c t i v i t y was found i n the w a t e r - s o l u b l e f r a c t i o n . T h e r e f o r e , i t appears that l i t t l e metabolism o f d i e l d r i n occurs in mosquitofish l i v e r s in e i t h e r p o p u l a t i o n . In a d d i t i o n , the degree o f i n t o x i c a t i o n i n S f i s h d i d not appear to a f f e c t the metabolism e i t h e r q u a l i t a t i v e l y or q u a n t i t a t i v e l y . DDT M e t a b o l i s m . - - The metabolism o f DDT has been studied i n R and S f i s h , f o l l o w i n g s i m i l a r protocols to c h l o r i n a t e d c y c l o diene metabolism: organic e x t r a c t i o n ( a c e t o n i t r i l e ) , t h i n l a y e r chromatography o f organic e x t r a c t s , and l i q u i d s c i n t i l l a t i o n counting o f the r e s u l t a n t spots ( 4 · ) . When S and R f i s h were exposed to 60 y g / l o f 1 4 C - l a b e l l e d _p_,£'-DDT f o r 4 h r , r a d i o a c t i v i t y was found in the spots which co-chromatographed with


9.


Organochlorine

155

Insecticides

DDT, DDD and DDE (Table X ) . There were no q u a l i t a t i v e q u a n t i t a t i v e d i f f e r e n c e s between the two p o p u l a t i o n s .

or

Table IX - R a d i o a c t i v i t y i n L i v e r s of S u s c e p t i b l e (S) F i s h E x h i b i t i n g Symptoms of Poisoning (s) and S and Resistant (R) F i s h not E x h i b i t i n g Symptoms of Poisoning ( a ) . Fish were Exposed to 30 y g / l 1 4 c - d i e l d r i n for 6 h r a

Dieldrin


Population

a

Water-soluble

ng d i e l d r i n e q u i v a l e n t s / ng d i e l d r i n mg protein % equivalents/mg protein

%

128.6

± 13.0(4)

97

3.9 ± 0.4(4)

3

Ss

162.7

± 14.5(4)

95

7.8 ± 0.9(4)

5

Ra

136.3

±

98

3.2 ± 0.3(5)

2

8.8(5)


From Watkins

(j3).

Table X - DDT and Metabolite Concentrations i n L i v e r s of S u s c e p t i b l e (S) and Resistant (R) F i s h Exposed to 60 y g / l 14c-p,jj'-DDT f o r 4 h r a

S ng DDT e q u i v a l e n t s / mg protein 624 + 36(6)

DDT

R

% 67.0

ng DDT e q u i v a l e n t s / mg protein

%

483 + 36(7)

74.1

DDD

75 ±

5(5)

8.0

42 ±

4(5)

6.5

DDE

109 ±

7(6)

11.7

51 +

6(5)

7.9

7.3

48 ±

8(6)

7.4

Water Soluble a

68 ± 10(6)

Data are expressed as mean ± SEM (N).

From Hamilton

(4).

Mixed-function Oxidase I n d u c t i o n . - - I n d i r e c t evidence of environmental induction of d e t o x i f y i n g enzymes i n the R f i s h has been observed as an increase i n the acute t o x i c i t y of parathion and a simultaneous decrease i n NADPH-dependent parathion


156

PESTICIDE AND XENOBIOTIC M E T A B O L I S M I N AQUATIC

ORGANISMS

d e a r y ! a t i o n in R f i s h with time held in the l a b o r a t o r y (17,18). However, d i r e c t attempts to induce mixed-function oxidase a c t i v i t y in mosquitofish have been d i f f i c u l t (19,20). Using DDT as a p o s s i b l e inducer r e s u l t e d in an i n c o n s i s t e n t degree of enzyme i n d u c t i o n , with some experiments y i e l d i n g no induction at all (19).


