The Pesticide Chemist and Modern Toxicology - American Chemical

19 Biochemical Aspects: A Summary

Downloaded by UNIV OF MASSACHUSETTS AMHERST on May 26, 2018 | https://pubs.acs.org Publication Date: August 10, 1981 | doi: 10.1021/bk-1981-0160.ch019

GINO J. MARCO CIBA-GEIGY Corporation, Biochemistry Department, 410 Swing Road, Greensboro, NC 27409

Historically, cancer has been associated with some kind of chemical exposure as was first documented from human exposure to environmental contaminants. Over 2 centuries ago, Percival Pott reported a high increase of scrotal cancers in young chimney sweeps. However, there have always been various incidences of certain cancers that had no clear association with a specific environmental contaminant. When I was in graduate school, biochemical studies devoted to understanding the mechanism of cancer were considered the "graveyard" for biochemists. It seems that many biochemical interpretations were based on minimal, if any, knowledge of the biological processes involved in cancer development. In the session's first paper, Dr. Laishes pointed to highly significant advances, armed with hard data, and in certain instances, left us with some exciting potential directions leading out of the "graveyard." The biochemistry of cancer development is far from understood and may differ in each target tissue. In seeking to solve this mystery, the biochemist, as a chemical Sherlock Holmes in this detective game, has been offered some interesting clues, for example, the demonstration that carcinogenesis can be divided, at least in some instances, into two qualitatively different biological processes, that is, initiation and promotion. Focus i s now on new e f f o r t s i n t o understanding, not only the molecular defects i n target c e l l s , but also the p h y s i o l o g i c m i l i e u necessary f o r the "promotion" of e a r l y a l t e r e d c e l l s to the development of frank, i n v a s i v e , and even metastatic carcinoma. The g e n e r a l i t y of the i n i t i a t i o n - p r o m o t i o n , two-step system i s one of our most readable d i r e c t i o n a l signs. The fascinating sequence r e l a t i o n s h i p s , that i s , the need to apply the i n i t i a t o r p r i o r to the promoter, provided a remarkable clue i n our understanding. Not only has the i r r e v e r s i b l e , a d d i t i v e concept surfaced again, but commonality of mechanisms i n many chemicals was seen. The common formation

0097-6156/81/0160-0323$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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of e l e c t r o p h i l i c reactants from s t r u c t u r a l l y diverse chemical carcinogens remains as one of the most powerful c o n t r i b u t i o n s of the past 20 years. With the concept of covalent binding to form carcinogenic adducts, we t r u l y were i n the realm of the chemist. Chemicals, with a n t a g o n i s t i c e f f e c t s on the process of c a r c i n o g e n e s i s , provided f u e l for the discovery of enzyme i n d u c t i o n i n mammals. As the concept of precarcinogenic compounds y i e l d i n g proximate and f i n a l l y ultimate carcinogens developed as metabolic processes, the biochemist moved c l o s e r to h i s areas of i n t e r e s t and e x p e r t i s e . F i n a l l y , what was c e r t a i n l y l o g i c a l , but required evidence, was the concept of r e p a i r mechanisms of carcinogenic DNA-adducts. We are now a c q u i r i n g the tools to allow us to d e l i n e a t e those a l t e r a t i o n s i n c e l l u l a r r e g u l a t o r y molecules, induced by chemical carcinogens, that are e s s e n t i a l to the biochemistry of cancer development. With our r e a l i z a t i o n that AAF, as a "complete" carcinogen, has both i n i t i a t i n g and promoting p r o p e r t i e s , the p o s s i b i l i t y of a simple straightforward mechanism of carcinogenesis i s more remote. In attempting to unravel some of these biochemical pathways having t o x i c o l o g i c a l consequences, Dr. G i l l e t t e i n d i c a t e d that t o x i c p o t e n t i a l between parent compound and i t s chemically stable metabolites was r e l a t i v e l y simple. I s o l a t i o n , i d e n t i f i c a t i o n , synthesis and t e s t i n g of these metabolites for t h e i r t o x i c e f f e c t s have been an e f f e c t i v e way to deal with them. However, other s t r a t e g i e s must be used for that e l u s i v e , o f t e n s p e c u l a t i v e , s h o r t - l i v e d chemically r e a c t i v e metabolite. Here, the d e t e c t i v e i n the chemist must again surface. While n u c l e i c acids are t a r g e t s leading to p o t e n t i a l serious consequences, other targets are equally of concern. I n t r a c e l l u l a r enzymes, p r o t e i n s i n general, c e l l membranes, and l o c a t i o n s of r e p a i r processes are some of the more important a d d i t i o n a l i n t e r a c t i o n s i t e s . However, d i r e c t covalent binding of a chemical i s not the only a l t e r a t i o n p o s s i b l e . As one example, i n d i r e c t attack can occur by free r a d i c a l s generated by the chemically r e a c t i v e intermediate. We now have t r u l y e l u s i v e mechanisms to sort out. The parent, stable metabolites, r e a c t i v e intermediates and i n d i r e c t r e a c t i v e e n t i t i e s provide us with the concepts to e l u c i d a t e the processes of a l l t o x i c o l o g i c a l phenomena, not s o l e l y c a r c i n o g e n e s i s . A complicating f a c t o r i s that a b i l i t y of the chemically r e a c t i v e metabolites to react with m u l t i p l e c e l l components, and proceeding at v a r y i n g r a t e s . Yet, the chemically r e a c t i v e metabolite may be scavenged by c e l l u l a r components with large numbers of n u c l e o p h i l i c groups leading to t h e i r p r e f e r e n t i a l attack. Also, these r e a c t i o n s might be developed a f t e r the toxic e f f e c t s of the parent have been expressed, p o s s i b l y leading our d e t e c t i v e down a wrong t r a i l .

