The Pesticide Chemist and Modern Toxicology - American Chemical

2 The Revolution in Toxicology: Real or Imaginary

Downloaded by STONY BROOK UNIV SUNY on May 21, 2018 | https://pubs.acs.org Publication Date: August 10, 1981 | doi: 10.1021/bk-1981-0160.ch002

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LEON GOLBERG Chemical Industry Institute of Toxicology, P.O. Box 12137, Research Triangle Park, NC 27709

Toxicology has traditionally been concerned with the effects of chemical or physical agents in bringing about alterations of structure, function or response of living organisms. The higher organisms used by the toxicologist are never devoid of spontaneous disease, especially as they age, so that it is against this background that toxic changes attributable to a test compound have to be gauged. As Salsburg (1) has pointed out: "When groups of animals are exposed to any biologically active substance over a long period of time, there will be a shift in patterns of lesions that will be dose related". The traditional task of the toxicologist has been to identify the nature of that shift in lesions, to characterize the dose-response relationships for each major change, and to elucidate the mechanism of toxic action - in other words, to determine the basis of that shift. An appropriate point of departure for considering toxic effects is the topic of homeostasis, the ensemble of defensive mechanisms that Nature has built into every organism. Homeostasis comprises the responses to changes, both external and internal, physiological adjustments (2, 3) that help to maintain what Claude Bernard termed "the stability of the internal medium", in other words the balance between the needs of the cell and the needs of the organism (4). Thus homeostasis can be considered in terms of three components, one concerned with the normal internal composition and function of the cell, another with the intercellular integration of function within a multicellular organism and the third being the gamut of compensating mechanisms that come into play when the organism is stressed by any of a multitude of physical or chemical agents such as hypoxia, extremes of temperature or the action of toxicants. The concept of homeostasis i s important to the t o x i c o l o g i s t 1

Current address: 2109 Nancy Nanam Drive, Raleigh, NC 27607. 0097-6156/81/0160-0007$05.00/0 © 1981 American Chemical Society Bandal et al.; The Pesticide Chemist and Modern Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 1981.


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because i t p r e s c r i b e s the l i m i t s w i t h i n which the body can a d j u s t to t o x i c e f f e c t s with no apparent d e v i a t i o n of normal f u n c t i o n , other than perhaps temporary p e r t u r b a t i o n s . From t h i s concept i s derived the s o - c a l l e d "No observed e f f e c t l e v e l " of exposure to a toxicant. In some instances the organism can meet the challenge and s t r e s s of t o x i c exposure by adaptations that i n v o l v e the development of tolerance, provided that time i s a f f o r d e d f o r the organism to change i n t h i s way. When exposure i s excessive i n degree or too abrupt or both, the p h y s i o l o g i c a l defense mechanisms prove inadequate and p a t h o l o g i c a l disturbances ensue. Even at t h i s p o i n t , however, when damage has been done to one or more t a r g e t organs, r e p a i r mechanisms are a v a i l a b l e that come i n t o play at many l e v e l s from DNA on up. Provided that the onslaught by the t o x i c a n t abates f o r a s u f f i c i e n t length of time to permit r e p a i r of s t r u c t u r e and r e s t o r a t i o n of f u n c t i o n to take p l a c e , the c o n d i t i o n of the organism may r e t u r n to apparent normality. Evidence on t h i s score w i l l be provided by long-term follow-up, or by f u r t h e r challenges with observation of the responses (2, J5, 6, T) . Over and above acute and subchronic e f f e c t s , there may be changes of more s u b t l e character, o c c u r r i n g e a r l y i n the course of exposure as s o - c a l l e d " s i l e n t " l e s i o n s but making themselves manifest much l a t e r i n the l i f e t i m e of the organism as frank p a t h o l o g i c a l changes, i n c l u d i n g n e o p l a s i a (8). The consequences of genetic t o x i c i t y a f f e c t i n g germinal c e l l s may only become apparent i n subsequent generations. F i n a l l y , the aging process i t s e l f may r e f l e c t the accumulation of t o x i c i n s u l t s , and f a i l u r e to achieve p e r f e c t i o n i n the r e s t o r a t i o n of damage, over the course of a l i f e t i m e . The p r o v i s i o n made to p r o t e c t c e l l s a g a i n s t oxygen t o x i c i t y i l l u s t r a t e s some of the p r i n c i p l e s mentioned above (9). The b i o l o g i c a l r e d u c t i o n of oxygen by the monovalent pathway proceeds through superoxide r a d i c a l s ( 0 2 ~ ) , hydrogen peroxide and hydroxyl r a d i c a l s ( 0 H ) , p o s s i b l y to s i n g l e t oxygen (Ϊ-Ο2). Hydroxyl r a d i c a l s are so dangerous to the c e l l that very e f f i c i e n t mechanisms e x i s t to l i m i t t h e i r formation by scavenging the superoxide r a d i c a l s , by means of superoxide dismutases, and d e s t r o y i n g H2O2 by c a t a l a s e s and peroxidases (10, 11_). Another and p a r t l y r e l a t e d t o x i c phenomenon i s l i p i d p e r o x i d a t i o n which i s capable of causing damage to c e l l membranes. The t o x i c e f f e c t s of many compounds are mediated, at l e a s t i n part, through l i p i d p e r o x i d a t i o n . Again, the c e l l possesses defenses i n the form of a n t i o x i d a n t s , super oxide dismutases, carotenoids and the enzymes glucose-6-phosphate dehydrogenase, g l u t a t h i o n e peroxidase and g l u t a t h i o n e reductase a c t i n g together (1_2, 13). Beyond i t s r o l e i n the a c t i o n of these l a s t two enzymes, g l u t a t h i o n e and kindred non-protein s u l f h y d r y l compounds a f f o r d b i o l o g i c a l p r o t e c t i o n against e l e c t r o p h i l e s , epoxides and other h i g h l y - r e a c t i v e p o t e n t i a l t o x i c a n t s through the a c t i o n of g l u t a t h i o n e S-transferases (14, L5> 16). e

