Skin Tumor Initiation Research Articles

7-Suffooxymethyl-12-methylbenz[a]anthracene is an electrophilic mutagen, but does not appear to play a role in carcinogenesis by 7,12-dimethylbenz[a]anthracene or 7-hydroxymethyl-12-methylbenz[a]anthracene

Although a bay-region dihydrodiolepoxide metabolite has been considered as a principal ultimate electrophilic and carcinogenic form of 7,12-dimethylbenz[a]anthracene (DMBA), other reactive metabolites might also play a role in the activation of this hydrocarbon in vivo. Earlier studies suggested the hydroxylation of a meso-anthracenic methyl group with subsequent formation of a benzylic ester bearing a good leaving group (e.g. sulfate) as a metabolic activation pathway for DMBA. In support of this hypothesis, the formation of an electrophilic and mutagenic sulfuric acid ester of 7-hydroxymethyl-12-methylbenz[a]anthracene (HMBA) by rat liver cytosolic sulfotransferase activity has previously been demonstrated, but no data have been reported on the carcinogenicity of this reactive ester. In the present study, we compared the carcinogenicity of chemically synthesized sodium 7-sulfooxymethyl-12-methylbenz[a]anthracene (SMBA) with that of the parent methyl and hydroxymethyl hydrocarbons. For this purpose, tests were made in several animal tumor models: induction of hepatomas in male B6C3F1 mice, lung adenoma induction in A/J mice, initiation of mouse skin tumors, development of sarcomas in rats at the injection sites, and initiation of preneoplastic enzyme-altered foci in rat liver. Data from all of these studies indicate that SMBA is not more carcinogenic than DMBA or HMBA. In addition, the carcinogenic activity of HMBA was not altered by dehydroepiandrosterone, a strong inhibitor of sulfotransferase activity for HMBA. DMBA produced only a low level of hepatic benzylic DNA adducts in rats when a relatively high dose was administered. These adducts constitute less than 5% of total DMBA residues bound to hepatic DNA. The rest of the adducts appear to be associated with other electrophilic intermediates including the dihydrodiol epoxide metabolites. Based on the results of our present study, it is unlikely that DMBA exerts its carcinogenic activity via metabolic activation to SMBA.

Protection against ultraviolet B radiation-induced photocarcinogenesis in hairless mice by green tea polyphenols

Our recent studies have shown that polyphenols present in green tea (GTP) possess significant antigenotoxic activity and afford protection against polycyclic aromatic hydrocarbon-induced skin tumor initiation in mice. In this study we assessed the effect of oral feeding and topical application of GTP on ultraviolet B (UVB) radiation-induced skin carcinogenesis in female SKH-1 hairless mice. Chronic oral feeding of GTP (0.1%, w/v) in drinking water resulted in significantly (P less than 0.01) lower tumor yield (percent of animals with tumors and number of tumors per mouse) and extended TDT50 (P less than 0.05), as compared to animals receiving normal drinking water. Topical application of GTP before UVB irradiation also afforded protection against photocarcinogenesis; however, the protective response was lower than that observed by oral feeding of GTP in drinking water. These results, in conjunction with our prior publications, suggest that consumption of green tea may reduce the risk of some forms of human cancer induced by both physical and chemical environmental carcinogens.

Novel coumarins as potential anticarcinogenic agents

The potential anticarcinogenic properties of several novel coumarin derivatives whose structures are based on polycyclic aromatic hydrocarbons (PAHs) were examined in the multistage model of mouse skin tumorigenesis. The test compounds were evaluated for their affinity to bind competitively with rat cytosolic Ah-receptor in rat hepatic cytosol, their effects on mouse epidermal aryl hydrocarbon hydroxylase (AHH) after topical application, and for their effects on the levels of hydrocarbon-DNA adducts formed in vivo. All compounds showed good correlations between cytosolic Ah-receptor binding and their ability to induce epidermal AHH activity. Among the derivatives evaluated the coumarin (8-methyl-9H-10-oxabenzo[a]pyren-9-one) exhibited the highest affinity for the Ah-receptor and was also the most potent inducer of epidermal AHH activity. This compound also effectively inhibited the covalent binding of 7,12-dimethylbenz[a]anthracene (DMBA) to epidermal DNA when given either 5 min or 24 h prior to application of [3H]DMBA. This novel coumarin derivative significantly inhibited skin tumor initiation by DMBA in SENCAR mice when given at a dose of 200 nmol, 5 min (69% inhibition) or 24 h (76% inhibition) prior to initiation. The results of these studies suggest that this class of compounds shows considerable promise for future development as potential inhibitors of PAH-mediated tumor initiation on mouse skin. Potential mechanism(s) for the anti-initiating action of these compounds are discussed.

