Medical and Pediatric Oncology 3: 15 1 - 157 ( 1 977)
Cocarcinogens As Environmental Hazards John D. Scribner, Ph.D. Pacific Northwest Research Foundation, Seattle, Washington
It has been known for about 30 years that certain substances which are not carcinogenic in themselves can enhance the effect of those which are. That is, these cocarcinogens can reduce the latent period of chemically induced tumors, increase the total number of tumors, or (usually) both. A variety of cocarcinogenic effects has been discovered through the use of the two-step regimen of inducing skin tumors in mice, in which a subcarcinogenic single dose of a carcinogen is administered to mice, followed by repeated topical treatment with a noncarcinogenic promoter. Eventually, tumors appear at the site of application of the promoter. In this regimen, the effect of the initiator has been found to be irreversible and additive, whereas the effect of the promotor is critically dependent on giving sufficiently large doses sufficiently close together. Here, the promoter fits the role of a cocarcinogen, and is what is usually meant by that less specific term. Now, let us examine several initiation-promotion systems, particularly the promoters used (Figs. 1-4). The initiation process itself can be enhanced or inhibited, and this fact has been used in experiments designed to investigate the mechanism of tumor initiation. Our concern, however, is with the cocarcinogenic agents themselves, for any substance which enhances tumor initiation must also be considered cocarcinogenic (Figs. 5,6). Some substances seem incapable of fully acting as promoters, but can continue the process once started. One of these is turpentine, which can continue the promotion process begun by a 6-week treatment with croton oil (Fig. 7). Finally, there have been many experiments published in which a noncarcinogen was given before or during a complete carcinogenic regimen, with the result being enhanced carcinogenicity. Some examples are shown in Figs. 8 and 9. A feature common to all of these cocarcinogenic influences is that they are likely reversible. Except for the cases of a single dose applied at a critical time relative to initiation, they also appear to require repeated administration in relatively large amounts to produce a statistically significant effect. In some strains of animals, certain tumors appear spontaneously, either in high or low frequency. Some of these strains are used as assay systems for chemical carcinogens. A sensitive and rapid system is the induction of pulmonary adenomas in the A-strain mouse, championed by Dr. Shimkin for many years. Figure 10 shows some examples of this assay.
Address reprint requests to John D. Scribner, Ph.D., Pacific Northwest Research Foundation, 1102 Columbia Street, Seattle, WA 98104.
151
0 1977 Alan R. Liss, Inc., 150 Fifth Avenue, New York, NY 10011
152
Scribner ~
All mice were treated with 13 pg 3-methylcholanthrene (MC) on the back Further treatment 0.2 ml acetone 2x/week 10 pg TPA in acetone 2Xlweek
Tumor yield at 12 weeks none
5 papillomas/mouse ~~
Fig. l a . Tumor promotion by 12-0 -tetradecanoylphorbol-17 acetate (TPA) (1).
All mice were treated once with 0.3% 7, 12-dimethylbenz(a)anthracene (DMBA)
Further treatment
Tumor yield at 12 weeks
benzene, 2xlweek 20% phenol 20% o-cresol 20% m-cresol 20% p-cresol
none 1.5 papillomas/mouse 1.4 papillomas/mouse 0.9 papillomas/mouse 0.6 papiIlomas/mouse
Fig. l b . Tumor promotion by various phenols (2).
All rats received 0.02% 2-acetamidofluorene in the diet for 18 days
Further treatment
Tumor yield
control diet
none (10 1 days) 0.6 hepatomas/rat (269 days) 0.6 hep./rat (101 days) 4.7 hep./rat (269 days)
0.05% phenobarbital in diet
Fig. 2. Promotion of hepatocarcinogenesis by phenobarbital (3).
There have been reservations about this type of assay for a long time. Several early experiments showed that nonspecific inflammation or bronchial hyperplasia did not increase the tumor incidence in these mice. However, other experiments suggested that this system would give a positive response to tumor promoters. Until there has been an adequate demonstration to the contrary, I will be concerned about this possibility.
