multistage carcinogenesis: implications for risk estimation

Cancer and Metastasis Reviews 7:5-18 (1988) (~) Kluwer Academic Publishers - Printed in the Netherlands

Multistage carcinogenesis: implications for risk estimation

Hiroshi Yamasaki Programme of Multistage Carcinogenesis, International Agency for Research on Cancer, 150, Cours Albert Thomas, 69372 Lyon Cedex 08, France

Key words: multistage carcinogenesis, tumor promotion, risk estimation

Abstract

In undertaking a quantitative estimation of carcinogenesis risk, it is essential to keep in mind that carcinogenesis is a multistage process, and that each stage can be affected by different classes of risk factors. Further- more, different mechanisms are involved in the various stages of carcinogenesis. Thus, a dose-response analysis of one given factor cannot provide an accurate estimation of carcinogenic risk.

Carcinogenic risk estimation is usually undertaken for a specific chemical or group of chemicals; however, the concept of multistage carcinogenesis is based on biological processes and not on the mechanisms of action of the agents involved. It is therefore important to consider three related, but different, factors involved in carcinogenesis: stage, agent, and activity of agent. This is especially important in developing a short-term test for stage-related risk factors, such as tumor-promoting agents. For this reason, carcinogens should not be classified according to only one chemical activity.

This article briefly reviews the cellular and molecular mechanisms involved in multistage carcinogenesis, and discusses their implications for risk estimation. Special consideration is given to the effect of treatment frequency on the response of tumor-promoting agents, as seen in long-term tests in experimental animals. It is proposed that exposure frequency be taken into account together with exposure dose.

Introduction

Since carcinogenesis is a multistage process, and each stage is influenced by a variety of endogenous and exogenous factors [1, 2], estimation of the risk presented by a single chemical compound may not adequately indicate the overall risk of the entire carcinogenic process. Even in the simplest example of multistage carcinogenesis, namely, two-stage carcinogenesis, one compound can clearly influence the carcinogenic potency of another: in the mouse skin model of two-stage carcinogenesis, for example, phorbol esters, which are only very weak- ly carcinogenic, can greatly enhance the production of tumors when applied after low doses of a variety of complete carcinogens [3-5]. Thus, the influence of compounds occurring simultaneously

with a given carcinogenic risk factor must also be considered. Although such considerations increase the complexity of carcinogenic risk estimation, such an approach is not only necessary for valid risk estimation but may also provide an efficient means of cancer prevention [6].

In order to estimate the risk of each stage of carcinogenesis, it is important to increase our knowledge of how these stages occur. In recent years we have seen rapid progress in the under- standing of the molecular and cellular mechanisms involved in multistage carcinogenesis. The mouse skin and rat liver two-stage carcinogenesis models [6-8], in particular, have elucidated the mechanisms of action of tumor-initiating and promoting agents. Such studies have suggested that initiating agents bind to DNA and cause genetic damage,

which appears to be irreversible [9], and recent studies suggest that cellular oncogenes are critical targets of initiating agents [10]. Tumor promoting agents, however, appear to act through epigenetic mechanisms, and their action appears to be reversible [9]. These findings are consistent with the idea that the process of initiation is irreversible, whereas the process of promotion is to some extent reversible. 'Reversibility' is thus one of the most important means of differentiating the risks of initiation and promotion in the process of carcinogenesis.

In estimating the risk of carcinogenic agents in terms of multistage carcinogenesis, only stage-related factors such as the initiating or promoting activity of an agent are usually taken into consideration. I would like to suggest that the stage, agent, and the activity of the agent involved in carcinogenesis be considered separately. This article presents my opinions on the subject, with special emphasis on reversibility, and discusses the importance of using the correct term for the correct concept. Unfortunately, our knowledge about multistage carcinogenesis is based largely on two- stage models of carcinogenesis, so the picture presented here is necessarily narrower than the actual process of multistage carcinogenesis.

Carcinogenic process, carcinogenic agents, and activity of carcinogens: The need for a clear separation of these terms

Terminology itself does not advance science; however, the misuse of terminology sometimes hinders the progress of science. In the study of multistage and multifactorial carcinogenesis, and especially in discussions of their implications for risk estimation, some confusion has been caused by the misuse of certain terms. Words used to define different stages of carcinogenesis are often applied to the agents that are involved. The stage-related activity of a chemical is often confused with its specific activity; for example, genotoxic or nongenotoxic activity, which could be defined more precisely by the way these chemicals interact with cells.

