Dose and frequency effect in mouse skin tumor promotion.

In order to study the influence of both dose and application frequency of tumor-promoting agents on tumor development, we conducted a large-scale mouse skin two-stage carcinogenesis experiment. The back skins of 1110 CD-1 mice were painted once with 50 micrograms benzo(a)pyrene. These mice were divided into 24 groups according to subsequent schedules of 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. Mice were treated with TPA at five different frequencies, i.e., daily, or every second, 4th, 8th, or 16th day, and six different TPA doses per application were used (0.1, 0.2, 0.4, 0.8, 1.6, or 3.2 micrograms), which allowed groups to be established with the same total dose of TPA applied per time unit. Six of the 30 frequency/dose combinations at extreme low or high frequency and dose were excluded. At each fixed frequency of TPA application, there was a good dose-response of TPA in mouse skin papilloma incidence. There was also a good application frequency-response relationship at fixed doses of TPA application. Within the set of groups in which animals received the same total dose of TPA per time unit, some variation was observed with respect to frequency of application. In general, TPA applications every 4th and 8th day tended to yield a small number of tumors.

Naphthofurans induced chromosomal aberrations detected in metaphase, anaphase and telophase V79 Chinese hamster cells.

The mutagenic activities of 5 newly synthesized naphthofurans were analysed in two in vitro cytogenetic assays: the metaphase chromosomal aberration assay and the anaphase telophase bridge-fragment assay. Both assays were conducted using V79 Chinese hamster cells. The compounds included: 2-nitro-7-methoxynaphtho[2,1-b]furan (A), 2-nitro-8-methoxynaphtho[2,1-b]furan (B), 2-nitro-naphtho[2,1-b]furan (C), 2-nitro-7-bromonaphtho[2,1-b]furan (D) and 7-methoxynaphtho[2,1-b]furan (E). The cells were treated with 3 concentrations (0.1, 0.2 and 0.4 microgram/ml) of each compound, in the dose range already tested in studies on the mutagenic properties of the same compounds realised with other systems. The highest concentration, only, was used in the anaphase-telophase assay. In the first approach, compounds A, B and C were active while compounds D and E did not increase significantly the aberration frequency above that of the DMSO controls. The results were confirmed in the second approach. They demonstrated that the two studies were complementary. Based on their genotoxic activities, the 5 compounds were ranked in the following decreasing order of potency: A congruent to B much greater than C greater than D congruent to E congruent to DMSO; which is comparable to the ranking order obtained in different in vitro mutagenic and carcinogenic assays. All these activities are closely related to the highly specific molecular structure of each compound, particularly to the nature and position of the different substituents introduced on the skeleton.

The in vitro micronucleus assay for detection of cytogenetic effects induced by mutagen-carcinogens: comparison with the in vitro sister-chromatid exchange assay.

The sensitivity of a cytogenetic assay, as expressed by the in vitro induction of micronuclei (MN), was compared to the in vitro induction of sister-chromatid exchanges (SCEs). Chinese hamster lung (V79) cells were exposed to 3 known alkylating agents: methyl methanesulphonate (MMS), ethyl methanesulphonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and to 5 newly synthesized naphthofurans: 2-nitro-7-methoxynaphtho[2,1-b]furan (A), 2-nitro-8-methoxynaphtho[2,1-b]furan (B), 2-nitronaphtho[2,1-b]furan (C), 2-nitro-7-bromonaphtho[2,1-b]furan (D) and 7-methoxynaphtho[2,1-b]furan (E). The induction of MN only was also analysed after exposure of the cells to 4 alcohols: ethanol, methanol, butanol and propanol. The lowest dose at which a significant effect could be observed was determined. In both assays, MNNG, MMS and EMS were equally active with the following order of potency: MNNG greater than MMS greater than EMS, the latter being a very weak inducer of MN and SCE. Compounds A and B were also very effective in both assays. Compound C was a more active inducer of SCE than MN. Compounds D and E were not active in either assay. None of the 4 alcohols induced MN. Our results are compared with the previously published data on in vitro and in vivo induction of SCE and MN. We conclude that the MN in vitro assay which detects clastogens as well as agents affecting the spindle apparatus, is a good indicator of genotoxicity, though slightly less sensitive than the in vitro SCE test. It could provide a rapid, simple and inexpensive complementary short-term test for the evaluation of potentially mutagenic chemicals.

