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.

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.

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.

Effect of serum concentration on the cytotoxicity and sister chromatid exchange induction by a chemical carcinogen and by a diesel particulate extract in V79 hamster cells.

The role of serum concentration on the cytotoxicity and on the sister chromatid exchange (SCE)-induction by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and by a diesel particulate extract (DPE), a complex mixture, has been carried out on V79 cells. An increase of the serum concentration in the medium decreases the toxicity of these chemicals, and especially when they are dispersed first in serum. Although no influence of serum concentration on the number of spontaneous SCEs occurring in control cells has been observed, the increase of serum concentration leads to a decrease in SCE's induction in treated cells. Our results show that serum can protect cells from the cytotoxic and mutagenic action of MNNG and diesel extract.

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.

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.