The Pesticide Chemist and Modern Toxicology - American Chemical

16 Metabolic Aspects of Pesticide Toxicology G. W A Y N E IVIE Veterinary Toxicology and Entomology Research Laboratory, Agricultural Research, Science and Education Administration, U.S. Department of Agriculture, College Station, T X S. KRIS B A N D A L

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Agricultural Products, 3M Company, St. Paul, M N

55101

At least 1500 organic and inorganic chemicals are used in a manner such that they can be called pesticides (1). These chemicals are indispensable in the management of a seemingly endless variety of pest organisms, including insects, weeds, fungi, bacteria, pest birds and mammals, and others. Pesticides are intentionally applied to many components of the environment, and they or their degradation products often move quite freely through the environment by mechanisms such as runoff, leaching, and volatilization. The production and use of pesticides on a world scale exceeds 3 billion pounds annually ( 1 ) , and it can safely be said that residues of various pesticides interact at some level with virtually all components of the environment. Pesticides by design are meant to be toxic! Although a major goal of the discipline of modern pesticide chemistry is to develop pesticides and consequent use patterns that confine pesticide toxicity to pest organisms, such a goal is seldom attained easily. All living organisms have much in common biochemically, and successful exploitation of the often relatively minor biochemical differences between pest and non-pest species is almost always difficult and is, in fact, sometimes impossible. Thus, i t is often necessary to use pesticides that are toxic not only to the pest species but to other organisms as well. Even when we succeed in developing what appear to be highly efficacious yet selective pesticides, we are always concerned that interactions of these chemicals or their transformation products with non-target species, particularly man, may result in some unforeseen toxic consequences. From t h e human p e r s p e c t i v e , t h e d i r e c t t o x i c o l o g i c a l i m p l i c a t i o n s o f p e s t i c i d e use t o o u r own s p e c i e s m e r i t t h e most t h o r o u g h and serious consideration. Most would a g r e e t h a t t h e j u d i c i o u s use of p e s t i c i d e s contributes i n a p o s i t i v e way t o many a s p e c t s o f human w e l f a r e , b u t we a l s o r e c o g n i z e t h a t t h e s e c h e m i c a l s h a v e g e n u i n e p o t e n t i a l f o r a d v e r s e human e f f e c t s . Therefore, i f the proposed use p a t t e r n s of a pesticide create a substantial l i k e l i h o o d t h a t i n t e r a c t i o n s w i t h man may o c c u r , i t i s p r u d e n t t o

0097-6156/81/0160-0257$07.00/0 © 1981 American Chemical Society In The Pesticide Chemist and Modern Toxicology; Bandal, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

T H E

258

PESTICIDE

C H E M I S T

A N D

M O D E R N TOXICOLOGY


define both the extent of these interactions and their toxicological significance. Our discussion w i l l center on the r o l e p l a y e d by m e t a b o l i s m i n t h e e x p r e s s i o n o f p e s t i c i d e t o x i c i t y and t h e e v a l u a t i o n o f t o x i c o l o g i c a l s i g n i f i c a n c e . We w i l l b r i e f l y d i s c u s s the importance of m e t a b o l i s m s t u d i e s i n d e v e l o p i n g more efficacious and selective pesticides. We will discuss the rationale and appropriate methodology used by metabolism s c i e n t i s t s i n t h e d e s i g n and e x e c u t i o n o f s u c h s t u d i e s . Finally, and most i m p o r t a n t l y , we w i l l a t t e m p t t o show how the metabolism o f p e s t i c i d e s may a f f e c t t h e i r t o x i c i t y , and how t h e d a t a from p e s t i c i d e m e t a b o l i s m s t u d i e s are used i n the p r o c e s s of e v a l u a t i n g t o x i c o l o g i c a l r i s k to man. The

Nature of

Metabolic

Reactions

Pesticides are transformed by living organisms through a great d i v e r s i t y of metabolic r e a c t i o n s . T h e s e r e a c t i o n s can be conveniently grouped into two categories, primary or phase I reactions, which are those that create or modify functional g r o u p s , and s e c o n d a r y or phase I I r e a c t i o n s , which are conjugations. A few e x a m p l e s a r e shown i n F i g u r e 1. Some a u t h o r s (2) feel that the terms phase I and phase II are not totally satisfactory because numerous e x a m p l e s a r e known o f phase II reactions preceding phase I r e a c t i o n s ( e . g . , d i r e c t conjugations of c h l o r i n a t e d phenols, Figure 1). Most p e s t i c i d e s , h o w e v e r , do not l e n d t h e m s e l v e s to phase I I r e a c t i o n s w i t h o u t p r i o r phase I modifications. Although i t is generally true that phase I metabolism of pesticides effects partial or complete detoxification, at least from an acute toxicity standpoint, m e t a b o l i c a c t i v a t i o n s do o c c u r and can be o f g r e a t t o x i c o l o g i c a l significance. Phase I I o r c o n j u g a t i o n r e a c t i o n s more o f t e n t h a n n o t s e r v e t o r e n d e r p e s t i c i d e s o r t h e i r m e t a b o l i t e s more p o l a r f o r more efficient excretion (e.g., in urine of mammals) or to f a c i l i t a t e transport for i n t e r n a l storage i n organisms that lack efficient excretory systems (e.g., plants). It is probably c o r r e c t t h a t most l i v i n g o r g a n i s m s can m e t a b o l i z e p e s t i c i d e s v i a b o t h p h a s e I and p h a s e I I m e t a b o l i c p a t h w a y s . The s c h e m a t i c shown i n F i g u r e 2 i s d e s i g n e d t o r e p r e s e n t the m a j o r m e t a b o l i c and d i s p o s i t i o n p a t t e r n s t h a t d i f f e r e n t p e s t i c i d e t y p e s might undergo i n h i g h e r animal systems. We have somewhat arbitrarily grouped pesticides into four categories, based on polarity. A very few p e s t i c i d e s , p r i m a r i l y some organochlorine i n s e c t i c i d e s and p a r t i c u l a r l y the i n s e c t i c i d e mirex, are highly l i p o p h i l i c , a r e q u i t e m e t a b o l i c a l l y s t a b l e , and t e n d t o be stored i n f a t w i t h m i n i m a l o r no m e t a b o l i s m . D i r e c t e l i m i n a t i o n through l i p i d c o n t a i n i n g a n i m a l b y p r o d u c t s ( m i l k o r eggs) t e n d s a l s o t o be an a p p r e c i a b l e t o m a j o r d i s p o s i t i o n mechanism f o r s u c h h i g h l y l i p o p h i l i c compounds. Most i n s e c t i c i d e s a r e l i p o p h i l i c , y e t are r a p i d l y m e t a b o l i z e d by b o t h phase I and phase I I r e a c t i o n s and are u l t i m a t e l y excreted from t h e b o d y . Some p e s t i c i d e s , i n c l u d i n g

In The Pesticide Chemist and Modern Toxicology; Bandal, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.


Phase II Metabolite

Figure L Examples of Phase I and Phase II metabolites of the carbamate insecticide carbanolate, the synthetic pyrethroid insecticide permethrin, and the wood preservative pentachlorophenol

PERMETHRIN

Pesticide

Phase I Metabolite


THE

260

PESTICIDE CHEMIST

A N D M O D E R N

TOXICOLOGY


phenolics, amines, e t c . , are r e a s o n a b l y polar compounds that generally have functionalities that permit direct conjugation reactions. O t h e r s , such as h e r b i c i d e s formulated as s a l t s , o r compounds that contain moieties that readily ionize at physiological pH, c a n be c o n s i d e r e d h y d r o p h i l i c and a r e o f t e n e x c r e t e d r a p i d l y w i t h o u t any m e t a b o l i s m a t a l l . Phase I a n d phase I I p e s t i c i d e m e t a b o l i t e s , and p o s s i b l y e v e n t h e p a r e n t p e s t i c i d e , may have t h e p o t e n t i a l f o r c h e m i c a l sequestration (i.e., covalent b i n d i n g , F i g u r e 2 ) w i t h t i s s u e components t h a t may u l t i m a t e l y l e a d t o the e x p r e s s i o n o f c h r o n i c t o x i c i t y . Most o r g a n i s m s , r e g a r d l e s s o f c o m p l e x i t y , s h a r e a number o f biochemical pathways f o r m e t a b o l i z i n g pesticides. Examples can r e a d i l y be f o u n d t o show t h a t many t y p e s o f p l a n t s and a n i m a l s m e t a b o l i z e p e s t i c i d e s by each of the four b a s i c types o f m e t a b o l i c changes: o x i d a t i o n , r e d u c t i o n , h y d r o l y s i s , and c o n j u g a t i o n (3_). Of course, species do d i f f e r i n the metabolism of p e s t i c i d e s , t h e s e d i f f e r e n c e s a r e sometimes q u i t e d r a m a t i c , and t h e y c a n be o f great significance in interpreting comparative toxicological effects. A l s o , s p e c i e s d i f f e r e n c e s i n p e s t i c i d e m e t a b o l i s m , once i d e n t i f i e d , q u i t e o f t e n p r o v i d e i m p e t u s t o t h e d e v e l o p m e n t o f more s e l e c t i v e p e s t - c o n t r o l agents. It i s not our purpose here to e x t e n s i v e l y review the l i t e r a t u r e on t h e m e t a b o l i s m o f i n d i v i d u a l p e s t i c i d e s b y a v a r i e t y of l i v i n g organisms. Numerous s u c h r e v i e w s a r e a v a i l a b l e , some a r e p e r i o d i c a l l y u p d a t e d , and we r e f e r t h e r e a d e r to several of t h e s e f o r an o v e r v i e w o f t h e v o l u m i n o u s l i t e r a t u r e i n t h i s field (4-14). Metabolic

