Effect of a 6-fluoro substituent on the metabolism and biological activity of benzo(a)pyrene.
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Animals↗
Biomedical subjects
T J Slaga
Publications and source records attributed to T J Slaga.
Host factors in the susceptibility of mice to tumour initiating and promoting agents.
It has been known for a long time that skin tumours can be induced by the sequential application of a subthreshold dose of a carcinogen (initiation stage) followed by repetitive treatment with a noncarcinogenic promoter (promotion stage). In mouse skin, the initiation stage requires only a single application of either a direct-acting carcinogen or a procarcinogen and is essentially an irreversible step, which, as data suggest, probably involves a somatic cell mutation. The promotion stage in mouse skin can be accomplished by a wide variety of weak or noncarcinogenic agents and is initially reversible, later becoming irreversible. Current information suggests that skin tumour promoters are not mutagenic but bring about a number of important epigenetic changes, such as epidermal hyperplasia and an increase in levels of polyamines, prostaglandins and dark basal keratinocytes as well as other embryonic conditions. Recently, tumour promotion in mouse skin was shown to consist of at least two stages, each of which can be accomplished by either a known promoter or a weak or nonpromoting agent. Some of the important characteristics of the first stage of promotion are: (1) only one application of a first-stage promoter, such as phorbol ester tumour promoters, calcium ionophore A23187, hydrogen peroxide and wounding, is needed; (2) it is partially irreversible; (3) increases in dark basal keratinocytes and prostaglandins are important; and (4) it can be inhibited by anti-inflammatory steroids and protease inhibitors. The second stage of promotion is initially reversible but later becomes irreversible. Polyamines and epidermal cell proliferation are important events in the second stage of promotion. A number of weak or nonpromoting agents, such as mezerein, are effective second-stage promoters, which can be counteracted by retinoic acid, anti-inflammatory steroids and polyamine synthesis inhibitors. Although skin tumour promotion has been studied extensively in mice, not all strains and stocks of mice are susceptible to phorbol ester tumour promoters. In this regard, C57Bl/6 mice appear to be fairly resistant to these tumour promoters. Not all species are equally susceptible to phorbol ester tumour promotion (mouse greater than rat greater than hamster, and the miniature swine is fairly resistant). It is not presently known if other experimental systems of carcinogenesis or the induction of human cancer go through a series of stages similar to that in the mouse skin.
Animals↗
Cellular and biochemical changes during multistage skin tumor promotion.
Skin tumor promotion in mice which may involve a free radical mechanism can be operationally and mechanistically further divided into at least two stages. The first stage, which is partially irreversible for 4 to 6 weeks, can be accomplished by a single application of 12-O-tetradecanoylphorbol-13-acetate (TPA) or by nonpromting agents such as 4-O-methyl-TPA, calcium ionophore A23187, and hydrogen peroxide, as well as by wounding. These agents plus wounding increase the number of dark basal keratinocytes, which suggest that these cells are important in the first stage of promotion. Recent data also suggest that the dark cells may be the critical target of skin tumor initiators. Prostaglandin E2 was found to specifically enhance stage I and increase the number of dark cells induced by TPA, whereas the protease inhibitor, tosyl phenylalanine chloromethylketone, specifically inhibited stage I of promotion and counteracted the TPA-induced dark cells. We have recently found that TPA and other first stage promoters can decrease the number of epidermal glucocorticoid receptors. Fluocinolone acetamide (FA) was found to inhibit both stages but was more effective in counteracting stage I of promotion. FA also prevents the TPA-induced dark cells and the decrease in glucocorticoid receptors caused by TPA. The second stage of promotion is initially reversible but later becomes irreversible. The weak promoting agent mezerein and the nonpromoting agent 12-deoxyphorbol-13-2,4,6-decatrienoate are effective stage II promoters. Polyamines, gene amplification and epidermal cell proliferation appear to be important events in stage II of promotion. Putrescine was found to specifically enhance stage II, whereas retinoic acid, difluoromethylornithine, and butylated hydroxyanisole specifically inhibited stage II of promotion and the mezerein-induced polyamine levels but not the mezerein-induced hyperplasia. The inhibition of stage II of promotion by antioxidants gives further support for the role of free radicals in tumor promotion. Mezerein was found to be much more effective in amplifying methotrexate resistance than TPA. Although, mezerein can not significantly decrease the number of glucocorticoid receptors or increase the number of dark cells after repetitive treatment, mezerein can maintain the TPA effect in a two-stage promotion protocol.