Biological and Molecular Aspects of Radiation Carcinogenesis in Mouse Skin

The process of mouse skin carcinogenesis can be operationally subdivided into at least three stages which have been termed initiation, promotion, and progression. Ionizing radiation has been found to be a weak initiator of malignant squamous cell carcinomas (SCCs) when radiation was followed by repeated treatments of the skin with the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Besides SCCs, ionizing radiation was found to induce, independent of tumor promoters, basal cell carcinomas (BCCs), a tumor histology not normally seen with chemical carcinogens and mouse skin. Fractionated doses of 1 MeV electrons were found to enhance the conversion of chemically induced benign papillomas to malignant SCCs. In addition to the biological studies, questions related to dominant transforming genes and differential gene expression in the radiation-initiated mouse skin tumors have been explored. Distinct non-ras dominant transforming gene(s) have been detected in radiation-initiated, TPA-promoted SCCs. Differences in the expression pattern of tumor-associated genes were seen in comparing chemically to radiation-induced benign and malignant skin tumors. Therefore, ionizing radiation has been shown to be active in the initiation of malignant skin tumors and progression of benign to malignant tumors in the mouse skin. The ability to divide the process of carcinogenesis into multiple stages in the mouse skin model has facilitated mechanistic studies that may elucidate the molecular pathways involved in radiation-versus chemically induced tumor development.

Mouse skin tumorigenicity studies of indoor coal and wood combustion emissions from homes of residents in Xuan Wei, China with high lung cancer mortality

The rural Xuan Wei County, Yunnan Province, China, has an unusually high lung cancer mortality rate that cannot be attributed to tobacco smoke or occupational exposure. The lung cancer rate is associated with 'smoky' coal, in contrast to wood or 'smokeless' coal burned in unventilated homes. This study was conducted to characterize and compare mouse skin tumorigenicity of the coal and the wood combustion emissions and to link the animal data to human lung cancer. Indoor air particles (less than 10 microns) were collected from a central commune where the lung cancer mortality rate is high and smoky coal is the major fuel used and also from a south-western commune where lung cancer mortality rate is low and where wood or smokeless coal are the major fuels used. The organic extracts of these indoor air particles from smoky coal, smokeless coal and wood combustion were analysed for polycyclic aromatic hydrocarbons and assayed for skin tumor initiation activity and complete carcinogenicity in SENCAR mice. The results showed that the organic extract of the emission particles from smoky coal combustion is the most active in tumor initiation among the three combustion emission samples followed by smokeless coal and then wood. The organic extract of the particles from smoky coal combustion was shown to be a potent complete carcinogen, whereas the wood extract was relatively inactive as a complete carcinogen. The extract of particles from the smokeless coal combustion was not tested for complete carcinogenicity because of inadequate supply. Eighty-eight percent of the mice treated with the smoky coal extract showed carcinomas, averaging 1.1 carcinomas per tumor-bearing mouse at the end of the 77 week study. These findings were in agreement with the epidemiological data, which showed that the Xuan Wei residents using smoky coal as a major fuel in homes had a high lung cancer mortality rate. This study demonstrates that the results of the tumorigenicity assays in mice were in agreement with human lung cancer data.

Metabolic activation of 9-hydroxymethyl-10-methylanthracene and 1-hydroxymethylpyrene to electrophilic, mutagenic and tumorigenic sulfuric acid esters by rat hepatic sulfotransferase activity

