Both (+/-)syn- and (+/-)anti-7,12-dimethylbenz[a]anthracene-3,4-diol-1,2-epoxides initiate tumors in mouse skin that possess -CAA- to -CTA- mutations at Codon 61 of c-H-ras.

We have determined the tumor-initiating activity of (+/-)syn- and (+/-)anti-7,12-dimethylbenz[a]anthracene-3,4-diol-1,2-epoxide (syn- and anti-DMBADE), the two metabolically formed bay-region diol epoxides of DMBA, and we have also analyzed mutations in the H-ras gene from tumors induced by these compounds. Using a two-stage, initiation-promotion protocol for tumorigenesis in mouse skin, we have found that both syn- and anti-DMBADE are active tumor initiators, and that the occurrence of papillomas is carcinogen dose dependent. All of the papillomas induced by syn-DMBADE (a total of 40 mice), 96% of those induced by anti-DMBADE (a total of 25 mice), and 94% of those induced by DMBA (a total of 16 mice) possessed a -CAA- to -CTA- mutation at codon 61 of H-ras. No mutations in codons 12 or 13 were detected in any tumor. Topical application of syn- and anti-DMBADE produced stable adducts in mouse epidermal DNA, most of which comigrated with stable DNA adducts formed after topical application of DMBA. Further analysis of the data showed that levels of the major syn- and anti-DMBADE-deoxyadenosine adducts formed after topical application of DMBA are sufficient to account for the tumor-initiating activity of this carcinogen on mouse skin. Previously, we showed that both the syn- and anti-DMBADE bind to the adenine (A182) at codon 61 of H-ras. Collectively, these results indicate that the adenine adducts induced by both bay-region diol epoxides of DMBA lead to the mutation at codon 61 of H-ras and, consequently, initiate tumorigenesis in mouse skin.

Altered glucocorticoid receptor expression and function during mouse skin carcinogenesis.

Glucocorticoids are the most potent inhibitors of tumor promotion in mouse skin, when applied with a promoting agent at the early stages of promotion. However, established skin papillomas become resistant to growth inhibition by glucocorticoids. Glucocorticoid control of cellular functions is mediated by the glucocorticoid receptor (GR), a well-known transcription factor. Here we present data on GR expression and function in mouse papillomas and squamous cell carcinomas. Tumors were produced in SENCAR mice by a 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol-13-acetate two-stage protocol. In early papillomas (after 15-20 wk of promotion), northern blotting revealed a decrease in the GR mRNA level that was confirmed by a binding assay. However, in late papillomas (after 30-40 wk of promotion), and especially in squamous cell carcinomas, the level of GR in both assays was similar to or higher than the GR level in normal epidermis. To test the functional capability of GR in tumors, we compared the effect of the synthetic glucocorticoid fluocinolone acetonide (FA) on keratinocyte proliferation and on expression of glucocorticoid-responsive genes in normal epidermis, hyperplastic skin surrounding tumors, and mouse skin papillomas. FA strongly inhibited DNA synthesis in keratinocytes in normal skin and tumor-surrounding skin but had no effect on DNA synthesis in papillomas. In addition, FA strongly induced metallothionein 1 expression and inhibited connexin 26 expression in skin but did not affect expression of these genes in tumors. These data suggest that alteration of both the expression and function of GR may be an important mechanism of tumor promotion in skin.

Connexin expression in epidermal cell lines from SENCAR mouse skin tumors.

Alteration of gap-junctional intercellular communication (GJIC) has long been proposed to be involved in carcinogenesis. Previously, we reported that the level of gap junctional intercellular communication in mouse skin carcinoma cell lines is significantly lower than in papilloma cell lines and normal mouse keratinocytes Klann et al., Cancer Res 49:699-705, 1989). Here, we present data on expression of the gap-junctional protein connexins (Cx) 26, Cx31.1, and Cx43 in a comprehensive panel of keratinocyte cell lines representing different stages of mouse skin carcinogenesis and the effect of different conditions of propagation on Cx phenotype. Northern and western blot analyses and immunostaining showed that all cell lines studied in vitro expressed Cx43 but most did not express Cx31.1 or Cx26. The abundance of Cx43 expression on plasma membranes correlated well with the level of GJIC. In vivo expression of Cx43 and Cx26 was strongly increased. Whereas none of tumorigenic cell lines expressed Cx26 gap junctions in culture, those growing as tumors in nude mice began to express Cx26 protein. The comparison of Cx expression on the keratinocyte membranes in three different groups of tumors (papillomas and squamous cell and spindle cell carcinomas) clearly revealed that the abundance of Cx43 and Cx26 expression directly correlated with the level of tumor differentiation. All studied tumors were Cx31.1 negative. These results suggest that both Cx expression and gap-junction permeability are gradually reduced during the tumor progression stage of mouse skin carcinogenesis.