Animals↗
Glucocorticoid receptor levels in mouse skin after repetitive applications of 12-O-tetradecanoylphorbol-13-acetate and mezerein.
Female Sencar mice were given twice weekly topical applications of 0.2 ml of acetone, 4 micrograms of 12-O-tetradecanoylphorbol-13-acetate (TPA), 2 micrograms of mezerein, or 4 micrograms of TPA followed by 2 micrograms of mezerein. Whole skin cytoplasmic glucocorticoid receptor content and dissociation constants were determined at various times after the last application of the agents. After four to 12 applications of acetone, the mean glucocorticoid receptor level was 0.49 +/- 0.02 pmol/mg cytosol protein, and the dissociation constant was 6.0 +/- 0.17 nM. The glucocorticoid receptor level was markedly reduced and remained low for at least 72 hr after repetitive applications of TPA. The mean receptor level was 0.113 +/- 0.011 (S.E.) pmol/mg cytosol protein, and the Kd was 2 to 4 times the control value. In contrast, the glucocorticoid receptor level was not significantly reduced after repetitive applications of mezerein. If four applications of mezerein were preceded by four applications of TPA, then the glucocorticoid receptor level was similar to that after TPA alone. It was also demonstrated that the glucocorticoid receptor level in papillomas was extremely low (0.022 pmol/mg cytosol protein) but was much higher in newborn skin (0.52 pmol/mg cytosol protein).
Administration, Topical↗
Studies on the mechanisms involved in multistage carcinogenesis in mouse skin.
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Animals↗
Contact-stimulated proliferation of cultured mouse epidermal cells by 3T3 feeder layers: inhibition of proliferation by 12-O-tetradecanoylphorbol-13-acetate (TPA).
Mouse epidermal cells can be subcultured at 31 degrees C onto an irradiated BALB/c 3T3 clone A31 feeder layer. A31 cells (supposedly derived from embryonic fibroblasts) were found to be specifically required for the optimal production of keratinizing epidermal colonies in secondary culture. This effect was not transmitted through the medium nor by the culture surface, since A31 cells plated on one end of a flask did not stimulate epidermal cell proliferation at the other end, even if the other end had previously held A31 cells. Epidermal cell contact with metabolizing A31 cells was probably necessary for the effect; fixed or freeze-thawed A31 cells were ineffective. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate, recently shown to interfere with contact-mediated transfer of label (metabolic cooperation) between Swiss 3T3 cells and cells of an established epidermal line in vitro, also blocked epidermal colony formation. The A31-epidermal cell interaction is apparently not a typical mesenchymal-epithelial interaction, since the basement membrane would prevent this contact in intact skin.
Animals↗
Effect of tunicamycin on receptors for tumor promoters.
A progressive decline in the specific binding of [20-3H]phorbol 12,13-dibutyrate ([3H]PDBu) and glycoprotein synthesis was observed following treatment of primary mouse epidermal cells with tunicamycin, a specific inhibitor of dolichol-mediated glycosylation. Following 18 h of treatment, the specific binding of [3H]PDBu was reduced to 33-56% of the control value. The total protein synthesis determined by leucine incorporation into acid-insoluble material was not altered by this antibiotic drug. These results suggest that the receptor for phorbol diesters is, or is functionally linked to, a glycoprotein on the cell surface.
Animals↗
alpha-Difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase, inhibits tumor promoter-induced polyamine accumulation and carcinogenesis in mouse skin.