Our previous studies on 7-hydroxymethyl-12-methylbenz[a]anthracene and 6-hydroxymethylbenzo[a]pyrene showed that cytosolic sulfotransferase activity plays a major role in the formation of hepatic benzylic DNA and RNA adducts by these carcinogens in rats. In the present study, we found similar sulfotransferase activity in rat liver cytosol which activates 9-hydroxymethyl-10-methylanthracene (HMA) and 1-hydroxymethylpyrene (HMP) to electrophilic sulfuric acid ester metabolites. Thus, incubation of these nonbay region hydrocarbons with calf thymus DNA in the presence of liver cytosol fortified with the sulfo-group donor, 3'-phosphoadenosine-5'-phosphosulfate (PAPS) produced benzylic DNA adducts that were chromatographically identical to those obtained by the reactions of the corresponding sulfuric acid esters with deoxyguanosine and deoxyadenosine. These adducts were also produced in the livers of infant rats injected i.p. with 0.25 mumol/g body wt of HMA or HMP. Administration of comparable doses of 9-sulfooxymethyl-10-methylanthracene (SMA) and 1-sulfooxymethylpyrene (SMP) resulted in much higher levels of hepatic benzylic DNA adducts than did the parent hydroxymethyl hydrocarbons. Both HMA and HMP induced His+ revertants in Salmonella typhimurium TA98 when preincubated with these bacteria in the presence of rat liver cytosol and PAPS. This sulfotransferase-mediated mutagenicity of HMA and HMP was reduced by dehydroepiandrosterone, an inhibitor of hepatic sulfotransferase activity for these hydrocarbons. SMA and SMP were directly mutagenic and their intrinsic bacterial mutagenicity was inhibited by glutathione (GSH) and GSH-S-transferase activity. Chloride ion at physiological concentrations enhanced the bacterial mutagenicity of SMA through the formation of 9-chloromethyl-10-methylanthracene as previously observed for SMP by Henschler et al. In contrast to the higher mutagenicity of 1-chloromethylpyrene (CMP) than SMP in bacteria, CMP formed smaller amounts of hepatic benzylic DNA adducts in rats than the sulfuric acid ester. SMA and SMP were weak skin tumor initiators in the mouse, but they were more active than HMA and HMP in this regard.

Tumor initiation in mouse skin by cutting oils

In order to study the skin tumor-initiating potential for fresh and used cutting oils, a chronic animal bioassay following an initiation-promotion protocol was performed. Tumor initiation was performed by topical application of a single dose of neat fresh/used cutting oil on the mouse skin. After a week, these animals were treated topically with 12-O-tetradecanoylphorbol 13-acetate (TPA), a potent skin tumor promoter. Repeated application of TPA resulted in the appearance of tumors almost at the same time (6-8 weeks) in the groups initiated with fresh or used cutting oil. After a prolonged application of TPA, for 28 weeks, it was observed that the cumulative number of tumors and tumor-bearing animals in the used oil initiated group was slightly less compared to that exposed to a fresh variety of oil. Histopathological examination of the tumors could not show any qualitative differences in the nature of tumors developed in both groups. It seems that both fresh and used cutting oils act as potent skin tumor initiators.

Inhibition of benzo[a]pyrene-7,8-diol formation in vitro by complex organic mixtures

Coal-derived complex organic mixtures [COM] with boiling points ⩾370° C (⩾700° F) are known to inhibit both mouse skin tumor initiation by benzo[ a]pyrene [BAP], and BAP-induced bacterial mutagenesis. We have examined the effects of 5 COM, with boiling points of 149–370°C (300–700°F), 370–398°C (700–750°F), 398–426°C (750–800°F), 426–454°C (800–850°F), and >454° C(>850° F), on both the rate and the route of BAP metabolism by rat liver homogenetes in vitro. When co-metabolized in 40:1 excess with BAP, all of the COM reduced the rate of BAP metabolism. The 149–370°C (300–700°F) COM reduced the initial rate of BAP metabolism to 34% of the rate for BAP alone, while the four higher-boiling COM reduced it to 6.3–9.3% of the rate for BAP alone. In addition, the 2 highest-boiling COM (426–454°C and >454°C boiling points) were found to reduce the percentage of BAP metabolized to BAP-7,8-diol, in comparison to incubations using BAP alone. The 370–398°C and 398–426°C COM did not alter the percentage of BAP metabolized to BAP-7,8-diol, while the 149–370°C COM increased it. Both the general inhibition of BAP metabolism (by all of the COM), and the specific inhibition of BAP-7,8-diol formation (by the highest boiling COM) may play a role in the inhibition of formation of BAP-induced skin tumors by these materials.

