The Pesticide Chemist and Modern Toxicology - ACS Publications

10 The Application of Fundamentals in Risk Assessment

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ALBERT C. KOLBYE, JR. Associate Bureau Director for Toxicology, Bureau of Foods, Food and Drug Administration, 200 C Street, SW, Washington, DC 20204 "Risk assessment" is a popular term that appeals to scientists and regulators concerned with the vexing problems associated with evaluating and estimating potential hazards to human health. My preference is to talk in terms of evaluating potential hazards to human health and to avoid using the term "risk assessment." This presentation will focus on aspects of evaluating chemical safety in relation to carcinogenesis, but the fundamental considerations are relevant to many other biological end-points of human disease. Extrapolation Models Risk assessment has too many different meanings depending upon the viewpoints of the scientists and non-scientists using the phrase. Today, one meaning of risk assessment concerns usage of various mathematical models to extrapolate dose-response relationships of toxicologic data observed by experimental or epidemiological techniques in order to project estimates of expected disease incidence from populations of animals to humans exposed to significantly smaller amounts of the chemical substance under investigation. These mathematical models vary in the premises assumed to apply to the shape of the dose-response curve as exposures are decreased to zero levels. Extrapolation models are frequently applied by statisticians examining biological dose-response data who have developed a significant volume of literature concerning theoretical considerations of such models. These models represent a very simplistic approach towards data that in reality reflect highly complex biological considerations not easily explained to non-toxicologists and regulators, nor readily understood by lay people, such as the ordinary citizen/consumer. E x t r a p o l a t i o n models are u s u a l l y a p p l i e d when c o n s i d e r i n g p o t e n t i a l l y carcinogenic chemicals from a regulatory and s o c i a l policy-making viewpoint. Many of the models adopt very cons e r v a t i v e premises that i n e f f e c t assume the hypothesis that

This chapter not subject to U.S. copyright. Published 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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there i s no "safe" exposure t o a carcinogenic chemical and that any exposure w i l l be a s s o c i a t e d with some d e f i n a b l e r i s k f o r cancer i n d u c t i o n i n the population at l a r g e . This philosophy implies that there are no threshold phenomena a s s o c i a t e d with cancer i n d u c t i o n . A recent comprehensive reference to e x t r a p o l a t i o n models can be found i n the Food Safety Council's F i n a l Report of the S c i e n t i f i c Committee, published June 1980.


Damage/Repair Balance Versus No-Threshold Premise The no-threshold hypothesis evolved i n r e l a t i o n t o various experimental and e p i d e m i o l o g i c a l s t u d i e s concerning the b i o l o g i c a l hazards r e l a t e d to penetrating i o n i z i n g r a d i a t i o n . I t has been c a l l e d the radiomimetic hypothesis, i . e . , that carcinogenic chemicals mimic the carcinogenic e f f e c t s of penetrating r a d i a t i o n . There are some data that i n d i c a t e that r e l a t i v e l y small increases i n exposure to penetrating r a d i a t i o n are a s s o c i a t e d with increases i n the incidence of various cancers. It i s appropriate to r e f l e c t that penetrating r a d i a t i o n by d e f i n i t i o n penetrates through t i s s u e s without r e s p e c t i n g many of the p h y s i o l o g i c a l b a r r i e r s such as membranes which have very important and complex f u n c t i o n s to regulate the entry and e x i t of chemicals i n c e l l s and m i c r o - c e l l u l a r o r g a n e l l e s . We should keep i n mind, however, that while penetrating r a d i a t i o n can and does induce damage to DNA, there are mechanisms e x i s t i n g i n the mammalian body to r e p a i r such b i o l o g i c a l damage. Unrepaired damage to DNA can occur e i t h e r by overwhelming p h y s i o l o g i c a l r e p a i r or i f the normal r e p a i r mechanisms cannot operate i n p a r t i c u l a r instances to r e p a i r c e r t a i n types of damage. We a l s o know that error-prone r e p a i r may occur and c o n t r i b u t e to the net r e s u l t i n g damage of DNA. When e v a l u a t i n g human exposures to penetrating r a d i a t i o n or to chemicals, we should consider the balance between b i o l o g i c a l damage and b i o l o g i c a l r e p a i r . I f r e p a i r i s complete, no permanent damage w i l l occur. I f r e p a i r i s incomplete, or p o t e n t i a t e s the damage because error-prone r e p a i r i s invoked, or i f normal r e p a i r i s overwhelmed by excessive damage, then adverse e f f e c t s relevant to the i n d u c t i o n of cancer and genetic damage are l i k e l y to occur, provided the somatic or germinal c e l l s involved survive. What Is A "Carcinogen"? We can now r e f l e c t on some of the present p h i l o s o p h i e s we c u r r e n t l y employ to detect and regulate "carcinogenic" chemicals. If exposing t e s t animals to a chemical i s a s s o c i a t e d with a s t a t i s t i c a l l y s i g n i f i c a n t increase i n the incidence of cancers i n the t e s t animals as compared to unexposed c o n t r o l s , our present p r a c t i c e i s to designate the chemical substance a "carcinogen."