Potent inhibitory effects of suicide inhibitors of P450 isozymes on 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene initiated skin tumors.

A single dose of 1-ethynylpyrene (EP), 1-vinylpyrene (VP) or 2-ethynylnaphthalene (EN) was applied to the skin of SENCAR mice 5 min before an initiating dose of 7,12-dimethylbenz[a]anthracene (DMBA) or benzo[a]pyrene (B[a]P) and the development of skin tumors then promoted with biweekly topical applications of 12-O-tetradecanoylphorbol-13-acetate (TPA). The application of EP strongly inhibited the formation of skin tumors initiated by either DMBA or B[a]P in a dose-dependent manner. Application of 44 pmol of EP inhibited tumor initiation by 10 nmol of DMBA approximately 25%; application of 440 nmol of EP inhibited tumor initiation by 200 nmol of B[a]P approximately 51%. A high single dose of EP (4.4-44 mumol) nearly eliminated skin tumor initiation by either 10 nmol of DMBA or 200 nmol of B[a]P. Application of VP also inhibited the formation of skin tumors initiated by either DMBA or B[a]P in a dose-dependent manner, but higher doses of VP than of EP were required to produce comparable inhibitions. Application of 44 nmol of VP inhibited tumor initiation by 10 nmol of DMBA approximately 30%; application of 4.4 mumol of VP inhibited tumor initiation by 200 nmol of B[a]P approximately 56%. Application of EN yielded contrasting results. EN inhibited the formation of skin tumors initiated by 10 nmol of DMBA, but the observed dose-dependence was minimal; tumors were decreased about 40% by 3.3 mumol of EN and only about 65% by 132 mumol of EN. A high single dose of EN (132 mumol) increased both the mean number of tumors per mouse and the percentage of mice that developed tumors after initiation by 200 nmol of B[a]P. Topical application of 4.4 mumol of EP, 22 mumol of VP or 33 mumol of EN to the skin of SENCAR mice 5 min before a single initiation dose of 2.5 mumol of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) had a minimal inhibitory effect (14-28%) on the development of skin tumors produced by subsequent biweekly promotion with TPA. A single dose of 44 mumol of EP or 132 mumol of EN followed by biweekly applications of TPA did not produce skin tumors; however, a dose of 44 mumol of VP followed by promotion with TPA produced a low but significant number of skin tumors.(ABSTRACT TRUNCATED AT 400 WORDS)

Induction of short-term markers of tumor promotion by organic peroxides.

Experiments from different laboratories have shown that benzoyl peroxide (BzPo) and other organic peroxides are effective tumor promoters in the mouse skin two-stage carcinogenesis system. In the present paper we have studied the short-term effect of six other organic peroxides, which have not been previously assayed as skin tumor promoters. These compounds were chosen for their molecular diversity, the type of radical predicted to be formed, solubility and availability. The parameters evaluated in this study include a series of short-term markers of tumor promotion, hyperplasia, induction of dark basal keratinocytes and induction of ornithine decarboxylase activity. After single applications the biological activity of the compounds was: m-chloroperoxybenzoic acid greater than di-m-methylbenzoyl peroxide greater than dicumyl peroxide greater than O,O-t-butyl-O-(2-ethylhexyl)mono-peroxycarbonate greater than benzoyl peroxide greater than di-m-chlorobenzoyl peroxide greater than di-t-butyl peroxide greater than t-butyl hydroperoxide. After multiple applications, the order of activity of the compounds was: dicumyl peroxide greater than di-m-methyl-benzoyl peroxide greater than O,O-t-butyl-O-(2-ethylhexyl)monoperoxy carbonate greater than m-chloroperoxybenzoic acid greater than di-m-chlorobenzoyl peroxide greater than t-butyl hydroperoxide greater than benzoyl peroxide greater than di-t-butyl peroxide. The difference of activity among the different compounds did not seem to correlate directly with the chemical stability of the compound; it is more likely that the activity depends on different factors such as percutaneous absorption, metabolism, and the rate of free radical formation in vivo. The data presented here further support the association between free radicals and tumor promotion since all of the compounds, with the exception of one, were active in inducing the short-term markers of tumor promotion. It will also establish conditions for future tumor experiments.

Inhibition of the binding of 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene to DNA in mouse skin epidermis by 1-ethynylpyrene.