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

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The Nature and Dimensions of T o x i c i t y Three aspects of t o x i c a c t i o n need to be defined as accuratel y as p o s s i b l e . In the f i r s t place the t o x i c p o t e n t i a l i t i e s of the t e s t compound should be explored with a view to p i n p o i n t i n g one or more target organs that are revealed i n the course of acute, subchronic, long-term, reproductive and other s t u d i e s . This information a f f o r d s a b i r d ' s eye view of the o v e r a l l landscape. Once the i n t r i n s i c capacity to cause i n j u r y to a s p e c i f i c t a r g e t organ or system has been c h a r a c t e r i z e d , some measure of the potency of the substance i s e s s e n t i a l , p r e f e r a b l y i n the form of dose-response data i n appropriate t e s t systems. Thus the potent i a l f o r n e u r o t o x i c i t y , m y e l o t o x i c i t y , mutagenicity or c a r c i n o g e n i c i t y i s s p e l l e d out i n terms of a s p e c i f i c bracket w i t h i n the range of 10? of p o s s i b l e potency. N a t u r a l l y t h i s d e f i n i t i o n a p p l i e s only to a given set of experimental circumstances: part i c u l a r species, s t r a i n , sex and age of animals derived from a p a r t i c u l a r stock at a p a r t i c u l a r source, housed under p a r t i c u l a r defined c o n d i t i o n s , and given a d i e t of s p e c i f i e d composition. A i r and water, i n common with many other d e t a i l s , r e q u i r e c l o s e a t t e n t i o n . Any one of these and numerous other minutiae of the t e s t i n g p r o t o c o l can i n f l u e n c e the outcome of the t e s t , and hence merits c l o s e a t t e n t i o n . Given a defined potency and a dose l e v e l at which no adverse e f f e c t i s observed ( i n comparison with cont r o l s ) , one i s i n a p o s i t i o n to draw a comparison with the a c t u a l or a n t i c i p a t e d l e v e l s of exposure of people or other species to the t e s t m a t e r i a l . Here we have a p o s s i b l e range of at l e a s t 10^; so that the product of potency and exposure (which are, of course, independent of each other) i s 1 0 ^ . For purposes of r i s k assessment the a l l - i m p o r t a n t question then i s : where, w i t h i n t h i s v a s t range, does a given chemical or p e s t i c i d a l i n g r e d i e n t l i e when i t i s used i n i t s intended a p p l i c a t i o n s ? Anyone tempted to adopt the popular expressions " t o x i c " or "non-toxic" should bear i n mind the f a c t that, l i k e sinners, none of us i s p e r f e c t : i t i s the nature and extent of our s i n s that matter. I n d i v i d u a l S u s c e p t i b i l i t y to Toxic E f f e c t s P e s t i c i d e s encounter s u s c e p t i b l e or r e s i s t a n t target s p e c i e s . The range of s u s c e p t i b i l i t y to t o x i c a c t i o n i s o f t e n very broad i n man and l a b o r a t o r y animals. Host s u s c e p t i b i l i t y i s predominantly determined by genetic background but may be profoundly i n f l u e n c e d a l s o by d i e t , human l i f e s t y l e ( i n c l u d i n g consumption of a l c o h o l , tobacco, drugs) age, sex, s t a t e of h e a l t h and numerous environmental f a c t o r s . Pregnancy and infancy are examples of c o n d i t i o n s i n which s p e c i a l s u s c e p t i b i l i t y may e x i s t . Genetic c o n t r o l of s u s c e p t i b i l i t y to t o x i c a n t s operates through a v a r i e t y of mechanisms. One of these i s metabolic. In animals, the murine Ah complex represents a " c l u s t e r " of genes e x e r c i s i n g temporal c o n t r o l on t i s s u e - s p e c i f i c r e g u l a t o r y genes


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c o n t r o l l i n g monooxygenase a c t i v i t i e s mediated by cytochrome P-450 (.17). In man and animals the pheno types determining a c e t y l a t o r (18, 19, 20, 21) and methylator (22) status have a powerful i n f l u e n c e on drug metabolism and t o x i c i t y . Human cancer s u s c e p t i b i l i t y i s based on "ecogenetics" of the i n d i v i d u a l ' s background and environmental exposures (23).


