SENCAR mouse skin tumorigenesis model versus other strains and stocks of mice.

The SENCAR mouse stock was selectively bred for eight generations for sensitivity to skin tumor induction by the two-stage tumorigenesis protocol using 7,12-dimethylbenz(a)anthracene (DMBA) as the initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as the promoter. The SENCAR mouse was derived by crossing Charles River CD-1 mice with skin-tumor-sensitive mice (STS). The SENCAR mice are much more sensitive to both DMBA tumor initiation and TPA tumor promotion than CD-1, BALB/c, and DBA/2 mice. An even greater difference in the sensitivity to two-stage skin tumorigenesis is apparent between SENCAR and C57BL/6 mice when using DMBA-TPA treatment. However, the SENCAR and C57BL/6 mice have a similar tumor response to DMBA-benzoyl peroxide treatment, suggesting that TPA is not an effective promoter in C57BL/6 mice. The DBA/2 mice respond in a similar manner to the SENCAR mice when using N-methyl-N-nitro-N-nitrosoguanidine (MNNG)-TPA treatment. The SENCAR mouse model provides a good dose-response relationship for many carcinogens used as tumor initiators and for many compounds used as tumor promoter. When compared to other stocks and strains of mice, the SENCAR mouse has one of the largest data bases for carcinogens and promoters.


Introduction
It is well known that skin tumors (papillomas, keratoacanthomas, and squamous cell carcinomas) can be induced in mice by a complete carcinogenesis protocol (carcinogen given repetitively) or by the sequential application of a subthreshold dose of a carcinogen (initiation stage) followed by repetitive treatment with a weak or noncarcinogenic tumor promoter (promotion stage). The initiation stage requires only a single application of either a direct or an indirect carcinogen at a subthreshold dose and is essentially irreversible, whereas the promotion stage is brought about by repetitive treatments after initiation and is reversible for a period of time but later becomes irreversible (1,2).
To provide a better understanding of the SENCAR mouse and its relative sensitivity to skin carcinogenesis when compared to other stocks and strains of mice, this paper first discusses the derivation of the SENCAR mouse.

Derivation
The SENCAR stock of mice was selectively bred for sensitivity to skin tumor induction by two-stage tumorigenesis. Consequently, the SENCAR mouse is extremely sensitive to two-stage carcinogenesis and coincidentally sensitive to complete carcinogenesis. The SENCAR mouse was derived from crossing Charles River CD-1 mice with skin tumor-sensitive mice (STS) *The University of Texas System Cancer Center, Science Park Research Division, P.O. Box 389, Smithville, TX 78957. and selected for sensitivity to 7,12-dimethylbenz-(a)anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) two-stage carcinogenesis for eight generations starting with the F1 cross as originally described by Boutwell (2). Figure 1 outlines the method used by Boutwell to select for the STS mice. A similar selection procedure was used by Boutwell and coworkers to derive the SENCAR mice as outlined in Figure  2. In both cases, the mice developing the earliest and most numerous papillomas after initiation-promotion treatment were selected for each breeding.
Comparison of SENCAR Mice to Other Stocks and Strains of Mice SENCAR mice are between 10 and 20 times more sensitive to DMBA tumor initiation than CD-1 mice (Table 1). However, as shown in Table 2, they are only between three and five times more sensitive to benzo(a)pyrene (B[a]P) tumor initiation than CD-1 mice (3). In addition, SENCAR mice are two to three times more sensitive to TPA promotion than CD-1 mice (3). BALB/c mice also appear to be consistently less sensitive to two-stage tumorigenesis than SENCAR mice (4).
Although several stocks and strains of mice have been used in skin tumor induction experiments very little dose-response data are available. In addition, with the exception of studies on SENCAR and C57BL/6 mice, very few comparative studies have been performed to determine the relative sensitivity of various stocks and strains of mice to carcinogens. An even greater differ- DMBA was applied topically once and followed 7 days later by twice weekly promoting applications of 8.5 nmole of TPA. The maximum percent standard deviation for all groups was 15%. b TPA-only group; the mice received twice weekly applications of 8.5 nmole of TPA for 25 wk. ence in the sensitivity to two-stage skin carcinogenesis is apparent between SENCAR and C57BL/6 mice than between the SENCAR and CD-1 mice. C57BL/6 mice are very refractory to two-stage skin carcinogenesis by B(a)P-TPA. As shown in Table 3, even high initiating doses of B(a)P (1600 nmole) and high promoting doses of TPA (10 ,ug) are quite ineffective in causing skin tumors in C57BL/6 mice. However, C57BL/6 mice do respond to complete carcinogenesis by B(a)P and, in this regard, they appear to be equal to or slightly more sensitive than SENCAR mice (5). This unequal susceptibility to complete and two-stage carcinogenesis within a stock or strain of mice strongly suggests that the promotional phases of complete and two-stage carcinogenesis are dissimilar. In addition, differences in sensitivity to initiation and promotion among mice may be caused by alterations in the promotional phase of twostage carcinogenesis. In this regard, we have recently found that benzoyl peroxide is an effective promoter in   C57BL/6 and SENCAR mice (5). For some reason, TPA is not an effective promoter in C57BL/6 mice.
Recently, DiGiovanni and coworkers (6) have found that the DBA/2 mice are much less sensitive to DMBA-TPA two-stage tumorigenesis than the SENCAR mice. If high initiating doses (400 nmole/mouse) of DMBA are used on the DBA/2 mice followed by TPA promotion, they do respond with a high tumor yield. When DBA/ 2 mice are compared with SENCAR mice following initiation with N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and TPA promotion, the DBA/2 and SENCAR mice respond with a nearly identical tumor response (6).