Basis

for Pesticide

Selectivity

In t h e use o f p e s t i c i d e s , a t t e m p t s a r e a l w a y s made t o d i r e c t t h e i r t o x i c a c t i o n s t o w a r d an i n d i v i d u a l o r g r o u p o f p e s t s p e c i e s , and i t i s a major goal of the p e s t i c i d e s c i e n t i s t to develop e f f i c a c i o u s p e s t i c i d e s and use p a t t e r n s such t h a t l i t t l e o r no t o x i c i t y to other l i f e forms o c c u r s . Such an a p p r o a c h i s c l e a r l y desirable from an e n v i r o n m e n t a l standpoint, but i t often has d e f i n i t e e c o n o m i c a d v a n t a g e s a l s o ( e . g . , p r o t e c t i n g p r e d a t o r s and parasites while controlling a pest insect). In some circums t a n c e s , a degree o f s e l e c t i v i t y i s a b s o l u t e l y e s s e n t i a l f o r the i n t e n d e d use ( e . g . , h e r b i c i d e s c a n n o t be l e t h a l t o t h e p r o t e c t e d crop) . Metabolism s t u d i e s i n the pest s p e c i e s , i n the s p e c i e s b e i n g p r o t e c t e d , and i n a s s o c i a t e d n o n t a r g e t o r g a n i s m s , c a n and o f t e n do p r o v i d e a w e a l t h o f u s e f u l i n f o r m a t i o n . Such s t u d i e s may lead t o a more thorough understanding o f t h e mechanisms o f p e s t i c i d a l a c t i o n , and t h i s knowledge o f t e n l e a d s i n t u r n t o t h e d e v e l o p m e n t o f more e f f i c a c i o u s , s e l e c t i v e , and environmentally acceptable pest c o n t r o l agents. W h i l e n o t a l w a y s s o , s e l e c t i v e t o x i c i t y c a n q u i t e o f t e n be attributed p r i m a r i l y i f not t o t a l l y to metabolic differences between s p e c i e s , e i t h e r i n the r a t e o f m e t a b o l i s m o r the n a t u r e o f


16.

iviE AND

products

Metabolic

BANDAL

formed.

The

w e l l - k n o w n example to to

a number

of pest

mammals.

i t i s very

(Figure 4 )

i s also

species, at

least

is

determines process Figure in

that 5)

to

serves

mammals by

to

these

to

Another

While

no

illustration.

an

N-hydroxylation

and

or

In

pig

any

species

the

guinea

appreciable

detoxification are

of

examples

influence the

with

T h i s compound

subsequent

pesticides

conjugation

to

lemming, N - h y d r o x y l a t i o n AAF

(AAF,

i s metabolized

and

and

on

metabolic

7-hydroxylation

extent,

to

Species

i s not

to

yield

inactive does

carcinogenic

I.

Differences in Its

% N-OH

Species pig

Lemming

of

0

72

trace

42

1-15

19-27

Rabbit

13-30

15-29

Hamster

15-20

35-39

Rat

Dog

5

Man

4-14 Smith

(Ref

(AAF)

Metabolism

Dose 7-OH

of

detoxification

metabolic

nature

occurs

examples

(Table I ) .

Related

From

ragweed,

by

Table

Guinea

of

and

in

C a r c i n o g e n i c i t y of 2 - A c e t y l a m i n o f l u o r e n e

Mammalian as

whereas

2-acetylaminofluorene

carcinogen

in

herbicide crop,

linuron

dramatic

as

rate

the

metabolic

the

the

slower

of

N-demethylation

of

mind,

that

monocarboxylic

rates

and

kind

toxicity

fact

i n metabolic

metabolic

involve

in

the

a

toxic

Carrot, a tolerant

by

rate

metabolites,

metabolites. occur

the

low

by

much

differences

Malathion

could

occurs.

carcinogenic not

which

toxicity

come r e a d i l y

to

linuron,

selectivity.

selective

a

nonherbicidal products,

(J_6 ) .

slowly in

due

at

is

i s highly

nontoxic

selectivity

linuron

s u s c e p t i b l e to

pesticides

The

a

i n some c a s e s .

metabolizes

more

to

occurs

(_1_5 ) .

linuron

which

explained

malathion

reaction

to

are

3)

(Figure

species, yet

between

much

malathion

insect

differences

261

Toxicology

Malathion

this

N-demethoxylation

Pesticide

selectivity.

insects

rapidly


insecticide

metabolize

whereas

susceptible

of

such

These

mammals r e a d i l y acid,

of

Aspects

Carcinogenicity

+ + + +

25-30

17).


262

T H E

PESTICIDE

PESTICIDAL HYDROPHILIC

POLAR

CHEMIST

A N D M O D E R N

TOXICOLOGY

CHEMICALS LIPOPHILIC

HIGHLY LIPOPHILIC (Metabolically Stable) -I

PHASE I

PHYSICAL

METABOLISM

SEQUESTRATION


11 (Fat)

(Ox., Red., Hydr.)

χ PHASE II

CHEMICAL

METABOLISM

SEQUESTRATION (Tissue Binding)

(Conjugation)

1

UNABSORBED DOSE

|

1 1

\

τ

BILIARY EXCRETION sENTEROHEPATIC

i

RENAL EXCRETION

|

CIRCULATION

MILK ^ /

URINE

FECES-

EGGS

Figure 2. Schematic of the major metabolic and disposition patterns of pesticides in higher animal systems. Pathways indicated by dashed lines are generally minor ones from a quantitative standpoint.

s H CCUI

o

S II

Q

H CO 3

hUCOvH H CO

15

I

3

2 H Ο

MALATHION Figure 3.

2 5

/

O M

J 2 H Ο

2 5

MALATHION «-MONOACID

Metabolic detoxification of the insecticide malathion


IVIE

AND

B A N D A L

Metabolic

Aspects of Pesticide

Toxicology

263


16.

Figure 5.

Mammalian metabolism of AAF to carcinogenic and noncarcinogenic metabolites


THE

264

PESTICIDE

CHEMIST

A N D M O D E R N

TOXICOLOGY

With the r a t , r a b b i t , hamster, and dog, however, AAF i s m e t a b o l i z e d t o a p p r e c i a b l e amounts o f t h e N - h y d r o x y m e t a b o l i t e , and AAF i s carcinogenic to these animals. Man likewise m e t a b o l i z e s AAF b y N - h y d r o x y l a t i o n , and w h i l e t h e c a r c i n o g e n i c i t y o f AAF t o man i s n o t c l e a r l y e s t a b l i s h e d , t h e i m p l i c a t i o n s a r e o b v i o u s (J_7 ) . More d e t a i l e d t r e a t m e n t s o f t h e m e t a b o l i c b a s i s f o r p e s t i c i d e s e l e c t i v i t y a r e a v a i l a b l e ( 4 , JJ5, VB_) .