Characterization and identification of 6 mutagens in opium pyrolysates implicated in oesophageal cancer in Iran.

Previous epidemiological studies have indicated an association between the ingestion of opium pyrolysates, dietary deficiencies, and a high incidence of oesophageal cancer in subjects in north-east Iran. Laboratory studies have shown that pyrolysates of opium and particularly of morphine, a major opium alkaloid, are highly mutagenic in bacteria and induce sister-chromatid exchanges in mammalian cells after metabolic activation. We now report the ability of these pyrolysates to transform Syrian hamster embryo cells in culture and present some evidence for their carcinogenicity in mice and hamsters following topical, subcutaneous, intratracheal and intragastric administration. 6 of the most abundant mutagenic compounds present in morphine pyrolysate were isolated and purified by high-performance liquid chromatography and characterized by gas chromatography/mass spectrometry and 1H-Fourier transform nuclear magnetic resonance spectroscopy. These hitherto unknown compounds, all containing a hydroxy-phenanthrene moiety, were identified as: 3-methyl-3H-naphth[1,2-e]indol-10-ol; 1,2-dihydro-3-methyl-3H-naphth[1,2-e]indol-10-ol; 6-methylaminophenanthren-3-ol; 2-methylphenanthro[3,4-d] [1,3]oxazol-10-ol; 2,3-dimethyl-3H-phenanthro[3,4-d]imidazol-10-ol and 2-methyl-3H-phenanthro[3,4-d]imidazol-10-ol. Mutagenicity in Salmonella typhimurium TA98 of these compounds increased in the order listed, the last compound being 35 times more active than benzo[a]pyrene. The mechanisms, by which these mutagens are formed and metabolically activated are discussed.

Two-stage (initiation-promotion) carcinogenesis in vivo and in vitro.

After a short review on the two-stage (initiation and promotion) chemical carcinogenesis in vivo, the conditions required for its demonstration have been reported with several experimental data, showing the role of two distince agents, the initiator and the promotor. The same phenomenon has also been demonstrated with cell cultures in vitro. Rat embryo cells treated with an initiator and then submitted to the continuous action of a promotor have been transformed more rapidly than the untreated cells. Transformation has been tested essentially by the tumorigenicity of the cells. The results indicate that tissue culture cells are an appropriate system to investigate the mechanism of two-stage chemical carcinogenesis and allow the detection of the initiating and promoting potentialities of various compounds.

Sensitivity of the skin of different mouse strains to the promoting effect of 12-0-tetradecanoyl-phorbol-13-acetate.

The sensitivity of the skin of Swiss DBA/2 and C57B1/6 mice was compared in two-stage carcinogenicity experiments. Groups of 30 mice were treated once with 7,12-dimethylbenz(a)anthracene (DMBA) (50 micrograms/mouse) which was applied to the shaved dorsal skin as initiator and, starting one week later, 12-0-tetradecanoyl-phorbol-13-acetate (TPA) was applied thrice-weekly at doses of either 0.04, 0.16 or 0.64 micrograms/mouse to the areas of initiated skin. Other groups of mice were similarly treated with TPA (0.64 micrograms) alone. The times at which papillomas and carcinomas first appeared were recorded. Overall the results show, in terms of the numbers of animals with tumours, the total numbers of tumours produced and the numbers of malignant tumours formed all in dose-response relationship, that the Swiss mice were the most sensitive and the B57B1/6 mice the least sensitive to the tumour-promoting effects of TPA with the DBA/2 mice occupying and intermediate position. This relationship also held in the control groups that were treated with TPA alone.