Much of this confusion could be avoided if we

realize that the terms describing the carcinogenic process - initiation, promotion, and progression - apply only to the carcinogenic process, and do not imply a mechanism of action of the chemicals involved in these stages. For example, agents involved in the initiation stage of carcinogenesis are often defined as 'initiators', and those with tumor- promoting activity in two-stage models are called tumor 'promoters'. These terms are not misleading when used within the correct context, but confusion occurs when the terms 'initiator' and 'promoter' are used to imply that initiating or promoting activity is the only activity of a chemical. It is important to realize that initiating or promoting activity may be only one characteristic of a chemical and that many carcinogens may have both activities.

The interchangeable use of the terms 'stage' and 'agent' has led some investigators to conclude that multistage carcinogenesis does not exist. For example, failure to find an example of a tumor promoter in human carcinogenesis led one investigator to conclude that there is no tumor promotion stage in human carcinogenesis [11], although it is clear from epidemiological and histological observations that human carcinogenesis is a multistage process [2, 6, 12, 13]. Although no exogenous or endogenous tumor promoter has been definitely identified as an agent in human carcinogenesis, some evidence suggests that such factors must occur in different stages of carcinogenesis. Several examples are listed in Table 1; for detailed information, readers are referred to our recent review [6]. Phorbol esters are prototypes of tumor-promoting agents for two- stage carcinogenesis in mouse skin, but these agents can also induce tumors in mice that are not exposed to initiating agents. This finding has also been used to argue against the two-stage model of carcinogenesis [14]. The argument is again based on the misconception that if phorbol esters are tumor-promoting agents they should act only as tumor promoters, whereas tumor promotion is only one of their activities. In practice, it is not important whether phorbol esters also have a weak complete carcinogenic activity; what is important is that they can dramatically promote the yield of tumors in mouse skin initiated by a low dose of carcinogen. Schulte-Hermann has recently ex- pressed a similar opinion [15].

Table 1. Examples of multistage, multifactorial carcinogenesis in humans.

Probable initiator Probable promoter References (early-stage carcinogen) (late-stage carcinogen)

Lung cancer Cigarette smoking Cigarette smoking 2, 74 Esophageal cancer ? Phorbol esters 75

Nitrosamines ? Pickled vegetables 76 (Roussin's red ?)

Nasopharyngeal cancer Epstein-Barr virus Phorbol esters 77 + n-butyrate

Burkitt's lymphoma Epstein-Barr virus Malaria infection 78 Endometrial cancer ? Conjugated estrogens 79 Colon cancer ? Dietary fat 80 Breast cancer ? Hormones, Dietary fat 13, 81 Prostate cancer ? ? 82 Bilateral retino-blastoma Heredity ? 12 Liver cancer Aflatoxin B~ Hepatitis B 83 Lung cancer Cigarette smoking + Asbestos exposure 84 Esophageal, laryngeal and oral cavity cancers Cigarette smoking + Alcohol consumption 85

From Yamasaki and Weinstein [6].

The confusion described above stems from a sim- plistic extension of initiation-promotion processes to describe the agents involved in two-stage carcinogenesis models. However , the stages of carcinogenesis do exist, even if pure initiating or promoting agents do not, and chemicals cannot be classified categorically on the basis of their activity in the two-stage model of carcinogenesis, since a given agent may have both initiating and promoting activity. These considerations are particularly important in assessing the risk due to chemicals involved in the different stages of carcinogenesis.

Another type of confusion derives from a care- less extrapolation of the results of mechanistic studies of two-stage carcinogenesis to the mechanisms of action of chemicals. Extensive studies with two-stage models of carcinogenesis, particularly in mouse skin and rat liver, provide convincing evidence that the dominant mechanisms involved in initiation are genotoxic, and that nongenotoxic mechanisms may be involved in tumor promotion [3-5]. These findings have been interpreted as indicating that chemicals involved in the different stages of carcinogenesis act by these same mechanisms. Thus, it is sometimes suggested that tumor- promoting agents are synonymous with nongeno-