Metabolism

S t u d i e s and S a f e t y E v a l u a t i o n

E v a l u a t i n g the t o x i c o l o g i c a l s i g n i f i c a n c e of p e s t i c i d e s to man i s seen t o be a h i g h l y complex a f f a i r when one c o n s i d e r s t h e v a r i o u s ways t h a t p e s t i c i d e s a r e u s e d , t h e r o u t e s b y w h i c h man may be e x p o s e d t o them a n d , p e r h a p s most i m p o r t a n t l y , t h e m u l t i t u d e o f chemical transformations that pesticides often undergo before man's e x p o s u r e t o them. Thus, while i t i s s u r e l y a p p r o p r i a t e to define the t o x i c o l o g i c a l interactions of a p e s t i c i d e ' s active i n g r e d i e n t i n e x p e r i m e n t a l a n i m a l s f o r e x t r a p o l a t i o n t o man, i t i s e n t i r e l y p o s s i b l e t h a t s u c h s t u d i e s may i n some c a s e s have l i t t l e relevance to real world human exposure. Because of the environmental i n s t a b i l i t y o f most o r g a n i c p e s t i c i d e s , i t seems reasonable and i n f a c t l i k e l y t h a t t h e g r e a t m a j o r i t y o f human exposure to p e s t i c i d e r e s i d u e s i s to products of t h e i r decompos i t i o n r a t h e r than to the p a r e n t m o l e c u l e . T h u s , n o t o n l y must we as m e t a b o l i s m s c i e n t i s t s d e l i n e a t e t h e b i o c h e m i c a l pathways o f p e s t i c i d e s i n e x p e r i m e n t a l a n i m a l s t h a t a r e r e p r e s e n t a t i v e o f man, we must as w e l l c l e a r l y d e f i n e t h e n a t u r e o f t h e i r environmental t r a n s f o r m a t i o n s , _ i f the products generated are l i k e l y t o i n t e r a c t w i t h man. While environmental t r a n s f o r m a t i o n s o f p e s t i c i d e s may o c c u r as t h e r e s u l t o f e i t h e r b i o c h e m i c a l ( m e t a b o l i c ) o r p h y s i c o chemical (e.g., photochemical) r e a c t i o n s , and b o t h have toxicological implications, our purpose here i s to consider only metabolic transformations. F o r any g i v e n p e s t i c i d e and use p a t t e r n , i t i s e a s i l y seen t h a t s e v e r a l t y p e s o f m e t a b o l i s m s t u d i e s may be needed t o p r o v i d e a framework f o r e v a l u a t i n g t h e t o x i c o l o g i c a l s i g n i f i c a n c e o f t h e compound t o man. As an e x a m p l e , we c a n c o n s i d e r a systemic i n s e c t i c i d e used as a s o i l - i n c o r p o r a t e d g r a n u l a r f o r m u l a t i o n f o r i n s e c t c o n t r o l on c o r n . B e c a u s e c o r n i s consumed b y b o t h man and h i s food a n i m a l s , s e v e r a l types o f metabolism s t u d i e s a r e approp r i a t e , i n c l u d i n g s t u d i e s o f t h e p e s t i c i d e i t s e l f i n one o r more l a b o r a t o r y monogastric mammals considered t o be human models. Metabolism s t u d i e s i n c o r n a r e needed t o determine t h e n a t u r e o f r e s i d u e s t o w h i c h man may be e x p o s e d t h r o u g h c o n s u m p t i o n o f c o r n from t r e a t e d c r o p s . S t u d i e s a r e a l s o needed i n food a n i m a l s t h a t a r e g i v e n c o r n i n t h e d i e t ( e . g . , c a t t l e , s w i n e , and p o u l t r y ) t o a s s e s s t h e e x t e n t t o w h i c h t h e p e s t i c i d e o r i t s m e t a b o l i t e s may a p p e a r i n meat, m i l k , p o u l t r y o r e g g s i n t e n d e d f o r human consumption. D a t a from a s o i l m e t a b o l i s m s t u d y m i g h t l i k e w i s e be needed if potentially toxic soil metabolites a r e a s s i m i l a t e d by the


i v i E AND BANDAL

16.

treated or

crop.

With

alternative

emphasized distinctions and

a r e from

must

means

study, or

of

question more

we

are attempting

changes

recognize

an e n d .

importantly,

here

standpoint

t o make u n d e r that

pesticide

265

additional I t must be

to

physicochemical

For the u l t i m a t e

segregate ones,

rather

field

such

arbitrary

conditions.

metabolism

organisms to

assess

value

plants, birds,

of data

toxicological

t o lower

Toxicology

studies

a s an end i n t h e m s e l v e s ; b u t r a t h e r , t h e y a r e

i s i t s yield

the

from

a toxicological

be i t i n m i c r o o r g a n i s m s ,

whatever,

ment


toward

of Pesticide

p e s t i c i d e s and use p a t t e r n s ,

may be d i f f i c u l t

always

c a n n o t be c o n s i d e r e d a

Aspects

s t u d i e s may be a p p r o p r i a t e .

although

or metabolic

i n any case We

other

metabolism

that,

biochemical

Metabolic

valuable

significance (i.e.,

of a

toward of

further

the

i t s environmental

toxicological

metabolism

l a b o r a t o r y mammals, assess-

pesticide

in

impact) o r ,

significance

t o man

himself. Methodology, Goals,

and R e g u l a t o r y

Considerations

A l t h o u g h t h e word " m e t a b o l i s m " ( G r . m e t a b o l e , c h a n g e ) h a s a rather limited connotation, a "pesticide metabolism study" i s u s u a l l y considered i n a broad s e n s e t o encompass n o t o n l y t h e metabolic a l t e r a t i o n s of the chemical i n question but also the a b s o r p t i o n , t r a n s p o r t , s t o r a g e , and e x c r e t i o n o r e l i m i n a t i o n o f t h e p a r e n t p e s t i c i d e and i t s m e t a b o l i t e s b y t h e e x p o s e d o r g a n i s m . The schematic i n F i g u r e 6 shows t h a t p e s t i c i d e " m e t a b o l i s m " c a n be considered a s more o r l e s s synonomous w i t h the toxokinetic phase of a pesticide/organism interaction. Of c o u r s e , a n y metabolic transformation that occurs i n the gut p r i o r to a b s o r p t i o n o f t h e p e s t i c i d e would be c o n s i d e r e d , and i s i n f a c t , metabolism. B e c a u s e p e s t i c i d e use p a t t e r n s o f t e n d i c t a t e t h a t m e t a b o l i s m s t u d i e s be c o n d u c t e d i n a number o f w i d e l y d i v e r g e n t l i f e forms, i t i s c l e a r t h a t no s i n g l e a p p r o a c h i s a p p r o p r i a t e f o r a l l c i r c u m stances. Thus, metabolism studies i n microorganisms, plants, mammals, e t c . , require specialized approaches based on t h e i n h e r e n t n a t u r e o f t h e o r g a n i s m and t h e g o a l s o f t h e s t u d y i t s e l f . Quite often t o o , the p o t e n t i a l use p a t t e r n s o f p e s t i c i d e s may d i c t a t e d i f f e r i n g m e t h o d o l o g i e s f o r s t u d i e s i n t h e same s p e c i e s . F o r e x a m p l e , m e t a b o l i s m s t u d i e s i n c a t t l e w i t h a p e s t i c i d e u s e d on f e e d g r a i n s o r f o r a g e c l e a r l y need be done o n l y w i t h o r a l administ r a t i o n , b u t i f a p r o d u c t i s t o be u s e d f o r e c t o p a r a s i t e c o n t r o l on c a t t l e a s a d e r m a l s p r a y , t h e d e r m a l r o u t e o f e x p o s u r e w o u l d a l s o be a p p r o p r i a t e . Given a s u i t a b l e experimental d e s i g n , what t h e n i s o u r g o a l as m e t a b o l i s m scientists i n conducting such a study? It i s , simply put, to define a c c u r a t e l y and t o t h e f u l l e s t extent p o s s i b l e t h e k i n e t i c and m e t a b o l i c b e h a v i o r o f t h e p e s t i c i d e u n d e r s t u d y i n an a p p r o p r i a t e o r g a n i s m u n d e r t h e c o n d i t i o n s c h o s e n . We want t o know how and a t what r a t e t h e p e s t i c i d e i s a b s o r b e d i n t o the living s y s t e m , t o what p r o d u c t s i t i s metabolized, and t o



