Thomas J. Slaga

Skin tumor initiating ability of benzo(a)pyrene 4,5- 7,8- and 7,8-diol-9,10-epoxides and 7,8-diol

The skin tumor initiating abilities of both K-region and non-K-region epoxides of benzo(a)pyrene(BP) were determined in mice using a two-stage system of tumorigenesis. BP-4,5-epoxide and BP-7,8-dihydrodiol-9,10-epoxide (anti) were found to be weak tumor initiators whereas BP-7,8-epoxide had about a third of the activity as the parent hydrocarbon, BP. However, the 7,8-dihydrodiol of BP was found to be approximately as potent as BP suggesting that it may be a proximate carcinogen.

Fluocinolone acetonide: a potent inhibitor of mouse skin tumor promotion and epidermal DNA synthesis.

Abstract The relationship between the inhibition of mouse skin tumor promotion and the inhibition of epidermal DNA synthesis by the steroidal anti-inflammatory agent, fluocinolone acetonide (FA), was investigated. Simultaneous doses of either 10, 1, or 0.1 μg of FA and phorbol ester tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), resulted in an almost complete inhibition of promotion, whereas 0.01 and 0.001 μg of FA resulted in inhibition rates of 82% and 15%, respectively. Likewise, simultaneous doses of 10 or 1 μg of fluclorolone acetonide (FCA) and TPA caused a nearly complete inhibition of promotion, whereas 0.1 μg of FCA decreased promotion by 62%. In general, as the dose of both steroids was increased, an increase in the tumor latency period was observed. With the exception of the borderline effect of 0.001 μg of FA, the above doses of FA inhibited epidermal DNA synthesis by at least 60% for a 24-h period. Topical treatment with 10 μg of FA resulted in an almost complete inhibition of DNA synthesis for 6 days. The administration of 10 μg of FA 24 h after TPA treatment brought about a maximal inhibition of DNA synthesis of 65%, as compared with a 98% inhibition in control mice whose DNA synthesis had not been prestimulated. That is, FA was not quite as effective on S-phase cells as on G-1 cells. There appears to be a relationship between the inhibition of tumor promotion and epidermal DNA synthesis.

The effects of weak or non-carcinogenic polycyclic hydrocarbons on 7, 12-dimethylbenz[a]anthracene and benzo[a]pyrene skin tumor-initiation*

Benzo[e]pyrene (B[e]P) inhibited 7,12-dimethylbenz[a]anthracene (DMBA) skin tumor-initiation in mice by 84%, whereas pyrene and fluoranthene inhibited DMBA initiation by 50 and 34%, respectively. However, B[e]P, pyrene and fluoranthene had either no significant effect or a slight enhancing effect on benzo[a]pyrene (B[a]P) skin tumor-initiation. In addition, B[e]P had essentially no effect on the initiating ability of (+/-)B[a]P-7 beta,8 alpha-diol-9 alpha,10 alpha-epoxide. As a tumor-initiator, B[e]P was found to have very weak activity at a 252 microgram/level (0.4 papillomas/mouse at 40 weeks) and no activity at 100 microgram. When given at a dose of 100 microgram twice weekly, B[e]P induced 2.1 papillomas/mouse at 30 weeks, and 25% of the mice had carcinomas at 40 weeks. However, B[e]P carcinogenic activity is weak when compared to B[a]P, which can induce a comparable tumor response at a dose of 5 microgram twice weekly. When B[e]P was tested as a tumor promoter at a dose of 100 microgram twice weekly after DMBA initiation, it induced 4.5 papillomas/mouse at 30 weeks and a 45% carcinoma incidence at 40 weeks, which was approximately twice as effective as B[e]P alone. The data show that B[e]P is a very weak tumor initiator, a weak complete carcinogen, a moderate tumor promoter, possibly a weak co-tumor-initiator when given with B[a]P, and a potent anit-tumor-initiator when given with DMBA. The anti-tumor initiating and co-tumor-initiating effects of B[e]P appear to be related to its ability to modify the conversion of the tumor initiator into an electrophilic intermediate(s) which are capable of covalently binding to DNA. In addition, B[e]P induced epidermal cellular proliferation which may be related to its promoting ability.

