Epidermal cell proliferation and promoting ability of phorbol esters.

Dose-response relationships on the abilities of several phorbol ester tumor promoters to promote skin tumors after 7,12-dimethylbenz[a]anthracene initiation and to bring about edema, inflammation, and epidermal hyperplasia were determined in female Charles River CD-1 mice. The promoting ability of the potent synthetic promoter, phorbol-12,13-dioctanoate (PdiC8), was determined over a dose range of 0.1-10 mug/application. Administration of PdiC8 two times weekly at dosages of 4, 6, 8, and 10 mug gave little variation in tumor response. A dose-dependent tumor response occurred at doses of 1-4 mug PdiC8. Only 1 papilloma was observed when PdiC8 was given twice weekly at a dose of 0.1 or 0.5 mug. A similar dose-response relation was observed for the ability of PdiC8 to stimulate epidermal hyperplasia. Investigations of other phorbol esters revealed an excellent correlation between their promoting ability and their ability to induce epidermal hyperplasia; however, that was not the case for compounds outside the phorbol ester series (i.e., acetic acid, cantharidin, and ethylphenylpropiolate).

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.

Effect of trichloropropene oxide on the ability of polyaromatic hydrocarbons and their "K-region" oxides to initiate skin tumors in mice and to bind to DNA in vitro.

The potent epoxide hydrase inhibitor, 1,1,1-trichloro-2,3-propene oxide (TCPO), enhanced the tumor-initiating ability of benzo[alpha]pyrene (BP) and 3-methylcholanthrene (MCA) but had no effect on 9,10-dimethyl-1,2-anthracene (DMBA) initiation in a two-stage system of tumorigenesis in female Charles River CD-1 mice. The tumor-initiating ability of dibenz[alpha,h]-anthracene (DBA) was decreased by prior topical treatment with 10 mumoles of TCPO. The tumor latency period of BP and MCA was decreased by TCPO but had no effect on DMBA or DBA. Topical treatment with 10 mumoles of TCPO did not initiate tumors in a two-stage system in mouse skin nor did it cause any histopathologic changes in the skin. The "K-region" epoxides of BP, DMBA, and MCA were weak tumor initiators when compared to the parent compounds. TCPO only slightly increased or had no effect on the tumor-initiating activity of the above epoxides. Pretreatment with Croton oil 18 hours prior to initiation with BP-4,5-epoxide also slightly enhanced the tumorigenic response in mouse skin. DBA-5,6-epoxide, when tested as a complete carcinogen at high doses (1 mg daily/10 days), was found to be a weak carcinogen but with activity comparable to that of DBA. TCPO only slightly increased the in vitro epidermally mediated covalent binding of the above parent polycyclic hydrocarbons to DNA.

Effects of antiinflammatory agents on mouse skin tumor promotion, epidermal DNA synthesis, phorbol ester-induced cellular proliferation, and production of plasminogen activator.

The antinflammatory ateroids fluocinoine acetonide, fluocinonide, and fluclorolone acetonide were found to be very effectiveinhibitory agents of mouse skin tumor promotion. These steroids also drastically inhibited epidermal DNA synthesis and epidermal cellular proliferation induced by a phorbal ester tumor promoter. In addition, these compounds were potent inhibitors, of plasminogen activator production in tumor cell cultures. The clinically used non-steroidal antiinflammatory agents oxyphenbutazone, indomethacin, and Seclazone also inhibite tumor promotion but were much less effective. Although these agents are useful against inflammatory disorders in general when given p.o., in our studies they had little effect on inflammation and epidermal cellular proliferation induced by a phorbol ester tumor promoter when given topically. The afore mentioned nonsteroidal antiinflammatory agents also had little effect on epidermal DNA synthesis. Oxyphenbutazone and indomethacin were less potent inhibitors of plasminogen activator production in tumor cells than were the antiinflammatory steroids, and Seclazone produced a negligible inhibition. There is, therefore, a general correlation in the potencies of a series of steroidal antiinflammatory agents for inhibition of tumor promotion and their ability to inhibit plasminogen activator production by tumor cell cultures and epidermal DNA synthesis.

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.

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.

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.

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.

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.

Comparison of the tumor-initiating activities of benzo(a)pyrene arene oxides and diol-epoxides.

