C57BL/6 mice are resistant to tumor promotion by full thickness skin wounding.

The present study demonstrates that C57BL/6 mice, previously shown to be relatively resistant to skin tumor promotion by phorbol esters as well as several other classes of tumor promoters, are resistant to skin tumor promotion by full thickness skin wounding. Two separate experiments were performed comparing female SENCAR and C57BL/6 mice for their sensitivity to skin tumor promotion by skin wounding following initiation with 7,12-dimethylbenz[a]anthracene (DMBA). In the first experiment, groups of mice were initiated with 25 nmol DMBA and then received full thickness skin wounds in the initiated skin 2 weeks later. Neither SENCAR nor C57BL/6 mice developed skin tumors during the 26 weeks following initial wounding. However, these groups were rewounded in week 27 and 14 weeks later the SENCAR mice had developed a significant tumor response (0.75 papillomas per mouse, 55% incidence). At this time, the C57BL/6 mice still did not have a tumor response significantly different from the acetone-initiated controls. A second experiment was performed using a 100 nmol initiating dose of DMBA. Fifteen weeks after initial wounding in this experiment, the group of SENCAR mice had 0.76 papillomas per mouse (41% incidence) whereas no tumors were present in the group of C57BL/6 mice, even 31 weeks after the initial wounding. The results demonstrate that C57BL/6 mice are resistant to an endogenous skin tumor promotion mechanism and strongly support a link between skin tumor promotion by several classes of chemical promoters and full thickness wounding.

Induction of epidermal ornithine decarboxylase activity in mouse skin exposed to biogenic silica fibers.

The present study demonstrates that biogenic silica fibers (BSF), previously shown to promote skin tumors in mice and more recently to promote the induction of mesotheliomas when injected into the pleural cavity of rats, rapidly induces epidermal ornithine decarboxylase (ODC) activity in SENCAR mice following topical application. The time course for induction of epidermal ODC by BSF was very similar to that observed following topical treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). Maximal ODC activity was observed 4-6 h following treatment with BSF. Cycloheximide (70 mg/kg i.p.) partially inhibited (61%) the induction of ODC by BSF at 5 h. In addition, retinoic acid (RA, 5 micrograms per mouse given 30 min before BSF) effectively inhibited BSF-induced ODC by 68%, while indomethacin (100 micrograms per mouse 2 h before BSF) had little or no effect. Copper(II) bis(diisopropylsalicylate) (2 mumol 30 min before BSF), an effective inhibitor of TPA-induced ODC activity and tumor promotion, also had little or no effect on BSF-induced ODC. The work described in this paper suggests that BSF induces epidermal ODC by a very specific mechanism that exhibits both similarities and differences with that of the phorbol ester, TPA. Nevertheless, this response strongly supports the conclusion that BSF is an effective tumor promoter in mouse skin and that ODC induction is an integral part of the mechanism of action of this environmental promoter.

Tumours of mesothelial origin in rats following inoculation with biogenic silica fibres.

Pleural implant experiments using Sprague-Dawley rats have shown that those injected with the carcinogen 15,16-dihydro-11-methylcyclopenta[a]phenenthren-17-one (11-methyl-17-ketone) intraperitoneally followed by silica fibres intrapleurally develop mesotheliomas. These tumours were histologically similar to those induced by crocidolite alone. The intraperitoneal injection of 11-methyl-17-ketone also induced leukaemia.

Synthesis and biological activity of some further cyclopenta[a]phenanthrenes.

16,17-Dihydro-11-methoxy-15H-cyclopenta[a]phenanthrene was synthesized by two different routes and tested for carcinogenicity in Tylers Original (TO) and SENCAR strain of mice by repeated application and initiation/promotion regime. 16,17-Dihydro-11-hydroxy-15H-cyclopenta[alpha]phenanthrene was also tested for carcinogenicity in the TO strain by repeated application. Surprisingly with both of these compounds, which lack conjugation in ring D, one of the ingredients essential for biological activity appears to be carcinogenic. It is tempting to speculate whether these compounds undergo metabolic oxidation in the skin at the benzylic C-17 position.

Structure-activity relationships for epidermal ornithine decarboxylase induction and skin tumor promotion by anthrones.