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


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Closer examination of t h i s premise as p r a c t i c e d suggests that every i n f l u e n c e upon the incidence of cancer exerted by various exposures to cancer substances, i f a p o s i t i v e i n f l u e n c e , would be designated as being "carcinogenic." Perhaps i t i s now appropriate to question the v a l i d i t y and s u f f i c i e n c y of that premise. (1_) As s c i e n t i s t s and r e g u l a t o r s , we tend to g e n e r a l i z e . Any g e n e r a l i t y w i l l have i t s exceptions, and frequently g e n e r a l i z a t i o n s are e i t h e r over-extended or attacked because some exceptions exist. In f a c t , any g e n e r a l i t y has i t s l i m i t a t i o n s . If we continue to designate "carcinogens" on the basis of whether or not a s t a t i s t i c a l l y s i g n i f i c a n t increase i n the incidence of cancer i s induced, l e t us examine f u r t h e r where that p r a c t i c e could lead us. There i s no question that some chemicals are strongly c a r cinogenic. A f t e r relevant experiments are performed, one can observe dramatic increases i n the incidence of c e r t a i n types of cancer as dose-related responses to the chemical exposure. But we should remember that a s t a t i s t i c a l l y s i g n i f i c a n t increase i n incidence does not n e c e s s a r i l y represent a dramatic or powerful increase i n incidence because, as the number of animals under t e s t increases, the a c t u a l d i f f e r e n c e s i n incidence patterns deemed to be s t a t i s t i c a l l y s i g n i f i c a n t w i l l grow smaller. Thus, a h i g h l y s i g n i f i c a n t s t a t i s t i c a l d i f f e r e n c e i n incidence may, i n a c t u a l i t y , be almost n e g l i g i b l e i n terms of p u b l i c h e a l t h importance, although obviously such i s not always the case. A p a r t i c u l a r l y relevant c o n s i d e r a t i o n i s whether the "normal" i n cidence of cancer has j u s t been s h i f t e d from one tumor type to another or merely represents an increased s u r v i v a l to o l d e r age of animals having an i n c r e a s i n g r i s k f o r cancer i n d u c t i o n with i n c r e a s i n g age. One a l s o wonders about the v a l i d i t y of i n t e r p r e t i n g e p i d e m i o l o g i c a l data concerning the apparently i n creased incidence of human cancer i n one country as compared to another when age s p e c i f i c incidence patterns f o r a l l diseases competing f o r m o r t a l i t y have not been completely accounted f o r and evaluated i n r e l a t i o n to each other. (2^) "Carcinogens" Versus Cancer Risk

Factors

A more fundamental question i s whether or not a l l i n f l u e n c e s on the incidence of cancer i n animals or humans are n e c e s s a r i l y r e l a t e d to d i r e c t "carcinogenic" a c t i o n per se ( i n the sense of e l e c t r o p h i l i c a c t i v i t y leading to covalent bonding with DNA). Many s c i e n t i s t s and r e g u l a t o r s concerned with the prevention of cancer are i n s u f f i c i e n t l y aware of a very extensive body of s c i e n t i f i c l i t e r a t u r e which documents the f a c t that many f a c t o r s can i n f l u e n c e the incidence patterns of cancer i n animals and humans. Many i n t e r a c t i o n s can and do take place that e i t h e r p o t e n t i a t e or ameliorate the e f f e c t i v e potency of other substances, endogenous or exogenous, that have p o t e n t i a l c a r c i n o g e n i c i t y . Other substances can d r a m a t i c a l l y i n f l u e n c e the b i o l o g i c a l r e s i s t a n c e of animals and humans to cancer


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i n d u c t i o n , thus a c t i n g to increase or decrease b i o l o g i c a l susc e p t i b i l i t y to cancer i n d u c t i o n . (_3, 4_) Obviously, a decrease i n b i o l o g i c a l r e s i s t a n c e to cancer i n d u c t i o n w i l l increase susc e p t i b i l i t y and, t h e r e f o r e , o v e r a l l r i s k f o r cancer. In t h i s p r e s e n t a t i o n , these substances are designated as "cancer r i s k factors."