Activation of the cellular harvey ras gene in mouse skin tumors initiated with urethane

Mouse skin tumors, benign papillomas, and squamous cell carcinomas (SCCs) were initiated by a single topical application of urethane followed by repeated promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). Using the NIH 3T3 focus forming assay, dominant transforming activity was detected in DNA isolated from SCC samples. Rearranged and amplified copies of the c-Ha-ras gene were detected in NIH 3T3 transformant cell lines, indicating that an activated Ha-ras gene had been transferred to the NIH 3T3 recipient cells. Analysis of p21ras from the transformant cell lines suggested that the activating ras mutation was present in codon 61. Ultimately, the Ha-ras gene was shown to be activated by a specific A----T transversion at the second position of codon 61. This mutation was detected in both benign papillomas and SCCs, suggesting the activation occurred early in tumor development. The results demonstrate a highly consistent activation of the Ha-ras oncogene by a specific point mutation, suggesting a functional role for an activated ras gene in the initiation of mouse skin tumors by urethane.

Protection against polycyclic aromatic hydrocarbon-induced skin tumor initiation in mice by green tea polyphenols.

Green tea is a popular beverage in China and Asia and has been shown to possess antipyretic, diuretic and several other pharmacological activities. The major constituents of green tea are polyphenols which have been found to possess antioxidant and antimutagenic properties. In this study green tea polyphenols (GTP) were evaluated as an anti-initiating agent against the skin tumorigenicity induced by polycyclic aromatic hydrocarbons (PAHs) in mice. In a complete skin tumorigenesis protocol using 3-methylcholanthrene the topical application of GTP to female BALB/c mice resulted in substantial protection against the onset and subsequent development of tumors. In the two-stage skin tumorigenesis protocol using 7,12-dimethylbenz[a]anthracene (DMBA) as the initiating agent followed by twice weekly applications of 12-O-tetradecanoylphorbol-13-acetate; (TPA) as tumor promoter, topical application of GTP to female SENCAR mice afforded significant protection against skin tumorigenicity. Oral feeding of GTP in drinking water to female SENCAR mice also protected against skin tumorigenesis in DMBA - TPA-treated animals. GTP when administered topically or orally significantly inhibited PAH - DNA adduct formation in epidermis after topical application of [3H]benzo[a]pyrene or [3H]DMBA. Our results suggest that GTP has substantial anti-skin-tumor-initiating activity against PAHs and could prove useful in protecting against some forms of human cancer.

Further investigations into the effect of non-benzo ring bay-region methyl substitutents on tumor-initiating activity of polycyclic hydrocarbons

The effect of a methyl substitutent at the non-benzo ring bay-region position of benzo[ e]pyrene (B[ e]P), cholanthrene (CA) and dibenz[ a,j]anthracene (DB[ a,j]A) on skin tumor-initiating activity was examined. A methyl substituent at the 1-carbon of B[ e]P enhanced tumor-initiating activity of the parent compound (0.18 vs. 2.4 papillomas/mouse for B[ a]P vs. 1-methyl-B[ e]P, respectively, with an 800 nmol initiating dose). A methyl substituent at the 7- and 14-carbons of DB[ a,j]A increased the activity of this weak initiator more than 13 times. The introduction of a methyl substituent at the non-benzo ring bay-region position of CA (i.e. carbon atom 6) dramatically increased tumor-initiating activity in SENCAR mice. 6-Methyl-CA was found to be a more potent skin tumor-initiator than 3-methyl-CA, and nearly as potent as 7,12-dimethylbenz[ a]anthracene, one of the most potent initiators yet tested in the 2-stage initiation-promotion mouse skin model. When taken together with previous results from our laboratories, the data further support the generality of the enhancing effect of a non-benzo ring bay-region methyl substituent on polycyclic hydrocarbon tumor-initiation.

Skin tumor initiating activities of the 9- and 10-fluoro derivatives of 7- or 12-methylbenz[a]anthracene and the 9- and 10-trifluoromethyl derivatives of 7,12-dimethylbenz[a]anthracene in SENCAR mice.