Cocarcinogens As Environmental Hazards
All rats received 2 mg of N-methyl-N-nitrosourea instilled into the bladder ~~~
~
~
~
Further treatment
Tumor yield
controls 2 g saccharinlkgl day
none 2 papilloma-bearing animals 2 carcinoma-bearing animals of 12 sampled within 50 weeks
Fig. 3. Promotion of bladder carcinogenesis by saccharin (4).
All fish (rainbow trout) received 20 ppb of aflatoxin B1 in the diet for one month Further treatment
Tumor yield, 9 months
control diet
31% 1.O hepatoma/fish 95% 20.0 hepatomas/fish
100 ppm methyl sterculate in diet
Fig. 4. Promotion of hepatocdrcinogenesis by methyl sterculate ( 5 ) .
~
All mice given single intraperitoneal injection of 25 mg urethane, then treated once/week with 0.25 ml of 0.5% croton oil Pretreatment
Tumor yield, 20 weeks
controls 0.25% ml of 20% acetic acid applied to back once 18 hr before initiation
39%, 0.8 papilloma/mouse 87%, 4.3 papillomas/mouse
Fig. 5a. Effect of acetic acid pretreatment on tumor initiation by urethane ( 6 )
~
~
~
All mice received a single topical dose of 0.25 ml of 0.3% DMBA; no promotion Other treatment
Tumor yield, 10 weeks
controls 0.25 ml 25% acetic acid once 5 min after initiation
0.09 papilloma/mouse 1.0 papilloma/mouse
Fig. 5b. Effect of acetic acid treatment on tumor initiation by DMBA (7).
153
154
Scribner ~~
~
All mice received 190 nmoles of DBA once topically, then 10 pg TPA 2x/week Other treatment
Tumor yield, 1 8 weeks
controls 3.67 pmoles benzoflavone once at time of initiation
4.0 papillomas/mouse 8.5 papillomas mouse
Fig. 6. Effect of 7, 8-benzoflavone on tumor initiation by dibenz(a,h) anthracene (DBA) (8).
All mice received 180 nmoles of DBA, once topically, then 0.5% croton oil 2x/week for 6 weeks
Further treatment
Tumor yield, 38 weeks after croton oil
controls turpentine IOx/week
12% skin carcinoma 56% skin carcinoma
Fig. 7. Tumor propagation by turpentine (9).
All hamsters received 1 mg DMBA applied to cheek pouch 3x/week for 6 weeks Other treatment
Tumor yield
controls 3,400 vitamin A palmitate, 3x/week for 10 weeks prior t o DMBA
none 100% carcinoma
Fig. 8. Cocarcinogenesis by vitamin A (10).
If all this were only of academic interest to those concerned with mechanisms of chemical carcinogenesis, there would be no reason for immediate concern or for this paper. Those of us in chemical carcinogenesis could debate in peace over the mechanism of promotion and the propagation of mouse lung adenomas. However, the Environmental Protection Agency (EPA) and the Health, Education, and Welfare Toxicology Subcommittee for Carcinogen Standards have decided to consider as a carcinogen any substance which induces an increased incidence of a tumor type normally seen, or causes such tumors to appear at an earlier time than would be otherwise expected. However, this is a definition of a cocarcinogen, and only of a cocarcinogen. Because of the extreme environmental precautions being demanded for industrial or laboratory uses of carcinogens, the necessity of applying caution to all substances intended for consumer use, and the often
Cocarcinogens As Environmental Hazards
155
paradoxical influences of substances which are cocarcinogenic, I believe we should restrict the definition of a carcinogen to any substance which induces a tumor type not usually observed. Of course, any substance which is active in the lung ademona or similar test should be tested immediately for its ability to induce new tumor types. It is reasonable to ask whether the end effect on the human target is not the same. Is a person affected any differently by developing a tumor directly caused by some compound or indirectly caused by promotion of a genetic predisposition? Of course not. However, it seems quite clear experimentally that compounds which are true carcinogens act irreversibly and additively, and require much less exposure than cocarcinogens, which apparently act reversibly and whose effects accordingly are not additive. Expressed differently, a person has a far lesser probability of getting cancer from exposure t o a cocarcinogen than from exposure to the same dose of a carcinogen. The difficulty of making definitions and acting on them is compounded by the gulf between zero exposure and callous disregard for human health. At what point on the
All mice received ‘‘local exposure” of unstated dose of MC to cervix for 9 weeks Other treatment
Tumor yield
controls
1 carcinoma of vaginaexocervix 5 carcinoma of endocervix 50 mice
subcutaneous implantation of 15-mg pellets of progesterone, one/three weeks)
15 carcinoma of vaginaexocervix 30 carcinoma of endocervix 50 mice
b
Fig. 9. Cocarcinogenesis by progesterone (11).