Carcinogenesis Involves Progressive Events and Is a M u l t i s t a g e Process A very s u b s t a n t i a l body of evidence leads to the c o n c l u s i o n that the i n d u c t i o n of cancer i s a multistage process i n v o l v i n g a progression of events leading to a formation of a colony of malignant c e l l s which then i s c a l l e d a malignant neoplasm. Cancer c e l l s are "malignant" because they do not respect normal p h y s i o l o g i c a l boundaries and do not accept b i o l o g i c a l c o n t r o l by the l a r g e r s o c i e t y of c e l l s comprising the organism such as the animal or human body. Thus, they p a r a s i t i z e the body, invade i n t o other t i s s u e s , and may seed new c o l o n i e s i n d i s t a n t t i s s u e s to form metastatic l e s i o n s . They do not u s u a l l y a t t a i n the malignant s t a t e immediately. Apparently passage through a d d i t i o n a l generations of c e l l s i s required i n order f o r the c e l l s to a t t a i n a s t a t e of r e l a t i v e autonomy. In i t s simplest form, the p r o g r e s s i v e events involved with the i n d u c t i o n of cancer have been r e f e r r e d to as the " i n i t i a t i o n " and "promotion" stages of c a r c i n o g e n e s i s . This two-stage model was f i r s t observed by s c i e n t i s t s such as Berenblum, Shubik, and Van Duuren who were i n v e s t i g a t i n g chemically-induced s k i n cancer i n rodents. They noted that s k i n c e l l s could be " i n i t i a t e d , " i . e . , s e l e c t i v e l y damaged i n such a way that they a t t a i n e d and r e t a i n e d a p o t e n t i a l f o r malignant conversion. If these e p i t h e l i a l c e l l s were then subsequently t r e a t e d with c e r t a i n chemicals capable of "promoting" the conversion of i n i t i a t e d c e l l s to malignant c e l l s (but incapable of inducing cancer by t h e i r own a c t i o n a l o n e ) , cancer would develop. While these phenomena are not p e r f e c t l y understood at the present time, much knowledge concerning e t i o l o g i c a l mechanisms has been developed i n the past s e v e r a l decades, which i s presented i n b r i e f as f o l l o w s : Genotoxicity and

Initiation

Usually, the f i r s t event i n t h i s multistage progression takes place when c e r t a i n types of damage to DNA are caused by v i r u s e s , r a d i a t i o n , or chemical i n s u l t . C5) The l a t t e r i n volves the c a p a b i l i t y of some e l e c t r o p h i l i c chemicals to react with DNA and c o v a l e n t l y bind to i t (such as by a l k y l a t i o n ) , and d i s r u p t normal sequencing of base p a i r s during r e p l i c a t i o n . Such chemicals have been described as being genotoxic, although



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t h i s term i s employed a l s o i n a broader sense of d e s c r i b i n g other mutagenic c a p a b i l i t i e s of the chemicals i n question, which may i n v o l v e d i f f e r e n t mechanisms l e a d i n g to genotoxic damage. When c e r t a i n types of genotoxic damage to DNA has occurred and other preconditions are f u l f i l l e d , the c e l l may proceed to the s t a t e of being " i n i t i a t e d " i n the sense of now having malignant potential. The p r e c o n d i t i o n s are that the c e l l s u r v i v e s the t o x i c i n s u l t to DNA, that the damage to DNA i s not s u f f i c i e n t l y r e p a i r e d to negate the damage, and that the " c r i t i c a l " unrepaired and damaged DNA can be encoded i n t o the r e p l i c a t i n g genome to p e r s i s t unrepaired i n future generations of c e l l s propagated from the one(s) o r i g i n a l l y i n c u r r i n g the c r i t i c a l genotoxic damage. If an i n i t i a t e d c e l l f o r some reason does not progress i n t o subsequent stages of events, and the s t a t e of i n i t i a t i o n does not revert back towards normality, the c e l l s w i l l r e t a i n t h e i r p o t e n t i a l f o r malignant conversion. There i s evidence from c e l l and t i s s u e c u l t u r e s t u d i e s that varying degrees of r e v e r s i o n towards normality appear to occur, but that i t may w e l l not be complete i n the sense that some i n i t i a t e d c e l l s are l i k e l y to r e p l i c a t e i n d e f i n i t e l y i n the f u t u r e , r e t a i n i n g t h e i r s t a t e of i n i t i a t i o n and thus t h e i r p o t e n t i a l f o r malignant conversion. Promotion Subsequently, a second stage i n the progression of events l e a d i n g to formation of malignant neoplasms i n v o l v e s h y p e r p l a s i a , r e p l i c a t i o n of c r i t i c a l l y damaged DNA i n the a c t i v e genome, increased DNA and i n c r e a s i n g degrees of abnormalities observed i n c e l l s t r u c t u r e and f u n c t i o n leading to autonomous behavior and the b i o l o g i c a l c h a r a c t e r i s t i c s a s s o c i a t e d with malignant neoplasms as described by many c y t o l o g i s t s and p a t h o l o g i s t s . The subsequent stage of carcinogenesis has been r e f e r r e d to as the "promotion" stage which has been e x t e n s i v e l y s t u d i e d o r i g i n a l l y i n the experimental i n d u c t i o n of s k i n cancer and l a t e r with respect to the i n d u c t i o n of other cancers. 03, 7) It was f i r s t n o t i c e d that some chemicals, at the doses given to s k i n , would not induce cancer by themselves or d i d so only a f t e r a prolonged latency period. However, other chemicals, i f a p p l i e d subsequently, "complete" the i n d u c t i o n process by inducing "promotional" phenomena and thus complete the prog r e s s i v e spectrum of events involved with the i n d u c t i o n of cancer. These phenomena have been reproduced experimentally or have been observed to occur not only i n s k i n cancer, but a l s o with respect to the i n d u c t i o n of malignancies i n l i v e r (8^ % 10), forestomach (11), lung (12), breast (L3), kidney (14), bladder (15, 16), and colon (13, 17, 18, 19, 20). Apparently such promoting substances may act i n part by i n f l u e n c i n g enzymes and inducing the synthesis of c e r t a i n polyamines, which




