Mechanism of mouse skin tumor promotion by chrysarobin.

The skin tumor-promoting ability of 1,8-dihydroxy-3-methyl-9-anthrone (chrysarobin) was compared with that of 12-O-tetradecanoylphorbol-13-acetate (TPA) and 1,8-dihydroxy-9-anthrone (anthralin) in SENCAR mice. Although dose-response comparisons indicated that chrysarobin was several orders of magnitude less potent than TPA for promoting papilloma formation, this anthrone was 1.5 to 2 times more potent than anthralin. Maximal papilloma responses were achieved by 15 weeks of promotion with TPA whereas at least 25 weeks of promotion were necessary to achieve maximal papilloma responses with chrysarobin or anthralin indicating marked differences in tumor latency between the two classes of compounds. Interestingly, at optimal promoting doses, chrysarobin gave a carcinoma response (22% with 0.3 carcinomas per mouse at 45 weeks) similar to that of TPA suggesting that this compound may be more efficient at promoting carcinomas than papillomas. In two-stage promotion experiments, chrysarobin was incapable of functioning independently as a Stage I or II promoter despite its complete promoting activity. Chrysarobin and TPA were compared at optimal promoting doses for their ability to induce: (a) skin edema, (b) epidermal hyperplasia, and (c) epidermal ornithine decarboxylase. In each case, distinct differences were noted between the two compounds. When taken together, the data support the hypothesis that anthracene-derived skin tumor promoters work at least in part by a mechanism different from the phorbol esters.

Formation and disappearance of benzo[a]pyrene DNA-adducts in mouse epidermis.

The rates of formation and disappearance of benzo[a]pyrene (B[a]P) DNA-adducts were analyzed in the epidermis of SENCAR mice over a 21-day time course. Mice were treated topically with 200 nmol/mouse of [3H]B[a]P at various times prior to sacrifice. The formation and disappearance of total adducts as well as individual adducts was determined and in addition, the rate of DNA turnover was monitored concurrently so that adduct disappearance could be expressed as a function of epidermal cell turnover. Under these experimental conditions, covalent binding of B[a]P to epidermal DNA reached a peak 24 h after treatment. Interestingly, between 24-48 h after application of the hydrocarbon there was a very rapid drop in the level of bound B[a]P to value approximately 50% of the maximum level at 24 h. Thereafter, the level of bound B[a]P disappeared at a much slower rate. In dual-label experiments, where the epidermal DNA was pre-labeled with [14C]thymidine, [3H]B[a]P DNA-adduct disappearance between 24-48 h was clearly more rapid than could be explained on the basis of epidermal DNA turnover. By 72 h and beyond, however, [3H]B[a]P DNA-adduct disappearance approximately paralleled DNA turnover. Examination of the rate of formation and disappearance of individual B[a]P DNA-adducts (nine individual adducts) suggested that some deoxyadenosine adducts were removed more rapidly than deoxyguanosine adducts. The results indicate that at least some epidermal cells have the capacity to repair B[a]P DNA-adducts. The data are discussed in relation to the process of tumor initiation in mouse skin.

DBA/2 mice are as sensitive as SENCAR mice to skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate.

Mice of the inbred strain DBA/2 responded to a two-stage, initiation-promotion tumorigenesis protocol when high initiating doses (400 nmol/mouse) of 7,12-dimethylbenz[a]anthracene were utilized. They also responded when N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was used as the initiating agent. The tumor response in both cases was characterized by a rapid rate of tumor development with the maximal tumor responses reached on or before the 15th week of promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). When DBA/2 mice were compared with SENCAR mice for promotion sensitivity following initiation with MNNG, the two mouse stocks responded with a nearly identical tumor response. C57BL/6 mice were essentially resistant to TPA promotion regardless of the initiator or the dose of initiator used. A preliminary study was conducted to determine how susceptibility to tumor promotion by TPA was inherited in F1 mice derived from DBA/2 (sensitive) and C57BL/6 (resistant) parents. The B6D2F1 mice were as sensitive as the DBA/2 parent, suggesting that susceptibility in these two inbred mouse strains is inherited as an autosomal dominant trait. The results show that these two inbred mouse strains may provide a model system for studying genetic factors controlling susceptibility to phorbol ester skin tumor promotion.

Formation of 7-hydroxymethyl-12-methylbenz(a)anthracene-DNA adducts from 7,12-dimethylbenz(a)anthracene in mouse epidermis.

The formation of DNA adducts from [3H]-7-hydroxymethyl-12-methylbenz(a)anthracene (7-OHM-12-MBA) and [3H]-7,12-dimethylbenz(a)anthracene (DMBA) in the epidermis of Sencar mice was analyzed. Comparison of Sephadex LH-20 chromatographic profiles of DNA samples isolated from mice treated with DMBA or 7-OHM-12-MBA suggested that the DMBA-treated animals contained DNA adduct(s) derived from the further metabolism of 7-OHM-12-MBA. Further analysis of DNA samples from DMBA-treated mice by high-pressure liquid chromatography demonstrated the presence of 5 DNA adducts which were chromatographically indistinguishable from the DNA adducts formed in 7-OHM-12-MBA-treated mice. Epidermal homogenates were utilized to catalyze the covalent binding of [3H]DMBA and [3H]-7-OHM-12-MBA to calf thymus DNA in vitro. Under conditions of limiting concentrations of [3H]DMBA, the majority of the DNA adducts formed chromatographed in regions where 7-OHM-12-MBA-DNA adducts eluted. A major DMBA-DNA adduct formed in this in vitro system eluted with the same retention time as did the major 7-OHM-12-MBA-DNA adduct formed in mouse skin in vivo. These results when coupled with the in vivo data suggest that 7-OHM-12-MBA is an intermediate for at least some of the binding of DMBA to epidermal DNA in Sencar mice.

Tumor initiating activity of 9- and 10-fluoro-7,12-dimethylbenz[a]-anthracene (DMBA) and the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on tumor initiation by monofluoro derivatives of DMBA in SENCAR mice.

We have determined the skin tumor initiating activity in SENCAR mice of 6 monofluoro derivatives of 7,12-dimethylbenz[a]anthracene (DMBA). 9-Fluoro-DMBA (9-F-DMBA) was approximately as active, and 10-F-DMBA was more active than the parent hydrocarbon, DMBA. The difference between DMBA and 10-F-DMBA was most dramatic at the highest initiating doses of 10-F-DMBA tested. 4-F-DMBA, which was only weakly active as an initiator, was also tested as a complete carcinogen on mouse skin; after 30 weeks of treatment, 50- and 100-nmol weekly doses failed to elicit papillomas or carcinomas. Animals treated with 50 nmol of DMBA weekly exhibited a 100% papilloma incidence and a 42% carcinoma incidence. Pretreatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) effectively inhibited tumor initiation with all of the monofluoro derivatives of DMBA tested. The ED50 (dose of TCDD producing half-maximal inhibition) for the inhibition of DMBA initiation in SENCAR mice was determined to be 1.8 X 10(-3) micrograms/mouse (5.6 pmol). The results indicate that the introduction of a fluorine atom in ring D of DMBA has no effect (positions 9 and 11) or enhances (position 10) tumor initiating activity. We believe 10-F-DMBA to be the first example of a hydrocarbon with a fluoro substituent giving rise to increased tumor initiating activity. The results also indicate that structural modifications that alter tumor initiating activity do not alter the ability of TCDD to inhibit tumorigenesis by DMBA.