The effects of 1-ethynylpyrene (EP), 1-vinylpyrene (VP) and 2-ethynlnaphthalene (EN) on the covalent binding of 7,12-dimethylbenz[a]anthracene (DMBA) and of benzo[a]-pyrene (B[a]P) to the epidermal DNA in mouse skin were investigated. When applied topically, 5 min before an initiating dose of 10 nmol DMBA or of 200 nmol B[a]P, EP was an effective inhibitor of the formation of the covalent complexes of these procarcinogenic polycyclic aromatic hydrocarbons (PAHs) with the epidermal DNA. VP, applied under the same conditions, was a significantly less effective inhibitor of the binding of DMBA to DNA and showed even weaker inhibition of the binding of B[a]P. EN was ineffective as an inhibitor of the binding of either DMBA or B[a]P. These results establish that both the pyrene nucleus and the ethynyl substituent of EP contribute to the effective inhibition of the binding of DMBA and B[a]P to the epidermal DNA of mouse skin. No significant changes in the ratios of the anti- to the syndiol epoxide-DNA adducts of DMBA or of B[a]P were produced by doses of EP that produced inhibitions of the binding to DNA. At doses of VP that inhibited covalent binding of both DMBA and B[a]P, no changes in DMBA-DNA adduct distributions were observed but changes in the relative proportions of several B[a]P-DNA adducts were noted. These data are discussed in terms of the potential of aryl acetylenes to act as suicide inhibitors (mechanism-based inactivators) of cytochrome P450-dependent monooxygenase isozymes.

Induction of terminal differentiation-resistant epidermal cells in mouse skin and in papillomas by different initiators during two-stage carcinogenesis.

Carcinogen treatment of normal mouse epidermal cells causes some cells, if cultured under the appropriate conditions, to continue to proliferate instead of terminally differentiate, forming foci at 37 degrees C in medium with a calcium level above 0.1 mM. We have examined these Calcium (Ca)-resistant cells formed in the skin of SENCAR mice after treatment with the carcinogen initiator 7,12-dimethylbenz[a]anthracene (DMBA) followed by tumor promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). Although in our previous studies TPA promotion initially increased the size but reduced the number of foci caused by the carcinogen initiator N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), TPA promotion of DMBA-treated mice increased the size but had no effect on the number of foci. Papillomas resulting from DMBA plus TPA treatment contained many rapidly growing Ca-resistant cells, corroborating our earlier results with MNNG. Permanent cell lines prepared from papilloma-derived foci formed squamous cell carcinomas in nude mice after relatively short periods in culture. These data provide further evidence that Ca-resistant cells may be papilloma (and perhaps carcinoma) precursors in vivo. In addition, since TPA tends to reduce the number of early Ca-resistant cells caused by MNNG but not by DMBA, this may at least partially explain why treatment with DMBA plus TPA is much more effective in producing papillomas in SENCAR mice than is treatment with MNNG plus TPA.

In vivo behavior of murine epidermal cell lines derived from initiated and noninitiated skin.

The in vivo behavior of cell cultures derived from normal and carcinogen-treated mouse epidermis was studied by implanting the cultures in a s.c. vascularized bed protected by a silicone chamber. Cells derived from normal adult mouse epidermis as well as cells derived from tumor-promoter-treated skin were unable to grow in these systems. Conversely, cell lines derived from skin initiated with single doses of N-methyl-N'-nitro-N-nitrosoguanidine or 9,10-dimethyl-1,2-benzanthracene proliferated in these chambers, reforming an epithelial structure. The type of structure in the chambers varied, ranging from formation of almost normal epithelia to atypical invasive behavior. The variable in vivo behavior among the different cell lines may be attributed to the initiation agent, the number of passages of the cultures, random genetic events, the strain of mouse, or a combination of these factors. Most of the cell types used in this study and all the cell lines that were able to grow in these chambers were selected for resistance to Ca-induced terminal differentiation. However, resistance to terminal differentiation according to the Ca2+ switch does not always correlate with the ability to grow in the chambers, since cell lines derived from spontaneous foci of resistance failed to grow in this system. These studies showed some of the possibilities of the SC silicone chambers to study the histogenic potential of cell lines derived from carcinogen-treated epidermis. This system also appears suitable to study the complex relationship between epidermal cells and specialized (dermal) stroma.

Terminal differentiation-resistant epidermal cells in mice undergoing two-stage carcinogenesis.