Discussion The uptake and d i s t r i b u t i o n of organochlorine i n s e c t i c i d e s has been studied under a v a r i e t y of c o n d i t i o n s . Although the r e s u l t s i n d i c a t e that f u r t h e r study i s needed on a c h a r a c t e r i z a t i o n of extraneous f a c t o r s that a f f e c t d i s p o s i t i o n , the studies c l e a r l y demonstrate the presence of a membrane b a r r i e r to i n s e c t i c i d e penetration in the R p o p u l a t i o n . This membrane b a r r i e r would a i d in the p r o t e c t i o n of t a r g e t s i t e s in the R f i s h from the i n s e c t i c i d e . This b a r r i e r i s f e l t to be an important f a c t o r in r e s i s t a n c e to organochlorine i n s e c t i c i d e s in mosquitofish. F u r t h e r , by v i r t u e of t h e i r l a r g e r l i v e r s , the R f i s h have a greater xenobiotic biotransformation p o t e n t i a l . However, the in vivo s t u d i e s show few c o n s i s t e n t d i f f e r e n c e s in metabolism between the two p o p u l a t i o n s . Biotransformation may be a major c o n t r i b u t o r y f a c t o r in mosquitofish r e s i s t a n c e to other p e s t i c i d e s , f o r example, organophosphorus and botanical i n s e c t i c i d e s , s i n c e the l e v e l of r e s i s t a n c e to these chemicals i s very low (4 f o l d or l e s s ) (18,20,21). However, biotransformation does not appear to play a major r o l e i n organochlorine i n s e c t i c i d e r e s i s t a n c e . The l e v e l s of r e s i s t a n c e which the mosquitofish demonstrate toward the c h l o r i n a t e d a l i c y c l i c i n s e c t i c i d e s (40 - 500 f o l d ) are the most i n t r i g u i n g of those s t u d i e d , yet they are impossible to e x p l a i n i n terms of d i s p o s i t i o n and biotransformation a l o n e . Although i n s e c t i c i d e metabolism cannot be completely d i s c o u n t e d , i t c o n t r i b u t e s l i t t l e to c h l o r i n a t e d a l i c y c l i c r e s i s t a n c e . B a r r i e r s to i n s e c t i c i d e penetration undoubtedly c o n t r i b u t e to chlorinated a l i c y c l i c resistance. However, we are led to conclude that these extremely high l e v e l s of r e s i s t a n c e are the r e s u l t of a postulated i n s e n s i t i v i t y of the t a r g e t s i t e which allows these f i s h to t o l e r a t e elevated i n t e r n a l l e v e l s of these t o x i c a n t s . We have, t h e r e f o r e , been able to i n d i r e c t l y assess the importance o f three f a c t o r s involved in c h l o r i n a t e d a l i c y c l i c i n s e c t i c i d e r e s i s t a n c e in m o s q u i t o f i s h : d i s p o s i t i o n , metabolism and t a r g e t s i t e s e n s i t i v i t y . In a highly p o l l u t e d environment in which mosquitofish have been placed under severe s e l e c t i v e pressures by chronic exposure to i n s e c t i c i d e s , the system of metabolism appears to be of l i t t l e s i g n i f i c a n c e in r e s i s t a n c e ; the


9.


Organochlorine

Insecticides

157

system of d i s p o s i t i o n with the development o f i n t e r n a l and external b a r r i e r s to i n s e c t i c i d e penetration i s the next most important; and the i m p l i c a t e d t a r g e t s i t e i n s e n s i t i v i t y i s p o t e n t i a l l y the most s i g n i f i c a n t f a c t o r i n the s u r v i v a l o f the population.

Abstract An insecticide-resistant (R) population of Gambusia affinis demonstrates a 5 to 500 fold resistance to organochlorine insecticides when the 48-hr LC values between the R population and a corresponding susceptible (S) population are compared. Uptake and disposition studies indicate that there is greater insecticide accumulation in tissues of S fish than in those of R fish. However, the difference in uptake between the two populations is not proportional to the degree of resistance for individual insecticides. Resistant fish can accumulate far greater body loads of insecticide without demonstrating symptoms of poisoning than can S fish.


50

Hepatic mixed-function oxidase activities demonstrated seasonal trends, with higher specific activities in the cold weather months in both populations with few differences in enzyme activities or cytochrome levels between the two populations. Metabolism of aldrin, dieldrin and DDT was similar between the two populations. R fish have larger relative liver size and, therefore, a greater potential for xenobiotic metabolism. However, biotransformation appears to be of minor importance in chlorinated alicyclic insecticide resistance in mosquitofish; barriers to penetration appear to be of greater importance; and an implied target site insensitivity appears to be the most important factor in resistance.


158

PESTICIDE AND XENOBIOTIC METABOLISM IN AQUATIC ORGANISMS

Acknowledgments This work was supported i n part by National I n s t i t u t e s of Health grants 5 ROI ES00412 and 5 ROI ES00840.