All compounds injected as aqueous dispersion (0.25 ml) once into tail vein of A strain male mice Compound
Tumor yield, 14 weeks
control
IS%, 1.0 tumor/mouse loo%, 28.2 tumors/mouse go%, 4.4 tumors/mouse 20%, 1.O tumor/mouse
0.25 mg DBA 0.25 mg benzo( a)pryene 0.25 mg benz(a)anthracene
Fig. 10a. Induction of lung adenomas as a carcinogenesis test system (12).
156
Scribner ~
All compounds injected as aqueous solution, intraperitoneally 3xlweeks for 4 weeks
Compound
Tumor yield, 20 weeks after injection
27 mmoles/kg urethane 23 mmoles/kg N-OH-urethan
19% 0.2 tumor/mouse 1OO%, 24.5 tumors/mouse loo%, 18.6 tumors/mouse
23 mmoles/kg propyl carbamate 32 mmoles/kg methyl carbamate
7570, 0.8 tumor/mouse 6%, O.l/tumor mouse
control
Fig. 10b. Induction of lung adenornas as a carcinogenesis test system ( 1 3 ) .
continuum between these extremes can the government reasonably act to protect the public? I believe that this question is made more difficult and more subject to political interference by the use of an inaccurate definition of a carcinogen. Known human carcinogens should be removed from traffic if possible, or reduced to the lowest exposure if not. Compounds which qualify only as experimental cocarcinogens fall into an entirely separate class, and should probably be considered on a case-by-case basis, certainly without any attempt to apply all available technology to achieve minimal exposure. Perhaps one should say that exposure to proven complete carcinogens should be reduced to the lowest possible level at the manufacturing and consumer level, and that all reasonable laboratory precautions should be required in research with such compounds. Exposure to cocarcinogens should be reduced to a level that is economically practical, but without any requirement to approach zero exposure. With this approach, regulatory agencies should, in the long run, have a better record of professional and public support for their actions than would be the case if they were to attempt to impose uneconomic, unpopular regulations on the use of carcinogens and cocarcinogens alike. NOTED ADDED IN PROOF
A valuable point was raised during the discussion regarding Fig. 8. Dr. Michael Sporn protested that the extreme toxicity of excessive doses of retinoids (regarding the experiment shown in Fig. 8) bears no meaningful relationship to the normal biologically necessary or therapeutic doses of such compounds. Dr. Scribner concurred with Dr. Sporn, and suggested that this instance is a good illustration of the type of cocarcinogenic influence which should be carefully weighed before considering any regulatory action. If the EPA criteria of carcinogenicity were to be made criteria for invocation of the Delaney clause, this single experiment would mandate removal of Vitamin A from the diet, an absurd action.