Carcinogens and/or Initiators Used With the SENCAR Mouse Model
The SENCAR mouse model currently has one of the largest data bases for carcinogens and/or initiators. All the carcinogens and/or initiators that have been tested in both the CD-1 and SENCAR mice have been found to be positive in both stocks of mice. The major difference is related to the increased sensitivity to and shorter latent period for tumor development in the SENCAR mice. As was the case in CD-1 mice, DMBA and B(a)P show a good dose-response relationship in SENCAR mice ( Table 4). As can be seen, a good correlation exists between the number of papillomas per mouse at 15 weeks and the final carcinoma incidence at 50 weeks. The percentage of mice with papillomas also has a reasonable correlation, but the dose response relationship is very narrow ( Table 4).
The carcinogenicity of many polycyclic aromatic hydrocarbons (PAHs), metabolites, and derivatives has been determined in the SENCAR mouse skin tumori-  (33). The mice were treated one week after initiation with twice weekly applications of 5 ,ug of TPA.
genesis model (4,. Besides the PAHs, a number of other classes of chemical carcinogens have been found to be positive in a dose-response relationship as skin carcinogens and/or initiators in the SENCAR mouse. These classes include sterig-matocystin, urethane, 1,3propanesultone, 4-nitroquinoline-N-oxide, MNNG, Nnitrosoethylurea (ENU), N-nitrosomethylurea, epichlorohydrin, and ,-naphthylamine (Slaga et al., unpublished data). In this regard, both MNNG and ENU have been found to be very potent skin carcinogens and tumor initiators, which suggests that these compounds will be useful, direct-acting carcinogens in the skin model (Slaga and Nesnow, unpublished data). Several other chemicals (dimethylnitrosamine, dimethylhydrazine, acetylaminofluorine, and 4-aminobiphenyl), which have been found to be positive as carcinogens in certain tissues, have been extensively studied in SENCAR mouse skin and found to be negative as carcinogens (Slaga et al., unpublished data). A possible explanation may be the lack of metabolic activation of these compounds in the skin. In addition, several compounds such as chloroform and malonaldehyde were also found to be negative as carcinogens in the SENCAR mouse skin tumorigenesis model (Slaga and coworkers, unpublished data).
A number of environmentally important complex mixtures have also been extensively studied for skin-carcinogenic and/or tumor-initiating activity in the SEN-CAR mouse (28)(29)(30)(31)(32). Of the complex mixtures tested, coke oven main and roofing tar both had strong skin carcinogenic and tumor-initiating activity, whereas the Nissan diesel exhaust sample was only a strong tumor initiator.

Promoters Used With the SENCAR Mouse Model
As in the case with carcinogens and/or tumor initiators, not all the various skin tumor promoters tested have been assayed in SENCAR mice. As shown in Table  5, a good dose-response relationship exists for TPA skin tumor promotion after DMBA initiation when considering either the number of papillomas per mouse at 15 weeks or the percentage of mice with squamous cell carcinomas at 50 weeks. The repetitive application of the promoter TPA without initiation by DMBA in general gives rise to a few tumors, but a dose-response relationship has never been noted ( Table 6). The maximum response observed after 50 weeks of treatment with TPA was a 22% incidence of mice with papillomas and 5% with carcinomas.
Although the initiation stage appears to be irreversible, the promotion stage is reversible, requiring a certain frequency of promoter application to induce tumors. Table 7 compares the promoting activity of various doses of TPA when given either three times, two times, or one time per week. In general, as the frequency of TPA application decreases, the promoting activity also decreases. It should be pointed out, however, that one application per week of TPA after initiation by 20 nmoles of DMBA is still fairly effective in tumor promotion. In constrast, high doses of TPA given once every 2 weeks or once every 3 weeks are ineffective in tumor promotion (33).
In terms of complex mixtures, diesel fuel was found to have a moderate skin-tumor-promoting activity, but was found to be negative as a carcinogen in SENCAR mice (Slaga et al., unpublished data).

Conclusion
The SENCAR mouse is, in general, one of the most sensitive stocks and strains of mice currently available for both complete and two-stage carcinogenesis studies. The SENCAR mouse skin tumorigenesis model has one of the largest data bases for skin carcinogens, tumor initiators, tumor promoters, and anticarcinogens (antiinitiators and antipromoters). The model provides excellent data on dose-response relationships when both pure substances and complex mixtures are tested. The two-stage tumorigenesis protocol can be considered a reliable and relatively short-term bioassay for carcinogens and promoters. This model has also been very important in determining the mechanism of action of carcinogens, promoters, and anticarcinogens.