toxic chemicals and initiating agents are synonymous with genotoxic chemicals. However, the fact that a chemical acts by genotoxic or nongenotoxic mechanisms does not restrict its activities. In addition, the terms 'genotoxic' and 'nongenotoxic' are derived, not from a stage-related concept of carcinogenesis, but from the type of interaction between chemicals and target cells, i.e., whether they react with target D N A or epigenetically [16-18]. The terms 'genotoxic' and 'nongenotoxic' should therefore not be used as synonyms for the stage- related nomenclature of chemicals, such as tumor- initiating or -promoting agents. It is advisable to avoid classifying carcinogens on the basis of only one of their activities; for example, phorbol esters have both 'genotoxic' and 'nongenotoxic' activity. In addition, various carcinogenic polycyclic hydrocarbons, which typically induce genotoxic effects, are also known to induce nongenotoxic changes, such as membrane receptor modulation, which are shared with phorbol esters [18a, 18b].

Mechanisms involved in multistage carcinogenesis- important aspects for risk estimation

Studies on the cellular mechanisms of multistage carcinogenesis, which use mostly mouse skin and rat liver models, suggest that in the initiation process agents interact with the genetic apparatus of cells, and in the promotion phase these latent initiated cells expand clonally over surrounding normal cells. Progression is a process in which benign tumors are converted into autonomous carcinomas; another genetic effect may be involved in this stage. There are numerous reports on the molecular and cellular mechanisms of multistage carcinogenesis and the biochemical mechanisms of action of initiating and promoting agents. I will not review this information, but I have selected information about the mechanisms of initiation and promotion that is important for risk estimation. The proceedings of several meetings on the models and mechanisms of tumor promotion are now available [19- 221 .

A single and very important difference between the mechanisms of initiation and promotion is their differential reversibility: initiation appears to be irreversible, while promotion is often reversible. For example, mice painted with a single dose of initiating agent develop similar papillomas whether they are painted with a tumor-promoting agent 1 year later or only a few days later [4]. However, when promoter treatment is discontinued after a short period there is no tumor formation; or if tumor-promoting agents are applied only with long intervals between applications, no tumor appears [4]. These differences in reversibility may reflect the mechanisms of action of the agents involved in the two stages.

The irreversibility of the initiation stage can be partially explained by the recent discovery that certain chemical carcinogens act by mutating specific genes. Thus, initiating agents .usually show genotoxic activity; recent studies on oncogene activation in specific tumors suggest that specific pro- to-oncogenes can be activated by initiating agents. For example, a single treatment of a rat with N- methyl-N-nitrosourea (MNU) induces mammary tumors, which contain an activated oncogene

shown to be a mutation of the H-ras proto-oncogene at the 12th codon [23]. Activation of this oncogene is due to a specific G to A transition; MNU is known to be a potent alkylating agent that preferentially induces G to A mutations by causing miscoding of a 6-methylguanine product [24]. In contrast, no such specific G to A transition was found at the 12th codon of the H-ras oncogene in tumors induced by 7,12-dimethylbenz(a)anthracene (DMBA), a carcinogen that binds predom- inantly to adenine residues [25]. Barbacid's group [23] found an A to T transversion at the 61st codon of H-ras in mammary tumors induced by DMBA.This model proposes that hormones play a role in tumor promotion. It is thus reasonable to conclude that specific oncogenes are activated by specific initiating stimulus, and that the activation of cellular oncogenes is involved in the initiation stage of carcinogenesis.

A series of experiments conducted by Balmain's group [26-29] and others [30] on the mouse skin two-stage carcinogenesis model provides further support for the idea that certain carcinogens induce a specific pattern of oncogene mutations, and that such mutations play an important role in the initiation of carcinogenesis. DNA from mouse skin papillomas and carcinomas produced by an initiation- promotion protocol had an A to T transversion at the 61st codon of the H-ras gene only when DMBA or dibenz(c, h)acridine was used as the initiator [28, 30]; when benzo(a)pyrene or N-methyl-N'-nitro- N-nitrosoguanidine was used as the initiator there was no such mutation at the 61st codon of H-ras,

although many tumors contained DNA that transformed NIH 3T3 cells [28, 30).