266

THE

PESTICIDE

CHEMIST

A N D

M O D E R N

TOXICOLOGY

where and t o what e x t e n t t h e s e p r o d u c t s a r e t r a n s p o r t e d , stored, and e x c r e t e d . Our most i m p o r t a n t and u s u a l l y most d i f f i c u l t t a s k i s , o f c o u r s e , t o d e f i n i t i v e l y c h a r a c t e r i z e the c h e m i c a l n a t u r e o f a s many o f the m e t a b o l i t e s as p o s s i b l e , g i v e n the l i m i t a t i o n s of our a n a l y t i c a l and spectrometric techniques and of our own scientific capabilities. I f the study i s d e s i g n e d to d e f i n e the m e t a b o l i s m of a p e s t i c i d e i n l a b o r a t o r y m o n o g a s t r i c mammals ( e . g . , the rat) for extrapolation to man, then a l l aspects of the pesticide's kinetics and metabolism are crucially important. Other studies may have aspects of various importance. For e x a m p l e , the c h a r a c t e r i z a t i o n o f low l e v e l s of r e s i d u e s in the seed of food crops (e.g., rice) is more significant than comparable identification of p o s s i b l y much h i g h e r residues in o t h e r , b u t i n e d i b l e , p o r t i o n s o f the p l a n t . F o r t h e same r e a s o n , r e s i d u e s r e t a i n e d by e d i b l e t i s s u e s o r s e c r e t e d i n t o the m i l k o r e g g s o f t r e a t e d f o o d a n i m a l s , s u c h as c a t t l e o r p o u l t r y , a r e of more t o x i c o l o g i c a l s i g n i f i c a n c e t h a n r e s i d u e s i n u r i n e o r f e c e s . One o f t h e b u r d e n s t h e m e t a b o l i s m s c i e n t i s t must b e a r i s t h a t the products of pesticide metabolism that are often of the g r e a t e s t p o t e n t i a l t o x i c o l o g i c a l s i g n i f i c a n c e (e.g., those i n the e d i b l e p a r t s o f many p l a n t s o r i n m i l k , e g g s , o r e d i b l e t i s s u e s o f f o o d a n i m a l s ) a r e o f t e n p r e s e n t o n l y i n e x c e e d i n g l y low c o n c e n t r a tions. Such p r o p e r t i e s of a p e s t i c i d e a r e , of c o u r s e , highly desirable ones t h a t more o f t e n than not represent accomplished g o a l s of p e s t i c i d e development. However, the c h a r a c t e r i z a t i o n o f such r e s i d u e s u s u a l l y demands the f u l l c a p a b i l i t i e s o f b o t h the scientist and his instrumentation, and in some cases these residues cannot be identified with the technology currently available· Of increasing importance to the design and execution of pesticide metabolism studies i s the impact of the regulatory requirements of pesticide-regulating agencies. In the United States, such r e g u l a t i o n s are issued by the U.S. Environmental P r o t e c t i o n A g e n c y , and t h e y must be c a r e f u l l y c o n s i d e r e d before i n i t i a t i n g most p e s t i c i d e m e t a b o l i s m s t u d i e s , p a r t i c u l a r l y t h o s e that have d i r e c t i m p l i c a t i o n s f o r human h e a l t h . In i t s most r e c e n t issuance of proposed g u i d e l i n e s f o r r e g i s t e r i n g p e s t i c i d e s in the United States (_19_) , t h e Agency states several major p u r p o s e s f o r mammalian m e t a b o l i s m s t u d i e s . These i n c l u d e : 1) t o i d e n t i f y and quantify s i g n i f i c a n t metabolites, 2) to determine p o s s i b l e b i o a c c u m u l a t i o n or b i o r e t e n t i o n of the t e s t p e s t i c i d e or i t s m e t a b o l i t e s , 3) t o d e t e r m i n e a b s o r p t i o n as a f u n c t i o n o f d o s e , 4) t o c h a r a c t e r i z e r o u t e s and r a t e s o f p e s t i c i d e e x c r e t i o n , 5) t o r e l a t e a b s o r p t i o n t o the d u r a t i o n of e x p o s u r e , and 6) t o e v a l u a t e the b i n d i n g of the t e s t p e s t i c i d e or i t s m e t a b o l i t e s i n p o t e n t i a l target organs. The p r o p o s e d r u l e s c o n t a i n r a t h e r g e n e r a l requirements f o r d o s a g e l e v e l s , d o s a g e r o u t e s , and o t h e r a s p e c t s o f s u c h s t u d i e s , i n c l u d i n g sample a n a l y s i s ( 1 9 ) .


i v i E AND

16.

Toxicity If

the

metabolism defined

appropriate

information

living

to

inherent

of

the

mammals


representative

of

i s often

toxicology

parent

usually

Far

subchronic

genicity the

ments

would

circumstances

due

such

separate

metabolites

every

nor

to

possible

man.

tution

they

represent

difficult

a

of

the

no

are

consequence

of as

being

relative

often

significance, "minor"

and

money

with

any

This expendi-

that require-

toxicity in

of

case

some o f

the

t o be

those studies

are

to

such of

pesticide under

most

either

least

predict biologic

of

may

in

toxicologic

"minor"

given been

t o be

of

factors.

fashionable

or

a

data,

present

system.

that

to

based A

"major"

greater p o t e n t i a l

r e g u l a t o r y sense,

than

However, i t seems c l e a r

that

has

essentially

related

differ

formed

consti-

become

has

i n the

chemical

Others

"major"

formed

interact

significant

i t has

of

possibly

detailed

are

appro-

behavior

preexisting

sort.

preclude

(V2_) .

selection

without

of

money

toxicologically

that

may

basis

any

closely

fact,

a p p e a r s t o be

of

classification

because

Thus, the

be the

construed

at

potentials that most

toxicological

distinction

category

significance

may

amounts

somehow a r e

semiquantitative

animal

carcinodone

It i s usually neither

limitations,

toxicological

for

in

chronic

i t s metabolites.

chronic

the

and

this

i n the

likely

and

by p e s t i c i d e

always

some w o u l d a r g u e

that

time

of

toxicological

reactive.

tests

rapid

evaluate

almost

with

on

hazard

problems

metabolites upon

logical

a

posed

to

pesticide that

judged,

such

usually

a

given

or

natural

such

toxicity

give

be

studies

justify

evaluate products

little

Perhaps because

metabolites

quantities for

acute

the

to

be

Limitations

consider

considered appropriate

are

the

the

can

synthetic

tests.

those

to

of

Some o f

that

be

the

(20^).

metabolite

with

of

tremendous time

of

the

assess-

accurately assess

s t u d i e s , but

difficult

the

l a b o r a t o r y mono-

can

hazards

this

of

products

not

the

use

the

effects

to

is

sufficient

h a z a r d s t h a t may

and

to

that

has

pesticide in

of

feeding

studies be

step

metabolites

acute

well, i.e.,

significance

synthesis

its to

job

products dog)

Minor V e r s u s Major M e t a b o l i t e s . priate

267

toxicologist

Comparative

the

chronic

is partly

for

of

pesticide

r e q u i r e d by

his

first

to provide

and

Full-scale other

the

studies.

estimate

parent

limitation

required

toxicological

and

of

the

rabbit,

more d i f f i c u l t

metabolites.

tures

Toxicology

a particular

does the

Chemical

pesticide

reliable

question.

rat,

man.

done

toxicological

toxicity

definitive the

the

Generally,

(e.g.,

metabolites

only

Pesticide

f a t e of

s y s t e m s , how

evaluate

ment

or

of

has

metabolic

generated?

gastric

scientist

the

metabolites

of

Aspects

Assessment

thoroughly the

Metabolic

BANDAL

by

chemicals

orders

of

significant

i n small "major"

toxicological foundation

(20,

often

metabolites

"minor"

have

are

in are

highly

metabolites

significance 21,

toxico-

magnitude;

amounts and

over

no

22).