The present study was designed to compare the skin tumor promoting and epidermal ornithine decarboxylase (ODC) inducing activities of various structural analogs of anthralin (1,8-dihydroxy-9-anthrone) and chrysarobin (1,8-dihydroxy-3-methyl-9-anthrone). Groups of 30 SENCAR mice each were initiated with 7,12-dimethylbenz[a]anthracene and 2 weeks later promoted with once- or twice-weekly applications of various doses of these anthrone derivatives. Carbon-10 (C10)-acyl derivatives of anthralin were active skin tumor promoters in the range of 25-440 nmol per mouse. 10-Acetylanthralin was significantly more active than 10-myristoyl-anthralin at low doses (e.g. 25 and 50 nmol per mouse) and nearly as potent as the unsubstituted compound. Higher doses (greater than or equal to 100 nmol per mouse) of this derivative were toxic, hence, reducing the final papilloma response. On a relative activity scale where anthralin is 1.0, these derivatives had activities that were approximately 0.7 and 0.2, respectively. 10,10-Dipropylanthralin was totally inactive at the doses tested. C6-Substituted derivatives of chrysarobin demonstrated diverse tumor promoting activities when tested in the range of 25-440 nmol per mouse. On a relative activity scale where chrysarobin is 1.0, 6-methoxychrysarobin (physcion anthrone) was approximately 0.9, whereas 6-hydroxychrysarobin (emodin anthrone) had no activity. Chrysophanic acid (1,8-dihydroxy-3-methyl-9,10-anthraquinone) was also inactive as a tumor promoter at the doses tested. In general, the tumor promoting activities of these anthrone derivatives correlated very well with their ability to induce epidermal ODC after a single topical application indicating an important role for this enzyme in skin tumor promotion by anthones. The ability of C10-substituted derivatives of anthralin to undergo base catalyzed oxidation in vitro correlated with both ODC inducing and tumor promoting activities. In addition, copper(II)bis(diisopropylsalicylate) was found to inhibit both ODC induction and skin tumor promotion by chrysarobin. These latter data, when taken together, suggest a role for oxidation at C10 in skin tumor promotion by anthrone derivatives.

Bay region distortions in cyclopenta[a]phenanthrenes.

Two newly synthesized cyclopenta[a]phenanthrenes, namely the 1-methyl (VIII) and 7,11-dimethyl (VII) derivatives of the parent ketone 15,16-dihydrocyclopenta[a]phenanthren-17-one (I), have been tested for their capacity to produce skin tumors in mice. The former (VIII) is essentially inactive, whereas the latter (VII) is very potent in both repeated application and two-stage tests. X-ray crystallographic structure analyses have been carried out on seven derivatives of (I), namely its 11-methyl (II), 11,12-dimethyl (III), 11-methoxy (V), 11-ethyl (VI) and 7,11-dimethyl (VII) analogues (carcinogens), the 1-methyl derivative (VIII), and 11,12,15,16-tetrahydro-11-methyl-17-oxocyclopenta[a]phenanthrene (IV) (both non-carcinogens). The detailed molecular structures resulting from these studies have shown the effects of steric interactions and substitutions on the bay-region geometry. The methyl group on C(11) causes distortions of the molecule in the bay region. Out-of-plane distortions in the bay regions of the 11-methyl derivatives (II, III, VII) are greater than for the 11-methoxy or the 11-ethyl derivatives (V, VI). Molecules (except for III and IV) are packed in the crystals with interactions that include C = O...H interactions; this packing is in layers that are nearly parallel to each other. A hydrogen atom of the 11-methyl group appears, from computer modeling, to interact sterically with the hydrogen atom of the bay-region expoxide group in the activated diol-epoxide; this steric interaction may force one conformer of the diol-epoxide to be the predominant form, thereby accounting for the importance of a bay-region methyl group. Further computer modeling has been used to analyze possible modes of interaction of the diol-epoxides of cyclopenta[a]phenanthrenes with DNA.

Comparison of the metabolism and DNA binding of the carcinogen 15,16-dihydro-11-methylcyclopenta[a]phenanthren-17-one by hamster embryo cells and the human hepatoma cell line HepG2.