The fact that an A to T transversion is found even in skin papillomas induced by painting with DMBA-12-0-tetradecanoylphorbol 13-acetate (TPA) suggests that this mutation is an early event [27]. In addition, the same specific mutation was observed when a tumor promoter different from TPA, i.e., chryserobin, was used [28], suggesting that the activation pattern is specific for initiating agents. We recently found a specific A to T transversion at the 61st codon of c-H-ras in nine of nine mouse skin carcinomas produced by transplacental initiation (DMBA) and postnatal promotion

(TPA); some of the papillomas contained the same mutation. These results suggest that DMBA can also induce an A to T transversion transplacentally [3l]. Specific mutation patterns at the 61st codon of H - r a s were also found in liver tumors induced by different carcinogens [32]: tumors induced by N- hydroxy-2-acetylaminofluorene had a C to A transversion at the first position on the 61st codon, whereas those induced by vinyl carbamate and 1-hydroxy-2',3'-dehydroestragole had predomin- antly an A to T transversion and an A to G transition respectively at the second position on the 61st codon [32].

Assuming that the main role of tumor promoters in promotion is to produce a clonai expansion of initiated cells, it is reasonable to think that activation of H - r a s oncogenes occurs during the initiation stage of carcinogenesis in this model. This assump- tion was directly supported by recent studies on retroviral ras oncogenes. When Balmain and his co-workers [29] applied Harvey murine sarcoma virus directly to mouse skin and subsequently administered phorbol esters a significant number of tumors were seen; Hsiao et al. [33, 33a] and others [33b] demonstrated that transformation of mouse or rat embryo fibroblasts containing ras oncogenes was enhanced by subsequent treatment with phorbol esters. These results are consistent with the idea that, since initiating agents cause specific structural alterations in proto-oncogenes, the initiation stage is irreversible.

As a reflection of the reversibility of the tumor promotion stage, many of the activities of tumor- promoting agents are also reversible [9]. Many known tumor-promoting agents do not cause mutation in cells and therefore appear to be nongenotoxic; however, as emphasized above, nongenotoxic chemicals should not be considered synonymous with tumor-promoting agents, since genotoxic activity and tumor promoting activity are defined differently and are not mutually exclusive.

During the tumor promotion stage, initiated cells expand clonally over surrounding normal cells; it is thus clear that an aberration of growth control is involved in promotion. It is also reasonable to assume that the cell differentiation pathway is altered in order to produce a tumor [34, 35].

Initiated cells must therefore have a selective growth advantage during tumor promotion, and tumor-promoting agents may provide this. For example, phorbol esters act like growth factors. These tumor promoters bind specifically to protein kinase C and activate it [36]. Since protein kinase C is considered to be involved in the control of cell growth and cell differentiation, its modulation may be a crucial step in phorbol ester-mediated tumor promotion. An interesting hypothesis is that protein kinase C activation is also involved in promotion triggered by non-phorbol ester-type tumor- promoting agents and during endogenous tumor promotion. This argument is based on the hypothesis that phorbol esters exert effects on initiated and surrounding non-initiated cells, and that only initiated cells respond differently, because of genetic information altered by initiating agents.

The long latent period between the time of exposure to a carcinogen and the manifestation of a tumor suggests that initiated cells are maintained in a dormant state by surrounding normal cells, probably through various forms of cell-cell interaction [37-39]. Cell-cell communication is considered to play an important role in controlling cell growth and differentiation [40]. In gap junctional intercellular communication in particular, cells form a specific structure (gap junctions) that joins the interior of one cell with that of another, through which they can exchange physiological molecules with a molecular weight of less than 1000 [40]. In normal tissue, cells connected by such gap junctions form an orderly society. It has been proposed that during promotion this society is destroyed, and initiated cells act like rebels [41]. Phorbol ester tumor promoters were first shown to inhibit gap-junctional communication, but many tumor-promoting agents have subsequently been found to have the same effect. Several lines of evidence suggest that inhibition of intercellular communication plays an important role in the promotion process p e r se.

(Interested readers are referred to the reviews in References 37, 41, and 42.)

These findings are consistent with the idea that tumor promotion involves nongenotoxic mechanisms. However, tumor-promoting agents can also have genotoxic effects; for example, phorbol esters

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have been shown to induce chromosomal aberra- tions [43], gene amplification [44], and aneuploidy [45]. It is difficult, however, to see these effects as essential mechanisms of tumor promotion, a generally reversible process, since some of these genetic effects are not readily reversible.

The role of reversibility in risk estimation - exposure interval as an important determinant

in dose-response

Is there a threshold for tumor promotion?