Cocarcinogens As Environmental Hazards
157
REFERENCES 1. Slaga, T. J., and Scribner, J. D. inhibition of tumor initiation and promotion by anti-inflammatory agents. J. Natl. Cancer Inst. 51:1723-1725, 1973. 2. Boutwell, R. K., and Bosch, D. K. The tumor-promoting action of phenol and related compounds for mouse skin. Cancer Res. 19:413-424, 1959. 3. Peraino, C., Fry, R. J. M., Staffeldt, E., and Kisieliski, W. E. Effects of varying the exposure to phenobarbital on its enhancement of 2-acetylaminofluorene-induced hepatic tumorigenesis in the rat. Cancer Res. 332701-2705, 1973. 4. Hicks, R. M., Wakefield, J. St. J., and Chowaniec, J. Co-carcinogenic action of saccharin in the chemical induction of bladder cancer. Nature 243:347-349, 1973. 5. Lee, D. J., Wales, J. H., and Sinnhuber, R. 0. Promotion of aflatoxin-induced hepatoma growth in trout by methyl malvalate and sterculate. Cancer Res. 31:960-963, 1971. 6. Pound, A. W. Further observations concerning the influence of preliminary stimulation by croton oil and acetic acid on the initiation of skin tumors in mice by urethane. Brit. J. Cancer 20:385398,1966. 7. Pound, A. W. The influence of preliminary irritation by acetic acid or croton oil on skin tumor production in mice after a single application of dimethylbenzanthracene, benzopyrene or dibenzanthracene. Br. J. Cancer 22:533-544, 1968. 8. Bowden, G. T., Slaga, T. J . , Shapas, B. G., and Boutwell, R. K. The role of aryl hydrocarbon hydroxylase in skin tumor initiation by 7, 12-dimethylbenz (a) anthracene and dibenz (a, h) anthracene using DNA binding and t h ~ r n i d i n e - ~incorporation H into DNA as criteria. Cancer Res. 34~2634-2642,1974. 9. Boutwell, R. K. Some biological aspects of skin carcinogenesis. Progr. Exp. Tumor Res. 4:207-250,1964. 10. Levij, I. S., Rwomushana, J. W., and Polliack, A. Enhancement of chemical carcinogenesis in the hamster cheek pouch by prior topical application of vitamin A palmitate. 3. Invest. Derm. 53:228231, 1969. 11. Reboud, S., and Pageant, G. Co-carcinogenic effect of progesterone on 20-methylcholanthrene induced cervical carcinoma in mice. Nature 241:398-399, 1973. 12. Andervont, H. B., and Shimkin, M. B. Biological testing of carcinogens. 11. Pulmonary-tumor induction technique. J. Natl. Cancer Inst. 1:225-239, 1940. 13. Shimkin, M. B., Wieder, R., McDonough, M., Fishbein, L., and Swern, D. Lung tumor response in strain A mice as a quantitative bioassay of carcinogenic activity of some carbamates and aziridines. Cancer Res. 29:2184-2190. 1969.
Cocarcinogens As Environmental Hazards John D. Scribner, Ph.D. Pacific Northwest Research Foundation, Seattle, Washington
It has been known for about 30 years that certain substances which are not carcinogenic in themselves can enhance the effect of those which are. That is, these cocarcinogens can reduce the latent period of chemically induced tumors, increase the total number of tumors, or (usually) both. A variety of cocarcinogenic effects has been discovered through the use of the two-step regimen of inducing skin tumors in mice, in which a subcarcinogenic single dose of a carcinogen is administered to mice, followed by repeated topical treatment with a noncarcinogenic promoter. Eventually, tumors appear at the site of application of the promoter. In this regimen, the effect of the initiator has been found to be irreversible and additive, whereas the effect of the promotor is critically dependent on giving sufficiently large doses sufficiently close together. Here, the promoter fits the role of a cocarcinogen, and is what is usually meant by that less specific term. Now, let us examine several initiation-promotion systems, particularly the promoters used (Figs. 1-4). The initiation process itself can be enhanced or inhibited, and this fact has been used in experiments designed to investigate the mechanism of tumor initiation. Our concern, however, is with the cocarcinogenic agents themselves, for any substance which enhances tumor initiation must also be considered cocarcinogenic (Figs. 5,6). Some substances seem incapable of fully acting as promoters, but can continue the process once started. One of these is turpentine, which can continue the promotion process begun by a 6-week treatment with croton oil (Fig. 7). Finally, there have been many experiments published in which a noncarcinogen was given before or during a complete carcinogenic regimen, with the result being enhanced carcinogenicity. Some examples are shown in Figs. 8 and 9. A feature common to all of these cocarcinogenic influences is that they are likely reversible. Except for the cases of a single dose applied at a critical time relative to initiation, they also appear to require repeated administration in relatively large amounts to produce a statistically significant effect. In some strains of animals, certain tumors appear spontaneously, either in high or low frequency. Some of these strains are used as assay systems for chemical carcinogens. A sensitive and rapid system is the induction of pulmonary adenomas in the A-strain mouse, championed by Dr. Shimkin for many years. Figure 10 shows some examples of this assay.