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i n t u r n stimulate h y p e r p l a s i a and DNA r e p l i c a t i o n , and induce changes with respect to c e l l c y c l i n g , c e l l u l a r d i f f e r e n t i a t i o n and maturation. Other changes may occur that are a s s o c i a t e d with membrane a c t i v i t y and f u n c t i o n . An important e f f e c t of a c c e l e r a t e d c e l l d i v i s i o n i s to a c c e l e r a t e the expression of f i x e d DNA damage i n the r e p l i c a t i n g genome. The i n t e r a c t i o n between b i o l o g i c a l r e p a i r of DNA damage and increased f i x a t i o n of such damage by a c c e l e r a t e d DNA r e p l i c a t i o n i s c r i t i c a l , because the timing and e f f e c t i v e n e s s of p h y s i o l o g i c a l DNA r e p a i r may be d i s t u r b e d by the increased m i t o t i c a c t i v i t y induced by h y p e r p l a s t i c t o x i c i t y . Error-prone r e p a i r can a l s o augment the degree to which damaged DNA i s propagated i n t o r e p l i c a t i n g DNA, causing c e l l u l a r " i n i t i a t i o n " which, i f "promoted," can progress to n e o p l a s t i c growth and the abnormal c h a r a c t e r i s t i c s a s s o c i a t e d with cancer. The c l a s s i c promoters are not c a r c i n o g e n i c per se, or only weakly so, since by themselves they u s u a l l y do not induce cancer, but when a p p l i e d to target c e l l s which have already been i n i t i a t e d by a cancer i n i t i a t o r , promoters w i l l f a c i l i t a t e , enhance, and p o t e n t i a t e the e f f e c t i v e potency of the i n i t i a t o r s to induce malignant transformations expressed as an increase i n tumor incidence and the e a r l i e r appearance of malignancies. D i r e c t - A c t i n g Complete

Carcinogens

D i r e c t - a c t i n g complete carcinogens have the a b i l i t y by themselves both to i n i t i a t e and promote tumor i n d u c t i o n so that i f c r i t i c a l doses are a t t a i n e d , the l e s i o n induced progresses to frank malignancy, i . e . , cancer. Other carcinogens may have l e s s promoting c a p a b i l i t i e s . Many p o t e n t i a l l y c a r c i n o g e n i c chemicals require metabolic a c t i v a t i o n before the metabolite has s u f f i c i e n t e l e c t r o p h i l i c biochemical a c t i v i t y to damage DNA and i n i t i a t e the s e r i e s of steps progressing to cancer i n d u c t i o n . (_5) Activation If a c t i v a t i o n of procarcinogens to e l e c t r o p h i l i c metabolites i s enhanced, the expectable r e s u l t i s an increase i n the number of c a r c i n o g e n i c a l l y a c t i v e molecules capable of i n i t i a t i n g cancer. Conversely, as d e a c t i v a t i o n or d e t o x i f i c a t i o n i n creases, one would expect l e s s e r amounts of p o t e n t i a l l y c a r cinogenic metabolites a v a i l a b l e to i n i t i a t e cancer i n d u c t i o n . Many compounds can enhance or i n h i b i t carcinogenesis without being carcinogenic themselves (or one carcinogen can enhance or i n h i b i t another) by inducing or i n h i b i t i n g microsomal enzymes. These metabolic pathways important to the a c t i v a t i o n or d e a c t i v a t i o n of p o t e n t i a l l y c a r c i n o g e n i c compounds may be d i s t o r t e d or shunted, thus markedly a f f e c t i n g the degree t o which a procarcinogen i s a c t i v a t e d to e l e c t r o p h i l i c s t a t u s .


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Many p o l y c y c l i c aromatic hydrocarbons have t h i s e f f e c t , as do the notable examples of p o l y c h l o r i n a t e d biphenyls and phénobarbital. In many i n s t a n c e s , they induce more d e a c t i v a t i o n than a c t i v a t i o n , but i n some instances they augment a c t i v a t i o n . These phenomena have been e x t e n s i v e l y described and documented i n the s c i e n t i f i c l i t e r a t u r e .


