Complete Carcinogen Research Articles

Characterization of a cloned ultraviolet radiation (UV)-induced suppressor T cell line that is capable of inhibiting anti-UV tumor-immune responses.

Ultraviolet radiation (UV) is a potent carcinogen for the induction of skin tumors. In this regard, UV represents a unique carcinogenic agent, in that depending on the dosage and conditions of administration it can function as either a complete carcinogen, a carcinogenic promoting agent, or an immunologic modulator of anti-tumor rejection responses. The immunologic modulatory activity of UV has been demonstrated in numerous studies. These studies have shown that subcarcinogenic doses of UV induce a population of suppressor T lymphocytes (Ts cells) that allow for the emergence and progression of UV-induced tumors. Although the phenotypic and functional properties of these cells have been established, it was unclear as to whether the UV-induced Ts cell population consisted of multiple Ts cell clones able to recognize a range of unique tumor antigens or a limited number of Ts cell clones with functional specificity directed toward a common tumor-associated antigen (TAA). To address this question, an interleukin 2-dependent, UV-induced cloned Ts cell line was derived, by limiting dilution without exogenous antigen stimulation, from the splenic T cell population of a C3H mouse that had been exposed to a subcarcinogenic dose of UV. This Ts cell line, designated UV2.10, was selected for its ability to suppress the in vitro differentiation of cytotoxic T cells from the draining lymph nodes of UV-induced tumor-immune mice. When transferred into non-UV-irradiated syngeneic mice, which normally reject a UV-induced tumor implant, the UV2.10 cells rendered their hosts susceptible to the growth of a battery of UV-induced tumors. Although capable of suppressing in vitro and in vivo UV-induced tumor-immune responses, UV2.10 cells did not inhibit the elicitation of contact hypersensitivity responses, the rejection of allogeneic skin grafts, responses, the rejection of allogeneic skin grafts, or the rejection of allogeneic UV-induced tumors. These data suggest that the cloned UV2.10 Ts cell line possesses functional antigenic specificity that may be limited to the regulation of immune responses that are directed toward the TAA expressed by syngeneic UV-induced tumors. Employing monoclonal antibodies and FACS analysis, the cell surface phenotype of the UV2.10 cell line was determined to be: Thy-1.2+, Lyt-1-, Lyt-2+/- (dim), L3T4a-, I-A/E-, and I-J+. This cell surface phenotype is indicative of a suppressor T cell. These data lend further support to the hypothesis that the UV-induced Ts cell population is clonal in nature and functions through its ability to recognize a common TAA(s) that appears to be expressed by virtually all UV-induced tumors.

Amplified response to phenobarbital promotion of hepatotumorigenesis in obese yellow Avy/A (C3H x VY) F-1 hybrid mice.

Mottled yellow Avy/A and agouti A/a (C3H x VY) F-1 hybrid male mice were fed untreated control diet or diet with a target dose of 500 p.p.m. sodium phenobarbital (PB) for 17-19 months. No differences in prevalence of hepatocellular adenomas or carcinomas were found between untreated yellow and agouti mice. PB treatment increased prevalence of adenomas but decreased prevalence of carcinomas. No difference in enhancement of adenoma formation by PB was observed between yellow and agouti mice bearing single adenomas. However, the proportion of PB-treated yellow mice bearing multiple adenomas (66%) was much greater than the proportion of analogous agouti mice (18%). Fatty changes in the periportal area of the liver and focal cytoplasmic vacuolization were induced to a much greater extent in PB-treated yellow mice than among treated agoutis. PB increased the prevalence and severity of focal areas of chronic inflammation in the liver considerably more in agouti than in yellow mice. The possible relation of this finding to the altered immune responses of obese yellow mice remains to be determined. The results of this study suggest that the use of yellow Avy/A and agouti A/a (C3H x VY) F-1 hybrid mice in carcinogenicity assays make make it possible to differentiate between weak and strong promoters as well as between promoters and complete carcinogens. Weak promoters should induce hepatocellular adenomas in yellow mice even if they fail to do so in agouti mice. Promoting substances which act similarly to PB may be identified in this system by simultaneously increasing adenoma prevalence and decreasing carcinoma prevalence. Complete carcinogens should increase carcinoma prevalence in the yellow mice even at low dose levels.