Metabolism of 15,16-dihydro-11-methylcyclopenta[a]phenanthren-17-one with hamster embryo cells and a human hepatoma cell line HepG2 is compared. Essentially, no metabolism was seen with hamster embryo cells but with the HepG2 cells, especially after induction with benzanthracene or Arochlor, this carcinogenic ketone was metabolized to give 1,2-dihydroxy-11-methyl-1,2,15, 16-tetrahydrocyclopenta[a]phenanthren-17-one, 3,4-dihydroxy-11-methyl-3,4,15,16-tetrahydrocyclopenta[a] phenanthren-17-one, 15,16-dihydro-15-hydroxy-11-methylcyclopenta[a]phenanthren-17-one, 15,16,-dihydro-16-hydroxy-11-methylcyclopenta[a]phenanthren-17- one, and three new metabolites, 16,17-dihydro-11-methyl-4,15,17-trihydroxy-15H-cyclopenta[a]phe phenanthrene, 15,16-dihydro-16,17-dihydroxy-11-methylcyclopenta[a]phenanthrene and 16,17-dihydro-11-methyl-15H-cyclopenta[a]phenanthren-17-ol. The reduction of the 17-ketone group and the formation of phenols with HepG2 cells appears to be the major difference between metabolism of 15,16-dihydro-11-methylcyclopenta[a]phenanthren-17-one with HepG2 cells and rat liver microsomes. The metabolic products of this ketone also bind to DNA. These results suggest that the component of the aryl hydrocarbon hydroxylase enzyme system that is responsible for the activation of cyclopenta[a]phenanthrenes is specific and not the same as that needed for polycyclic aromatic hydrocarbons, and that HepG2 cells contain a reductase which is specific for the 17-ketone function in the cyclopenta[a]phenanthrenes.

Comparison of the binding of some carcinogenic and non-carcinogenic cyclopenta[a]phenanthrenes to DNA in vitro and in vivo.

After microsomal activation in vitro, both the strong carcinogen 15,16-dihydro-11-methylcyclopenta[a]phenanthren-17-one and its inactive 12-methyl isomer bind covalently to added DNA, in the ratio approximately 6:1 (1595 and 254 mu mol/mol of DNA phosphorus, respectively). Over a thousand times less binding was observed when DNA was isolated from the skin of groups of mice that had received a topical dose of 1000 nmol of these two compounds, and of the unsubstituted ketone (non-carcinogen) and its 11,12-dimethyl derivative (carcinogen), 48 h previously; the binding ratios were 458, 155, 19 and 974 nmol/mol DNA phosphorus, respectively. Covalent binding of the 11-methyl compound to mouse skin DNA, measured 48 h after topical application, was linear with dose over the range 50 - 1000 nmol. It has previously been demonstrated for groups of mice initiated with 200 or 400 nmol of this carcinogen and promoted by repeated application of croton oil that skin tumour incidences were 50 and 70%, respectively. For a topical dose of 1000 nmol of the inactive 12-methyl isomer DNA binding in skin falls in this range, equivalent to DNA binding given by approximately 340 nmol of the carcinogen. Thus although the inactive unsubstituted parent compound essentially fails to bind to skin DNA after topical application, there does not seem to be a consistent relationship between extent of DNA binding in vivo and carcinogenicity among these cyclopenta[a]phenanthrenes. However, loss of the 12-methyl adducts from skin followed a logarithmic course over the first 10 days following application, with a half life of 3.5 days. In contrast, it was previously shown that removal of the 11-methyl adducts could not be measured above the normal rate of DNA turnover for this mouse tissue (half life, 6-7 days). It is suggested that active DNA repair of the 12-methyl lesions may contribute to the lack of activity of this isomer.

The biological activity and activation of 15,16-dihydro-1,11-methanocyclopenta[a]phenanthren-17-one, a carcinogen with an obstructed bay region.

The proposal that an unobstructed bay region is a prerequisite for tumorigenic activity in cyclopenta[a]phenanthrene-17-ones is not supported by the observation of the tumorigenicity of 15,16-dihydro-1,11-methanocyclopenta[a]phenanthrene-17-one towards the skin of T.O. mice. The title compound is oxidised in vitro by a mixed function oxidase to produce, inter alia, a trans-3,4-dihydrodiol, postulated as the proximate tumorigen. Unequivocal identification of a second metabolite as a trans-1,2-dihydrodiol derivative demonstrates the potential for enzymatic oxidation within the obstructed bay region and supports the proposal that the ultimate tumorigen is a trans-3,4-dihydrodiol-anti-1,2-oxide. This is further substantiated by the chromatographic behaviour of the major hydrocarbon-nucleoside adduct derived from mouse skin treated with the parent compound in vivo. The structures of certain others of the metabolites produced in vitro are also considered.