Tumor Promoters/Potentiators As discussed e a r l i e r , other chemicals are capable of i n ducing a s e r i e s of changes i n the same target c e l l s that are s u s c e p t i b l e to i n i t i a t i o n of cancer, but these chemicals are only capable of a c t i n g as tumor promoters or tumor p o t e n t i a t o r s . Examples of these chemicals range from the croton o i l d e r i v a t i v e s , such as c e r t a i n phorbols, on through a broad spectrum of other n a t u r a l l y - o c c u r r i n g or man-made compounds. Many of these compounds have been designated already as p o t e n t i a l c a r cinogens by observing s t a t i s t i c a l l y s i g n i f i c a n t increases i n the i n c i d e n c e of cancer a s s o c i a t e d with exposures to these chemicals. I t may be of i n t e r e s t to suggest that chloroform and carbon t e t r a c h l o r i d e have tumor promoting or p o t e n t i a t i n g a c t i v i t y , as do saccharin, DDT, PCBs, c e r t a i n phenols, phénob a r b i t a l and, b e l i e v e i t or not, such substances as ethanol, b i l e a c i d s , c i t r u s o i l , and others, i n c l u d i n g hormones suth as e s t r a d i o l . (21) Modifying Factors In s i m i l a r fashion, but now on a broader b i o l o g i c a l s c a l e , the s e n s i t i v i t y to cancer i n d u c t i o n of an organism, be i t a c e l l , t i s s u e , organ, or animal, can be s u b s t a n t i a l l y i n f l u e n c e d by other "modifying f a c t o r s . " These i n d i r e c t l y act to i n fluence the m i l i e u i n which the target c e l l s e x i s t and modify b i o l o g i c a l resistance. I f one immuno-suppresses an animal and oncogenic v i r u s e s are endogenously present, an i n c r e a s e i n the r i s k f o r cancer i n d u c t i o n w i l l l i k e l y be the r e s u l t . I f one a l t e r s the hormonal s t a t u s , profound changes i n the b i o l o g i c a l a c t i v i t y of many c e l l s w i l l occur, a f f e c t i n g cancer r i s k . Genetic f a c t o r s a l s o i n f l u e n c e r i s k f o r cancer. Excesses or d e f i c i e n c i e s of c e r t a i n m i c r o - n u t r i e n t s such as vitamins or minerals or macro-nutrients, such as p r o t e i n or f a t , a l s o can have dramatic i n f l u e n c e s upon the s u s c e p t i b i l i t y of animals to the i n d u c t i o n of cancer. Such m o d i f i e r s appear t o act by modifying or i n t o x i c a t i n g enzymes, c o - f a c t o r s , s u b s t r a t e s , and membranes important to the maintenance of normal homeostatic p h y s i o l o g i c a l f u n c t i o n and b i o l o g i c a l defenses a g a i n s t cancer induction. Some b i o l o g i c a l defense mechanisms against t o x i c i n s u l t s , i n c l u d i n g those from carcinogens, i n v o l v e substances l i k e Vitamins A, C, and E. Glutathione and other s u l f h y d r y l compounds can a l s o d e a c t i v a t e carcinogens.



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Hyperplastic Toxicity The term " h y p e r p l a s t i c t o x i c i t y " i s used i n t h i s presentat i o n to describe t o x i c i t y - i n d u c e d c e l l p r o l i f e r a t i o n a s s o c i a t e d with increased m i t o t i c a c t i v i t y , increased DNA (as judged by increased d e n s i t y of nuclear chromatin s t a i n i n g or i n d i c i a of increased DNA s y n t h e s i s ) and other changes a s s o c i a t e d with enzyme and membrane f u n c t i o n s which are not of a malignant nature per se. H y p e r p l a s t i c t o x i c i t y i s a very common c e l l u l a r r e a c t i o n that can occur i n almost a l l mammalian t i s s u e s i n response to a v a r i e t y of t o x i c i n s u l t s . The c e l l s p r o l i f e r a t e , i n c r e a s i n g i n number by reproducing f a s t e r . The i n f l u e n c e of t o x i c i t y and h y p e r p l a s i a on cancer i n d u c t i o n has been the subject of much i n t e r e s t and some controversy f o r many years. The controversy centered on the nature and extent of the p r e c i s e r o l e of hyperp l a s i a i n r e l a t i o n to c a r c i n o g e n e s i s . One question was somewhat over-emphasized i n the minds of many cancer researchers: was the i n d u c t i o n of h y p e r p l a s i a a necessary p r e c o n d i t i o n f o r i n d u c t i o n of cancer? (For many c e n t u r i e s , p h y s i c i a n s observed that the onset of cancer appeared to be a s s o c i a t e d with chronic i r r i t a t i o n and inflammation of t i s s u e s , such as s c r o t a l cancer i n chimney sweeps, s k i n cancer i n c e r t a i n occupations where s k i n i r r i t a t i o n was observed i n a s s o c i a t i o n with exposure to chemical substances, and i n t h i s century the a s s o c i a t i o n of lung cancer i n people with chronic b r o n c h i t i s induced by i n h a l i n g tobacco smoke or other i r r i t a t i n g fumes.) Beginning e a r l y i n t h i s century and c o n t i n u i n g to t h i s day, many experiments were performed to provide data to c l a r i f y the r e l a t i o n s h i p s between h y p e r p l a s i a and cancer. The answer to t h i s p a r t i c u l a r question seems to be the f o l l o w i n g : Observable h y p e r p l a s t i c t o x i c i t y i s not a necessary p r e r e q u i s i t e f o r the i n d u c t i o n of each and every type of cancer, i f by i n d u c t i o n of cancer you mean i n i t i a t i o n of cancer. But a l l cancers are forms of malignant h y p e r p l a s i a . Hyperp l a s i a , metaplasia, and d y s p l a s i a are observed and documented progressive stages i n the development of malignant neoplasms, i . e . , those new growths of malignant t i s s u e c e l l s we c a l l cancer. H y p e r p l a s t i c t i s s u e responses to many t o x i c agents i n v o l v e abnormal a c c e l e r a t i o n of c e l l r e p l i c a t i o n which i n turn i n v o l v e s a marked increase i n m i t o t i c a c t i v i t y , i n c l u d i n g an increase of DNA. Not a l l compounds which induce h y p e r p l a s i a can act as tumor promoters, thus while the phenomena a s s o c i a t e d with tumor promotion i n c l u d e h y p e r p l a s t i c changes, h y p e r p l a s i a per se i s not p r e c i s e l y the same phenomenon as promotion. However, c e r t a i n patterns of h y p e r p l a s t i c t o x i c i t y appear to be i d e n t i c a l and c o i n c i d e n t a l with c e r t a i n b i o l o g i c a l phenomena observed to occur when the c l a s s i c a l tumor promoters are administered to