Address reprint requests to John D. Scribner, Ph.D., Pacific Northwest Research Foundation, 1102 Columbia Street, Seattle, WA 98104.
151
0 1977 Alan R. Liss, Inc., 150 Fifth Avenue, New York, NY 10011
152
Scribner ~
All mice were treated with 13 pg 3-methylcholanthrene (MC) on the back Further treatment 0.2 ml acetone 2x/week 10 pg TPA in acetone 2Xlweek
Tumor yield at 12 weeks none
5 papillomas/mouse ~~
Fig. l a . Tumor promotion by 12-0 -tetradecanoylphorbol-17 acetate (TPA) (1).
All mice were treated once with 0.3% 7, 12-dimethylbenz(a)anthracene (DMBA)
Further treatment
Tumor yield at 12 weeks
benzene, 2xlweek 20% phenol 20% o-cresol 20% m-cresol 20% p-cresol
none 1.5 papillomas/mouse 1.4 papillomas/mouse 0.9 papillomas/mouse 0.6 papiIlomas/mouse
Fig. l b . Tumor promotion by various phenols (2).
All rats received 0.02% 2-acetamidofluorene in the diet for 18 days
Further treatment
Tumor yield
control diet
none (10 1 days) 0.6 hepatomas/rat (269 days) 0.6 hep./rat (101 days) 4.7 hep./rat (269 days)
0.05% phenobarbital in diet
Fig. 2. Promotion of hepatocarcinogenesis by phenobarbital (3).
There have been reservations about this type of assay for a long time. Several early experiments showed that nonspecific inflammation or bronchial hyperplasia did not increase the tumor incidence in these mice. However, other experiments suggested that this system would give a positive response to tumor promoters. Until there has been an adequate demonstration to the contrary, I will be concerned about this possibility.
Cocarcinogens As Environmental Hazards
All rats received 2 mg of N-methyl-N-nitrosourea instilled into the bladder ~~~
~
~
~
Further treatment
Tumor yield
controls 2 g saccharinlkgl day
none 2 papilloma-bearing animals 2 carcinoma-bearing animals of 12 sampled within 50 weeks
Fig. 3. Promotion of bladder carcinogenesis by saccharin (4).
All fish (rainbow trout) received 20 ppb of aflatoxin B1 in the diet for one month Further treatment
Tumor yield, 9 months
control diet
31% 1.O hepatoma/fish 95% 20.0 hepatomas/fish
100 ppm methyl sterculate in diet
Fig. 4. Promotion of hepatocdrcinogenesis by methyl sterculate ( 5 ) .
~
All mice given single intraperitoneal injection of 25 mg urethane, then treated once/week with 0.25 ml of 0.5% croton oil Pretreatment
Tumor yield, 20 weeks
controls 0.25% ml of 20% acetic acid applied to back once 18 hr before initiation
39%, 0.8 papilloma/mouse 87%, 4.3 papillomas/mouse
Fig. 5a. Effect of acetic acid pretreatment on tumor initiation by urethane ( 6 )
~
~
~
All mice received a single topical dose of 0.25 ml of 0.3% DMBA; no promotion Other treatment
Tumor yield, 10 weeks
controls 0.25 ml 25% acetic acid once 5 min after initiation
0.09 papilloma/mouse 1.0 papilloma/mouse
Fig. 5b. Effect of acetic acid treatment on tumor initiation by DMBA (7).
153
154
Scribner ~~
~
All mice received 190 nmoles of DBA once topically, then 10 pg TPA 2x/week Other treatment
Tumor yield, 1 8 weeks
controls 3.67 pmoles benzoflavone once at time of initiation
4.0 papillomas/mouse 8.5 papillomas mouse
Fig. 6. Effect of 7, 8-benzoflavone on tumor initiation by dibenz(a,h) anthracene (DBA) (8).
All mice received 180 nmoles of DBA, once topically, then 0.5% croton oil 2x/week for 6 weeks
Further treatment
Tumor yield, 38 weeks after croton oil
controls turpentine IOx/week
12% skin carcinoma 56% skin carcinoma
Fig. 7. Tumor propagation by turpentine (9).