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


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By use of various inducers and i n h i b i t o r s of the metabolism of the toxicant and the emphasis on covalent binding to p r o t e i n , as an i n d i r e c t measure of the concentration-time exposure of the r e a c t i v e metabolite to the t a r g e t , we are provided with a strategy p e r m i t t i n g the use of k i n e t i c s already well defined i n the protein-enzyme f i e l d . By conduct i n g a sequence of i n v i v o and i n v i t r o experiments using the stated s t r a t e g y , a case can be made for the involvement of a chemically r e a c t i v e metabolite i n a given t o x i c i t y . This approach c e r t a i n l y h i g h l i g h t s the m u l t i - d i s c i p l i n e d and d i v e r s e methodological approaches r e q u i r i n g the philosophy of the s c i e n t i f i c g e n e r a l i s t and i n t e r d i s c i p l i n a r y a c t i o n i n experimental design. The r e p a i r mechanism ideas developed i n the previous papers encourage one to b e l i e v e that an absolute t h r e s h o l d e x i s t s for chemical i n d u c t i o n of cancer. I t i s the concept of one i r r e v e r s i b l e molecular event leading to the i n d u c t i o n of cancer that provides the stimulus arguing against a t h r e s hold. Implied i n the concept of threshold i s the a b i l i t y to q u a n t i t a t i v e l y estimate the carcinogenic r i s k at low l e v e l s of exposure. Dr. Ramsey addressed t h i s question which brings to bear the e l u c i d a t i o n of the shape of dose-response curve. He discussed the pharmacokinetic c h a r a c t e r i s t i c of a chemical since they are i n t r i c a t e l y l i n k e d to i t s t o x i c response. The clues to unravel i n t h i s d e t e c t i v e ' s game are how b i o l o g i c a l r a t e processes vary with chemical concentration. We saw that the t r a n s i t i o n from l i n e a r to non-linear k i n e t i c s , as the dose l e v e l increased, c o n s t i t u t e s the pharmacokinetic t h r e s hold; and t h i s t r a n s i t i o n was a gradual one. The extrapol a t i o n made from data obtained above or below the pharmac o k i n e t i c threshold dose were shown to be of major concern when attempting to p r e d i c t t o x i c e f f e c t s at low exposure levels. In assessing r i s k by use of models, the main d i f f e r ences are i n the r a p i d i t y with which zero exposure i s approached. But, few make p r o v i s i o n for an absolute threshold for carcinogenic response. There i s absolute dependence on concentration of the carcinogenic e n t i t y being d i r e c t l y p r o p o r t i o n a l to dose or parent chemical. Thus, p r e d i c t i o n s based on dose l e v e l s alone can lead to a f a l l a c i o u s c o n c l u s i o n . The speaker i n d i c a t e d that the r e l a t i o n s h i p between steady state concentrations and administered dose l e v e l s to be c r u c i a l i n i n t e r p r e t i n g and p r e d i c t ing any t o x i c response as a f u n c t i o n of exposure l e v e l . Thus, we have added mathematical t o o l s to our chemical ones in order to expose a chemical of t o x i c o l o g i c a l concern. There i s another complicating threshold concept. The c y t o t o x i c threshold i s considered to be the r e s u l t of the f i n i t e c a p a c i t y of the c e l l to t o l e r a t e i n j u r y to the m u l t i p l i c i t y of c e l l u l a r components other than c r i t i c a l parts o f the genome before the c e l l i t s e l f sustains i n j u r y . Recurring