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the same target c e l l s . If the t o x i c i t y induced by t o x i c challenges r e s u l t s i n the i n d u c t i o n of c e r t a i n polyamines or other f a c t o r s that appear to be capable of s t i m u l a t i n g c e l l u l a r and DNA h y p e r p l a s i a and changes i n membrane and enzyme f u n c t i o n s , then a tumor promoting a c t i o n i s l i k e l y to r e s u l t which w i l l f a c i l i t a t e and enhance cancer i n d u c t i o n provided that the t a r g e t c e l l s have already been i n i t i a t e d by the same or d i f f e r ent chemicals. (22) Having seen that h y p e r p l a s i a i n one form i s an i n t e g r a l part of the expression of an i n i t i a t e d c e l l i n t o a neoplasm, and that other forms of h y p e r p l a s t i c t o x i c i t y are co-equal with tumor promotion, we can now ask whether or not p r e - e x i s t i n g h y p e r p l a s t i c t o x i c i t y enhances the b i o l o g i c a l s u s c e p t i b i l i t y of p a r t i c u l a r t i s s u e s to the i n d u c t i o n of cancer? The answer to t h i s question i s yes, at l e a s t i n many instances, since a s u b s t a n t i a l body of evidence again i l l u s t r a t e s many s i t u a t i o n s where p r e - e x i s t i n g t o x i c i t y and h y p e r p l a s i a r e s u l t e d i n an increase i n the b i o l o g i c a l s u s c e p t i b i l i t y of target t i s s u e s to cancer i n i t i a t i o n and f u r t h e r promotion. (14, 23, 24) I t would appear that one f a c t o r may i n v o l v e DNA r e p l i c a t i o n with an increase i n the amount and surface area of DNA a v a i l a b l e as a target f o r a l k y l a t i o n and m i s p a i r i n g . Other f a c t o r s that may be involved i n c l u d e : increased permeability of membranes to t o x i c agents, d i s t o r t i o n and impairment of enzymes, and r e l a t e d c o - f a c t o r s and substrates. These are important f o r maintenance of normal p h y s i o l o g i c a l c e l l functions and b i o l o g i c a l defense against t o x i c i n s u l t s i n c l u d i n g those from e l e c t r o p h i l i c c a r cinogens. The d e a c t i v a t i n g a c t i v i t i e s of the endoplasmic r e t i c u l u m and microsomal enzymes a l s o protect against c a r c i n o gens. I f these p r o t e c t i v e metabolic pathways are f u n c t i o n a l l y d i s t o r t e d o r impaired, b i o l o g i c a l s u s c e p t i b i l i t y t o cancer i n d u c t i o n can be increased. Dose-Response

Considerations

As mentioned e a r l i e r , complete carcinogens can both i n i t i a t e and promote cancer by themselves, but d i f f e r e n c e s i n dose-response have been observed and documented, which suggest that the promoting a c t i o n of complete carcinogens i s r e l a t e d to higher and repeated dosages of the same chemical, whereas i n i t i a t i o n of malignant transformation may occur a t lower doses. This implies then that the r i s k f o r cancer i n d u c t i o n by a complete carcinogen w i l l be increased as exposure to that carcinogen increases, because not only w i l l a greater amount of c e l l u l a r i n i t i a t i o n be e f f e c t e d , but the promoting a c t i o n of the compound w i l l be more e f f e c t i v e l y expressed by repeated exposures and thus these d r i v i n g forces w i l l r e s u l t i n the f a s t e r i n d u c t i o n of cancer.