in

man

THE

268


Toxicological

Significance

PESTICIDE

CHEMIST

A N D

M O D E R N

TOXICOLOGY

of Pesticide Metabolites

Detoxification and Activation Reactions» From an acute toxicity standpoint, the metabolism of pesticides by most organisms usually results i n their c o n v e r s i o n to p r o d u c t s of lesser biological activity. T h e r e a r e s e v e r a l r e a s o n s why such w o u l d be e x p e c t e d , n o t t h e l e a s t o f w h i c h i s t h e f a c t t h a t t h e d e t o x i f i c a t i o n s y s t e m s o f l i v i n g o r g a n i s m s have e v o l v e d f o r j u s t such a purpose. Certainly, too, structure-activity relationships a r e u s u a l l y so c r i t i c a l t h a t t o x i c i t y , e s p e c i a l l y i n the acute sense, i s often greatly reduced or t o t a l l y eliminated as the result of e s s e n t i a l l y any chemical transformation. Numerous examples of m e t a b o l i c r e a c t i o n s l e a d i n g to m o r e - o r - l e s s complete p e s t i c i d e d e t o x i f i c a t i o n c o u l d be c i t e d , b u t t h e ο - d e e t h y l a t i o n o f chlorfenvinphos and the ester hydrolysis of carbaryl, both insecticides, are shown as somewhat representative examples (Figure 7). W h i l e most m e t a b o l i c r e a c t i o n s r e s u l t in total or nearly t o t a l d e t o x i f i c a t i o n s , some do n o t , and i t i s such t r a n s f o r m a t i o n s t h a t most c o n c e r n t h o s e who a t t e m p t t o e v a l u a t e t h e t o x i c o l o g i c a l significance of p e s t i c i d e metabolites. Classical examples of m e t a b o l i c a c t i v a t i o n are the o x i d a t i v e d e s u l f u r a t i o n o f phosphorot h i o n a t e s and the N - h y d r o x y m e t h y l a t i o n of schradan (Figure 8 ) . W h i l e p a r a t h i o n and s c h r a d a n p e r s e a r e e s s e n t i a l l y n o n t o x i c , t h e indicated metabolic reactions convert them to potent a n t i c h o l i n e s t e r a s e s , and t h u s m e t a b o l i s m i s o b l i g a t o r y to their toxicity. O t h e r p e s t i c i d e m e t a b o l i t e s o f t e n have d e g r e e s o f a c u t e t o x i c i t y t h a t a r e o n l y m o d e r a t e l y above o r b e l o w t h o s e o f t h e p a r e n t compounds. Examples of moderate a c t i v a t i o n i n c l u d e the s u l f o x i d a t i o n o f m e t h i o c a r b and the 5 - h y d r o x y l a t i o n o f p r o p o x u r t o yield metabolites that are 8to 10-fold more active as a n t i c h o l i n e s t e r a s e a g e n t s {23_, F i g u r e 9 ) . An example o f m e t a b o l i c transformations that lead to moderate detoxification is the N - h y d r o x y m e t h y l a t i o n o f N - m e t h y l c a r b a m a t e s s u c h as m e x a c a r b a t e t o p r o d u c t s t h a t a r e somewhat l e s s a n t i c h o l i n e r g i c (23^ F i g u r e 1 0 ) . I t s h o u l d be e m p h a s i z e d t h a t even i f the p r o d u c t s of p e s t i c i d e metabolism retain partial or full inherent toxicity, the s t r u c t u r a l a l t e r a t i o n s t h a t r e s u l t from m e t a b o l i s m may facilitate r a p i d e l i m i n a t i o n from t h e body o r f u r t h e r m e t a b o l i s m t o n o n t o x i c p r o d u c t s which, of course l e a d s to g r e a t l y reduced toxicological potential· As an example, a r o m a t i c h y d r o x y l a t i o n of a given pesticide may not always diminish inherent t o x i c i t y , (e.g., p r o p o x u r ) but the p r e s e n c e of the h y d r o x y l group i n the m o l e c u l e w o u l d be e x p e c t e d t o l e a d t o r a p i d c o n j u g a t i o n and e x c r e t i o n by mammals. Pesticide Conjugates. A l t h o u g h the p r i m a r y m e t a b o l i s m of p e s t i c i d e s does n o t n e c e s s a r i l y r e s u l t i n a d i m i n u t i o n o f a c u t e t o x i c i t y , secondary or c o n j u g a t i v e r e a c t i o n s almost always do. P e s t i c i d e conjugates are u s u a l l y h i g h l y polar (e.g., g l u c o s i d e s ,


16.

iviE A N D BANDAL

Metabolic


(G.I. Tract, Skin, Lungs)

TOXODYNAMIC PHASE

ABSORPTION

DISSOLUTION OF

Available for Absorption

DISTRIBUTION

Available

METABOLISM

for Action

PESTICIDE/METABOLITE RECEPTOR

Aspects of pesticide-organism interactions

. Vc-o-< W

O C

CI

TOXIC "EFFECT

INTERACTION

EXCRETION

Figure 6.

a-f

269

Toxicology

TOXOKINETIC PHASE

EXPOSURE PHASE

PESTICIDE


CI

CHCI O Vc-o-p;

o w

H

2 5 CI

CHLORFENVINPHOS

Figure 7.

Examples of metabolic detoxification of the insecticides chlorfenvinphos and carbaryl


270

THE

\

2

/

X

OC H 2

PESTICIDE

CHEMIST

A N D M O D E R N

\ = /

2 5

TOXICOLOGY

OC H 2

5

PAR Α Τ Η I O N

H C

/CH H X /


J

Ν

Ο \ | | ^ P - O - P

3

ν /CH~ N

3

C /

0

2

N

O

\ j |

Ο

Y /

N

;P-O-PC

C H . 3

Ν

3

C H

/CH OH

N

HX/ N.

H

3

H C/

/CH N

3

C H

3

0

SCHRADAN

Figure 8.

Figure 9.

Metabolic activation of the insecticides parathion and schradan

Metabolic transformations leading to moderate activation of the insecti cides methiocarb and propoxur


16.

iviE AND

glucuronides, excreted acute

by

biological

studies plant

shown

metabolite


significantly offer

a It

may

not

be

the

i s well reactive

case

compounds may Bound conducted the

be

in

occur, can

such

be

of as

made

least

of

of

to

be

gastric

The

(_25^

and

mutagenic

of or

to

or

aspect the

carcinogenic considered

is

beyond

papers

of

exocon

always

represent but

(vide

conjugates

i n some

as

thus

innocuous

circumstances. studies

frequently at

a

removal

regarding dietary

formed

infra);

metabolism and

animal

such

the

are

portion

from

the

toxicological

I f bound

exposure

is

residues likely

t i s s u e s , some

to

estimation

t o x i c o l o g i c a l s i g n i f i c a n c e , or by

mammalian

several from

most

their

feeding

p e s t i c i d e s have

the

digestive

i t may

be

that

pesticides will

gauge

pesticide events

effects,

carcinogenicity

scope

of

reviews in this

to

be

this of

an

paper.

these

the

e f f e c t s on

carcinogenic

molecular

generally

other

or

21) , and

of

is

the

not

presumably

s u l f a t e conjugates are

naturally arise.

accurately

and

published

is

is

been

tract such

have

at

studies. of

found mono-

chemically

little

or

no

Carcinogenicity

mutagenicity

to

would

and

toxicological effects.

attempts

human

absorbed

particularly

important

discussion

all

potential

produce mutagenic

most

sulfate

significance.

ability

pesticides,

major

extensively

standpoint,

carcinogens

pesticide

plant

from

The

a

1-naphthol

almost

pesticide

that

26^,

is

or

acute

techniques,

residues

is

i n c a s e s where t h e

chronic

or

some

bioavailability,

residues

Mutagenicity

can

their

mammals

toxicological

of

residues

edible

bound

an

Questions

appreciably

unidentified

from

defies

such

in

their

Fortunately, not

such

matrices

exist.

which

reactions

inappropriate

study.

be and

l i b e r a t e d 1-naphthol

acid

conjugates

Most

radioactivity under

the

metabolite,

indeed

Although

their

radiotracer

always

significant.

similar

Re s i due s .

readily

significant may

carbaryl,

protection

glucuronide

totally

using

significance occur

of

regarding

of

does

example,

reconjugation

are

of

conjugate

toxic

glucuronic

(.24 ) ·

intermediates

consideration

matrix

with

pesticide

known t h a t

for

they

course,

However, t h e

t o x i c o l o g i c a l hazard

It

of

that

a

insecticide 11).

degree

of

potential

is toxicologically

as the

the

t o x i c , such

i s true

reduced

this

urine

significant

(aglycone)

regenerate

(Figure

the

is,

271

Toxicology

devoid

innocuous

1-naphthol,

reconjugated

in

Pesticide

usually

There

to

of

of

acids, etc.),

are

otherwise

of

in rats

rapidly

they

that

conjugate

excreted

a

an

cleaved

have

hydrolyzed

and

effects.

that

metabolically glucoside

Aspects

s u l f a t e s , mercapturic mammals,

possibility

is

Metabolic

BANDAL

chronic

hereditary

responses

toxicity material

i n mammals, i s

toxicological evaluation.

leading or

of

(and

up

the

also

acute

to

expression

the A of

r e l a t i o n s h i p s between teratogenicity,

which

t o x i c o l o g i c a l phenomenon)

Rather, the

subjects

the

of that

(28,

reader 29 ) and

i s referred to

volume.


several

272

THE

PESTICIDE

CHEMIST

A N D M O D E R N

TOXICOLOGY

Ο II

0-C-NHCH OH


2

MEXACARBATE Figure 10.

Metabolic N-hydroxymethylation leading to moderate detoxification of N-methylcarbamate insecticides such as mexacarbate

Figure 11.