All hamsters received 1 mg DMBA applied to cheek pouch 3x/week for 6 weeks Other treatment
Tumor yield
controls 3,400 vitamin A palmitate, 3x/week for 10 weeks prior t o DMBA
none 100% carcinoma
Fig. 8. Cocarcinogenesis by vitamin A (10).
If all this were only of academic interest to those concerned with mechanisms of chemical carcinogenesis, there would be no reason for immediate concern or for this paper. Those of us in chemical carcinogenesis could debate in peace over the mechanism of promotion and the propagation of mouse lung adenomas. However, the Environmental Protection Agency (EPA) and the Health, Education, and Welfare Toxicology Subcommittee for Carcinogen Standards have decided to consider as a carcinogen any substance which induces an increased incidence of a tumor type normally seen, or causes such tumors to appear at an earlier time than would be otherwise expected. However, this is a definition of a cocarcinogen, and only of a cocarcinogen. Because of the extreme environmental precautions being demanded for industrial or laboratory uses of carcinogens, the necessity of applying caution to all substances intended for consumer use, and the often
Cocarcinogens As Environmental Hazards
155
paradoxical influences of substances which are cocarcinogenic, I believe we should restrict the definition of a carcinogen to any substance which induces a tumor type not usually observed. Of course, any substance which is active in the lung ademona or similar test should be tested immediately for its ability to induce new tumor types. It is reasonable to ask whether the end effect on the human target is not the same. Is a person affected any differently by developing a tumor directly caused by some compound or indirectly caused by promotion of a genetic predisposition? Of course not. However, it seems quite clear experimentally that compounds which are true carcinogens act irreversibly and additively, and require much less exposure than cocarcinogens, which apparently act reversibly and whose effects accordingly are not additive. Expressed differently, a person has a far lesser probability of getting cancer from exposure t o a cocarcinogen than from exposure to the same dose of a carcinogen. The difficulty of making definitions and acting on them is compounded by the gulf between zero exposure and callous disregard for human health. At what point on the
All mice received ‘‘local exposure” of unstated dose of MC to cervix for 9 weeks Other treatment
Tumor yield
controls
1 carcinoma of vaginaexocervix 5 carcinoma of endocervix 50 mice
subcutaneous implantation of 15-mg pellets of progesterone, one/three weeks)
15 carcinoma of vaginaexocervix 30 carcinoma of endocervix 50 mice
b
Fig. 9. Cocarcinogenesis by progesterone (11).
All compounds injected as aqueous dispersion (0.25 ml) once into tail vein of A strain male mice Compound
Tumor yield, 14 weeks
control
IS%, 1.0 tumor/mouse loo%, 28.2 tumors/mouse go%, 4.4 tumors/mouse 20%, 1.O tumor/mouse
0.25 mg DBA 0.25 mg benzo( a)pryene 0.25 mg benz(a)anthracene
Fig. 10a. Induction of lung adenomas as a carcinogenesis test system (12).
156
Scribner ~
All compounds injected as aqueous solution, intraperitoneally 3xlweeks for 4 weeks
Compound
Tumor yield, 20 weeks after injection
27 mmoles/kg urethane 23 mmoles/kg N-OH-urethan
19% 0.2 tumor/mouse 1OO%, 24.5 tumors/mouse loo%, 18.6 tumors/mouse
23 mmoles/kg propyl carbamate 32 mmoles/kg methyl carbamate
7570, 0.8 tumor/mouse 6%, O.l/tumor mouse
control
Fig. 10b. Induction of lung adenornas as a carcinogenesis test system ( 1 3 ) .