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Dosing Test Animals at S u b s t a n t i a l Levels Our current p r a c t i c e i s to dose t e s t animals at s u b s t a n t i a l l e v e l s to determine whether or not a compound induces cancer. If such dosing r e s u l t s i n c e l l damage such as h y p e r p l a s t i c t o x i c i t y (25), which may invoke tumor promoting or p o t e n t i a t i n g a c t i v i t y , the f a c t that endogenous i n i t i a t o r s (such as n i t r o s amines) e x i s t i n the mammalian body implies that a p o s i t i v e i n d u c t i o n of cancer i n a population of t e s t animals w i l l not d i f f e r e n t i a t e between i n i t i a t i o n and promotion. (1, _3) Theref o r e we w i l l not know from t h i s evidence alone whether the "carcinogen" i s an i n i t i a t o r or a promoter, or both. I f the compound when tested at s u b s t a n t i a l l e v e l s can p o t e n t i a t e the e f f e c t i v e c a r c i n o g e n i c potency of endogenous carcinogens by other mechanisms, or i f i t acts to decrease b i o l o g i c a l susc e p t i b i l i t y to cancer i n d u c t i o n from endogenous carcinogens, or from other carcinogens that may e x i s t environmentally, again we w i l l not be able to t e l l what category of c a r c i n o g e n i c a c t i v i t y we are dealing with unless we ask the relevant questions i n the f i r s t place and develop the data to provide relevant answers. Strategy f o r Preventing Chemically-Induced Cancer i n Humans The second c o n s i d e r a t i o n concerns our strategy f o r p r e venting cancer i n humans by c o n t r o l l i n g exposures to chemicals and s e t t i n g p r i o r i t i e s f o r t e s t i n g , r e g u l a t i n g , and other forms of p u b l i c h e a l t h a c t i o n . Since c e r t a i n forms of t o x i c i t y c l e a r l y can enhance the i n d u c t i o n of cancer, should not one of our highest p r i o r i t i e s i n cancer prevention be to prevent a l l p o t e n t i a l l y t o x i c exposures to chemicals (not only f o r cancer prevention but obviously to prevent a l l forms of t o x i c i t y ) ? By preventing t o x i c i t y per se, l i k e l y we w i l l prevent a s i g n i f i c a n t amount of cancer. Let us remember that many of the human cancers a s s o c i a t e d with human exposures to most o c c u p a t i o n a l and c e r t a i n environmental carcinogens were w e l l w i t h i n t o x i c doseresponse ranges and that t i s s u e damage i n c l u d i n g h y p e r p l a s t i c t o x i c i t y were frequent concomitants. The most dangerous carcinogens are l i k e l y to be those that have the a b i l i t y to i n i t i a t e and/or promote cancer at subtoxic doses not l i k e l y to a t t r a c t much a t t e n t i o n per se u n t i l i d e n t i f i e d as such. Should we not place emphasis as a f i r s t p r i o r i t y on d e t e c t i n g and c o n t r o l l i n g the worst carcinogens f i r s t , i . e . , those which are e f f e c t i v e i n i t i a t o r s and promoters at r e l a t i v e l y low subtoxic doses l i k e l y to be w e l l w i t h i n the range of a n t i c i p a t e d human exposure? I f we are i n t e r e s t e d i n p r a c t i c i n g that philosophy, then we need to re-examine our procedures and p r a c t i c e s f o r d e t e c t i n g carcinogens, modify our t e s t i n g p r o t o c o l s , and evaluate p o t e n t i a l hazards to human h e a l t h from a d i f f e r e n t p e r s p e c t i v e than we do now.


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F i r s t and Second Order Cancer Induction We then should consider c l a s s i f y i n g the i n d u c t i o n of cancer i n t o two c l a s s e s : F i r s t Order, those having the capab i l i t y to induce cancer at subtoxic doses, e i t h e r because they are e f f e c t i v e i n i t i a t o r s or complete carcinogens; and Second Order, those that act to i n f l u e n c e the i n d u c t i o n of cancer by inducing various forms of h y p e r p l a s t i c t o x i c i t y by a c t i v a t i n g endogenous e l e c t r o p h i l e s , or by decreasing b i o l o g i c a l r e s i s t a n c e to other independently operating patterns of cancer i n d u c t i o n . (26, 27) The v a l i d i t y of t h i s approach can be tested without going to the extremes of an ED01 experiment such as that performed with 2AAF a t the N a t i o n a l Center f o r T o x i c o l o g i c a l Research. That experiment showed that 2AAF c a r c i n o g e n i c i t y involved two patterns of i n d u c t i o n operating at d i f f e r e n t dose-response curves, one of which was operating w i t h i n the lower doses given. Simply s t a t e d , we can determine from 90-day i n vivo studies whether or not a t e s t substance induces h y p e r p l a s t i c t o x i c i t y i n various target organs, and, i f so, a t what doses. Serial s a c r i f i c e s and i n t e r r u p t e d dosing schedules are needed to determine the progressive nature of the l e s i o n s noted and the extent of b i o l o g i c a l r e p a i r . Within the range of inducing h y p e r p l a s t i c t o x i c i t y , one group of animals i s then c a r r i e d for l i f e t i m e dosing. Well below the range of inducing hyperp l a s t i c t o x i c i t y , other groups are c a r r i e d through t h e i r l i f e t i m e s with s e r i a l s a c r i f i c e s being conducted to note prog r e s s i v e l e s i o n s . Another cohort of animals are subjected to i n t e r m i t t e n t or i n t e r r u p t e d dosing at subtoxic l e v e l s . A d d i t i o n a l i n v e s t i g a t i o n s could be conducted such as using phénob a r b i t a l and p o l y c h l o r i n a t e d biphenyls to induce microsomal enzymes and to promote h y p e r p l a s t i c t o x i c i t y i n organs such as l i v e r i n order to determine the e f f e c t s of added s t r e s s . I f conducted p r o p e r l y , we should have enough i n vivo data to determine whether we are dealing with a f i r s t or second order carcinogen i f cancer i n f a c t i s induced. Other sources of b i o l o g i c a l data may be h e l p f u l i n t h i s regard, e s p e c i a l l y i n v i t r o mutagenicity and transformation data. E v a l u a t i n g Cancer Risk Factors As discussed above, there i s a need to go beyond genera l i z a t i o n s that a substance does or does not "induce" cancer. We need to determine whether or not p a r t i c u l a r f a c t o r s can p o t e n t i a l l y i n f l u e n c e cancer i n d u c t i o n , and, i f so, how and under what circumstances? (26, 28) We need to evaluate i n i t i a t o r s and "complete carcinogens" i n one category as F i r s t Order carcinogens. Second Order compounds should not be c a l l e d "carcinogens", even though under some circumstances and at