We have used an in vivo-in vitro approach to investigate the cellular aspects of two-stage skin carcinogenesis. Female SENCAR mice initiated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were promoted twice weekly with 12-O-tetradecanoylphorbol-13-acetate (TPA). Epidermal cultures from untreated or TPA-treated mice had few focus-forming cells resistant to calcium-induced terminal differentiation. Cultures from mice treated with MNNG alone formed numerous foci. Brief promotion (four TPA treatments) of MNNG-treated mice produced fewer but statistically larger foci, suggesting that TPA was selecting against more slowly growing cells. MNNG plus TPA-treated mice with very early papillomas produced more and larger foci than those due to MNNG treatment alone, suggesting that the papillomas may have comprised calcium-resistant cells. These cells may indeed be initiated cells since a permanent cell line arising after MNNG plus brief TPA treatment eventually formed histological papillomas in vivo. If calcium-resistant cells are initiated, then there were many more initiated cells in the skin (with or without TPA treatment) than papillomas expected, implying that either some initiated cells never formed papillomas, or that a significant accumulation of initiated cells had already occurred in the skin within 2 weeks of MNNG treatment. Subsequent TPA promotion of these cells apparently produced a toxic response that passively selected for more rapidly growing initiated cells, which eventually accumulated into papillomas.

The growth of cultured human foreskin keratinocytes is not stimulated by a tumor promoter.

The tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA) does not stimulate the growth of human epidermal cells in foreskin explant cultures; a dose-dependent inhibition is seen at doses higher than 10(-5) micrograms/ml. TPA also inhibits epidermal growth factor-stimulated growth and does not induce ornithine decarboxylase activity or increase polyamine levels. This is not due to the rapid breakdown of TPA, since TPA is not metabolized to any appreciable extent.

Benzo(a)pyrene metabolism in primary cultures of mouse epidermal cells and untransformed and transformed epidermal cell lines.

The metabolism of [3H]benzo(a)pyrene [B(a)P] by cultures of primary mouse epidermal cells and untransformed and transformed epidermal cell lines was investigated. All three cell types effectively metabolized [3H]B(a)P. The major organic solvent-extractable metabolites found intracellularly in primary cultures were trans-7,8-dihydro-7,8-dihydroxybenzo(a)pyrene and 3-hydroxybenzo(a)pyrene, although quantities of 9-hydroxybenzo(a)pyrene, trans-9,10-dihydro-9,10-dihydroxybenzo(a)pyrene, and quinones also were present. The major organic solvent-soluble metabolites found in the extracellular medium were trans-9,10-dihydro-9,10-dihydroxybenzo(a)pyrene and trans-7,8-dihydro-7,8-dihydroxybenzo(a)pyrene, with smaller quantities of unconjugated phenols and quinones. The major water-soluble metabolites found in the extracellular medium were conjugated with glucuronic acid [primarily 3-hydroxybenzo(a)pyrene and several quinones]. No sulfate conjugates of [3H]B(a)P metabolites were detected. [3H]B(a)P metabolism was similar in cultures of untransformed and transformed epidermal cell lines but differed from the primary cultures. The major intracellular and extracellular organic solvent-soluble metabolites were diols. Little or no unconjugated phenols were detected. Both the untransformed and transformed epidermal cell lines converted [3H]B(a)P to water-soluble metabolites, primarily glucuronide conjugates. In contrast to the primary cells, a major pathway of trans-7,8-dihydro-7,8-dihydroxybenzo(a)pyrene metabolism in the untransformed and transformed cell lines was a glucuronide conjugate. Primary mouse epidermal cells provide an important model system for studying factors affecting the activation and detoxification of hydrocarbon carcinogens.

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.

Improved conditions for murine epidermal cell culture.

An improved method for cultivating newborn mouse epidermal cells has been developed that increases the longevity, epithelial nature and efficiency of cell-line establishment. The use of Super Medium, an enriched Waymouth's formulation, increased proliferation for long periods of time, as did incubation at 31 degrees C rather than 37 degrees C. The fetal bovine serum requirement was found to be reduced at the lower temperature. An increase in labeling indices was seen when epidermal growth factor (EGF) or the cyclic nucleotides were added and the presence of EGF receptors was determined. Of the prostaglandins (PG) examined, PGE1 and PGE2 produced the greatest increase in DNA synthesis. The PG precursors, arachidonic and 8,11,14-eicosatrienoic acid, were also greatly stimulatory. The use of a lethally irradiated 3T3 feeder layer at 31 degrees C proved superior in maintenance of an epithelial morphology. Subculturable cell lines were established much more readily and reproducibly in carcinogen-treated cultures grown under the improved conditions.

Biotransformation of 7,12-dimethylbenz[alanthracene by mouse epidermal cells in culture.