Toxic

Interactions

Beside the i n f l u e n c e of inadvertent exposures to environmental toxicants at home or i n the workplace, the d e l i b e r a t e use of therapeutic agents, " s t r e e t " drugs, s o l v e n t " h u f f i n g " and other sources of a m u l t i p l i c i t y of t o x i c agents may impinge on the e f f e c t s of p e s t i c i d a l exposure, i n the f i e l d or elsewhere. While the Washington Post (June 20, 1980) may have gone too f a r i n d e s c r i b i n g Agent Orange as " j u s t one g a r n i s h i n a t o x i c c o c k t a i l " , a t t e n t i o n does need to be d i r e c t e d to the p o s s i b i l i t i e s of a d d i t i v e , s y n e r g i s t i c or a n t a g o n i s t i c i n t e r a c t i o n s between s e v e r a l chemicals a c t i n g simultaneously or s e q u e n t i a l l y . This issue was addressed by the Mrak Commission (24) under three headings: i n h i b i t i o n of esterases, a l t e r a t i o n of microsomal enzyme a c t i v i t y , and t a r g e t - l e v e l i n t e r a c t i o n s . Also taken i n t o account were the i n f l u e n c e s exercised by t i s s u e storage of p e r s i s tent compounds, and by exogenous p h y s i c a l f a c t o r s such as d i e t , temperature and r a d i a t i o n . Much more i s now known about each of these t o p i c s , p a r t i c u l a r l y the i n d u c t i o n of hepatic mixed f u n c t i o n oxidase a c t i v i t y (25, 26) or i t s i n h i b i t i o n by exposure to heavy metals such as cadmium (27). There i s a prevalent tendency to emphasize the p o s s i b i l i t y of a d d i t i v e and s y n e r g i s t i c t o x i c (esp e c i a l l y carcinogenic) e f f e c t s of simultaneous exposures, but not to mention the well-documented f a c t that a n t a g o n i s t i c i n t e r a c t i o n s between the b i o l o g i c a l e f f e c t s of the components may render a mixture l e s s t o x i c or even non-carcinogenic (2, _5). One of the important spheres of i n t e r a c t i o n l i e s i n p o s s i b l e m o d i f i c a t i o n of the immune status and responses of t e s t organisms, i n c l u d i n g man. The f i e l d of immunotoxicology, l i k e that of b e h a v i o r a l t o x i c o l o g y , i s s t i l l i n i t s infancy. A penetrating a n a l y s i s of the problems inherent i n premature e f f o r t s to pres c r i b e mandatory t e s t s i n t h i s area (28) concludes as f o l l o w s : "There i s no way of knowing what t e s t s are more s e n s i t i v e , r e p r e s e n t a t i v e of e f f e c t s , and would provide c o n s i s t e n t conclusions i f a number of t e s t chemicals were examined. Toxicology i s becoming more and more a r e g u l a t o r y d i s c i p l i n e and the trend of looking f o r new t e s t s that would evaluate untoward h e a l t h e f f e c t s seems strong. In t h i s p e r s p e c t i v e we should r e a l i z e that adding more t e s t s i n t o x i c i t y t e s t i n g schedules, p a r t i c u l a r l y with respect to immunotoxicity e v a l u a t i o n , may not o f f e r much advantage. "The need of b a s i c science i n v e s t i g a t i o n s i n t o x i c o l o g i c



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research needs no f u r t h e r emphasis. This might be the time to d i v e r t our a t t e n t i o n i n t o looking more f o r the mechanisms r a t h e r than merely the e f f e c t s of chemicals on the immune system. Only then can we make more o b j e c t i v e judgments on the r i s k s and b e n e f i t s of environmental chemicals, p a r t i c u l a r l y when the chemical exposures are low but prolonged, and the system i n question i s the one that g e n e r a l l y expresses i t s d e f i c i e n c i e s o r m o d i f i c a t i o n s only when challenged by an unwanted invader. Only a f t e r we have a b e t t e r understanding of these mechanisms can we a p p r o p r i a t e l y understand the species d i f f e r e n c e s , mechanisms of immune tolerance, i f any, and even the t o x i c o l o g i c e f f e c t that might be mediated v i a immune m o d i f i c a t i o n s . " S e l e c t i o n of Test M a t e r i a l The foundations of e f f e c t i v e t o x i c o l o g i c a l assessment may be undermined i f i n s u f f i c i e n t a t t e n t i o n i s devoted to a v a r i e t y of chemical aspects of the problem. The d e c i s i o n whether to study a t e c h n i c a l product or a p u r i f i e d m a t e r i a l (and, i f so, what degree of p u r i t y ) i s , of course, fundamental and o f t e n very d i f f i c u l t . Beyond that, one has to r e a l i z e that the s p e c i f i c a t i o n of a compound i s u s u a l l y drawn up f o r t e c h n i c a l purposes rather than as a b a s i s f o r t o x i c o l o g i c a l i n v e s t i g a t i o n (29). Consequently, a number of c r i t i c a l s a f e t y issues may be overlooked. Time and again, much t o x i c o l o g i c a l e f f o r t has been set a t nought by f a i l u r e to pay a t t e n t i o n to what appeared to be unimportant " t r a c e " impurit i e s o r added s t a b i l i z e r s i n commercial products. There i s a long h i s t o r y of mistakes, and current concerns about the presence of d i o x i n i n 2,4,5-T, pentachlorophenol, hexachlorophene, and a v a r i e t y of other c h l o r i n a t e d compounds i l l u s t r a t e the p r i n c i p l e . On the other hand, the search f o r traces of trace i m p u r i t i e s i n the p u r s u i t of an explanation f o r the a l l e g e d c a r c i n o g e n i c i t y of s a c c h a r i n has gone to i n c r e a s i n g extremes (30). One has a l s o to take i n t o c o n s i d e r a t i o n the changes which the compound may undergo before i t f i n a l l y enters the body of the i n d i v i d u a l of i n t e r e s t . F o r example: i n t e r a c t i o n with food components; degradation during the course of formulation or storage or i n the environment, i n c l u d i n g photochemical o x i d a t i o n ; and b i o t r a n s f o r m a t i o n i n a v a r i e t y of organisms, from b a c t e r i a to p l a n t s and animals, i n c l u d i n g the i n t e s t i n a l f l o r a of man. P a r t i c u l a r l y with a t e c h n i c a l product comprising numerous components and i m p u r i t i e s , the o b j e c t i o n i s o f t e n r a i s e d that i n v e s t i g a t i o n s of the s o r t recommended i n v o l v e an extraordinary e f f o r t which i s not j u s t i f i e d . Obviously, a reasonable balance has to be maintained between e f f o r t involved and s i g n i f i c a n c e of the r e s u l t s . A d e c i s i o n on how much e f f o r t should be necessary w i l l i n p a r t be based on the l e v e l s of exposure to be used i n t o x i c i t y t e s t s . I f these are high, then i t may w e l l happen that t r a c e i m p u r i t i e s can assume considerable importance i n determining the b i o l o g i c a l outcome.