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some doses they may be powerful d r i v i n g f o r c e s that can subs t a n t i a l l y i n f l u e n c e cancer i n d u c t i o n . Second order substances may a c t i v a t e c e r t a i n f i r s t order compounds to e l e c t r o p h i l i c i n i t i a t o r s of DNA damage important both to carcinogenesis and mutagenesis. Other second order compounds may promote or p o t e n t i a t e cancer i n d u c t i o n by inducing h y p e r p l a s t i c t o x i c i t y or invoking other mechanisms. Those compounds that i n t e r f e r e with normal p h y s i o l o g i c a l status by d i s t u r b i n g p r o t e c t i v e enzymes, s u b s t r a t e s , vitamins, n u t r i e n t s , hormones, immune mechanisms, e t c . , are l i k e l y to increase r i s k f o r cancer i n duction caused by independently operating f i r s t order substances e i t h e r manufactured endogenously i n the body (such as n i t r o s amines) or e n t e r i n g i n t o bodily contact from environmental sources. The r o l e of " t o x i c i t y " per se should not be underestimated as a p o t e n t i a l l y powerful f o r c e that can s u b s t a n t i a l l y i n f l u e n c e b i o l o g i c a l s u s c e p t i b i l i t y to cancer i n d u c t i o n by decreasing b i o l o g i c a l r e s i s t a n c e . Such cancer r i s k f a c t o r s should be evaluated using a v a i l a b l e techniques (from the f i e l d s of t o x i c o l o g y , pharmacology, and n u t r i t i o n ) to study dose-response phenomena a s s o c i a t e d with t h e i r modes of a c t i o n , metabolism (both normal and abnormal), and e x c r e t i o n . Newer techniques f o r studying i n t e r a c t i o n s without having to r e s o r t to super-scale l i f e - t i m e bioassays are becoming a v a i l a b l e every year. In v i t r o techniques can supplement short-term i n vivo s t u d i e s , but we should not overemphasize i n v i t r o approaches, nor under-estimate the value of i n vivo b i o l o g i c a l data s i n c e i t i s extremely important to estimate the t o x i c i t y of compounds i n the context of the b i o l o g i c a l defense-mechanisms a v a i l a b l e to the mammalian body. (29, 30) In t h i s regard, e p i d e m i o l o g i c a l s t u d i e s can provide much needed information concerning b i o l o g i c a l s u s c e p t i b i l i t y and r e s i s t a n c e f a c t o r s , p a r t i c u l a r l y i n r e l a t i o n to human metabolism, n u t r i t i o n , and genetic f a c t o r s which are extremely important. Last, but by no means l e a s t , the mode and amount of exposures to various substances should be kept i n f u l l c o n s i d e r a t i o n at a l l times. We have tended to over-emphasize micro-exposures to substances we suspect of having unusual patterns of b i o l o g i c a l a c t i v i t y , at the expense of having ignored the macro-exposures to the universe of n a t u r a l compounds present i n the foods we eat. Just because they may be n u t r i e n t s and Nature-produced does not mean that they cannot and do not exert powerful i n fluences upon cancer i n d u c t i o n . They can and do, both to protect against or to enhance cancer i n d u c t i o n , but that i s another topic f o r a d i f f e r e n t time.


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1.

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26. Hecker, Ε., Cocarcirtogens or Limited Cancer Inducing Agents. Important New Aspects of the Etiology of Human Tumors and the Molecular Mechanisms of the Origin of Cancer. NaturwissenSchaften, 1978, 65, 640-648. 27. Berenblum, I., A Re-evaluation of the Concept of Cocarcinogenesis, Progr. Exp. Tumor Res, 1969, 11, 21-20. 28. Berenblum, I., Carcinogenicity Testing for Control of Environmental Tumor Development in Man, ISR. J. Med. Sci., 1979, 15(6), 973-479. 29. Kolbye, A.C., J r . , Regulatory Considerations Concerning Muta genesis, J . Soc. Cosmet. Chem., 1978, 29, 727-732. 30. Kolbye, A.C., J r . , "Impact of Short-Term Screening Tests on Regulatory Action, Applied Methods in Oncology #4", Elsevier Biomedical Scientific Publishing Company, Amsterdam, 1980. (In Press). RECEIVED

February 12, 1981.


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