Because tumor promotion is reversible, it has long been argued that there should be a threshold below which tumor promotion does not occur. This idea is based primarily on data from the mouse skin two- stage carcinogenesis model, in which no promotion is seen when promoting agents are applied at long intervals [4]. In other words, tumor-promoting activity cannot be accumulated, unless the agents are applied at short intervals. This also implies that there may be a linear accumulation of promoting activity if tumor-promoting agents are applied continuously. It is important to note that the threshold so described is quite different from the classical concept, which is based only on the exposure dose. A threshold based on exposure interval is related not only to exposure dose but, more importantly, to exposure interval. These two ideas should be clearly differentiated; one is a 'dose threshold', the other is a 'frequency threshold' [46]. In classical dose-response studies, chemicals are applied as a single dose or as a continuous exposure, e.g., by feeding. In the initiation-promotion protocol, mouse skin is painted with promoting agents only at certain intervals, and never continuously.

The idea of a cumulative dose based on dose and frequency of exposure to initiating and promoting agents is presented schematically in Fig. 1. It is clear that when the action is genotoxic, or irreversible, the interval from exposure is less important, whereas the exposure interval plays a crucial role in the cumulative effective dose of chemicals with a reversible action [46].

"Dose-time interval of application" response

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Fig. 1. Schematic diagram of relationship between cumulative effect and dose schedule (see text for details). Left, irreversible effect; right, reversible effect.

Is there a threshold for tumor initiation?

In initiation-promotion models of carcinogenesis, initiation is regarded as a dormant stage: a single treatment with an initiating agent should not induce a tumor, although the effect of the agent remains in the cells so that a tumor-promoting agent can transform them into a tumor. Although this phenomenon is not usually linked with a threshold, I think it can be concluded that there is a biological threshold for initiating agents as carcinogens. One study clearly shows the existence of a threshold dose of initiating agents, below which no tumor appears unless a tumor-promoting agent is applied (see Reference 47 and below).

Although we know little about the dose-responses of initiating and promoting agents, there appears to be a dose threshold for initiating agents

and a frequency threshold for promoting agents. Whatever the dose-response curves of tumor-initiating and -promoting agents are, it is clear that these agents potentiate each other's capacity for carcinogenesis. Therefore, it is important to regard both initiating and promoting agents as risk factors in carcinogenesis. Moreover, with regard to cancer prevention, if initiating agents produce only a dormant form of tumor (initiated cells) and this dormant tumor is not manifested as a tumor unless promoting agents are applied, exposure to initiating agents may not represent as great a risk as tumor promoting. Exposure to tumor promoters is thus the major risk for cancer, and if the action of tumor-promotion agents is indeed reversible, a rapid way of preventing cancer would be to elim- inate tumor-promoting agents from our environ- ment. This point should be taken into account when one attempts an intervention study in a specific human population. It is reasonable to include anti-tumor-promoting agents in such a trial so that a rapid effect is expected.

Dose-response of initiating agents and promoting agents in the initiation-promotion model

In multistage carcinogenesis, initiating (early- stage) agents and promoting (late-stage) agents themselves cannot be considered as completely carcinogenic factors; the activity of initiating agents can only be assessed when promoting agents are applied, and the activity of promoting agents can only be estimated when initiating agents have been used. Therefore, it is inevitable that the dose- respose of initiating agents is influenced by tumor- promoting agents, and the dose-response of tumor- promoting agents is influenced by the presence of initiating agents.

As discussed earlier, treatment frequency appears to be an important factor in the tumor-promoting activity of agents. It is also possible that treatment frequency influences the initiation stage as well because of the time for metabolism, in- ducibility of the P450 systems, DNA repair, etc., required for various initiating agents. There is evidence for this with radiation in induction of

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C3H10T1/2 cell transformation; however, the radiation in these studies was used as a complete carcinogen rather than an initiating agent [47a].

Few studies have dealt with dose-response in terms of multistage carcinogenesis. Selected examples in which the influence of tumor-promoting and initiating agents on each other has been studied are given below.