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P r e d i c t i v e t o x i c i t y i n v o l v e s much more than s t u d i e s i n a n i mals. A l o g i c a l h i e r a r c h i c a l approach to the e v a l u a t i o n of hazard presented by a t e s t m a t e r i a l i s i l l u s t r a t e d i n Table I. The sequence i s not intended to imply a s e r i e s of w a t e r t i g h t compartments: the a r t of modern toxicology l i e s i n the s k i l l f u l deployment of the most appropriate procedures, s e v e r a l l y or i n comb i n a t i o n , to answer s p e c i f i c questions.


Table I.

1. 2. 3. 4. 5. 6. 7.

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Structure-activity correlation P h y s i c a l and chemical p r o p e r t i e s In v i t r o and other short-term t e s t s Screening procedures Animal s t u d i e s Human s t u d i e s Risk assessment

A n a l y s i s of q u a n t i t a t i v e 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 (QSAR) has become i n c r e a s i n g l y important as a means of p r e d i c t i n g l i k e l y b i o l o g i c a l a c t i v i t y on the b a s i s of the v a s t s t o r e of e x i s t i n g information on SAR. The t r a d i t i o n a l approach has been Hansch a n a l y s i s , i n c o r p o r a t i n g independent v a r i a b l e s and physicochemical parameters, and i n v o l v i n g r e g r e s s i o n a n a l y s i s of p a r t i t i o n c o e f f i c i e n t s , e l e c t r o n i c e f f e c t s of s u b s t i t u e n t s (Hammett sigma parameter), s t e r i c parameters (Taft s t e r i c constants, Verloop parameters) and i n d i c a t o r v a r i a b l e s (31). More r e c e n t l y , p a t t e r n r e c o g n i t i o n techniques have come to the f o r e , i n which a computer generates, on the b a s i s of the s t r u c t u r e of the compound, molecular s t r u c t u r e d e s c r i p t o r s to be used f o r mathematical analys i s of QSAR. Remarkable p r e d i c t i v e accuracy has been achieved, f o r instance w i t h v a r i o u s c l a s s e s of carcinogens (32, 33). Two problems e x i s t : the r e l i a b i l i t y of the data base, and the need to i n c o r p o r a t e metabolic information. As to the f i r s t , the weaknesses of the standard carcinogenesis bioassay are not as w e l l recognized as they should be 02, 5), but some e f f o r t i s at l a s t under way to t r y to overcome them (34). I n t r o d u c t i o n of metabolic information has to be very s e l e c t i v e , concentrating on metabolic a c t i v a t i o n r a t h e r than the m u l t i p l i c i t y of d e t o x i c a t i o n products. In a document e n t i t l e d "Proposed System f o r Food Safety Assessment", the S c i e n t i f i c Committee of the Food Safety Council (35) has attempted to d e l i n e a t e the steps by which d e c i s i o n s on s a f e t y or t o x i c i t y are a r r i v e d at. What i s i n t e r e s t i n g about t h i s approach i s the departure from the t r a d i t i o n a l sequence of t e s t s