Mammalian metabolism of the glucoside conjugate of 1-naphthol, a major plant metabolite of the insecticide carbaryl


16.

iviE AND

Metabolic

BANDAL

Carcinogenicity chronic

effects

potential for

of

at

countries. (usually

the

least rat

and

mouse),

over

the

expected,

there

effects

of


to

make

direct

the

only

do

pesticide

pervading

likely

of

of p e s t i c i d e s

most

developed species

involve

doses

literature

of

chronic the

As

on

species,

test

would

the

be

carcino-

mostly

mammals,

However, s i n c e

long-term

invariably

done

with

the

i t i s usually

parent

impossible

c a r c i n o g e n i c i t y , when

se,

a

i t

occurs,

from

under

endogenous

of

itself

be

which

or

likely

be

to

vivo

proper

metabolites

the

appear

likely

significance,

to

and

be

that

of

adequate

circumstances,

of

that

origin

that the

as

from

enter

would might

that are

novel

the

pesticide study

for

not be

are

generate pesticide

in structure,

potential

human

a

more

circumstance

separate

they

is

of

not

parent

a

the

i t

safety

could

Examples

considerable

could

than

negatives"

the

be

"false

"false

require

compound. animal

likely

i s i n i n s t a n c e s where humans

metabolites

parent

of p l a n t or

metabolite

give

only appropriate

might

effects

the would

more

studies

the

extrapolation,

could

about

be

compound

such

studies

seem t h a t

what

or

to

pesticide

different

learned

from

to

of

then

toxicological

totally

In

be

obtained

from

metabolites

30).

could

carcinogenic

by

otherwise

How

mutagenic

parent

would,

vivo,

patterns

the

studies

c o n c e i v a b l y even

metabolites

mammalian

in

metabolite

exposed

a

of

in

such

pesticide

mutagenic

of

If

excess

{20_,

I t would

that

data

likewise

appear

chronic

far

from

(20) ·

the

that

compound

anything

pesticide

of

for

would

also,

disposition

metabolite

there

from

is

tests.

evaluated

humans

overwhelming

that

reliably

doses

m e c h a n i s m s , and

consequence doubtful

in

i t

a l l pesticide

appropriate.

parent

by

or

cases

consideration

in

the

most

experimental

mutagenicity

properly

separate

nor

metabolite

probably

protective

in vitro be

direct

metabolites,

consideration

metabolites

positives"

that

that

obtain

pesticide

In

necessary is

be

formed

in

and

high

(28).

i f ever

or

hazard?

of

neither

metabolite

in

of

s u b j e c t most

JLn v i v o

logic

behavior

given

seldom

metabolites

carcinogenic

per

we

to r o u t i n e l y

a b a t t e r y of

can

is

correlations

approval

potential

carcinogenic

(18-24 m o n t h s ) .

various

almost

of

generally

i t s metabolites,

c a r c i n o g e n i c i t y of

impractical to

of

in

of the

o r more mammalian

they

reviewed

are

any

the

States

i n two

volume

feared

metabolism. Not

on

tests

not

to

273

Toxicology

assessment

relatively

pesticides been

most

lifespans

large

has

carcinogenicity

with

a

Pesticide

an

and

to

normal

is

some o f t h i s and

required

animals

their

the

and United

be

of

of

prior

the

may

exposure

pesticide

in

Tests

pesticide genic

is certainly pesticides,

i s usually required

use,

and

Aspects

toxicological

food

chain

through

most

logical

contaminated f o o d s t u f f s . Certainly, means

of

hazards

of

chemical

most

best

prevalent

judgments

pesticide structures

carcinogens. the

the

making

This

procedure

about

metabolites to

process

and the is

perhaps simply

those

of

may

imprecise,

available

be for

the

mutagenic by

recognized but

determining

or

carcinogenic

relating mutagens i t is what


their or

probably pesticide

THE

274

PESTICIDE

CHEMIST

A N D

M O D E R N

TOXICOLOGY

metabolites merit concern or more detailed study. Even i f t a r g e t e d m e t a b o l i t e s give p o s i t i v e r e s u l t s i n i n v i v o or i n v i t r o tests for mutagenicity, i t must c o n t i n u a l l y be remembered t h a t s u c h f i n d i n g s can o n l y be c o n s i d e r e d , a t m o s t , s u g g e s t i v e e v i d e n c e o f a p o t e n t i a l m u t a g e n i c o r c a r c i n o g e n i c h a z a r d t o man. Further, c o n s i d e r a t i o n of the mutagenic potency of the m e t a b o l i t e s , the probable extent of human exposure to them, and other considerations, may often indicate that a mutagenic or c a r c i n o g e n i c hazard t o man, even i f i t e x i s t s , i s exceedingly low.


Metabolic

Aspects

of P e s t i c i d e

C a r c i n o g e n i c i t y and

Mutagenicity

In recent years, i t has become evident that for many well-studied chemical carcinogens, metabolic activation to a reactive intermediate in the host i s required i n order for r e a c t i o n w i t h DNA and o t h e r c e l l u l a r m a c r o m o l e c u l e s t o o c c u r (31, 32 ) . T h u s , many c a r c i n o g e n s a p p e a r t o be p r e c a r c i n o g e n s , which are metabolized i n v i v o to t h e i r r e a c t i v e forms, or ultimate carcinogens. The u l t i m a t e c a r c i n o g e n s i d e n t i f i e d o r p o s t u l a t e d so f a r , a l t h o u g h t h e y o f t e n have no common s t r u c t u r a l f e a t u r e s p e r s e , c o n t a i n r e l a t i v e l y e l e c t r o n - d e f i c i e n t atoms t h a t can react c o v a l e n t l y , without the a i d of enzymes, w i t h electron-rich or nucleophilic atoms i n c e l l u l a r components, e s p e c i a l l y in such m a c r o m o l e c u l e s as the n u c l e i c a c i d s and p r o t e i n s (32.)· Thus, c a r c i n o g e n i c p o l y c y c l i c aromatic hydrocarbons are m e t a b o l i z e d to several carcinogenic electrophiles, including epoxides, radical cations, and dihydroxy epoxides (Figure 12). Carcinogenic aromatic amines, amides, and nitro compounds appear to be subjected to N - h y d r o x y l a t i o n , then conjugation with glucuronic a c i d o r s u l f a t e t o a more r e a c t i v e s p e c i e s ( F i g u r e 1 3 ) . With nitroso compounds, some o f w h i c h are potent carcinogens, the ultimate alkylating species is likewise thought to be an e l e c t r o p h i l i c m e t a b o l i t e , probably a diazonium or carbonium i o n ( F i g u r e 14). On t h e b a s i s o f s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s among known c a r c i n o g e n s , some g e n e r a l i z a t i o n s can be made r e g a r d i n g t h e t y p e s of reactive f u n c t i o n a l i t i e s i n p e s t i c i d e s or t h e i r metabolites t h a t might convey mutagenic or c a r c i n o g e n i c p o t e n t i a l · Because electrophilicity i s a s s o c i a t e d w i t h many u l t i m a t e mutagens and carcinogens, any pesticide transformation to an electrophilic s p e c i e s c o u l d be o f p o t e n t i a l s i g n i f i c a n c e . However, upon r e v i e w o f the m u l t i t u d e of mechanisms t h r o u g h which v a r i o u s p e s t i c i d e s are, or can be metabolized, one quickly realizes that the generation of potentially reactive species, or of their precursors, is rather commonplace. Aromatic and aliphatic e p o x i d a t i o n s , N - h y d r o x y l a t i o n s , the g e n e r a t i o n of amines t h a t can f o r m n i t r o s a m i n e s , and o t h e r r e a c t i o n s o f p o t e n t i a l significance a r e w e l l documented i n the p e s t i c i d e literature, yet there i s l i t t l e i n d i c a t i o n t h a t most p e s t i c i d e s c o n s t i t u t e any significant



16.

i v i E A N D BANDAL

Figure 12.

Metabolic

Aspects

of Pesticide

Toxicology

275

Examples of metabolic activation of polycyclic aromatic hydrocarbons to reactive electrophiles

DNA

Figure 13.