One of the best examples of the clear effect of treatment frequency with tumor-promoting agents is probably an experiment by Boutwell in 1964 [4]. He applied a total dose of 1500/xg croton oil to mouse skin in single doses of 125 tzg at intervals of 1 week, 2 weeks, or 4 weeks after initiation with a single application of 75/zg DMBA. He found that, although the total dose of croton oil was the same, no papilloma was produced with the longest interval. He also observed that when he applied croton oil once a week 1.6 papillomas were induced per mouse, whereas only 1.1 papillomas per mouse were obtained when the same amount was applied once every 2 weeks. These results are consistent with the conclusion that the effect of tumor-promoting agents cannot be cumulated when the application interval is too long. In the same experiment [4], Boutwell also fractionated the doses of the initiating agent, DMBA, applying a total of 1/~g either as a single dose, as four doses at intervals of twice a week, or as four doses once every 2 weeks; then all mice were painted with croton oil for 32 weeks. He found that the tumor yields were very similar (Fig. 2). This result is again consistent with the idea that the effect of an initiating agent can be accumulated whatever the interval of application.

Burns et al. [47] confirmed these findings in a more detailed and larger scale experiment. The initiating agent, benzo(a)pyrene, was painted weekly for a total dose of 4, 8, 16, 32, 64 or 128/xg in 2, 4, 8, 16, or 32 weekly fractions, in order to compare the dose-responses of benzo(a)pyrene with different intervals of applications. Fraction- ation of the doses of initiating agent did not alter the yield of papillomas and carcinomas when the mice were subsequently painted with TPA. Papil- loma production at different doses of initiating agent at different fractionation times is summa- rized in Fig. 3. A linear, cumulative effect of initia-

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o. 25/2 t o. 25,u 1 4 t i m e s 4 t i m e s

once/2weeks twice/week

A B

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Fig. 2. Effect of 'application frequency' of initiating (DMBA) and promoting (croton oil) agents on mouse skin tumor yield. A. after a single application of 75 ug DMBA, a total of 1500 ug croton oil was painted at different intervals. B. a total of 1.00 ug DMBA was painted at different intervals as indicated, and then all mice were painted with croton oil. (Taken from Ref. 4, with permission of the publishers and authors.)

ting agents on tumor formation was thus confirmed.

The effect of application interval of tumor-promoting agents, originally shown by Boutwell [4], was also confirmed in an experiment conducted by Van Duuren et al. [48]. Mouse skin was given a single initiating painting with DMBA, and papillomas were produced by painting TPA at doses of 0.02, 0.1, 0.5, 2.5, 5.0, and 25.0/zg per mouse, either once a week or three times a week. By

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Fig. 3. Log-log plot of the yield of papillomas per mouse as a function of dose per fraction. The line was fitted by regression analysis and indicates a very good linear fit (exponent, 0.9). (Reproduced from Ref. 47, with the permission of the publishers and authors.)

analyzing the tumor yield, the authors concluded that the frequency of application is more important than the total dose of TPA. For example, when they applied 0.5/zg three times a week to give a total dose of 78#xg, they obtained more tumors than when they applied 2.5/zg once a week for a total dose of 130/zg. We have also carried out a large scale experiment using benzo(a)pyrene as the initiator and TPA as the promoter, and have confirmed that the frequency of TPA treatment is an important factor in the final tumor yield (unpub- lished results).

Although the above experiments suggest that the frequency of application of a tumor-promoting agent can alter the yield of tumors on mouse skin, there was generally a good dose-response effect when the effect of different doses of TPA at a fixed interval of application on yield of tumors was examined [47-49]. This suggests that there is no usual dose-related threshold or any drastic difference in the dose-response of tumor promoters in compari- son to that of initiating agents.

The influence of promoting agents on the dose- response of initiating agents is clearly demonstrated by experiments carried out by Burns et al. [47]. Mice were treated with different doses of benzo(a) pyrene with and without the tumor-promoting agent, TPA. In the presence of TPA, the dose- response to benzo(a)pyrene was very clear in terms of papilloma and carcinoma production, but when the mice were not painted with TPA there ap-

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Fig. 4. Dose-effect at 350 days for carcinoma induction in mouse skin exposed to a weekly dose of benzo(a)pyrene on Monday, with or without 5.0 ug TPA on Wednesday and Friday. Doses refer to the amount of benzo(a)pyrene given per week. Treat- ments were started at 56 days of age. (Taken from Ref. 47, with the permission of the publishers and authors.)

peared to be a relatively high threshold dose. Ben- zo(a)pyrene itself would therefore appear to be tumorigenic, although the presence of TPA can drastically alter the dose-response to an initiating agent with regard to the formation of both papillomas and carcinomas (Fig. 4). We obtained essen- tially the same results in a similar experiment. In addition, using two-stage in vitro transformation of C3H 10T1/2 cells, a linear dose-response to X-rays was seen only in the presence of the tumor-promoting agent, TPA [50].