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c y t o t o x i c i n j u r y may lead to an increased rate of tumor production. While the c y t o t o x i c threshold i s not e a s i l y e x p r e s s i b l e i n the formal mathematical terms of the pharmac o k i n e t i c threshold, i t nevertheless comprises a range of dose l e v e l s above which the rate of chemically induced cancer may be d i s p r o p o r t i o n a t e l y much greater than that at lower levels. Dr. Ivie proceeded to show the complexities seen i n how b i o l o g i c a l systems deal with a x e n o b i o t i c , with emphasis on pesticides. Since p e s t i c i d e s by design are meant to be t o x i c and a l l l i v i n g things have much i n common b i o c h e m i c a l l y , the t o x i c o l o g i c a l consequences to man must be considered i n t h e i r use. Yet, the j u d i c i o u s use of p e s t i c i d e s contributes i n p o s i t i v e ways to human welfare. Studies i n p e s t i c i d e metabolism not only show the r o l e i n the expression of p e s t i c i d e t o x i c i t y but aid i n the e v a l u a t i o n of t o x i c o l o g i c a l s i g n i f i cance of these metabolic products. Studies leading to an understanding of the mechanisms of p e s t i c i d a l a c t i o n aid i n appropriate s e l e c t i o n of pest c o n t r o l agents with minimal environmental consequences. The e v a l u a t i o n of 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 must include i t s metabolites since much of human exposure i s r e l a t e d to the decomposition products of the p e s t i c i d e . Yet, p e s t i c i d e metabolism studies cannot be considered as an end to themselves, but rather are a means to an end. That i s , they are intended to gain data of value toward assessment 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 the p e s t i c i d e . As the speaker showed, the metabolism i s composed of complex, multi-stepped r e a c t i o n s , leading to complicated, d i f f i c u l t to i d e n t i f y metabolites, o f t e n present i n extremely small concentrations. This places the chemist working i n metabolism research at the knife-edge of modern technology and at the razor's edge of i n t e r p r e t a t i o n . For example, what i s a major or minor metabolite and of what s i g n i f i c a n c e i s a bound residue? An added wrinkle i s the impact of r e g u l a t o r y requirements on the why, what and how of metabolism research. As posed i n t h i s paper, can p e s t i c i d e metabolism studies 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, e s p e c i a l l y with more t o x i c o l o g i c a l relevance to the ultimate b i o l o g i c a l system, man? P o s s i b l y , the d i r e c t use i n man of r e l a t i v e l y safe techniques, such as heavy isotopes, could be one of several ways i n future s t u d i e s . Dr. Wright continued the look into newer biochemical s t r a t e g i e s for understanding p e s t i c i d e t o x i c o l o g y . The focus was on approaches to improve the q u a l i t y of human r i s k assessment based on q u a n t i t a t i v e dose-response data generated in experimental animals. The nature and magnitude of the target dose can be a prime determinant of the nature and amount of key l e s i o n s . In t h i s approach, DNA i s considered the key ( i . e . , primary, c r i t i c a l ) target of most chemical