We have determined the skin tumor initiating activity in SENCAR mice of several 9- and 10-substituted mono- and dimethylbenz[a]anthracene derivatives. 9-fluoro-7-methylbenz[a]anthracene (9-F-7-MBA) was approximately as active as 7-MBA, whereas 10-F-7-MBA was considerably more active as a skin tumor initiator than the parent compound. Initiating doses of 200 and 400 nmol per mouse of 10-F-7-MBA yielded 14.17 +/- 0.16 and 22.47 +/- 1.64 papillomas per mouse after 18 weeks of promotion with 12-O-tetradecanoylphorbol-13-acetate, whereas comparable doses of 7-MBA yield 2.13 +/- 0.12 and 4.73 +/- 0.68 papillomas per mouse respectively. The effect of fluoro substituents at positions 9 and 10 of 12-MBA, a less potent tumor-initiator than 7-MBA, was also examined. 9-F-12-MBA was only slightly more active than 12-MBA, whereas 10-F-12-MBA was again considerably more active than the parent compound. Doses of 400 and 800 nmol per mouse of 10-F-12-MBA yielded 24.97 +/- 2.18 and 22.20 +/- 6.47 versus 1.13 +/- 0.80 and 3.00 +/- 0.17 papillomas per mouse respectively for comparable initiating doses of 12-MBA. The effect of introducing a trifluoromethyl (CF3) group at the 9 and 10 positions of 7,12-dimethylbenz[a]anthracene was also examined. A CF3 group at either of these positions essentially eliminated the tumor initiating activity of DMBA at the doses tested. These results when taken together with previous results from our laboratory suggest that electron donating substituents at positions 9 and 10 of the benz[a]anthracene nucleus either have no effect or enhance skin tumor initiating activity, whereas electron withdrawing groups at these positions dramatically reduce or abolish tumor initiating activity.

The intracellular fate of [3H]benzo[a]pyrene diol epoxide diastereomers localized in vivo in SENCAR mouse epidermis: a quantitative electron microscopic study

The intracellular localization of topically applied anti and syn diastereomers of racemic benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE) was investigated in SENCAR mouse epidermis using conventional electron microscopic (EM) autoradiography. The shaved backs of adult mice were treated in vivo with the active skin tumor initiator anti [3H]BPDE or with its stereoisomer syn [3H]BPDE, which is inactive as an initiator. After 3, 12 and 24 h of exposure, thin sections of the treated skin were prepared for high resolution autoradiography to determine the intracellular distributions of radioactivity bound within the epidermis. For each treatment and exposure time, the interfollicular area of epidermis examined by EM encompassed 350-700 keratinocytes, and extended from the basal lamina to the innermost keratinizing layer at a magnification that permitted basal and suprabasal cell populations to be distinguished in situ following autoradiography. Statistical analysis of the distributions of silver grains localized over keratinocytes at 3-, 12- and 24-h timepoints revealed that 27.2% of the carcinogenic anti [3H]BPDE grains occurred over epidermal nuclei, which was significantly more (P less than 0.001) than 20.0% of the non-carcinogenic syn [3H]BPDE grains associated with nuclei at all timepoints examined. In the basal cell population at the 3-h time point 33% of both racemic diastereomers were found within nuclei; at 12 and 24 h of exposure the nuclear association of anti [3H]BPDE grains increased (P less than 0.01) to 39% and 47% respectively, while the nuclear-associated syn [3H]BPDE grains remained at 29% and 33%. The localization of [3H]BPDE observed in our experiments suggests that the epidermal cell nucleus may be an important intracellular target for anti BPDE.

Tumor-initiating activity of the 9,10-dihydrodiol- and 9,10-dihydrodiol-7,8-epoxide of 3-methylcholanthrene in SENCAR mice

The skin tumor-initiating activities of several bay-region metabolites of 3-methylcholanthrene (3-MCA) were determined in SENCAR mice. 3-MCA- anti-9,10-diol-7,8-epoxide possessed weak tumor-initiating activity when tested at 100 and 200 nmol/mouse doses (0.27 and 0.67 papillomas per mouse after 18 weeks of promotion with 12- O-tetradecanoylphorbol-13-acetate (TPA)). 3-MCA- trans-9,10-diol at initiating doses of 50 and 100 nmol/mouse was as active as 3-MCA. 3-MCA- trans-9,10-diol was also tested for mutagenic activity toward V79 cells in cell-mediated assays and found to be ∼ 2-times more potent than 3-MCA. The data suggest that 3-MCA- trans-9,10-diol is a proximate carcinogen for mouse skin.

Effects of ellipticine, flavone, and 7,8-benzoflavone upon 7,12-dimethylbenz[a]anthracene, 7,14-dimethyldibenzo[a,h]anthracene and dibenzo[a,h]anthracene initiated skin tumors in mice.