Frequency of sister chromatid exchanges in human lymphocytes cultivated with a human hepatoma cell line as an indicator of the carcinogenic potency of two cyclopenta[a]phenanthrenes.

We demonstrate here that the carcinogen 15,16-dihydro-11-methylcyclopenta[a]phenanthren-17-one can cause sister chromatid exchange in human lymphocytes as well as it can cause mutation in bacterial cells and in V79 hamster cells. The non-methylated parent compound which has no tumorigenic action and yet significantly mutates both Salmonella typhimurium TA 100 and hamster V79 cells, has no effect on the frequency of sister chromatid exchange. These results support the idea that sister chromatid exchanges are a valuable additional indicator of tumorigenic potential.

Mutagenesis in Chinese hamster cells by cyclopenta(a)phenanthrenes activated by a human hepatoma cell line.

The cyclopenta(a)phenanthrene, 15,16-dihydro-11-methyl-cyclopenta(a)phenanthren-17-one, had potent mutagenic activity in cell-mediated mutation assays with V79 Chinese hamster cells as targets, and cells of the human hepatoma line HepG2 as mediators of activation. The compound was inactive when low-passage hamster embryo cells were used as activators. When the mutagenic activity of a series of cyclopenta(a)phenanthrenes was compared in mutation assays with HepG2 cells as activators, there was a good correlation between mutagenic activity in this system and carcinogenic activity in mouse skin in vivo. One exception was a noncarcinogenic compound, which is mutagenic in the Ames' test, and was also mutagenic in the mammalian cell assay.

Carcinogenicity and mutagenicity of some alkoxy cyclopenta[a]phenanthren-17-ones; effect of obstructing the bay region.

Six 11-O-alkoxy derivatives of the phenol 15,16-dihydro-11-hydroxycyclopenta[a]phenanthren-17-one were prepared and tested for their ability to initiate skin tumours on mouse skin after topical application, followed by promotion with croton oil. The 11-methoxy derivative was the most active, but was less so than the strong carcinogen 15,16-dihydro-11-methyl-cyclopenta[a]phenanthren-17-one (Ib). The 11-ethoxy derivative was somewhat less active as a tumour initiator than the 11-methoxy compound, and the 11-n-propoxy and 11-n-pentoxy derivatives were inactive. The 11-iso-propoxy and, surprisingly, the 11-n-butoxy compound possessed weak initiating activity. The phenol, which is too insoluble inorganic solvents to be tested in this way, proved to be about half as active as a skin tumour initiator compared with the 11-methyl derivative (Ib) when these compounds were injected s.c. in oil, and this was followed by topical promotion of dorsal skin remote from the site of injection. 15,16-Dihydro-15-methoxy-cyclopenta[a]phenanthren-17-one essentially lacked tumour initiating activity on mouse skin, but its 11-methyl homologue was moderately active. The carcinogenicity of the 11-alkoxy compounds was in general paralleled by their mutagenicity to Salmonella typhimurium TA100 in the Ames' test, with the exception of the 11-phenol which was not a mutagen under these conditions (plate assay).

High skin tumour initiating activity of the metabolically derived trans-3,4-dihydro-3,4-diol of the carcinogen 15,16-dihydro-11-methylcyclopenta[a]phenanthren-17-one.

Eight main metabolites of the parent carcinogen 15,16-dihydro-11-methylcyclopenta[a]phenanthrene-17-one (I) were assayed for their ability to initiate skin tumours in T.O. mice after topical application in two-stage experiments with croton oil used as the promoter. All were less active than 1 with the exception of the trans-3,4-dihydro-3,4-diol which was more than ten times as active. This diol is therefore confirmed as the proximate carcinogen, a conclusion reached previously on different evidence.

Co-carcinogenicity of promoting agents.

An experiment is described to investigate whether coadministration of a promoting agent would enhance the carcinogenicity of a repeatedly administered complete carcinogen. Topical application of the strong carcinogen 15,16-dihydro-11-methylcyclopenta[a]phenanthren-17-one (I) in toluene containing 1% v/v croton oil is about five times more effective than applications in toluene alone, as judged from the respective mean latent periods. A similar effect is also apparent for benzo[a]pyrene.