continuum between these extremes can the government reasonably act to protect the public? I believe that this question is made more difficult and more subject to political interference by the use of an inaccurate definition of a carcinogen. Known human carcinogens should be removed from traffic if possible, or reduced to the lowest exposure if not. Compounds which qualify only as experimental cocarcinogens fall into an entirely separate class, and should probably be considered on a case-by-case basis, certainly without any attempt to apply all available technology to achieve minimal exposure. Perhaps one should say that exposure to proven complete carcinogens should be reduced to the lowest possible level at the manufacturing and consumer level, and that all reasonable laboratory precautions should be required in research with such compounds. Exposure to cocarcinogens should be reduced to a level that is economically practical, but without any requirement to approach zero exposure. With this approach, regulatory agencies should, in the long run, have a better record of professional and public support for their actions than would be the case if they were to attempt to impose uneconomic, unpopular regulations on the use of carcinogens and cocarcinogens alike. NOTED ADDED IN PROOF
A valuable point was raised during the discussion regarding Fig. 8. Dr. Michael Sporn protested that the extreme toxicity of excessive doses of retinoids (regarding the experiment shown in Fig. 8) bears no meaningful relationship to the normal biologically necessary or therapeutic doses of such compounds. Dr. Scribner concurred with Dr. Sporn, and suggested that this instance is a good illustration of the type of cocarcinogenic influence which should be carefully weighed before considering any regulatory action. If the EPA criteria of carcinogenicity were to be made criteria for invocation of the Delaney clause, this single experiment would mandate removal of Vitamin A from the diet, an absurd action.
Cocarcinogens As Environmental Hazards
157
REFERENCES 1. Slaga, T. J., and Scribner, J. D. inhibition of tumor initiation and promotion by anti-inflammatory agents. J. Natl. Cancer Inst. 51:1723-1725, 1973. 2. Boutwell, R. K., and Bosch, D. K. The tumor-promoting action of phenol and related compounds for mouse skin. Cancer Res. 19:413-424, 1959. 3. Peraino, C., Fry, R. J. M., Staffeldt, E., and Kisieliski, W. E. Effects of varying the exposure to phenobarbital on its enhancement of 2-acetylaminofluorene-induced hepatic tumorigenesis in the rat. Cancer Res. 332701-2705, 1973. 4. Hicks, R. M., Wakefield, J. St. J., and Chowaniec, J. Co-carcinogenic action of saccharin in the chemical induction of bladder cancer. Nature 243:347-349, 1973. 5. Lee, D. J., Wales, J. H., and Sinnhuber, R. 0. Promotion of aflatoxin-induced hepatoma growth in trout by methyl malvalate and sterculate. Cancer Res. 31:960-963, 1971. 6. Pound, A. W. Further observations concerning the influence of preliminary stimulation by croton oil and acetic acid on the initiation of skin tumors in mice by urethane. Brit. J. Cancer 20:385398,1966. 7. Pound, A. W. The influence of preliminary irritation by acetic acid or croton oil on skin tumor production in mice after a single application of dimethylbenzanthracene, benzopyrene or dibenzanthracene. Br. J. Cancer 22:533-544, 1968. 8. Bowden, G. T., Slaga, T. J . , Shapas, B. G., and Boutwell, R. K. The role of aryl hydrocarbon hydroxylase in skin tumor initiation by 7, 12-dimethylbenz (a) anthracene and dibenz (a, h) anthracene using DNA binding and t h ~ r n i d i n e - ~incorporation H into DNA as criteria. Cancer Res. 34~2634-2642,1974. 9. Boutwell, R. K. Some biological aspects of skin carcinogenesis. Progr. Exp. Tumor Res. 4:207-250,1964. 10. Levij, I. S., Rwomushana, J. W., and Polliack, A. Enhancement of chemical carcinogenesis in the hamster cheek pouch by prior topical application of vitamin A palmitate. 3. Invest. Derm. 53:228231, 1969. 11. Reboud, S., and Pageant, G. Co-carcinogenic effect of progesterone on 20-methylcholanthrene induced cervical carcinoma in mice. Nature 241:398-399, 1973. 12. Andervont, H. B., and Shimkin, M. B. Biological testing of carcinogens. 11. Pulmonary-tumor induction technique. J. Natl. Cancer Inst. 1:225-239, 1940. 13. Shimkin, M. B., Wieder, R., McDonough, M., Fishbein, L., and Swern, D. Lung tumor response in strain A mice as a quantitative bioassay of carcinogenic activity of some carbamates and aziridines. Cancer Res. 29:2184-2190. 1969.