Metabolic activation of an aromatic amine that ultimately can lead to the formation of a reactive electrophile and alkylation of DNA


THE

276

PESTICIDE

CHEMIST

A N D

M O D E R N

TOXICOLOGY


mutagenic or c a r c i n o g e n i c h a z a r d . C l e a r l y , t h e mere g e n e r a t i o n o f reactive metabolites does not assure that an expression of toxicity will follow. Subsequent r a p i d d e t o x i c a t i o n of r e a c t i v e metabolites no doubt occurs i n many i n s t a n c e s , the reactive s p e c i e s may f o r m a d d u c t s w i t h n o n c r i t i c a l m a c r o m o l e c u l e s o r o t h e r b o d y c o n s t i t u e n t s , and even i f r e a c t i v e m e t a b o l i t e s do alkylate e s s e n t i a l c e l l u l a r m a c r o m o l e c u l e s , subsequent e v e n t s , such as DNA r e p a i r m e c h a n i s m s , may n e g a t e any p o t e n t i a l t o x i c e f f e c t s ( 3 3 ) . I n most i f n o t a l l c a s e s i n w h i c h p e s t i c i d e s have i n f a c t been shown t o be carcinogenic (2j8 ) , t h e r e has b e e n no clear d e f i n i t i o n o f the r o l e t h a t m e t a b o l i s m t o r e a c t i v e i n t e r m e d i a t e s may o r may n o t have p l a y e d i n c a u s i n g such e f f e c t s . On t h e b a s i s of our current understanding of the mechanisms of chemical carcinogenicity, metabolism of at least some carcinogenic p e s t i c i d e s to r e a c t i v e e l e c t r o p h i l e s i n v i v o may occur as an a c t i v a t i o n step. A l t e r n a t i v e l y , i t may be t h a t most c a r c i n o g e n i c pesticides are epigenetic carcinogens rather than genotoxic carcinogens, i.e., they are cancer promoters rather than a l k y l a t i n g agents. I t i s g e n e r a l l y a c c e p t e d t h a t some c h e m i c a l s may i n d u c e tumor f o r m a t i o n w i t h o u t d i r e c t l y i n i t i a t i n g n e o p l a s t i c changes in any cell. Thus, chemicals that depress immune responses o r a l t e r the h o r m o n a l b a l a n c e in a particular tissue might provide the appropriate c o n d i t i o n s f o r the preferential g r o w t h o f p r e e x i s t i n g tumor c e l l s ( 3 2 . ) · Further, chemicals that induce or i n h i b i t the a c t i o n o f d r u g m e t a b o l i z i n g enzymes may promote cancer by enhancing the activation or inhibiting the d e t o x i f i c a t i o n of other chemical carcinogens. It i s therefore possible t h a t m e t a b o l i s m t o r e a c t i v e e l e c t r o p h i l e s may not be i n v o l v e d a t a l l i n the e x p r e s s i o n o f c a r c i n o g e n i c a c t i o n o f many o r most c a r c i n o g e n i c p e s t i c i d e s . One o r more o f s u c h p r o m o t i o n m e c h a n i s m s m i g h t e x p l a i n the c a r c i n o g e n i c i t y o f t h e insecticide m i r e x , w h i c h i s r e p o r t e d t o be a hepa t o c a r c i n o g e n i n m i c e ( 3 4 ) , even though t h e r e i s s t r o n g evidence t h a t l a b o r a t o r y r o d e n t s are unable to metabolize t h i s i n s e c t i c i d e ( 3 5 , 3 6 ) . Pesticide

M e t a b o l i t e s and

the

Regulatory

Process

All pesticides can be considered to present at least a p o t e n t i a l t o x i c o l o g i c a l h a z a r d to man, and c e r t a i n l y t h e primary g o a l i n the r e g u l a t i o n o f t h e s e chemicals i s to minimize such risks as much as p o s s i b l e . Because r i s k t o man is clearly a function of exposure, risks are g e n e r a l l y minimized by the r e g u l a t i o n of exposure. T h i s i s done t h r o u g h the setting of tolerances. Tolerances represent maximum limits (expressed u s u a l l y i n p a r t s p e r m i l l i o n ) of a p e s t i c i d e , i t s m e t a b o l i t e s , o r b o t h , t h a t may l e g a l l y a p p e a r i n human f o o d s t u f f s , a n i m a l feeds, etc., as a result of pesticide use. The determination of w h e t h e r a t o l e r a n c e w i l l be g r a n t e d and a t what l e v e l i t w i l l be s e t can be a c o m p l i c a t e d p r o c e s s , b u t s e v e r a l f a c t o r s a r e u s u a l l y involved. These i n c l u d e t h e i n h e r e n t t o x i c i t y o f t h e pesticide


16.

iviE AND

and/or

its

proposed or

metabolites,

use

human

of

the

food),

sources,

the

of

In

man. Depending

pesticide

low

tolerance

significant likely

be

P=S

(37,

P=0

Figure

considered

from

equivalent

to

minimal

of

existing

the

tolerance

is

mutagenic

actions

15).

in

sulfur

The

esters

quite or

both

The

for

any

possible

and

phenols, and the

toxicological

effect

Extrapolation

t o Man:

To

t a k e n by

our

use

most

types

of

insecticide

animal

systems

hydrolysis

analogs activity

of

the

and

are

toxicologically

other

hand,

included

form

or

for

knowledge,

a regulatory

pesticide

i s seen w i t h The

both

particularly could

pesticide

a

the not

retain

will

are

of

under

the

toxicological characteristics

behavior,

of

on

a of

inclusion may

ester

be

be

commodity. that

for

to

are

carcinogenic

been

and

and

sulfone

standpoint

metabolites,

toxicity

of

of

to

therefore

plant

risk

pesticide

their Others

organophosphate

pesticide

have y e t

that

oxidation,

the other

sufficiently

judged

Examples

sulfoxide

registrations.

demonstrated

at

retain anticholinesterase

regulatory

registration

be

and

a l l

and

components

unnecessary.

the

i s metabolized

sulprofos.

individual

denying

in

from

significant

as

importance

t o x i c o l o g i c a l importance

sulprofos It

seen

set of

may

feed

application,

significance,

metabolites

tolerance.

conversion,

phosphate

are

the

animal

exposure

absence

properties

action,

as

methods,

included

i s deemed

under

be

which

to

38,

intact

can

the

be

toxicological

included

sulprofos,

Some

toxicological

metabolites by

not

limits

human

toxicological

may

toxic

(e.g.,

pesticide

tolerances

assure

their

or

tolerance.

sufficiently within

upon

may

of

277

Toxicology

their

analytical

a l l cases,

to

of

proposed

available

l e v e l s t o , presumably,

Pesticide

commodity

extent

the

low

metabolites

nature

likely

for

the

considerations.

the

Aspects of

contaminated

the

need

sensitivity


Metabolic

BANDAL

the

Problem of

demonstrated

the

however,

a g e n c y on

metabolite, parent Species

basis

for

revocation the

unless

no

of such

basis the

of

same

compound. Variations

The primary purpose of evaluating the metabolic and t o x i c o l o g i c a l b e h a v i o r of p e s t i c i d e s i s to a s s e s s the r i s k to man that may result from their use and subsequently to take appropriate regulatory steps to minimize such r i s k s . Obvious ethical and other considerations prevent direct studies of p e s t i c i d e s i n humans e x c e p t i n most u n u s u a l c i r c u m s t a n c e s , t h u s e x t r a p o l a t i o n s t o man must u s u a l l y be made on the b a s i s o f d a t a obtained with monogastric laboratory mammals. Unfortunately, laboratory research animals are generally chosen more for convenience than f o r r a t i o n a l , s c i e n t i f i c r e a s o n s . The handling and housing requirements, incidence of disease, supply and, p e r h a p s most i m p o r t a n t , c o s t , a r e among the f a c t o r s c o n s i d e r e d in choosing a species for research (39.)· p e s t i c i d e metabolism s t u d i e s , t h e r a t a n d / o r mouse i s u s u a l l y t h e s p e c i e s o f choice. We q u i t e w i l l i n g l y assume, p e r h a p s b e c a u s e no o b v i o u s a l t e r n a t i v e s F

o

r


278

THE

R-H C 2

R-hLC

N

/

PESTICIDE

OH I R-HC

N-NO

CHEMIST

A N D M O D E R N

TOXICOLOGY

R-C

N-NO

N-NO

R'-H C

R-H C

2

2


DIALKYLNITROSAMINE

R-H C

N-NO

2

R—H C

/

R-H C

/

2

ALKYLATED DNA

R-H C-N^N 2

,N=N

.OH

2

Figure 14. Metabolic activation of a dialkylnitrosamine leading to the generation of reactive electrophiles and ultimately to the alkylation of DNA

(O) / ~ ~ \

ll/O-C Η

(O) C

ft f~\

Î

H

"/°- ? 5

N/°- 2 5 S-C H

f~\

C

3

SULPROFOS

SULPROFOS

OH

Figure 15.

SULPROFOS

PHENOL

SULFOXIDE

SULFONE

•OH

•OH

\_/ PHENOL

SULFOXIDE

H

3

PHENOL

SULFONE

Structures of the insecticide sulprofos and its major plant and animal metabolites


7


16.