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by i n s e r t i n g a t an e a r l y stage i n the i n v e s t i g a t i o n s two elements of p a r t i c u l a r importance: t e s t s of genetic t o x i c i t y , and metab o l i c and pharmacokinetic s t u d i e s . The sequence of the main groups of i n v e s t i g a t i o n s may vary according to the nature of the t e s t m a t e r i a l and the purposes f o r which i t i s intended. The emphasis on these two groups of s c i e n t i f i c procedures i s a l l the more welcome because of the r e l u c t a n c e on the p a r t of both Indust r y and Regulatory a u t h o r i t i e s to accept the key r o l e and fundamental importance of metabolism and pharmacokinetics, i n r e l a t i o n to the c o n t r i b u t i o n that such data can make to the design of p r o t o c o l s , as w e l l as to the understanding of e f f e c t s and o v e r a l l i n t e r p r e t a t i o n of t o x i c o l o g i c a l data. The d e c i s i o n - t r e e approach (35) makes l i m i t e d p r o v i s i o n f o r the study of t r a n s p l a c e n t a l , p r e n a t a l and p o s t n a t a l events and omits d e t a i l e d c o n s i d e r a t i o n o f b e h a v i o r a l and immunological aspects of t o x i c a c t i o n . ( I t i s not intended to cover environmental c o n s i d e r a t i o n s . ) The outlook i s pragmatic. I n e v i t a b l y there i s no expressed i n t e r e s t i n e l u c i d a t i n g the mechanism of t o x i c a c t i o n of the t e s t compound. Nevert h e l e s s , the i n f o r m a t i o n intended to be gathered i n the course of the study of metabolism and pharmacokinetics and genetic t o x i cology, when s k i l l f u l l y combined, may w e l l throw considerable l i g h t on the b a s i c b i o l o g i c a l p r o p e r t i e s of the compound. One d i f f i c u l t y i n achieving such understanding i s the f a c t that, i f the d e c i s i o n - t r e e approach i s adhered to r a t h e r r i g i d l y , t o x i c o l o g i c a l p r o p e r t i e s and target organs w i l l not have been revealed a t the time that the i n v e s t i g a t i o n s on metabolism and pharmacokinetics are being c a r r i e d out. While whole-body autoradiography i s a u s e f u l guide i n d i r e c t i n g such i n v e s t i g a t i o n s , there i s no s u b s t i t u t e f o r knowledge of the s i t e ( s ) and doseresponse r e l a t i o n s h i p s of t o x i c a c t i o n . In view of the d e t a i l e d a t t e n t i o n that w i l l be p a i d to metabol i s m l a t e r i n the Conference, metabolic a c t i v a t i o n w i l l be the main focus of d i s c u s s i o n here, s i n c e i t gives r i s e to e l e c t r o p h i l i c a l k y l a t i n g or a r y l a t i n g intermediates capable of inducing damage to c r i t i c a l c e l l u l a r macromolecules. A diagrammatic view i s provided i n F i g . 1 of the o b s t a c l e course faced by such an e l e c t r o p h i l e i n reaching a n u c l e o p h i l e a t the t a r g e t s i t e . Of p a r t i c u l a r importance i n determining t o x i c i t y i s the d e l i c a t e balance between, on the one hand, e l e c t r o p h i l e production and, on the other, e l e c t r o p h i l e d e s t r u c t i o n , o r other b i o t r a n s f o r m a t i o n s that serve the purpose of d e a c t i v a t i o n (36). A host of s p e c i e s s p e c i f i c and o r g a n - s p e c i f i c f a c t o r s e x e r c i s e t h e i r i n f l u e n c e on t h i s balance. The a v a i l a b i l i t y of r a p i d t e s t s of mutagenic p o t e n t i a l has f a c i l i t a t e d the d e t e c t i o n of a c t i v a t e d metabolites. An elegant demonstration of the use of the Ames t e s t f o r t h i s purpose i s provided by the work of Casida and h i s colleagues (37, 38> 39 > who tracked down the formation of a mutagenic a c t i v a t i o n product, 2 - c h l o r o a c r o l e i n , from S - c h l o r o a l l y l t h i o c a r b a m a t e h e r b i c i d e s ( d i a l l a t e , t r i a l l a t e and s u l f a l l a t e ) . Metabolic a c t i v a t i o n by



14


METABOLISM •Topography • Specificities • Activities Activation Protective | " mechanisms • memJ branesi ^Detoxkation C

Figure 1.

Repair Systems Target dose / TARGET \ • Efficiency Cxt / Microenvironment ιi · Capacity • Fidelity Physicochemical properties ,

e

Modulating Foctors

Diagram of the chain of events attendant upon metabolic activation


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i n t e s t i n a l b a c t e r i a and by p l a n t e x t r a c t s has a l s o been shown to occur (41).