Persistent suppression of humoral and cell-mediated immunity in mice following exposure to the polycyclic aromatic hydrocarbon 7,12-dimethylbenz[a]anthracene

Carcinogen-induced immunosuppression has been implicated as an epigenetic mechanism in promoting the outgrowth and metastasis of neoplastic cells. It has previously reported that the complete carcinogen 7,12-dimethylbenz[a]antracene (DMBA) suppresses both humoral immunity (HI) and cell-mediated immunity (CMI) 3–5 days following exposure. Since persistent systemic immunosuppression may be more relevant in tumor outgrowth, assays quantitating HI and CMI, including those functions involved in tumor resistance, were performed 4 and 8 weeks following exposure to tumorigenic doses of DMBA. Adult B6C3F1 female mice were administered DMBA dissolved in corn oil subcutaneously at 5, 50 and 100 μg/g body weight in ten equal doses over 2 weeks (corn oil = vehicle control). The number of splenocytes producing IgM antibody to the T-dependent antigen, sheep erythrocytes, was suppressed up to 95% and 98% at 4 and 8 weeks, respectively. The IgG response was similarly depressed 75% and 98% at 4 and 8 weeks, respectively. Lymphoproliferation of splenocytes in response to the mitogens LPS, PHA and Con A were depressed up to 88%, 78% and 83% at 4 weeks and 63%, 63% and 67% respectively, at 8 weeks. In addition, alloantigen-induced proliferation of splenocytes in a one-way mixed lymphocyte culture was suppressed up to 90% at 8 weeks. The ability to generate cytotoxic T-lymphocytes (CTL) in vitro against P815 tumor cells was depressed at both time periods (88% and 60%, respectively) as was natural killer (NK) cell cytolysis of YAC-1 tumor targets (84% and 55%, respectively). The immunosuppression noted in these parameters was similar to that observed within 3–5 days following DMBA dosing. The persistent immunosuppression induced by the PAH carcinogen DMBA, including CTL and NK cell tumoricidal functions, may represent an important epigenetic mechanism contributing to tumor outgrowth or metastasis by this class of agents.

Tumor-initiating activity of major in vivo metabolites of indeno[1,2,3-cd]pyrene on mouse skin.

Indeno[1,2,3-cd]pyrene is a ubiquitous environmental pollutant which is active as a tumor initiator and complete carcinogen on mouse skin and is carcinogenic in rat lung. The major metabolites of indeno[1,2,3-cd]pyrene as formed in vivo in mouse skin have been identified. 8-Hydroxyindeno[1,2,3-cd]pyrene is the most abundant metabolite identified. 9-Hydroxyindeno[1,2,3-cd]pyrene and trans-1,2-dihydro-1,2-dihydroxyindeno[1,2,3-cd]pyrene are also major in vivo metabolites in mouse skin. Several minor metabolites were also identified. Among these are trans-1,2-dihydro-1,2,8-trihydroxyindeno[1,2,3-cd]pyrene, trans-1,2-dihydro-1,2,9-trihydroxyindeno[1,2,3-cd]pyrene, indeno[1,2,3-cd]pyrene-1,2-dione, and 10-hydroxyindeno[1,2,3-cd]pyrene. The tumor-initiating activity of several of the major in vivo metabolites of indeno[1,2,3-cd]pyrene has been investigated on mouse skin. Trans-1,2-dihydro-1,2-dihydroxyindeno[1,2,3-cd]pyrene and 1,2-dihydro-1,2-epoxyindeno[1,2,3-cd]pyrene both produced an 80% incidence of tumor-bearing mice at a total initiating dose of 1.0 mg. The activity of this K-region dihydrodiol and K-region oxide was, however, less than that of the parent hydrocarbon. These data suggest that 1,2-dihydro-1,2-epoxyindeno[1,2,3-cd]pyrene, which is an ultimate mutagenic metabolite of indeno[1,2,3-cd]pyrene, is not the ultimate tumorigenic metabolite on mouse skin. 8-Hydroxyindeno[1,2,3-cd]pyrene, which is mutagenic when assayed in the presence of a microsomal activation system, exhibited only weak tumor-initiating activity. These results indicate that the principal metabolic activation pathways associated with the mutagenic activity of indeno[1,2,3-cd]pyrene are not related to its tumor-initiating activity on mouse skin.

Choline deficiency and chemical carcinogenesis.