Inhibition of chemically-induced skin carcinogenesis in mice by peanut oil preparations.

Epidermal hyperplasia and sebaceous gland destruction - good indicators of carcinogenic potential - were studied in short-term mouse skin experiments following application of BaP and TPA dissolved either in a peanut oil mixture or in acetone. Subsequently, the carcinogenicity of BaP and DMBA alone or in association with TPA was dissolved in the same vehicles, and determined in mouse long-term skin tests. In parallel, ODC activity and binding to DNA, RNA and proteins were examined in epidermal cells after exposure to TPA and BaP respectively. When the peanut oil excipient was used as a solvent, a complete inhibition of BaP and TPA activities was observed in short-term skin tests, as well as a complete inhibition of BaP, DMBA and TPA carcinogenicity in long-term tests. TPA-induced ODC activity was suppressed by the peanut oil mixture while BaP binding to nucleic acids and proteins of epidermal cells was only slightly inhibited. These results indicate that the excipient possesses anti-carcinogenic potentials for epidermal cells. The persistence of BaP binding to macromolecules in epidermal cells without tumor development suggests that the carcinogenic action of BaP may include both genotoxic and epigenetic mechanisms.

[Chemical carcinogenesis in tissue culture: criteria and transformation tests]. / Cancérogénèse chimique en culture de tissus: criteres et test de transformation

We belive that there are four criteria for the evaluation of cell transformation in culture: development of transformed colonies, appearance of altered foci when cells sensitive to contact inhibition are used, formation of colonies in agar, and the capacity to induce tumors in animals (tumorigenic potentiality). The formation of transfomred colonies provides a short term (t. of BERWALD-SACHS) to determine the transformating activity of chemical carcinogens. Judging by the published results there is a good correlation between this in vitro test and in vivo results. Nevertheless this test does not seem to me appropriate to studies on the activity of complex mixtures. But the use of a short term test to monitor transformation in the course of long term tests does seem justified and useful. The formation of altered foci is the basis of two intermediate term tests (t. of CHEN-HEIDELBERGER, and t. of HUEBNER'S group). The value of these two tests have been experimentally proven, but their only disadvantage is the necessity of using cells that are not readily available. Formation of colonies in agar is a criterion useful only for the control of cell transformation during long term tests. Tumorigenicity constitutes a long term test. It is the absolute proof of cell transformation. We propose a long term experimental system combined with periodic checks of transformation by cell cloning in liquid medium (criteria of the transformed colonies or altered foci), and by cloning in agar. The reported results show that it is possible to determine cellular transformation accurately without recourse to the animal. This combined long term test permits the classification of treatments based on their transformating capacity.

In vitro induction of sister-chromatid exchanges after G0 exposure of human lymphocytes to five naphthofurans.

The induction of sister-chromatid exchanges (SCE) was studied in cultured human lymphocytes of two donors treated in G0 by five naphthofurans. The results showed that 2-nitro-7-methoxynaphtho(2,1-b)furan and 2-nitro-8-methoxy-naphtho(2,1-b)furan were very potent inducers of SCE, 2-nitronaphtho(2,1-b)furan was moderately active, while 2-nitro-7-bromonaphtho(2,1-b)furan and 7-methoxynaphtho(2,1-b)furan did not induce any SCE. These data corroborate the mutagenic, clastogenic and carcinogenic activities already evaluated in other in vitro and in vivo assays. They also show that some naphthofurans, but not all, are able to induce long-lived DNA lesions, like most alkylating agents.

Metabolism and cytotoxicity of 7,12-dimethylbenz[a]anthracene by hamster, rat and rabbit embryo cell cultures.