Varying doses of ellipticine (EL), flavone (FL), or 7,8-benzoflavone (78BF) were applied to mouse skin 5 min before an initiating dose of 10 nmol 7,12-dimethylbenz[a]anthracene (DMBA), 47.5 nmol 7,14-dimethylbenzo[a,h]anthracene (DDBA), or 200 nmol dibenzo[a,h]anthracene (DBA) and the development of skin tumors in the mice then promoted by topical applications of 2 micrograms 12-O-tetradecanoylphorbol-13-acetate (TPA). As expected, treatment with 78BF (37 nmol or 370 nmol) markedly inhibited the skin tumor initiation by DMBA (greater than 70%). High doses of FL (4500 nmol) or EL (410 nmol) also inhibited DMBA tumorigenesis (52% and 82%, respectively) but lower doses of FL (450 nmol) or EL (4.1 nmol) stimulated DMBA tumorigenesis (greater than 40%). As was the case with DMBA initiation, the higher doses of FL or EL inhibited DDBA skin tumorigenesis and the lower doses of these two modifiers stimulated the DDBA tumorigenesis. In contrast with the results with DMBA initiation, treatment with 78BF (370 nmol or 3700 nmol) slightly enhanced DDBA tumorigenesis (22% and 6%, respectively). Treatment with EL and FL at all doses tested stimulated DBA tumorigenesis (range 4-51%), while treatment with 370 nmol 78BF slightly stimulated DBA tumorigenesis (19%) and treatment with 3700 nmol slightly inhibited DBA tumorigensis (9%). The effects of a range of 78BF doses upon skin tumor initiation by 40 nmol DMBA were also investigated. While all doses of 78BF tested (0.37-370 nmol) inhibited the DMBA tumorigenesis, the dose response was not linear; treatment with 3.7 nmol 78BF resulted in more papillomas per mouse (12.20) than did treatment with either 0.37 nmol 78BF (8.70) or 37 nmol 78BF (5.97). It is concluded that modifiers such as 78BF, FL and EL may have a variable, dose-dependent effect upon skin tumor initiation by carcinogenic polycyclic arylhydrocarbons. Some implications of this proposal are discussed.

Chapter 7: MULTISTAGE SKIN CARCINOGENESIS: A USEFUL MODEL FOR THE STUDY OF THE CHEMOPREVENTION OF CANCER

Skin carcinogenesis can be operationally and mechanistically divided into at least three stages; tumor initiation, stage I and stage II of promotion. Current information suggests that reactive intermediates of skin tumor initiators are mutagenic and bind convalently to DNA of epidermal stem cells (dark basal keratinocytes) leading to some irreversible alteration in the differentiation capacity of these cells. Inhibitors of skin tumor initiation by polycyclic aromatic hydrocarbons (PAH) decrease the level of the PAH diol-epoxide bound to specific DNA adducts. The tumor promoters have been shown to have many cellular and biochemical effects in the skin. Recent data suggests that free radicals may be important in skin tumor promotion. The first stage of promotion is partially irreversible and can be accomplished by a single treatment of a tumor promoter such as TPA or by non-promoting agents such as 4-0-methyl-TPA, calcium ionophore A23187, and hydrogen peroxide, as well as wounding. These agents increase the number of dark basal keratinocytes, which suggest that these cells are important in the first stage of promotion. Prostaglandin E2 was found to specifically enhance stage I of promotion whereas the protease inhibitor tosyl phenylalanine chloromethylketone (TPCK) specially inhibited stage I of promotion and the TPA-induced dark basal keratinocytes. The second stage of promotion is initially reversible but later becomes irreversible. The weak promoting agent mezerein is an effective stage II promoter. Polyamines 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 (RA), diflouromethylornithine (DFMO), and butylated hydroxyanisole (BHA) specially inhibited stage II of promotion and the mezerein-induced polyamine levels. Floucinolone acetonide (FA) was found to inhibit both stages but was more effective in counteracting stage I of promotion. Although, TPA can cause a decrease in the number of glucocorticoid receptors during promotion, FA can effectively prevent this loss. Recent data suggest that skin tumor promotion can be effectively inhibited by a combination of stage I and II inhibitors. Furthermore, skin carcinogenesis can be counteracted by a combination of low and nontoxic doses of BHA, TPCK, DFMO and vitamin E.