Mutagenic and carcinogenic metabolites of the carcinogen 15,16-dihydro-11-methylcyclopenta[a]phenanthren-17-one.

Microsomal metabolites of the carcinogen 15,16-dihydro-11-methylcyclopenta[a]phenanthren-17-one (Structure I) were separated by high-pressure liquid chromatography, and their structures were established on the basis of their ultraviolet and mass spectra, together with considerations of their general chemical properties. This was assisted by comparisons with metabolites formed in the same way from the synthetic 15-hydroxy (Structure III), 16-hydroxy (Structure II), and 11-hydroxymethyl (Structure IV) derivatives, which themselves occur as metabolites of Structural I. Products derived from attack at the two benzo-ring double bonds occurred, but no K-region products were found. Only metabolites having a non-bay region 3,4-dihydrodiol system were mutagenic and bound to DNA after in vitro microsomal activation, and it was concluded that the 3,4-dihydro-3,4-diol (Metabolite e) was the main form and that the 3,4-diols of the monools (Structure II to IV) were minor proximate forms of this carcinogen. In a two-stage experiment, the synthetic 16-ol (Structure II) was shown to be almost as carcinogenic as was Structure I itself in mice; the 15-ol (Structure III) and 11-hydroxymethyl derivative (Structure IV) were much less active. The same order was also observed in the mutagenicity of these compounds in the Ames test.

The carcinogenicity of 15,16-dihydro-11-methyl-cyclopenta[a]phenanthren-17-one.

Direct comparison of skin-tumour induction by 15,16-dihydro-11-methylcyclopenta[a]phenanthren-17-one (I) and by benzo[a]pyrene on mouse skin, both by repeated application or by initiation with a single dose followed by promotion with croton oil, demonstrated that these two carcinogens have similar potency. After repeated application of (I) the mean latent period for skin-tumour induction was linearly related to the logarithm of the dose over a 10-fold dose range. Under these conditions, application of the aryl-hydrocarbon-hydroxylase inhibitor 7,8-benzoflavone together with (I) inhibited tumour induction by about 40%. By contrast, in the 2-stage experiment, little effect on tumour incidence or latent period was observed when this inhibitor was applied with the single initiating dose of (I). Co-administration of the epoxide-hydratase inhibitor 1,1,1-trichloropropene oxide caused enhancement by shortening the latent period. After s.c. injection of (I) into mice, a similar number of tumours was induced on skin remote from the site of injection by promotion with corton oil begun either one week or 6 months after initiation. Gastric instillation of (I) into female rats induced mammary adenocarcinomas.

The relationship between metabolism, DNA binding, and carcinogenicity of 15,16-dihydro-11-methylcyclopenta(alpha)phenanthren-17-one in the presence of a microsomal enzyme inhibitor.

The mean latent period for skin tumor production by the carcinogen 15, 16-dihydro-11-methylcyclopenta [alpha] phenanthren-17-one (Compound IVb) in the mouse was 30 weeks for a dose of 60 mug/week and about 45 weeks for 60 mug/week, while at 0.6 mug/week, no tumors were observed during 100 weeks. Simultaneous administration of the closely related noncarcinogen (IVa) (54 mug/week) together with the carcinogen at 60 mug/week had no effect on the mean latent period. Simultaneous administration of a threefold quantity of the microsomal enzyme inhibitor 7, 8-benzoflavone (I) with the carcinogen at the highest dose increased the mean latent period to 38 weeks, while at the intermediate dose it completely suppressed tumor formation. Neither ketone IVa nor IVb bound covalently to calf thymus DNA in vitro without prior metabolic activation. After incubation with rat liver microsomes and NADPH in the presence of air, both ketones bound covalently to added DNA in vitro, the noncarcinogen (IVa) about four times more extensively than the carcinogen (IVb), roughly in proportion to the overall extents to which these ketones were metabolized. In contrast, overall metabolism of the carcinogen (IVb) was somewhat increased by the addition of a threefold quantity of the inhibitor (I) to the incubation mixture, but binding to added DNA was almost completely prevented. These results are discussed in connection with the hypothesis that cellular DNA is the target of the carcinogen (IVb) for tumor initiation.