1. The metabolism of 7,12-dimethylbenz[a]anthracene (DMBA) by rat, hamster and rabbit fibroblasts has been studied and compared with the metabolism of the hydrocarbon by liver homogenates. The metabolsim of DMBA by cell cultures and by liver homogenates was very similar. The ethyl acetate-soluble metabolites identified included dihydrodiols, phenols and hydroxymethyl derivatives. Other more polar metabolites and unidentified water-soluble metabolites were also formed. 2. High yields of phenols and other more polar metabolites, which may be tetrahydrotetrols, were produced by rabbit fibroblasts. 3. Kinetic studies showed that the metabolic activity of rabbit fibroblasts was high but that the conversion of DMBA into water-soluble metabolites was lower than with hamster and rat fibroblasts. 4. 7,12-Dimethylbenz[a]anthracene-induced cytotoxicity was inversely related to the conversion of metabolized DMBA to water-soluble derivatives.

Two-stage carcinogenesis with rat embryo cells in tissue culture.

Transformation of rat embryo fibroblasts in vitro has been investigated using initiation with either benzo(a)pyrene (BaP), 7,12-dimethylbena(a)anthracent (DMBA) or benzo(e)pyrene (BeP) and promotion with either phorbol ester (TPA) or croton oil (Cr.Oil). The criteria used to assess in vitro transformation were (a) the efficiency of cloning in liquid medium, (b) abnormal cellular morphology and (c) the development of malignant tumours following s.c. inoculation of newborn rats. The results show that the cloning efficiency, which remained low in the control cells, was increased to a variable extent in the treated groups. Transformation occurred in all groups, but occurred earliest in cells that were initiated and promoted. Initiation with DMBA or BaP and promotion with TPA or Cr.Oil led to the earliest acquisition of malignancy. Correlations were found between the transformation of cells in vitro and the acquisition of malignant potential, and between the carcinogenic action of the compounds in vitro and their action in vivo, but cloning efficiency was not a reliable indicator of in vitro transformation or of malignancy. In most cases in vitro transformation appeared to precede the acquisition of malignancy, but in two cases it occurred later. The studies also show that BeP, which is a tumour initiator in vivo, also acts in this way in vitro. The conclusion drawn from a discussion of these results and of two-stage carcinogenesis in vivo is that two-stage carcinogenesis can be reproduced in tissue culture; this model may be useful in studies of those mechanisms of chemical carcinogenesis that involve the processes of initiation and promotion.

[Carcinogenic effect of 7,12-dimethylbenz(a)anthracene 5,6-oxide in mice]. / Action concérogène du 7,12-dimethylbenz(a)anthracène 5,6-oxyde chez la Souris

The comparative carcinogenic activities of 7,12-dimethylbenz(a)anthracene 5,6-oxide (DMBA-5,6-oxide) and 7,12-dimethylbenz(a)anthracene (DMBA) to skin and subcutaneous tissue were studied. It was found that DMBA-5,6-oxide was less carcinogenic to both the skin and subcutaneous tissue, than DMBA. Moreover it does not show any significant initiating acitivity. The results are discussed.

Benzo (a) pyrene uptake by normal and by tumour cells in vitro.

A method for determining the quantitative uptake of benzo(a)pyrene/B(a)P/by cells in culture is described. B(a)P uptake is very rapid and increases with concentration. The amount of B(a)P taken up by normal or by tumour cells depends on the cell type, and also, on the species from which they were derived.

Use of the orthogonal design method to study the synergistic effects of asbestos fibres and 12-O-tetradecanoylphorbol-13-acetate (TPA) in the BALB/3T3 cell transformation system.

An orthogonal design method was used to study the transforming effects of chrysotile and crocidolite asbestos fibres in BALB/3T3 cells. Three experiments, designed by tables L9 (3(4)), L8 (2(7)) and L6 (3(1) x 2(2)) of the orthogonal method respectively, were performed separately. The results indicate exponential reductions in survival of treated cells concomitant with a linear increase in exposure concentrations from 0.1 to 10.0 micrograms/cm2, and that chrysotile was more toxic than crocidolite; the total transformation frequency was significantly increased with both chrysotile and TPA concentrations. There was synergism between chrysotile and TPA in sequential treatment, which suggests that chrysotile is an initiator and has a complete transforming effect at 10.0 micrograms/cm2. Crocidolite only has an initiating-like effect within the dose range of 0.1-10.0 micrograms/cm2, and no synergistic effect when associated with TPA.