Human Studies Studies c a r r i e d out i n human v o l u n t e e r s , or with t i s s u e s of human o r i g i n have great p o t e n t i a l value (42). The c o n t r i b u t i o n of e p i d e m i o l o g i c a l s t u d i e s i s discussed l a t e r i n t h i s Conference by Dr. M. W. Palshaw. In c e r t a i n instances, such as a n t i c h o l i n e s t e r a s e s , only human experience can serve to d e f i n e n o - e f f e c t and minimum-effect l e v e l s f o r man. In other s i t u a t i o n s where exposure i s low, i n d i c e s of e f f e c t may be hard to come by. The a l k y l a t i o n of h i s t i d i n e and c y s t e i n e i n the g l o b i n moiety of hemoglobin has been suggested as an index of e f f e c t s of a l k y l a t i n g agents (43) but has not found general a p p l i c a t i o n . In c o n t r a s t to these problems, evidence of exposure to p e s t i c i d e s i s o f t e n much more r e a d i l y a v a i l a b l e by a n a l y s i s of excreta, body f l u i d s and expired a i r (44). The power of modern a n a l y t i c a l procedures, a t o p i c to be addressed l a t e r i n t h i s Conference, i s exemplified by the c h a r a c t e r i z a t i o n of 115 organic compounds i n samples of breath from 54 subjects (44)· Exhaled ethane and n-pentane i n mice, r a t s and monkeys (45) has proved to be a u s e f u l index of l i p i d p e r o x i d a t i o n , these gases being derived from ω 3and ωβ-fatty a c i d hydroperoxides (12, 13). Non-invasive measures of drug metabolizing capacity have been developed, using ^Cphenacetin or -^C-aminopyrine; hepatic d y s f u n c t i o n can be assessed i n an analogous manner (46, 47, 48, 49). On the h o r i z o n i s the e x c i t i n g promise of the a p p l i c a t i o n of nuclear magnetic resonance to monitor metabolite concentrations, n o n - i n v a s i v e l y , i n human subjects (50). F i n a l l y , human t i s s u e s are f i n d i n g i n c r e a s i n g use f o r meta b o l i c and other s t u d i e s . A human l i v e r bank has been e s t a b l i s h e d i n Sweden f o r storage of l i v e r microsomal suspensions (51)· Human lymphocytes, monocytes and f i b r o b l a s t s have found extensive a p p l i c a t i o n , p a r t i c u l a r l y i n mutagenic s t u d i e s (52, S3, 54, 550. Summing up, while the p r i n c i p a l emphasis i n t h i s Conference w i l l n e c e s s a r i l y be placed on animal s t u d i e s , the t o x i c o l o g i s t should s e i z e every opportunity to secure human data, f o r which animal r e s u l t s are at best an imperfect s u b s t i t u t e . A Revolution i n Toxicology? Advances i n t o x i c o l o g i c a l methodology i n recent years, notably b e t t e r a n a l y t i c a l methods and the procedures made a v a i l a b l e by molecular b i o l o g y and genetic t o x i c o l o g y are making p o s s i b l e an understanding of the mechanisms of t o x i c a c t i o n . This i n i t s e l f i s an a u t o c a t a l y t i c process: as we gain b e t t e r and b e t t e r under standing of such mechanisms i t becomes e a s i e r to deal with the next problem i n the same category. In the process of under standing mechanism, one has to take i n t o account the i n f l u e n c e of




16

exposure to the compound on homeostatic processes o f the body, and of the defensive and adaptive l i m i t s which the body can a t t a i n i n response to t o x i c exposure. Thus there evolves a comprehension of the dose-response r e l a t i o n s h i p f o r that p a r t i c u l a r compound under the c o n d i t i o n s of t e s t i n g . E v a l u a t i o n of s a f e t y i n v o l v e s the conceptual i n t e g r a t i o n and i n t e r p r e t a t i o n of the information gained from p h y s i c a l measurements, knowledge of chemical s t r u c t u r e and p r o p e r t i e s , and the study of b i o l o g i c a l e f f e c t s i n r e l a t i o n to doses used. Thus an i n t e l l e c t u a l a c t i v i t y enters i n t o e v a l u a t i o n of s a f e t y which transcends the mere assembly of data. Interp r e t a t i o n of r i s k assessment i n v o l v e s f u r t h e r a thorough knowledge and understanding of the nature, uses and exposure l e v e l s ( e x i s t ing or a n t i c i p a t e d ) of a chemical or mixture of chemicals i n a product. The new concepts, techniques and approaches that a r e c r e a t i n g a ferment i n Toxicology, taken together with the impetus f u e l l e d by a c c e l e r a t i n g advances i n the b a s i c sciences, b i d f a i r to r e v o l u t i o n i z e the p r a c t i c e of r i s k assessment. Whether t h i s very r e a l promise w i l l be t r a n s l a t e d i n t o concrete achievements i n terms of greater safety depends on the freedom and encouragement a f f o r d e d to the t o x i c o l o g i s t to p a r t i c i p a t e i n and advance the r e v o l u t i o n . In the short term, the prospects do not appear f a v o r a b l e ; but H i s t o r y teaches us that powerful f o r c e s working f o r change do u l t i m a t e l y f i n d expression, despite bureaucratic defense of the s t a t u s quo.