We have reviewed the current status of our knowledge concerning the biologic effects of dietary choline (lipotrope) deficiency in modifying chemical carcinogenesis in experimental animals and discussed its possible mechanisms. Choline deficiency produces various pathologic lesions, involving virtually every organ of the body, as a result of a decrease in phospholipid and acetylcholine synthesis and in the supply of labile methyl groups. The liver is the only organ in which a relationship has been consistently demonstrated between choline deficiency and chemically induced tumors. The deficient diet enhances the initiating potency of several carcinogens and acts as a strong cocarcinogen. Diet also exerts a strong promoting effect, though the possibility that it is a complete carcinogen cannot be ruled out. Phase I enzymes of the carcinogen metabolizing system are uniformly depressed by choline deficiency, but very little information is available regarding the effects of diet on Phase II enzymes that detoxify carcinogen metabolites. Possible modifications of carcinogen-induced DNA damage and their repair processes have not been adequately scrutinized. Solid evidence suggests that feeding a choline-deficient diet leads to enhanced liver cell proliferation, an inadequate supply of methyl groups for transmethylation reactions, and membrane lipid peroxidation. Induced cell proliferation and hypomethylation of DNA may alter the state of gene expression, including that of specific cellular oncogenes. Lipid peroxidation may alter the structure and function of membrane receptors related to liver cell growth or may directly damage cellular DNA. Thus these alterations, individually or in combination, could play a critical role in the diet-induced modification of chemical carcinogenesis.

Models of hepatocarcinogenesis in the rat--contrasts and comparisons.

A variety of approaches for the induction of altered hepatic foci, hyperplastic nodules, and hepatocellular carcinomas in rat liver have been developed. These protocols, aided by the appearance of preneoplastic lesions during the carcinogenic process, have proven to be very useful for examining many of the characteristics of events involved in rat hepatocarcinogenesis. A number of models have demonstrated distinct steps or stages in the progression of the carcinogenic process. These protocols are currently employed in the classification and distinction of agents effecting hepatocarcinogenesis at one or more of its stages. This review presents an overview of the present-day multistage hepatocarcinogenesis model systems in the rat, with contrasts and comparisons of these systems. The potential uses of the model systems in the identification of complete carcinogens, initiating agents, and promoting agents devoid of initiating action are discussed.

TPA(12-O-tetradecanoyl-phorbol-13-acetate) as a carcinogen for mouse skin

In order to study a possible dose/response relationship in the tumorigenic effect of 12-O-tetradecanoyl-phorbol-13-acetate (TPA), groups of hairless mice were treated topically on the back skin with different doses and treatment schedules of TPA in acetone. A group of 104 mice (56 M/48 F) received a single application of 20 nmol TPA; 80 mice (32 M/48 F) received 2 X 20 nmol TPA at 3 day intervals; 95 mice (48 M/47 F) received five applications; and 32 mice (16 M/16 F) received 50 applications of 20 nmol TPA twice weekly. Two groups of 32 mice (16 M/16 F) were painted topically on the back skin twice weekly with doses of 17 and 10 nmol TPA, respectively, in each application for 60 weeks or until the animals died. All applications were given in 0.1 ml reagent grade acetone. A control group was treated twice weekly with acetone alone for 60 weeks. Long-term application of 17 nmol TPA was toxic to the animals, and only 20% survived 10 months. Doses of 10 nmol TPA twice weekly were less toxic, and 80% of the animals survived 14 months. The other doses were fairly non-toxic. The results were assessed statistically by accepted methods for tumor rates and yields, respectively. There was a significant tumor incidence after the higher doses of TPA and a clear dose-response relationship. This further strengthens the evidence that TPA is a complete carcinogen, and not merely a promoter.

Methyl groups or additional aromatic groups donate tumour promoting activity.

There are many examples in chemical carcinogenesis in which the introduction of methyl groups or aromatic rings increased the potency of polycyclic hydrocarbons and aromatic amines. Examination of the data in the light of the multistage process of carcinogenesis indicates that these groups convert initiators into complete carcinogens. As complete carcinogens have both initiating and promoting action, the added groups may be considered to have donated tumour promoting activity to the molecule. Such groups were once called auxocarcinogens.1

Tumour initiators, promoters, and complete carcinogens.

Tumour initiators, promoters, and complete carcinogens.

Preneoplastic lesions after complete and incomplete aromatic amine liver carcinogens in initiation-promotion experiments

Preneoplastic lesions after complete and incomplete aromatic amine liver carcinogens in initiation-promotion experiments

Prooxidant states and tumor promotion.

There is convincing evidence that cellular prooxidant states--that is, increased concentrations of active oxygen and organic peroxides and radicals--can promote initiated cells to neoplastic growth. Prooxidant states can be caused by different classes of agents, including hyperbaric oxygen, radiation, xenobiotic metabolites and Fenton-type reagents, modulators of the cytochrome P-450 electron-transport chain, peroxisome proliferators, inhibitors of the antioxidant defense, and membrane-active agents. Many of these agents are promoters or complete carcinogens. They cause chromosomal damage by indirect action, but the role of this damage in carcinogenesis remains unclear. Prooxidant states can be prevented or suppressed by the enzymes of the cellular antioxidant defense and low molecular weight scavenger molecules, and many antioxidants are antipromoters and anticarcinogens. Finally, prooxidant states may modulate the expression of a family of prooxidant genes, which are related to cell growth and differentiation, by inducing alterations in DNA structure or by epigenetic mechanisms, for example, by polyadenosine diphosphate-ribosylation of chromosomal proteins.