The role of ornithine decarboxylase (OrnDCase, EC 4.1.1.17) and of the polyamines [putrescine (Put), spermidine (Spd), and spermine (Spm)] in mouse skin tumor promotion was investigated by the use of alpha-difluoromethylornithine (CHF2-Orn), an enzyme-activated irreversible inhibitor of OrnDCase. 12-O-Tetradecanoylphorbol 13-acetate (TPA), mezerein, and ethyl phenylpropiolate (EPP) were employed as complete, stage II specific, and nonpromoting agents, respectively. TPA and mezerein, but not EPP, provided for a dose-dependent increase in tissue Put accumulation. The Put level in papillomas developed by TPA (2 micrograms) treatment was approximately equal to 15-fold higher than that of the surrounding skin tissue; Spd accumulation was 2- to 3-fold greater in the papillomas. Put administered (intraperitoneally) with TPA greatly enhanced papilloma yield. CHF2-Orn, given orally or intraperitoneally, abolished the TPA-induced OrnDCase activity and Put accumulation in mouse epidermis. The reduction of polyamine accumulation by CHF2-Orn was directly proportional to reduction of tumor size. CHF2-Orn administered in a two-stage (TPA-mezerein) promotion protocol [Slaga, T. J., Fischer, S. M., Nelson, K. G. & Gleason, G. L. (1980) Proc. Natl. Acad. Sci. USA 77, 3659-3663; Slaga, T. J., Klein-Szanto, A. J. P., Fischer, S. M., Weeks, C. E., Nelson, K. & Major, S. (1980) Proc. Natl. Acad. Sci. USA 77, 2251-2254] reduced tumor size, inhibited by 65-70% the number of papillomas per mouse, and decreased by 40% the percentage of mice with tumors when given with the stage II agent mezerein. CHF2-Orn provided considerably less effect on tumorigenesis when administered with the TPA portion of the protocol, and CHF2-Orn did not inhibit the induction of dark basal keratinocytes by TPA. Based on our results with CHF2-Orn, we suggest that regulation of polyamine biosynthesis, particularly Put, is a critical factor in stage II promotion.
Alkynes↗
Comparison of levels of benzo[a]pyrene diol epoxide diastereomers covalently bound in vivo to macromolecular components of the whole epidermis versus the basal cell layer.
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7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
Inhibition of mouse skin tumor promotion by several inhibitors of arachidonic acid metabolism.
12-O-Tetradecanoylphorbol-13-acetate promotion of skin tumors in mice can be inhibited by topical application of either the phospholipase A2 inhibitor dibromoacetophenone or the cyclooxygenase-lipoxygenase inhibitors 5,8,11,14-eicosatetrayonic acid or 1-phenyl-3-pyrazolidinone. The phospholipase A2 inhibitors in particular appear to be among the most potent inhibitors of skin tumor promotion known. These results support the hypothesis that at least some of the products of arachidonic acid transformation are essential for tumor promotion.
5,8,11,14-Eicosatetraynoic Acid↗
Effect of tumor promoters on lipid peroxidation in mouse skin.
Products of lipid peroxidation were measured in mouse epidermis. These were shown to increase with advancing age. Conversely, a decline in these products was observed on treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent tumor promoting agent. A decline in lipid peroxidation occurred within 4 h after application of 2 micrograms TPA and the maximum effect was seen at 22-24 h. A lesser active tumor promoter, phorbol dibenzoate; and ethyl phenylpropiolate, a purely hyperplastic agent, also lowered lipid peroxidation; while phorbol, a non-promoter, did not show any significant effect. Mezerein, a resiniferonal derivative with weak promoting activity but a potent stage II promoter, appeared to be more potent than TPA in lowering the basal levels of peroxidation. The TPA-induced decrease in lipid peroxidation could be prevented by fluocinolone acetonide, a potent antipromoting and antimitotic agent, but not by retinoic acid and tosylamino-2-phenylethylchlorimethyl-ketone which are relatively potent antipromoting agents but lack antimitotic activity, suggesting that the decrease of lipid peroxidation by tumor promoting agents is possibly related to their mitotic activity. Furthermore, skin papilloma and carcinoma contain lower levels of lipid peroxidation compared to epidermis from the same mice.
Age Factors↗
Effects of inhibitors of tumor promotion on 12-O-tetradecanoylphorbol-13-acetate-induced keratin modification in mouse epidermis.
In order to investigate the role that hyperplasia plays in the induction of the keratin modifications by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), the effects of several inhibitors of tumor promotion and cycloheximide on the TPA-induced protein changes were studied. The antiinflammatory steroid fluocinolone acetonide and cycloheximide, which prevent the induction of hyperplasia, were found to prevent the appearance of the TPA-induced keratin modifications. Retinoic acid and tosylphenylalanylchloromethyl ketone, which have minor influence on hyperplasia, were found to have essentially no effect on these protein changes. These results provided further evidence that the TPA-induced epidermal keratin changes were associated with the induction of hyperplasia by TPA and not necessarily related to promoting ability.