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7. Golberg, L. (1979). Implications for human health. Environ. Health Perspect. 32:273-277. 8. Hard, G. C., King, H., Borland, R., Stewart, B. W. and Dobrostanski, B. (1977). Length of in vivo exposure to a carcinogenic dose of dimethylnitrosamine necessary for subsequent expression of morphological transformation by rat kidney cells in vitro. Oncology 34:16-19. 9. Ciba Foundation Symposium 65. (1979). Oxygen Free Radicals and Tissue Damage. Excerpta Medica, New York. 10. Fridovich, I. (1979). Superoxide dismutases: defence against endogenous superoxide radical. In Oxygen Free Radicals and Tissue Damage. Excerpta Medica, New York, pp. 77-94. 11. Flohé, L. (1979). Glutathione peroxidase: Fact and fiction. In Oxygen Free Radicals and Tissue Damage. Excerpta Medica, New York, pp. 95-122. 12. Tappel, A. L. (1980). Measurement of and protection from in vivo lipid peroxidation. In Free Radicals in Biology, Vol. IV. Ed. W. A. Pryor, Academic Press, New York, pp. 1-47. 13. Bus, J. S. and Gibson, J. E. (1979). Lipid peroxidation and its role in toxicology. In Reviews in Biochemical Toxicology 1. Eds. E. Hodgson, J. R. Bend and R. M. Philpot, Elsevier/North-Holland, New York, pp. 125-149. 14. Reed, D. J. and Beatty, P. W. (1980). Biosynthesis and regulation of glutathione: Toxicological implications. In Reviews in Biochemical Toxicology 2. Eds. E. Hodgson, J. R. Bend and R. M. Philpot, Elsevier/North-Holland, New York, p. 213. 15. Chasseaud, L. F. (1979). The role of glutathione and glutathione S-transferases in the metabolism of chemical carcinogens and other electrophilic agents. In Advances in Cancer Research, Vol. 29. Eds. G. Klein and S. Weinhouse, Academic Press, New York, pp. 175-274. 16. Berrigan, M. J . , Gurtoo, H. L., Sharma, S. D., Struck, R. F. and Marinello, A. J. (1980). Protection by N-acetylcysteine of cyclophosphamide metabolism - related in vivo depression of mixed function oxygenase activity and in vitro denaturation of cytochrome P-450. Βiochem. Biophys. Res. Commun. 93:797-803. 17. Kahl, G. F., Friederici, D. E., Bigelow, S. W., Okey, A. B. and Nebert, D. W. (1980). Ontogenetic expression of regulatory and structural gene products associated with the Ah locus. Dev. Pharmacol. Ther.1:137-162. 18. Timbrell, J. A. (1979). The role of metabolism in the hepatotoxicity of isoniazid and iproniazid. Drug Metab. Rev. 10:125-147. 19. Reece, P. Α., Cozamanis, I. and Zacest, R. (1980). Kinetics of hydralazine and its main metabolites in slow and fast acetylators. Clin. Pharmacol. Ther. 28:769-778.



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20. Shepherd, A.M.M., Ludden, Τ. Μ., McNay, J. L. and Lin, M.S. (1980). Hydralazine kinetics after single and repeated oral doses. Clin. Pharmacol. Ther. 28:804-811. 21. Tannen, R. H. and Weber, W. W. (1980). Inheritance of acetylator phenotype in mice. J. Pharmacol. Exp. Ther. 213:480-484. 22. Weinshilboum, R. M. (1980). 'Methylator status' and assess ment of variation in drug metabolism. Trends in Pharmacol. Sci. 1:378-380. 23. Harris, C. C., Mulvihill, J. J., Thorgeirsson, S. S. and Minna, J. D. (1980). Individual differences in cancer susceptibility. Ann. Intern. Med. 92:809-825. 24. Report of the Secretary's Commission on Pesticides and their Relationship to Environmental Health (1969). "Inter actions". U. S. Department of Health, Education, and Wel fare, Washington, D.C., pp. 509-564. 25. Fabacher, D. L., Kulkarni, A. P. and Hodgson, E. (1980). Pesticides as inducers of hepatic drug-metabolizing enzymes—I. Mixed function oxidase activity. Gen. Pharmac. 11:429-435. 26. Kulkarni, A. P., Fabacher, D. L. and Hodgson, E. (1980). Pesticides as inducers of hepatic drug-metabolizing enzymes— II. Glutathione S-transferases. Gen. Pharmac. 11:437-441. 27. Chadwick, R. W., Faeder, E. J., King, L. C., Copeland, M. F., Williams, K. and Chuang, L. T. (1978). Effect of acute and chronic Cd exposure on lindane metabolism. Ecotoxicol. Environ. Safety 2:301-316. 28. Sharma, R. P. and Zeeman, M. G. (1980). Immunologic altera tions by environmental chemicals: Relevance of studying mechanisms versus effects. J. Immunopharmacol. 2:285-307. 29. Scientific Committee, Food Safety Council. (1978). The importance of specifications for substances in their safety evaluation in foods. In "Proposed System for Food Safety Assessment". Fd Cosmet. Toxicol., Suppl. 2, 16:17-24. 30. National Academy of Sciences Committee Saccharin Report No. 1. (1978). "Saccharin: Technical Assessment of Risks and Benefits". National Research Council/National Academy of Sciences, Washington, D. C. pp. 3-44 to 3-61. 31. Stuper, A. J . , Brügger, W. E. and Jurs, P. C. [Eds.] (1979). Computer Assisted Studies of Chemical Structure and Biological Function. John Wiley & Sons, New York, pp. 2-14. 32. Jurs, P. C., Chou, J. T. and Yuan, M. (1979). Computer-assisted structure-activity studies of chemical carcinogens. A heterogeneous data set. J. Medicinal Chem. 22:476-483. 33. Jurs, P. C., Chou, J. T. and Yuan, M. (1979). Studies of chemical structure-biological activity relations using pattern recognition. In Computer-Assisted Drug Design. Eds. E. C. Olson and R. E. Christoggersen, American Chemical Society, Washington, D.C., pp. 103-129.