An approach to the development of a short‐term whole‐animal bioassay to distinguish initiating agents (incomplete carcinogens), promoting agents, complete carcinogens, and noncarcinogens in rat liver

With increased knowledge of the stages of initiation and promotion in the natural history of neoplastic development and the multiple observations that such stages are likely to be ubiquitous in the natural history of all histogenetic types of neoplasms, it is becoming increasingly important to distinguish chemical agents that act selectively during one or the other of these stages. Utilizing data obtained from the published literature as well as further observations, the experimental findings reported in this paper serve to indicate an approach to the development of a short-term whole-animal bioassay capable of distinguishing agents that act at one or the other or both of the stages of initiation and promotion in rat liver. Because of the tissue specificity for both complete carcinogens and promoting agents, this bioassay system is not likely to identify the initiating and promoting capabilities of all carcinogenic agents and thus at present is not designed to replace the standard chronic bioassay systems. However, in view of the diverse metabolic capabilities of hepatic tissue, the system proposed in this paper may represent a model capable of distinguishing the stages of carcinogenesis at which many, if not most, specific carcinogenic chemicals exert their primary action.

Interaction of epidermal growth factor with initiators and complete carcinogens in the C3H10T1/2 cell culture system.

Unlike 12-O-tetradecanoylphorbol-13-acetate, epidermal growth factor (EGF) could not promote the appearance of type III foci from initiated C3H10T1/2 cells. At appropriate concentrations, EGF induced the formation of type II colonies in the absence of any initiator. At higher concentrations, EGF suppressed the induction of both type II and type III colonies elicited by methylcholanthrene.

Studies on the promoting and complete carcinogenic activities of some oxidizing chemicals in skin carcinogenesis

Six oxidizing chemicals were tested for promoting and complete carcinogenic activities in skin carcinogenesis using female Sencar mice. In the promotion tests, the chemicals were applied twice a week for 51 weeks after initiation with dimethylbenzanthracene (DMBA). In the tests for complete carcinogenic activities, the chemicals alone were applied for 51 weeks. Benzoyl peroxide was found to be a potent promoter as reported previously. Moreover, possible complete carcinogenic action of this chemical was found in this study. Potential promoting effect was suspected in sodium chlorite. Potassium bromate, ammonium persulphate, hydrogen peroxide and sodium hypochlorite were inactive either as a promoter or a complete carcinogen.

Cytophotometric manifestation of the biochemical changes in various skin constituents induced by a single subcutaneous administration of 7,12-dimethylbenz(a)anthracene in rabbit

The effect of a single subcutaneous administration of 7,12-dimethylbenz(a)anthracene, the powerful complete carcinogen, under the skin was studied in the rabbit. The study reports an ordered sequential biochemical and cytophotometric changes induced by the carcinogen. While the biochemical studies comprised of sequential quantitative estimations of DNA, RNA and protein per mg of skin, the cytophotometric studies consisted of the estimation of the level of macromolecules in a cell/nucleus, in different skin constituentsviz. epidermis, hair follicle shaft region and hair follicle bulb region. Biochemical results indicate an initial rise in the level of DNA and RNA and reduction in protein upto 20 days. From 40 to 60 days treatment duration there was a ‘steady-state’ showing a constant level of all the parameters while the highest peak was observed on the 80th day. The site of these biochemical changes among different skin constituents was determined with the help of cytophotometer which indicates the highest level of nucleic acids in epidermis region right from the initial stage (i.e. 10th day) to the 90th day of treatment in comparison to two other regions hair follicle shaft and hair follicle bulb regions. Histological studies, on the other hand, reveal a greatly, though gradual, increased nuclear area and the highest rate of proliferation only in hair follicle bulb region, thus suggesting a definite role of this region of the skin in the carcinogenesis. All these results suggest that the important event in the initiation phase of 7,12-dimethylbenz(a)anthracene mediated skin carcinogenesis in rabbit might be associated with epidermal region but the role of hair follicle bulb region should also be considered as of an equal significance during the process.

Irreversibility of liver tumors in C3H mice.