Indomethacin enhancement of TPA tumor promotion in mice

Skin tumor promotion in mice by 12-O-tetradecanoylphorbol-13-acetate (TPA) is characterized by hyperplasia and inflammation. Based on the inhibitory effect of the steroidal anti-inflammatory drugs, non-steroidal agents, such as indomethacin, were also expected to show some degree of inhibition; however, repeated tumor experiments demonstrate that indomethacin enhances TPA promotion in a dose-response manner. A time-of-application effect was evident such that indomethacin given 2 h prior to TPA resulted in the greatest enhancement. Flurbiprofen was also observed to enhance promotion slightly.

Separation of epidermal cells by density centrifugation: a new technique for studies on normal and pathological differentiation*

Murine keratinocytes, isolated by flotation trypsinization of skin, can be separated into five groups by centrifugation through Percoll, a colloidal silica gradient. Within each group a good correlation was found between density, plating efficiency, morphological appearance, DNA synthesis, and degree of keratinization/cornification. This method can be applied equally well to fetal, newborn, or adult keratinocytes and should be useful in a variety of studies including isolation of subpopulations of pathological cell types, work on chalones and hyperplastic diseases such as psoriasis, and in vitro transformation studies.

Studies with chlorinated dibenzo-p-dioxins, polybrominated biphenyls, and polychlorinated biphenyls in a two-stage system of mouse skin tumorigenesis: potent anticarcinogenic effects

The chlorinated dibenzo-p-dioxins and chlorinated dibenzo-furans are among the most potent teratogens and toxins that are found as environmental contaminants. The polychlorinated biphenyls and polybrominated biphenyls are not nearly as toxic as the dioxins, but they are environmentally more prevalent than the dioxins. Both classes of compounds are inducers of cytochrome P/sub 450/-mediated microsomal monooxygenases and of aminolevulinic acid synthetase. The dioxins induce aryl hydrocarbon hydroxylase, the enzyme system responsible for converting polycyclic aromatic hydrocarbons to active and inactive metabolites. PCBs and PBBs induce the monooxygenase system with properties similar to both phenobarbital and 3-MC. Timing is important in the application of PCB to the diets of laboratory animals. Treatment with PCBs prior to the administration of carcinogen results in reduction of tumor response whereas treatment subsequent to application of the carcinogen results in enhancement of the tumorogenic response. The two-stage system of mouse skin tumorigenesis allows one to evaluate critically the initiation and promotion phases of carcinogenesis individually. (DLS)

Benzo(a)pyrene metabolism in mouse epidermis. Analysis by high pressure liquid chromatography and DNA binding

Mouse epidermal homogenates contain an inducible aryl hydrocarbon hydroxylase (AHH) complex that catalyzes the formation of benzo(a)pyrene-7,8-dihydrodiol from benzo(a)pyrene (BP) as assessed by high pressure liquid chromatography (HPLC). 5,6-Benzoflavone (5,6-BF), 7,8-benzoflavone (7,8-BF) and 17-beta-estradiol decreased and butylated hydroxytoluene (BHT) enhanced oxidative metabolism of BP when added in vitro. Epoxide hydrase activity (hydration of benzo(a)pyrene-4,5-epoxide) (BP-4,5-epoxide) was enhanced by 17-beta-estradiol, 5,6-BF, and 7,8-BF. BHT exhibited no significant effect and 1,2-epoxy-3,3,3-trichloropropane (TCPO) inhibited hydrase activity. The capacity of epidermal homogenates to catalyze the covalent binding of BP to DNA indicated that addition of both 5,6-BF and 7,8-BF decreased binding. BHT and TCPO did not significantly affect DNA-binding.