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34. National Institute of Environmental Health Sciences. Request for Research Grant Applications: RFA (NIH-NIEHS-EP-811). NIH Guide for Grants and Contracts, Vol. 9, No. 12, October 10, 1980. 35. The Scientific Committee, Food Safety Council. (1978). "Pro posed System for Food Assessment". Fd Cosmet. Toxicol., Suppl. 2, 16:1-136. 36. Wright, A. S. (1980). The role of metabolism in chemical mutagenesis and chemical carcinogenesis. Mutat. Res. 75:215-241. 37. Schuphan, I., Rosen, J. D. and Casida, J. E. (1979). Novel activation mechanism for the promutagenic herbicide diallate. Science 205:1013-1015. 38. Schuphan, I. and Casida, J. E. (1979). S-chloroallyl thiocarbamate herbicides: Chemical and biological formation and rearrangement of diallate and triallate sulfoxides. J. Agric. Food Chem. 27:1060-1067. 39. Rosen, J. D., Schuphan, I., Segall, Y. and Casida, J. E. (1980). Mechanism for the mutagenic activation of the her bicide sulfallate. J. Agric. Food Chem. 28:880-881. 40. Rosen, J. D., Segall, Y. and Casida, J. E. (1980). Muta genic potency of haloacroleins and related compounds. Mutat. Res. 78:113-119. 41. Wildeman, A. G., Rasquinha, I. A. and Nazar, R. N. (1980). Effect of plant metabolic activation on the mutagenicity of pesticides. Amer. Ass. Cancer Res., Abstract No. 357, p. 89. 42. Golberg, L. (1975). Safety evaluation concepts. J. Ass. Official Analyt. Chem. 58:635-644. 43. Calleman, C. J., Ehrenberg, L., Jansson, B., Osterman-Golkar, S., Segerback, D., Svensson, K. and Wachtmeister, C. A. (1978). Monitoring and risk assessment by means of alkyl groups in hemoglobin in persons occupationally exposed to ethylene oxide. J. Environ. Path. Toxicol. 2:427-442. 44. Krotoszynski, Β. Κ., Bruneau, G. M. and O'Neill, H. J. (1979). Measurement of chemical inhalation exposure in urban population in the presence of endogenous effluents. J. Analyt. Toxicol. 3:225-234. 45. Dumelin, E. E., Dillard, C. J. and Tappel, A. L. (1978). Breath ethane and pentane as measures of vitamin Ε protection of Macaca radiata against 90 days of exposure to ozone. Environ. Res. 15:38-43. 46. Desmond, P. V., Branch, R. Α., Calder, I. and Schenker, S. (1980). Comparison of [ C]phenacetin and amino[ C]pyrine breath tests after acute and chronic liver injury in the rat. Proc. Soc. Exp. Biol. Med. 164:173-177. 47. Roots, I, Nigam, S., Gramatzki, S., Heinemeyer, G. and Hildebrandt, A. G. (1980). Hybrid information provided by the C-aminopyrine breath test studies with C-monomethylaminoantipyrine in the guinea pig. Naunyn-Schmiedeberg's Arch. Pharmacol. 313:175-178. 14

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48. Henry, D. Α., Sharpe, G., Chaplain, S., Cartwright, S., Kitchingman, G., Bell, G. D. and Langman, M.J.S. (1979). The [ C] -aminopyrine breath test, a comparison of different forms of analysis. Br. J. clin. Pharmac. 8:539-545. 49. Sotaniemi, Ε. Α., Pelkonen, R. O. and Puukka, M. (1980). Measurement of hepatic drug-metabolizing enzyme activity in man. Eur. J. Clin. Pharmacol. 17:267-274. 50. Griffiths, J. R. and Iles, R. A. (1980). Nuclear magnetic resonance - a 'magnetic eye' on metabolism. Clin. Sci. 59:225-230. 51. Von Bahr, C., Groth, C.-G., Jansson, H., Lundgren, G., Lind, M. and Glaumann, H. (1980). Drug metabolism in human liver in vitro: Establishment of a human liver bank. Clin. Pharmacol. Ther. _27:711-725. 52. Albertini, R. J. (1980). Drug-resistant lymphocytes in man as indicators of somatic cell mutation. Teratogenesis, Carcinogenesis, and Mutagenesis 1:25-48. 53. Lake, R. S., Kropko, M. L., McLachlan, S., Pezzutti, M. R., Shoemaker, R. H. and Igel, H. J. (1980). Chemical carcinogen induction of DNA-repair synthesis in human peripheral blood monocytes. Mutat. Res. 74:357-377. 54. Yang, L. L., Maher, V. M. and McCormick, J. J. (1980). Errorfree excision of the cytotoxic, mutagenic N-deoxyguanosine DNA adduct formed in human fibroblasts by (±)-7β,8α-dihydroxy9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene. Proc. Natl. Acad. Sci. USA 77:5933-5937. 55. Vigfusson, Ν. V. and Vyse, E. R. (1980). The effect of the pesticides, dexon, captan and roundup, on sister-chromatid exchanges in human lymphocytes in vitro. Mutat. Res. 79:53-57.


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