The formation of cell- and medium-associated metabolites of 7,12-dimethylbenz[a]anthracene (DMBA) by primary mouse epidermal cells was examined using high-pressure liquid chromatography. Cells were cultured in the presence of 14C DMBA for various time periods prior to harvesting. Ethyl acetate/acetone (2:1) extractable metabolites found associated with cells cochromatographed with 7-hydroxymethyl-12-methylbenz[a]anthracene (7-OHM-12-MBA), 12-hydroxymethyl-7-methylbenz[a]anthracene (12-OHM-7-MBA), (+/-)-trans-3,4-dihydro-3,4-dihydroxy-7,12-dimethylbenz[a]anthracene ((+/-)-trans-DMBA-3,4-diol) and phenols. The major metabolite(s) found within cells cochromatographed with DMBA-phenol(s). Ethyl acetate/acetone extractable metabolites found in the medium cochromatographed with 7-OHM-12-MBA, 12-OHM-7-MBA, (+/-)-trans-DMBA-3,4-diol, (+/-)-trans-8,9-dihydro-8,9-dihydroxy-7,12-dimethylbenz[a]anthracene ((+/-) -trans-DMBA-8,9-diol) and phenols. The major ethyl acetate/acetone soluble metabolite found in the medium cochromatographed with (+/-)-trans-DMBA-8,9-diol. This metabolite is rapidly excreted unchanged from the cells into the medium. In addition, primary epidermal cells rapidly converted 14C DMBA to water soluble metabolites that could not be extracted from the medium with ethyl acetate/acetone. Approximately 50% of these water soluble metabolites were extractable with organic solvent upon treatment of the medium with beta-glucuronidase. Phenolic metabolite(s) represented 75-85% of the total beta-glucuronidase releasable material. The results indicated that primary mouse epidermal cells in culture rapdly converted DMBA to a variety of hydroxylated products some of which were conjugated with glucuronic acid. In addition, the formation of (+/-)-trans-DMBA-3,4-diol and its retention within the cells provides additional support for an important role for this metabolite in carcinogenesis by DMBA.

Metabolic conversion of 12-O-tetradecanoylphorbol-13-acetate in adult and newborn mouse skin and mouse liver microsomes.

Tritiated 12-O-tetradecanoylphorbol-13-acetate (TPA) was applied to adult mouse skin; at specified time intervals the mice were killed, and the labeled phorbol was extracted and subjected to separation and quantitation by high-pressure liquid chromatography. After 24 hr, TPA comprised greater than 96% of the recovered label from the skin, and its apparent half-life was 17.8 hr. Pretreatment of adult skin with TPA for 4 weeks before treatment with labeled TPA resulted in an increase in the clearance rate of TPA from the skin. Skin from newborn mice was capable of converting TPA into monoesters and phorbol, but the clearance rate in the adult was about 12 times more rapid than it was in the newborn. Epidermal homogenates converted TPA into 12-O-tetradecanoylphorbol, phorbol-13-acetate, and phorbol. Hepatic homogenates were able to convert TPA to monoesters and phorbol at rates 14 to 15 times faster than were epidermal homogenates. Attempts to isolate any previously undescribed metabolites of TPA by use of liver homogenates were unsuccessful, and mixed-function oxidation did not contribute to the metabolism of TPA. From inhibitor studies it was judged that esterases were implicated in the conversion of TPA to monoesters and phorbol. The results support the hypothesis that the tumor-promoting activity of TPA is directly related to its concentration in a specific tissue and that conversion of TPA to an active metabolite probably does not occur.

Lack of involvement of 6-hydroxymethylation in benzo[a]pyrene skin tumor initiation in mice.

The skin tumor-initiating activities of benzo[a]pyrene (BP), 6-hydroxymethylbenzo[a]pyrene (6-OH-CH2-BP), and 6-methylbenzo[a]pyrene (6-CH3-BP), as well as the effects of 7,8-benzoflavone (7,8-BF), quercetin, and 1-benzylimidazole on their activity, were determined in outbred female CD-1 mice by use of a two stage system of tumorigenesis. The skin tumor-initiating activity of 6-OH-CH2-BP and 6-CH3-BP was 12.5 and 20%, respectively, of the activity of BP, 7,8-BF had little effect on the skin tumor-initiating activity of 6-OH-CH2-BP and 6-CH3-BP. However, a dose-dependent inhibition of BP tumorigenesis by 7,8-BF was noted. Quercetin and 1-benzylimidazole also inhibited BP skin tumor-initiating activity. These findings indicated that direct hydroxymethylation of BP is not an important pathway in the activation of BP in mouse skin tumor initiation.