Tumor initiating activities of 1-nitropyrene and its nitrated products in SENCAR mice

The environmental mutagens, 1-nitropyrene, 1,3-dinitropyrene, 1,6-dinitropyrene and 1,8-dinitropyrene were evaluated for their ability to initiate skin tumors in male and female SENCAR mice. 1-Nitropyrene (greater than 99.5% purity) did not induce papilloma formation over a dose range of 0-3.0 mg/mouse after 30 weeks of promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). Systemic administration of 1-nitropyrene by i.p. injection over a dose range of 0-8.0 mg/mouse followed by topical treatment with TPA also did not induce papilloma formation. A mixture of dinitropyrenes (1,3-dinitropyrene, 1,6-dinitropyrene and 1,8-dinitropyrene (1:1.94:1.95] (99% purity) was dermally applied to SENCAR mice over a dose range of 0-2.0 mg/mouse and the mice subsequently promoted with TPA. A significant induction of papillomas was observed at 30 weeks (0.37-0.39 papillomas/mouse, 26-29% of the mice bearing tumors) at 2.0 mg/mouse. Comparison of this data with that obtained from the same mouse strain using diesel exhaust particulate extracts containing 1-nitropyrene and its nitrated products indicate that these nitrated polynuclear aromatics do not significantly contribute to the mouse skin tumorigenic activity of diesel exhaust particulate extracts.

Comparison of Complete Carcinogenesis and Tumor Initiation and Promotion in Mouse Skin: The Induction of Papillomas by Tumor Initiation-Promotion a Reliable Short Term Assay

Skin tumors can be induced by the sequential application of a subthreshold dose of a carcinogen (initiation phase), followed by repetitive treatment with a weak or noncarcinogenic tumor promoter. Using a two-stage system to induce tumors in Senear mice there is a very good dose-response relationship between the induction of the number of papillomas per mouse at early times (10 to 20 weeks) and the final carcinoma incidence after longer latency (20 to 50 weeks). This system can be used not only to determine the tumor initiating and promoting activities of a compound but if the agent is given repeatedly by itself one can also determine if it is a complete carcinogen, i.e., if it has both tumor initiating and promoting activity. With the exception of a few pure tumor initiators, there is in general a good qualitative and quantitative correlation between the ability of a polycyclic aromatic hydrocarbon (PAH) to act as a complete carcinogen and to act as a tumor initiator in mouse skin. In addition, if the agent is given concurrently with a known complete carcinogen or a tumor initiator one can also determine if the agent has co-carcinogenic or co-tumor initiating activity or even possibly anticarcinogenic activity. Likewise, if the agent is given concurrently with a known tumor promoter one can determine if the agent has co-promoting or anti-pro-moting activity. There is a good correlation between the tumor-initiating activities of PAH and their abilities to bind covalently to DNA. In addition, various inhibitors of PAH tumor initiation show a strong correlation with their abilities to inhibit the binding of the PAH to DNA and their anti-tumor initiating activities. There is also a good correlation between the promoting abilities of phorbol esters to promote tumors and their abilities to induce ornithine decarboxylase (ODC), cell proliferation and dark basal keratinocytes. When other nonpromoting hyperplastic agents are used, only dark cell induction correlates with promotion. Certain polyamines and prostaglandins can enhance phorbol ester tumor promotion. Anti-inflammatory steroids, retinoids, and protease inhibitors are potent inhibitors of tumor promotion. They inhibit tumor promotion by inhibiting either the 12-0-tetradecanoylphorbol-13-acetate (TPA) induced cell proliferation, ODC and/or dark basal keratinocytes. Certain weak promoters such as mezerein which mimics TPA in many biochemical and morphological effects are potent second step promoters in a two-stage promotion regimen.

2,3,7,8-tetrachlorodibenzo-p-dioxin: potent anticarcinogenic activity in CD-1 mice

Abstract Topical pretreatment with non-toxic doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin, a contaminant formed in the commercial synthesis of the herbicide 2,4,5-trichlorophen-oxyacetic acid, strongly inhibited the initiation of skin tumors by 7,12-dimethylbenz(a)-anthracene and benzo(a)pyrene in female CD-1 mice. 2,3,7,8-tetrachlorodibenzo-p-dioxin also produced marked induction of epidermal monooxygenase enzymes functional in the conversion of 7,12-dimethylbenz(a)anthracene to a variety of hydroxylated products. The time course of anticarcinogenic effects resulting from pretreatment with the dioxin correlated with the magnitude of induction as well as with a singnificant reduction in the quantity of 7,12-dimethylbenz(a)anthracene metabolites covalently bound in vivo to epidermal DNA and RNA but not protein.