All the experiments described above were carried out using mouse skin as the target tissue. It is obviously more difficult to determine dose-responses with respect to internal tumors, since the animals must be killed in order to count the tumor yield accurately. Several studies have examined the dose-response to tumor-promoting agents of the rat liver. Some reports have claimed that there was an apparent threshold for tumor-promoting doses. For example, Goldworthy et al. [51] used enzyme- altered loci in the liver as the endpoint for an initiation-promotion protocol in rats. After initiation with intragastric administration of N-nitroso- diethylamine (DEN) and feeding of the promoting agent, phenobarbital, at different doses, they found a good dose-response with 0.001%-0.05% phenobarbital in the diet but no effect at less than

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0.001%, indicating an apparent threshold for promotion by phenobarbital. However, it is apparent from their experimental design and results that it would not have been possible to detect an effect of phenobarbital at a dose of less than 0.001%, since only a few enzyme-altered foci were to be expected at this level, which could well be hidden at the background level. There is still no clear-cut experiment in which a threshold for tumor-promoting agents has been shown in rat liver.

Pitot etal. [51a] have recently reported that when phenobarbital or TCDD were administered as tumor promoting agents in the rat liver two-stage carcinogenesis model (DEN initiation), there were fewer and smaller enzyme-altered foci produced than in animals initiated by DEN only. Although these data can be used as evidence for the presence of a threshold dose for promoting agents in carcinogenesis, it is difficult to interpret these data since the promoting agents showed inhibitory effect, rather than no effect, on initiated liver. It is interesting to note that Williams [51b] also reported a similar effect with butylated hydroxyanisole on the promotion of forestomach and glandular stomach tumors in MNNG-initiated rats.

Good experimental data suggest that the frequency of application of tumor-promoting agents can influence the yield of rat liver enzyme-altered foci. Pitot et al. [52] have shown that when phenobarbital administration is interrupted there is a de- crease in foci yield as compared to a group in which phenobarbital administration is not interrupted, although the total doses of administered phenobarbital were approximately the same in the two groups. These results and those from mouse skin studies described above are consistent with the idea that the promoting effect of a chemical is not simply cumulative when the administration interval is long.

These results are presented in order to give an idea of the kind of data that are available for estimating the dose-responses of initiating and tumor- promoting agents. From the point of view of public health, it is clear that the data base is not sufficient for a firm conclusion, especially concerning tumors of internal organs. Because dose-response is of fundamental importance for estimating the risk

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posed by environmental carcinogens, more studies on dose-response in terms of multistage carcinogenesis must be done, although it is recognized that such experiments will involve much manpower and funding.

The present status of short-term tests for stage- related carcinogenic risk factors

I have thus far discussed risk estimation in terms of multistage carcinogenesis, mainly on the basis of stage-related mechanisms. In reality, however, we must estimate the risk presented by each chemical involved in carcinogenesis. Considering the number of chemicals we are exposed to, it is obvious that we cannot rely on rodent bioassays, especially when we would like to have a dose-response for each chemical in combination with an initiating or promoting agent. Therefore, it is important to develop a short-term test that reflects the stages of carcinogenesis and that can be used to assess individual chemicals in a short time.

Previous mechanistic studies of carcinogenesis concentrated on the mechanisms of action of carcinogens; to my mind, there has been insufficient attention paid to the mechanisms of the different stages of carcinogenesis. Studies on the mechanisms of action of individual chemicals may not reveal what is essential for the stages of carcinogenesis. In order to develop short-term tests for chemicals involved in different stages, it is therefore essential to accelerate the accumulation of information about these stages. Information at cellular and tissue levels should be accumulated; recent rapid developments in molecular biology may be helpful.

The available data indicates, as discussed earlier, that mutation may be involved in the initiation stage of carcinogenesis. On that basis, mutation assays should be useful as short-term tests for initiating agents. Tests in which the endpoint is chromosomal damage are less obviously classifiable in terms of their relevance to different stages of carcinogenesis.