IVIE

A N D B A N D A L

Metabolic


Toxicology

279

e x i s t , t h a t r e s u l t s from m e t a b o l i s m s t u d i e s w i t h t h e s e a n i m a l s a r e i n f a c t p r e d i c t i v e o f what w i l l happen i n man, o r a t l e a s t t h a t a n y d i f f e r e n c e s w i l l n o t be t o x i c o l o g i c a l l y " s i g n i f i c a n t . " Yet t h e r e a r e c l e a r i n d i c a t i o n s t h a t , i n metabolism as w e l l as other toxicological phenomena, considerable species differences do i n d e e d e x i s t (2J_, 40_). L a b o r a t o r y r o d e n t s , i n f a c t , appear t o be poor metabolic p r e d i c t o r s f o r man! In a comparison o f t h e m e t a b o l i c p a t h w a y s f o r 21 d r u g s and o t h e r compounds i n t h e r a t and man (4Y), t h e r a t p r o v i d e d a "good" m e t a b o l i c model f o r man w i t h o n l y 4 compounds and was a " p o o r " o r " i n v a l i d " model ( m e t a b o l i c p a t h w a y s q u i t e d i f f e r e n t ) w i t h 15 o f t h e compounds s t u d i e d ( T a b l e II). However, t h e r h e s u s monkey o r marmoset p r o v i d e d "good" m e t a b o l i c m o d e l s f o r man w i t h 16 o f t h e 21 compounds. Iti s reasonable t o assume t h a t s i m i l a r results w o u l d be s e e n with various pesticides, and t h u s many o f t h e m e t a b o l i s m s t u d i e s c u r r e n t l y used as a b a s i s f o r e x t r a p o l a t i n g t o x i c o l o g i c a l results w i t h p e s t i c i d e s t o man may be o f l i m i t e d p r e d i c t i v e v a l u e · The potential toxicological consequences o f t h i s a r e , of course, unknown·

Table I I . C o m p a r i s o n o f L a b o r a t o r y R o d e n t s and Sub-human P r i m a t e s a s M e t a b o l i c M o d e l s f o r Man METABOLIC SIMILARITY TO M A N MONKEY

RAT

COMPOUND Amphetamine Chlorphentermine 4-Hydroxy-3,5-diiodobenzoic acid Indolylacetic acid Norephedrine*

Invalid

Good

Poor

Good

Fair

Good

Τ

τ Good j_

Phenmetrazine* Phenylacetic acid Sulphamethomidine 1 - Naphthylacetic

acid

Sulphadimethoxine Sulphadimethoxypyridine

1

Halofenate Methotrexate Sulphasomidine Hydratropic acid Diphenylacetic acid Indomethacin Morphine Oxisuran 2 - Aceta m idof luorene Phencyclidine From Smith and Caldwell (Ref 41 ).

Good J_ Poor

Fair

Fair

Fair

Good J_

Fair

Poor

Poor

Marmoset, all others rhesus

_L

monkey


280

THE


P e s t i c i d e Metabolism:

PESTICIDE CHEMIST AND

Prospects and

MODERN TOXICOLOGY

Problems

P e s t i c i d e metabolism studies are, without question, very important components i n the evaluation of the toxicological s i g n i f i c a n c e of p e s t i c i d e s to man. The r a t e , extent, mechanisms, and products of metabolism are i n e v i t a b l y l i n k e d to the expression of t o x i c a c t i o n , and a c l e a r d e f i n i t i o n of p e s t i c i d e b i o t r a n s f o r mation is often a necessary prerequisite to understanding mechanisms of t o x i c i t y and to the formulation of approaches for assessment and management of p o t e n t i a l l y undesired t o x i c e f f e c t s . What does the future hold? Can p e s t i c i d e metabolism studies and the data they generate be more e f f e c t i v e l y used i n the safety e v a l u a t i o n process? Can these studies be made more p r e d i c t i v e and thus more t o x i c o l o g i c a l l y r e l e v a n t to man? I t i s , of course, d i f f i c u l t i f not impossible to foresee the future a c c u r a t e l y . We w i l l , however, make a few observations on these and other matters · Only a few years ago, a p e s t i c i d e metabolism study was considered successful i f only the major metabolites were c h a r a c t e r i z e d , and t h i s was often done s o l e l y by chromatographic means -- without s p e c t r a l confirmation of s t r u c t u r e . Today i t i s not uncommon to see r e p o r t s i n which most i f not a l l of the detected metabolites of a p e s t i c i d e i n a given system are f u l l y and unequivocally characterized by s p e c t r a l means. Several f a c t o r s have contributed to such advancements, i n c l u d i n g the f a c t that many of us now have a v a i l a b l e i n our research l a b o r a t o r i e s a f u l l complement of up-to-date, often s t a t e - o f - t h e - a r t a n a l y t i c a l , chromatographic, and spectrometric instrumentation. Advances i n our c a p a b i l i t i e s to c h a r a c t e r i z e organic compounds, p a r t i c u l a r l y advances in microspectrometric techniques such as GLC-mass spectroscopy, FT-NMR, and FT-IR make p o s s i b l e the i d e n t i f i c a t i o n of many metabolites at the microgram l e v e l . The versatility, a c c e s s i b i l i t y , and o v e r a l l importance of r a d i o t r a c e r techniques to the metabolism s c i e n t i s t have never been greater. Stable isotopes (e.g., ^H, ^C, ^N) are beginning to find more use in p e s t i c i d e metabolism s t u d i e s , and with mass spectroscopy or NMR, s t a b l e isotopes can be very u s e f u l t o o l s f o r both metabolite c h a r a c t e r i z a t i o n and mechanistic studies (42_) · In the metabolism study of the f u t u r e , there w i l l continue to be, and r i g h t l y so, great emphasis placed on definitive characterization of a l l metabolites p o s s i b l e . Hopefully, we w i l l see i n the future continuing advances i n our c a p a b i l i t i e s to more f u l l y c h a r a c t e r i z e p e s t i c i d e conjugates and "bound" residues, because these products often comprise the bulk of the total residue and their t o x i c o l o g i c a l s i g n i f i c a n c e , p a r t i c u l a r l y chronic e f f e c t s , i s far from clear· Species v a r i a t i o n s that may s e r i o u s l y a f f e c t the v a l i d i t y of l a b o r a t o r y animal metabolism studies as p r e d i c t i v e models for man are a problem without apparent s o l u t i o n . For proper e v a l u a t i o n of the t o x i c o l o g i c a l s i g n i f i c a n c e of p e s t i c i d e s to man, metabolism


281

16. IVIE AND BANDAL Metabolic Aspects of Pesticide Toxicology

studies

i n humans

totally should

be

f a r more

acceptable Because to

and and

subhuman


studies

with

destructive proper

of

pesticides

levels,

number

present

some

enzyme

contrast

to

other

the

will to

Historically, specific believe

that

such

a pattern

metabolism methodology this

field

However,

requirements be

groups a period

such

metabolism

need

of

Such

philosophy

primates

an

approach

and

could

induction

of

would

possibly

of

to

pesticide

us

quite

yield

could

o f many y e a r s f o r

i t seems

likely

the

be

will

that

the

minor

data

i n that

there that in

chemistry,

toxicological

required

and

to

in

of

much

have

even the

i s concern further

on

moves

metabolism and

more

and

pesticide

part

toward

studies

may

innovative

to Few

agencies i n

support the

more

reason

i n the f u t u r e .

to

as of

future.

more

is little

of regulatory

studies

imaginative

in

become

there

as well

evaluation

become

agencies

not continue

or p r o p r i e t y

metabolism

scientists

may

totally

be

realistically,

progressed,

t h e wisdom

counterproductive

be

However,

and

over

requirements

has

registrations.

may

not

small

regulatory

regulatory

detailed

respect

toxicological

use o f r a d i o i s o t o p e s

low l e v e l

in

doubt

pesticide

time

question

respect

t o man.

involved no

more

resource,

merits

studies

However,

are,

as

requiring

as

with

pesticide

of p e s t i c i d e metabolism

disciplines

responsive

i n vivo

chemicals.

advantages—the

discipline

pesticides,

would

such

p r e d i c t i v e value

The

or data

systems.

disadvantages

primates

these

i n regulatory

problems,

metabolizing potential

single

of p e s t i c i d a l

adjustments

to

as

metabolism.

limited that

are

primates

present

animals

very

the j u d i c i o u s

metabolic

require

at

treasure

using

because

With

a

clearly

pesticide

a l l circumstances.

apply

primates

of

as

subhuman

reasons, conventional

even

not

invaluable

large

scientific

or

than

models

represent

For these

of l i f e .

dosage

provide

are a

should

just

p o s i t i o n of these

they

i n most

restrictions

yet

used

human

use.

investigations

better

effectively

accurate

primates

the wisest

needed,

I t seems, h o w e v e r , t h a t

because

inappropriate

a

clearly

o f the e v o l u t i o n a r y

man,

and

are

inappropriate.

of

some

specific well

research

be in

discouraged.

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RECEIVED

J . Agric. Food

February 2, 1981.


The Pesticide Chemist and Modern Toxicology - American Chemical

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