The ability of arene oxides, and diol epoxides of benzo(a)pyrene to initiate skin tumors in mice was determined by using a two-stage system of tumorigenesis. (+/-)-7beta,8alpha-Dihydroxy-9alpha, 10alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene was a more effective tumor initiator than was (+/-)-7beta,8alpha-dihydroxy-9beta,10beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene when applied topically to CD-1 mice and then followed by twice-weekly applications of the promotor 12-O-tetradecanoylphorbol-13-acetate. (+/-)-7beta,8alpha-Dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene was approximately 20 to 30% as active as benzo(a)pyrene was as a tumor initiator. (+/-)-7beta,8alpha-Dihydroxy-7beta,8beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, benzo(a)pyrene, 9,10-oxide, and benzo(a)pyrene 11, 12-oxide, possessed about 1, 2, and 10%, respectively, of the tumor-initiating activity of benzo(a)pyrene.

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.

Skin tumor initiating ability of benzo(a)pyrene 4,5- 7,5- 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.

Skin-tumor-initiating ability of benzo(a)pyrene-7,8-diol-9,10-epoxide (anti) when applied topically in tetrahydrofuran.

The skin-tumor-initiating abilities of various metabolites of benzo(a)pyrene (BP) were determined in mice by using a two-stage system of tumorigenesis. We previously reported that BP-7,8-dihydrodiol (+/- trans) was approximately as potent as BP, suggesting that it may be a proximate carcinogen, but the alleged ultimate carcinogen of BP [BP-7,8-dihydrodiol-9,10-epoxide (anti)] was a weak tumor initiator (Cancer Lett.2: 115, 1976). Because of its high reactivity, the tumor-initiating ability of the BP-7,8-dihydrodiol-9,10-epoxide (anti) was determined by using acetone, benzene, and tetrahydrofuran (THF) as the solvent vehicles. The 'diol-epoxide' of BP was found to be an effective tumor initiator when applied topically in THF. The effectiveness of the various vehicles for the 'diol-epoxide' was as follows: THF greater than benzene greater than acetone; however, acetone was the best solvent for BP tumor initiation. The BP-9,10-dihydrodiol and BP-3-hydroxy were found to be weak tumor initiators. BP-3-hydroxy was also tested for tumor-promoting ability and was found to be inactive in this capacity.

The effects of benzoflavones on polycyclic hydrocarbon metabolism and skin tumor initiation.

The effects of benzoflavones on skin tumor initiation by polycyclic hydrocarbons and epidermal aryl hydrocarbon hydroxylase were investigated. 7,8-Benzoflavone (7,8-BF) was found to be a potent inhibitor of the inhibition of skin tumors by 3-methylcholanthrene (MC) as well as 7,12-dimethylbenz(a)anthracene (DMBA). 5,6-Benzoflavone(5,6-BF) inhibited tumor initiation by MC and DMBA, but to a lesser degree than 7,8-BF. Dose-response studies of the capacity of 7,8-BF to inhibit DMBA tumor initiation revealed that 7,8-BF was an effective inhibitor at 2.5 microgram and a maximum inhibition of 90% occurred at 100 microgram of 7,8-FB. The tumor initiating ability of 7-hydroxymethyl-12-methylbenz(a)anthracene (7-OHMe-12MeBA) was not inhibited by 7,8-BF. Epidermal aryl hydrocarbon(benzo(a)pyrene hydroxylase(AHH) was increased by 5,6-BF and either had no effect or was slightly inhibited by 7,8-BF when given either topically or i.p. Both flavones when added directly to the assay tubes inhibited the in vitro epidermal AHH activity from control and MC pretreated mice by greater than 75%. When added in vitro, 7,8-BF and 5,6-BF inhibited epidermally mediated covalent binding of radioactive DMBA and dibenz(a,h)anthracene to DNA by 50% or more. The inhibition of skin tumor initiation by 7,8-BF and 5,6-BF appears to be partially related to its ability to inhibit the formation of electrophilic intermediates.

The effects of anti-inflammatory agents on skin tumor initiation and aryl hydrocarbon hydroxylase.

The effects of various clinically used anti-inflammatory agents on mouse skin tumorigenesis and aryl hydrocarbon hydroxylase (AHH) were investigated. Oxyphenbutazone, a nonsteroidal anti-inflammatory agent, inhibited 3-methylcholanthrene (MC) tumor initiation but was less effective than the steroidal anti-inflammatory agent, dexamethasone. Oxyphenbutazone was not found to induce AHH activity in mouse epidermis, whereas indomethacin and Seclazone had a slight inducing effect. When these agents were added directly to the in vitro AHH assay, they did not inhibit AHH activity. However, additional experiments have shown a decreased epidermally mediated covalent binding of MC to DNA in vitro when the epidermal homogenates were isolated from mice pretreated with either dexamethasone or oxyphenbutazone and MC at 3 or 12 hr before killing.

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.