Despite abundant information about the mechanisms of action of phorbol ester tumor promoters,

we know little about the promotion stage of carcinogenesis. One testable hypothesis involves the blocking of intercellular communication [37, 38, 46]. As mentioned above, the hypothesis is that initiated cells remain dormant under the control of surrounding normal cells, and tumor-promoting agents block intercellular communication so that the initiated cells become free to expand clonally. Several lines of evidence suggest that this is not only an effect of tumor-promoting agents, but is also involved in the promotion stage [37, 41, 42]. An assay to measure intercellular communication is now being tested as a possible short-term test for tumor-promoting agents. The available data suggest that this assay detects not only phorbol ester- type tumor-promoting agents, but also several other types [53-64]. There are several ways of mea- suring gap-junctional intercellular communication [40]. A metabolic cooperation assay is the most popular one, probably because of its technical sim- plicity. Our laboratory uses the dye transfer assay, in which the dye is injected into a cell and its spread into neighboring cells through gap junctions is measured [65]. This technique requires a little more skill. We have used it to test 10 known tumor- promoting agents; several gave positive results, but others were negative [64]. Further data to validate this test for detection of tumor-promoting agents is awaited.

Another possible test for tumor-promoting agents is the two-stage model of cell transformation. Two-stage in vitro cell transformation systems were developed to simulate two-stage in vivo carcinogenesis; the cellular and molecular mechanisms involved may be similar [66-71]. There are at least three different cell transformation systems that can be used to screen for tumor-promoting agents: these are Syrian hamster embryo cell transformation [70], and transformation of C3H 10T1/2 [66] and BALB/c 3T3 [69, 71] cell lines. However, the data base is still too small for the test to be validated as a screening assay.

Since tumor promotion appears to be a process that involves not only interactions between chemicals and cells, but also systemic reactions, it may not be possible to develop an in vitro short-term test that can detect all classes of tumor-promoting

agents. Considering also the possible tissue speci- ficity of tumor-promoting agents, it would probably be easier to develop in vivo, rather than in vitro, short-term tests. An in vivo short-term test for tumor-promoting agents should have an endpoint that can be detected before tumors are produced, e.g., preneoplastic lesions [52]. Several attempts have been made to use the production of preneoplastic lesions as a short-term in vivo assay for carcinogens and tumor-promoting agents [72, 73]. This kind of assay should be further developed and validated.

Conclusions

In order to estimate risk in terms of multistage carcinogenesis, it is important to distinguish three different, but related, factors involved in carcinogenesis, i.e., stage, agent, and activity of agent.

It is recommended that environmental chemical carcinogens should not be classified on the basis of only one of their activities, such as genotoxic, initiating, or promoting activity.

Because their action is reversible, tumor-promoting agents may exhibit different dose-responses when applied at different intervals. How- ever, it is important to distinguish these responses from classical dose-response effects. It is strongly recommended that both dose and frequency of application be considered when estimating risk in terms of multistage carcinogenesis.

When an agent is acting as either an initiating or a promoting agent, its dose-response is greatly modified by the presence of other environmental factors, which may act as tumor-promoting, initiating, or cocarcinogenic agents.

There is currently no validated in vitro short- term test to detect the tumor-promoting activity of environmental chemicals. Tests to measure blocking of intercellular communication and two-stage cell transformation are being validated. In vivo short-term tests using preneoplastic lesions as end- points should be further exploited.

Given the role of tumor-promoting (late-stage) carcinogens as the rate-limiting factor in carcinogenesis, their elimination would be a rapid means of cancer prevention.

15

Acknowledgements

I would like to thank many colleagues for their helpful discussion and advice: Drs L. Tomatis, R. Montesano, M. Hollstein, J. Kaldor, E. Johnson and E. Cardis, International Agency for Research on Cancer; Dr H. Pitot, McArdle Laboratory for Cancer Research, Madison, WI; Dr I.B. Wein- stein, Columbia University, New York, N.Y.; Dr G.M. Williams, Naylor Dana Institute for Disease Prevention, Valhalla, N.Y.; Dr J.C. Barrett, Na- tional Institute of Environmental Health Sciences, Research Triangle Park, N.C.; Dr B.N. Ames, University of California, Berkeley, CA; Dr T. San- her, Institute for Cancer Research, Oslo; and Dr G. Astrup, Elkem a/s, Oslo. I also thank Ms. Fu- chez for her secretarial help and Ms. Heseltine for her editing of the manuscript.

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