Transforming activities of sodium fluoride in cultured Syrian hamster embryo and BALB/3T3 cells.

The transforming activity of sodium fluoride was studied in the SHE and the BALB/3T3 cell culture systems. Initiating and promoting activities were then investigated by means of the orthogonal methodology. Sodium fluoride was found to induce morphological transformation of SHE cells seeded on a feeder layer of X-irradiated cells at high concentrations (75-125 micrograms/ml). When the cells were seeded in the absence of a feeder-layer, the transformation frequencies increased in a dose-dependent manner with the concentrations of sodium fluoride ranging from 0 to the highly toxic concentration of 200 micrograms/ml. In the BALB/3T3 cell system, sodium fluoride was negative in the standard Kakunaga procedure, while through the experiment designed by table L8 (2(7] of the orthogonal method, an initiating-like effect and a weak promoting activity were detected within the concentrations ranging from a 25 micrograms/ml to a 50 micrograms/ml concentration which is highly toxic for BALB/3T3 cells. From these results, it is suggested that, besides a genetic mode of action, sodium fluoride could possibly act through a non-genotoxic mechanism.

Morphological transformation in three mammalian cell systems following treatment with 6-nitrochrysene and 6-nitrobenzo[a]pyrene.

Two nitroaromatics, 6-nitrobenzo[a]pyrene (6-N-BaP) and 6-nitrochrysene (6-N-CRY), and the corresponding parent hydrocarbons, benzo[a]pyrene (BaP) and chrysene (CRY), were studied in in vitro transformation assays with Syrian hamster embryo (SHE) cells, BALB/3T3 and C3H10T1/2 mouse cell lines. The three cell systems showed different sensitivities to the transforming effects of the chemicals studied, SHE cells being the most efficient, followed by 3T3 cells and the last being C3H10T1/2 cells. In the SHE cell system all compounds were active. Considering the concentrations (in microM) and the transformation frequency BaP was the most active, followed by 6-N-BaP, 6-N-CRY and CRY. In the BALB/3T3 standard assay and in the C3H10T1/2 assay only BaP was clearly active. When used as initiators 6-N-BaP and 6-N-CRY were inactive in the C3H cell system. In conclusion 6-N-BaP appears less active in in vitro systems than the parent compound BaP; 6-N-CRY is probably negative since it is questionable in vitro and negative in mouse skin.

Use of an orthogonal design method to study two-stage chemical carcinogenesis in BALB/3T3 cells.

An orthogonal design method was used to study two-stage chemical carcinogenesis in BALB/3T3 cells. Four factors were studied: different concentrations of the initiator N-methyl-N-nitro-N'-nitrosoguanidine (MNNG); different concentrations of the promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA); different concentrations of fetal calf serum (FCS) and different times at which TPA was added after cell initiation. From the results of three experiments designed by Table L9(3(4)), L8(2(7)) and L16(4(5)), 0.3 microgram/ml MNNG was the highest possible initiating concentration and 0.25 microgram/ml TPA was the minimum effective concentration for promoting activity. There is synergy between MNNG and TPA, the optimum combination in sequential treatment being 0.3 microgram/ml MNNG and 0.25 microgram/ml TPA. The 10% FCS standard concentration was the optimal one; however, below 5% few foci appeared. The time at which TPA was added had little effect on cloning efficiency and transformation frequency. So the use of this orthogonal design in cell culture has many advantages: several factors can be tested simultaneously; it is easy to find the optimal protocol conditions and the dose-response relationship is stable, which enables the reproducibility to be improved. In addition, the different tables proposed may help to reveal unexpected problems.