Animals↗
Anticarcinogenic and cocarcinogenic effects of benzo[e]pyrene and dibenz[a,c]anthracene on skin tumor initiation by polycyclic hydrocarbons.
In the present study, we have examined the effects of benzo[e]pyrene (B[e]P) and dibenz[a,c]anthracene (DB[a,c]A) on the skin tumor-initiating activities of methylated and non-methylated polycyclic aromatic hydrocarbons (PAH). B[e]P, when applied 5 min prior to initiation with seven different PAH skin carcinogens, effectively inhibited the tumor-initiating activities of 7,12-dimethylbenz[a]anthracene (DMBA) and dibenz[a,h]anthracene (DB[a,h]A) but had little or no effect on the tumor-initiating activities of 3-methyl-cholanthrene (MCA), 7-methylbenz[a]anthracene (7-MBA), 12-methylbenz[a]anthracene (12-MBA), and 5-methyl-chrysene (5-MeC). B[e]P potentiated the tumor-initiating activity of benzo[a]pyrene (B[a]P) by approximately 30%, DB[a,c]A, when applied 5 min prior to initiation, inhibited the tumor-initiating activities of DMBA, MCA, and DB[a,h]A but had little or no effect on the tumor-initiating activities of B[a]P, 7-MBA, 12-MBA, and 5-MeC. DB[a,c]A, when applied 12, 24, or 36 h prior to initiation with B[a]P, which allowed time for induction of epidermal monooxygenase enzymes, inhibited tumor initiation. The covalent binding of DMBA and B[a]P to epidermal DNA was examined under the influence of B[e]P. Doses of 20 and 200 nmol B[e]P given 5 min prior to 10 nmol [3H]DMBA reduced binding to 47 and 22%, respectively, of the control value. In contrast, doses of 200 or 2000 nmol B[e]P given 5 min prior to 200 nmol [3H]B[a]P had little or no effect on total binding. The data indicate that one cannot predict anti and cocarcinogenic effects of B[e]P and DB[a,c]A on the basis of a presence or absence of a methyl substituent. In addition, fundamental differences exist in the processing and metabolism of DMBA and B[a]P by mouse epidermal cells.
9,10-Dimethyl-1,2-benzanthracene↗
Tumor-initiating activity of 4-fluoro-7,12-dimethylbenz[a]anthracene and 1,2,3,4-tetrahydro-7,12-dimethylbenz[a]anthracene in female SENCAR mice.
We have determined the skin tumor-initiating activity in SENCAR mice of two A-ring derivatives of 7,12-dimethylbenz[a]anthracene (DMBA). 4-Fluoro-7,12-dimethylbenz[a]anthracene at a dose of 200 nmol per mouse exhibited weak activity, producing 0.6 papillopmas per mouse; doses of 10 and 20 nmol per mouse had no activity. A derivative of DMBA with the A-ring reduced, 1,2,3,4-tetrahydro-7,12-dimethylbenz[a]anthracene (1,2,3,4,-H-DMBA), had substantial tumor-initiating activity when compared with the parent hydrocarbon. In one experiment, doses of 10 and 100 nmol per mouse gave rise to 1.6 and 9.5 papillomas per mouse, respectively; similar results were obtained in 3 additional experiments. Although the tumor-initiating activity of 1,2,3,4,-H4-DMBA was approximately one-tenth that of DMBA, this derivative was slightly (17%) more active than benzo[a]pyrene. 1,2,3,4-H4-DMBA was tested for the ability to induce mutations to 6-thioguanine-resistance in Chinese hamster V79 cells. In the absence of feeder cells capable of metabolizing polycyclic hydrocarbons, it was not mutagenic. However, in a cell-mediated mutation assay with secondary hamster embryo cells as activators, this derivative produced mutations in a dose-dependent manner and was approximately one-tenth as active as DMBA. These results indicate that metabolism of DMBA at positions 1-, 3-, 2- and 4- is important for biological activity and that for certain derivatives (i.e., 1,2,3,4-H4-DMBA), alternate pathways of metabolic activation may also be important.