Mouse skin tumor-initiating activity of benz[ e]aceanthrylene and benz[ l]aceanthrylene in SENCAR mice

Benz[ e]aceanthrylene (B[ e]A) and benz[ l]aceanthrylene (B[ l]A), cyclopenta-fused derivatives of benz[ a]anthracene, have been reported to be active bacterial cell and mammalian cell gene mutagens. In this study they were evaluated as skin tumor initiators in both male and female SENCAR mice. Both polycyclic aromatic hydrocarbons (PAHs) induced papilloma formation in the range of 50–1000 nmol/mouse. B[ l]A was the most active, being approximately 4 times as active as benzo[ a]pyrene (B[ a]P) while B[ e]A had activity approximately equivalent to B[ a]P. These results are in contrast to those reported for the air pollutant, cyclopenta[ cd]pyrene, another cyclopenta-fused PAH, which is a weak mouse skin tumor initiator. We postulate that these cyclopenta-PAHs are formed by pyrosynthetic routes similar to other environmental cyclopenta-PAHs and that they may be of importance as contributors to air pollution carcinogenesis.

Mutagenic activity of methyl- and fluoro-substituted derivatives of polycyclic aromatic hydrocarbons in a human hepatoma (HepG2) cell-mediated assay

Several series of methyl- and fluoro-substituted polycyclic aromatic hydrocarbon (PAH) derivatives were tested for mutagenic activity in cell-mediated assays with cells of the human hepatoma cell line, HepG2, as PAH activators. The mutagenic activity of dibenz[ a, h]anthracene [DB(a,h)A] increased progressively with the substitution of a methyl group at one or both non-benzo bay-region sites. At a concentration of 0.25 μg/ml, the mutation frequencies induced by DB(a,h)A, 7-MeDB(a,h)A and 7,14-diMeDB(a,H)A were 1.3, 13.1 and 59.0 6-thioguanine-resistant colonies/10 5 viable V79 cells, respectively. Methyl groups at non-benzo bay-region sites in 3-methylcholanthrene and benzo[ e]pyrene had little effect on mutagenic activity at the highest concentration which could be tested (2 μg/ml). The presence of a fluorine atom on the bay-region A-ring of 7,12-dimethylbenz[ a]anthracene (DMBA) drastically reduced mutagenic activity. At a concentration of 1.0 μg/ml, the mutation frequencies induced by DMBA, 1-fluoro-DMBA and 4-fluoro-DMBA were 50.5, 6.8 and 1.6, respectively. On the other hand, the mutation frequency was increased 6-fold when the fluoro substituent was in the 10-position of the D-ring of DMBA. Thus, for the most part, the relative mutagenic activities of these compounds in the HepG2 cell-mediated assay paralleled their skin tumor-initiating activity in SENCAR mice reported earlier (DiGiovanni et al., 1982, 1983a, b). These studies demonstrate the value of the HepG2 cell line as an exogenous, intact human cell activation system in short-term assays designed to evaluate the genotoxic effects of PAHs.

Influence of 13- cis-retinoic acid on mouse skin tumor initiation and promotion

Tumor initiation in CD-1 mice by benzo[ a]pyrene (BaP) or N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) was unaffected by topical pretreatment with 13- cis-retinoic acid (13- cis-RA). Likewise, anthralin-induced tumor promotion in SENCAR mice was unaffected by pretreatment with 13- cis-RA. These results suggest that the action of retinoids in preventing either tumor initiation or promotion is very carcinogen or cocarcinogen specific.

Mutagenesis and skin tumour initiation by shale- and coal-derived oils and their distillation fractions

Crudes and distillation fractions of three shale- and four coal-derived liquids and petroleum were screened for mutagenic activity in Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100, and for tumour initiating activity in mouse skin. Selected analytical tests were also conducted to identify and characterize the crudes and fractions. Low boiling (naphtha) fractions of all crudes showed no biological activity. The higher boiling fractions, the residues and the crude oils were mutagenic and initiated tumours. Some coal-derived liquid fractions were significantly more active by either test than similar boiling range fractions of petroleum and shale-derived oils. The mutation frequencies (revertants μg -1) as well as the total number of tumours were also related to the C/H weight ratios of the various fractions, supporting the view that aromatic compounds may be responsible for some of those activities. In most cases, the expression of mutagenic activity required an activation system, although a few fractions of shale- and coal-derived oils were weakly active without activation. In general, there was a strong positive relationship between the mutation data and the initiation-promotion data. Two linear relations were observed between the total number of skin tumours and the mutation frequencies in strains TA1538, TA98, TA1537 and TA100: one is associated with the medium boiling fractions while the other includes the residues and crude liquids.