Literature Cited

1. Rehwoldt, R.E., Kelley, E. and Mahoney, M. Investigations into acute toxicity and some chronic effects of selected herbicides and pesticides on several fresh water fish species. Bull. Environ. Contam. Toxicol. (1977) 18:361-365. 2. Culley, D.D. and Ferguson, D.E. Patterns of insecticide resistance in the mosquitofish. J. Fish Res. Bd. Can. (1969) 26:2395-2401. 3. Ferguson, D.E., Ludke, J.L. and Murphy, G.G. Dynamics of endrin uptake and release by resistant and susceptible strains of mosquitofish. Trans. Amer. Fish Soc. (1966) 95:335-344. 4. Hamilton, M.L. "Uptake and metabolism of DDT by insecticide-susceptibleand -resistant mosquitofish, Gambusia affinis." M.S. Thesis, Mississippi State University (1976) 48 p. 5. Watkins, J.H. "A comparative study of the uptake and metabolism of aldrin and dieldrin in insecticide-resistant and -susceptible mosquitofish (Gambusia affinis)." M.S. Thesis, Mississippi State University (1974) 39 p. 6. Hunsinger, R.N. "Uptake and disposition of endrin in insecticide-susceptible and -resistant mosquitofish (Gambusia affinis)." M.S. Thesis, Mississippi State University (1978) 26 p. 7. Scales, E.H. "Comparative studies of endrin uptake in insecticide-resistant and -susceptible mosquitofish (Gambusia affinis)." M.S. Thesis, Mississippi State University (1973) 32 p. 8. Omura, T. and Sato, R. The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J. Biol. Chem. (1964) 239:2370-2378. 9. Strittmatter, P. NADH-cytochrome b5 reductase. "Methods of Enzymology, X." (Ed. by Estabrook, R.W. and Pullman, M.E.) p. 561-565. Academic Press. New York. 1967 10. Ernster, L., Siekevitz, P. and Palade, G.E. Enzyme-structure relationships in the endoplasmic reticulum of rat liver. J. Cell. Biol. (1962) 15:541-562. 11. Masters, B.S.S., Williams, C.H., Jr., and Kamin, H. The preparation and properties of microsomal TPNH-cytochrome c reductase from pig liver. "Methods in Enzymology, X." (Ed. by Estabrook, R.W. and Pullman, M.E.) p. 565-573. Academic Press. New York. 1967.



9.

YARBROUGH

AND

CHAMBERS

Organochlorine

Insecticides

159

12. Mackler, B. Microsomal DPNH-cytochrome c reductase. "Methods in Enzymology, X." (Ed. by Estabrook, R.W. and Pullman, M.E.) p. 551-553. Academic Press. New York. 1967. 13. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. (1951) 193:265-275. 14. Dewaide, J.H. and Henderson, P. Th. Seasonal variation of hepatic drug metabolism in the roach, Leuciscus rutilus L. Comp. Biochem. Physiol. (1969) 32:489-497. 15. Wells, M.R., Ludke, J.L. and Yarbrough, J.D. Epoxidation and fate of [14C] aldrin in insecticide-resistant and -susceptible populations of mosquitofish (Gambusia affinis). J. Agric. Food Chem. (1973) 21:428-429. 16. Chambers, J.E. and Yarbrough, J.D. Aldrin metabolism in insecticide-resistant and susceptible mosquitofish. Federation Proc. (1978) 37:693. 17. _. Organophosphate degradation by insecticide -resistant and -susceptible populations of mosquitofish (Gambusia affinis). Pestic. Biochem. Physiol. (1973) 3:312-316. 18. _. Parathion and methyl parathion toxicity to insecticide-resistant and -susceptible mosquitofish (Gambusia affinis). Bull. Environ. Contam. Toxicol. (1974) 11:315-320. 19. Chambers, J.E. Induction of mixed-function oxidase activity by DDT in the mosquitofish. Federation Proc. (1976) 35:375. 20. Fabacher, D.L. and Chambers, H. Apparent resistance to pyrethroids in organochlorine-resistant mosquitofish. Proc. 26th Ann. Conf. Southeastern Game Fish Comm. (1972) p. 461-464. 21. _. Rotenone tolerance in mosquitofish. Environ. Pollut. 3:139-141. RECEIVED

January 2, 1979.


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