9,10-Dimethyl-1,2-benzanthracene↗
Cell-mediated mutagenesis and tumor-initiating activity of the ubiquitous polycyclic hydrocarbon, cyclopenta[c,d]pyrene.
The ubiquitous polycyclic aromatic hydrocarbon (PAH), cyclopenta[c,d]pyrene (CPP), was tested to determine its mutagenicity for 6-thioguanine and ouabain resistance in Chinese hamster V79 cells and its tumor-initiating activity in the skin of the tumor susceptible Sencar mice. The potent carcinogen/mutagen, benzo[a]pyrene (BP), was included for comparison. Inasmuch as V79 cells do not metabolize PAHs, mutagenesis was tested both in the presence and in the absence of X-irradiated golden hamster embryo fibroblasts capable of metabolizing PAH. Neither CPP nor BP showed mutagenicity for V79 cells in the absence of the embryo cells. In the presence of these cells (in the cell-mediated assay) both PAHs elicited, in a dose dependent manner, a cytotoxic and mutagenic response in V79 cells. CPP was however less active than BP in inducing both of these responses. At the optimal expression time and at the dose range of 0.1-1 microgram/ml, CPP induced 2-8 6-thioguanine resistant mutants per 10(5) colony forming cells compared to 9-50 mutants induced by BP. Similarly, these doses of CPP induced 1-9 ouabain resistant mutants per 10(6) colony forming cells compared to 7-75 mutants induced by BP. CPP was also active in initiating skin tumors in approximately 60% of the mice at 200 micrograms, the highest dose tested. BP was more efficient in tumor initiation and yielded a similar response with 10 micrograms. These results indicate that CPP and BP elicit, in the cell mediated assay, a mutagenic response similar to the activity of these PAH in the skin of Sencar mice.
Animals↗
Selective modification of nuclear proteins by polycyclic aromatic hydrocarbons and by benzo[a]pyrene diol epoxides.
Metabolites of benzo[a]pyrene (B[a]P) have been shown to modify chromosomal proteins with great specificity. Using the (+) and (-) enantiomers of anti-B[a]P diol epoxide to label isolated nuclei we found a remarkable difference in the capacity of these two compounds to modify histones H2A and H3. The (+) enantiomer modified histones H2A and H3, while the (-) enantiomer, which was shown to modify mainly histone H2A, had a much lower affinity for histone H3. We have also examined the selective, modification of chromosomal proteins by different polycyclic aromatic hydrocarbons and it was observed that 7,12-dimethylbenz[a]-anthracene (DMBA), 3-methylcholanthrene (3-MC) and B[a]P showed qualitative similarities in terms of their protein binding. This suggests that stereospecific interactions leading to binding of reactive metabolites of DMBA, B[a]P and 3-MC to chromosomal proteins share common features.
7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
The down-modulation of receptors for phorbol ester tumor promoter in primary epidermal cells.
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Animals↗
Effects of peroxides on rodent skin: epidermal hyperplasia and tumor promotion.
Free radical generating peroxides are potent skin irritants. After a single topical application of either 10, 20, or 40 mg of lauroyl peroxides or benzoyl peroxide on the dorsal skin of Sencar mice, the epidermal thickness increased markedly. No major inflammatory or vascular alterations were noted. On the other hand, 15 or 30% hydrogen peroxide produced an extensive epidermolysis, as well as inflammation and vascular injury, followed by quick regeneration and epidermal hyperplasia. Both lauroyl peroxide- and benzoyl peroxide-induced hyperplasias were characterized by a sustained production of dark basal keratinocytes, which constituted approximately 10% of the basal cell population during the first week after single topical application. Hydrogen peroxide-induced epidermal hyperplasias also exhibited numerous dark cells, but their presence was less sustained. Although all these peroxides were inactive either as initiators or as complete carcinogens, lauroyl peroxide was as effective as benzoyl peroxide when used as a skin tumor promoter in a two-stage carcinogenesis protocol. In a similar experimental protocol, hydrogen peroxide proved to be a very weak skin tumor promoter.
Administration, Topical↗