The present study was designed to test the possibility that spontaneous regression of hepatocellular tumors might be observed in mice. This problem was studied by sequential liver biopsies in C3H male mice that had been treated with dieldrin (CAS: 60-57-1) as well as in animals treated with N-diethylnitrosamine (CAS: 55-18-5) and in untreated control mice. Adenomas were seen in some animals at the second laparotomy when there had been no tumor at the first laparotomy. In a few mice there was histologic progression from adenoma to carcinoma. A change in predominant cell type in adenomas from clear to basophilic or eosinophilic was also observed in some cases. Additional hepatocellular carcinomas were observed in some animals necropsied at 2 years of age. These observations suggest that spontaneous hepatic tumors and tumors in mice treated with either complete carcinogens or nongenotoxic compounds have a strong tendency to progress. Tumor regression in mice appears to be unusual. No consistent relationship of histologic grade of hepatocarcinoma to the type of chemical employed was observed.

Some actual aspects of tumor induction and promotion.

Quantitative data on chemical carcinogenesis illustrate how difficult it is to decide whether there are thresholds for complete carcinogens. With tumor promoters there probably are safe levels, but these are difficult to determine. The results recorded in the experiments of Fibiger, in which tumors of the stomach were seen, could have been caused by the tumor-promoting effects of a biological agent.

State of the art: Summary and commentary

Cancer Biology The process of carcinogenesis is viewed as a multistage one, involving initiation, promotion, and progression: initiation involving a change in DNA; promotion, a change in the expression of the altered DNA; and progression, a perpetuation of genetic change but with the potential for further genetic instability. Initiation is regarded as irreversible, with a threshold determined by the number of DNA molecules that have been appropriately altered. A wide variety of substances have been incriminated as promoters in different experimental systems, but they can only promote carcinogenesis in DNA that has already been initiated. Elucidation of various biochemical steps involved in transformation in certain experimental systems has revealed possibilities for the development of interventional strategies for interrupting specific events in the process. How and to what extent oncogenes fit into the multistage concepts of carcinogenesis remain to be fully clarified. Within the constraints of existing evidence, proto-oncogenes may be regarded as constituting normal endogenous DNA sequences, the oncogenic expression of which may be influenced by the DNA alterations consequent to the action of initiators and possibly promoters. In the latter circumstance, promoters may appear to be complete carcinogens by facilitating expression of oncogenes. Understanding the role of oncogenes promises to continue at an explosive pace. That a single mutation in a gene codingFor p21 protein generates a consistent and identifiable difference in the gene product that distinguishes normal from transformed cells is an exciting possibility. Metastasis is directly or indirectly the principal cause of death in patients with cancer. Biologically, the process of metastasis is a multiphase and sequential one involving a number of steps, interruption of

Active oxygen acts as a promoter of transformation in mouse embryo C3H/10T1/2/C18 fibroblasts.

There is much evidence from in vivo and in vitro carcinogenesis studies that active oxygen species play a role in tumor promotion. We tested directly whether superoxide produced extracellularly by xanthine-xanthine oxidase (X-XO) has the capacity to promote initiated mouse embryo C3H/10T1/2 fibroblasts. Cell cultures initiated with either 137Cs gamma-rays or benzo[a]pyrene diol epoxide I were found to transform 3-30 times more effectively when subsequently treated daily for 3 weeks with nontoxic doses of X-XO. Scavengers of active oxygen radicals such as superoxide dismutase or superoxide dismutase in combination with catalase reduced the frequency of appearance of transformed foci by 3-25 times when compared to cultures receiving X-XO alone. These results show that active oxygen species such as superoxide and H2O2 can act in a promotional manner that mimics the effects of the mouse skin promoter phorbol 12-myristate 13-acetate in this system. X-XO also acted as a weak complete carcinogen.

The tumorigenicity of 5-azacytidine in the male Fischer rat

5-Azacytidine was administered to young adult male Fischer rats. Tumors were found in 31 out of 70 rats that had received 5-azacytidine and survived 18 months from the start of the experiment. Several rats had multiple primary tumors. In the rats that were tested for complete carcinogenicity a variety of tumor types was found. These included acute leukemia and malignant reticuloendotheliosis, and tumors of the testis, skin, and bronchus. No hepatic tumors were found in the group that was tested for hepatic tumor initiation. Hepatocellular carcinomas were found only in the group that was examined for hepatic tumor promotion by receiving a prior initiating dose of diethylnitrosamine. No tumors were found in the age controls. Thus, in these initial experiments, 5-azacytidine appeared to be a complete carcinogen, inducing tumors in several organs, and a tumor promoter but not a complete carcinogen for the liver.