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mutagens. Thus, the target dose of such chemicals i s DNAdose estimated by determining the nature of the adducts o f r e a c t i o n of the ultimate mutagen with DNA and measuring the amounts of these adducts. Reaction rate constants and b i o l o g i c a l h a l f - l i v e s of the adducts as well as duration of exposure are needed for the c a l c u l a t i o n of target dose. Provided that target dose can be accurately measured i n humans and i n the experimental model, then the exposure values i n the e x t r a p o l a t i v e models for r i s k assessment can be s u b s t i t u t e d by estimates of target dose. S u b s t i t u t i o n of exposure values by target dose should improve the q u a l i t y of r i s k assessment by emphasizing the f a c t o r s that i n f l u e n c e the nature and the concentration of the toxicant at i t s c r i t i c a l target. It was emphasized that the appropriateness of the metabolizing system of the model to f i n d these adducts needs to be experimentally e s t a b l i s h e d , not simply assumed. However, human primary c e l l c u l t u r e s could e f f e c t i v e l y mimic i n v i v o metabolism. The major p r a c t i c a l and conceptual problems associated with the target dose approach center around the determination of target dose (DNA-Dose) i n humans. While r a d i a t i o n t e c h n i ques are u s e f u l i n experimental animals, t h i s approach i s not a p p l i c a b l e to humans. The use of haemoglobin as a dosemonitor for DNA-adducts was discussed with i n d i c a t i o n s that t h i s technique may not always be appropriate. The use of immunochemistry i n assessing target dose was suggested with ample opportunity for chemists and immunologists to develop a j o i n t endeavor by the d e t e c t i o n and assay of p r o t e i n and n u c l e i c acid adducts. The target dose approach i s designed to take i n t o account d i f f e r e n c e s between the b i o l o g i c a l model and human f a c t o r s for determining the rate of formation of key l e s i o n s . But, the r i s k model takes no account of d i f f e r ences between test system and humans i n f a c t o r s to determine progression of key l e s i o n s into overt b i o l o g i c a l e f f e c t s . Future work should i d e n t i f y the relevant species d i f f e r e n c e s i n f a c t o r s determining the progression phase. The target dose approach for assessing genetic r i s k i n man i s yet to be applied to the p e s t i c i d e f i e l d except for determining the relevance of b a c t e r i a l mutation test data for p r e d i c t i o n of genetic r i s k i n mammals. However, t h i s i s a new strategy worth c o n s i d e r i n g . Every s c i e n t i f i c d i s c i p l i n e has i t s problems and p i t f a l l s and biohemistry i s no exception. Most of those discussed by Dr. Waggoner may not seem new. Maybe that i s a reason they are s t i l l problems and p i t f a l l s . One p i t f a l l i s the often lack of coordinate planning, e s p e c i a l l y between the f i e l d s of chemistry and t o x i c o l o g y . An example of t h i s i s the minor e f f o r t i n d e f i n i n g the mechanism of t o x i c o l o g i c a l action. Is the pressure to meet regulatory needs a cause of this? Such pressure could even be the reason for minimal



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amounts of comparative biochemical s t u d i e s . While animal metabolic studies are a way to quick data, i s t h i s d e t r a c t i n g from more i n t e n s i v e development of i n v i t r o biochemical studies? The route of exposure c e r t a i n l y provides problems and p i t f a l l s i n a l l aspects (methodologically and i n t e r p r e t a t i v e l y ) , e s p e c i a l l y i n o r a l vs. dermal vs. i n h a l a t i o n treatments. Separation of the three i n a meaningful way i s a r e a l challenge. Metabolite i d e n t i f i c a t i o n i s c o n t i n u a l l y i n a s t a t e - o f - t h e - a r t world simply because new t o o l s and t e c h n i ques open approaches not p r e v i o u s l y a c c e s s i b l e . As h i g h l y complex metabolites are i s o l a t e d at lower concentrations, problems magnify, i n c l u d i n g those of contamination and stability. What do the studies of metabolism of unknown metabolite mixtures r e a l l y show? Since a l l the metabolites i n a t i s s u e may not be ingested, how are the r e s u l t s i n t e r preted, e s p e c i a l l y when low l e v e l s g e n e r a l l y must be fed? P o s s i b l y the l a r g e s t problem and p i t f a l l , r e l a t e d to a l l of science as w e l l , i s the r e s i s t a n c e to change and o p p o s i t i o n to n o n - t r a d i t i o n a l approaches. So where do we now stand i n the biochemical area? Biochemical studies to understand carcinogenic a c t i o n c e r t a i n l y are no longer a graveyard. In f a c t , the area i s very much a l i v e with new t o o l s , methodology and concepts. New informat i o n i s c o n t i n u a l l y s u r f a c i n g , p r o v i d i n g many new ideas about the carcinogenic processes. Rather than a graveyard, i t i s more l i k e a s i x - l a n e expressway with r e s u l t s and conclusions speeding, with d i r e c t i o n , toward a r a t i o n a l e l u c i d a t i o n of these complex i n t e r a c t i o n s . R E C E I V E D February 9,

1981.


The Pesticide Chemist and Modern Toxicology - American Chemical

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