Formation and persistence of DNA adducts in epidermal and dermal mouse skin exposed to benzo(a)pyrene in vivo.

Tritiated benzo(a)pyrene was applied topically to 35 old male Swiss mice that received 250 nMole (0.63 mCi) per mouse in 150 microliters acetone. At each time point of study, 1, 3 and 7 days, 10 animals were killed, the skin was removed and the susceptible epidermic cells were separated from resistant dermis. The initial level of the total BaP-DNA adduct after 1 day of test was 2 fold higher in epidermic cells; in addition, the concentration of the individual modified deoxyribonucleoside adducts was 4 fold greater in epidermis. However, the nature and the repartition of the modified nucleosides analyzed by high performance liquid chromatography were similar. They were 5 fold more in epidermic cells, except for the (+/-) SynBaP- diolepoxide - deoxyguanosine adduct which was 2 fold more in dermic cells. The major DNA adduct formed in both types of cells was the (+) antiBaP diolepoxide - deoxyguanosine = (+) anti BPDE-dG with 75% of total adduct in epidermic and 55% in dermal cells. The decreased amount of BaP bound to DNA of epidermic and dermic cells may be similar and 90% of (+) anti BPDE-dG was removed after a week of treatment; in addition, a minority that bound with 9OH-BaP was also shown to be persistent. Although the persistence of adduct was 7 fold more important in susceptible epidermal cells than in the resistant dermic population, the nature of adduct repartition and the similar type of excision suggest that other mechanisms are also involved in the biologically different response of epidermis versus dermis to the carcinogenic BaP when it is applied topically.

[Specific induction by phorbol ester of the gene transcription and of the activity of ornithine decarboxylase in two control and transformed epithelial cell lines. Modulator effect of anti-inflammatory agents]. / Induction spécifique par l'ester de phorbol de la transcription du gène et de l'activité d'ornithine décarboxylase dans deux lignées de cellules épithéliales témoins et transformées. Effet modulateur des agents anti-inflammatoires.

The mechanism of ornithine decarboxylase (ODC) induction by phorbol ester (TPA) has been studied in two permanent epithelial cell lines, a control (Ctr) and a Benzo (a) pyrene transformed line (BaP-tr); the degree of ODC gene expression (ODC-mRNA) was evaluated in comparison to the ODC activity. A small dose of TPA (4 x 10(-8) M) highly induced ODC activity in these cells. The induction levels differed however, corresponding respectively to 4:1 (induced: basal ODC) in Ctr cells and to 2:1 in BaP-tr cells. This difference reflected the variation of ODC gene expression; the ODC-mRNA induction was 6:1 in Ctr cells and 3:1 in BaP-tr cells. Repetitive TPA treatment decreased the ODC induction in these cells, as compared to that resulting from a single TPA treatment. Studies of ODC modulation were performed in presence of anti-inflammatory agents. In the two cell lines, Indomethacin (anti-cyclooxygenase) did not change the level of ODC induction by TPA. Nordihydroguaiaretic acid (NDGA, anti-lipoxygenase) inhibited this induced ODC. These results differed from that obtained in vivo in mouse skin. Dexamethasone (DXME, anti-phospholipase A2) showed different action according to treatment time. Used together with TPA (t0), DXME inhibited ODC induction by the carcinogen; with three hours delay after TPA (t3), DXME treatment stimulated ODC in the cells. This divergent action may be reproduced by Actinomycin D, while Cycloheximide only exhibited constant inhibition. Studies now in progress suggested that the inhibition of TPA induced ODC by DXME may reflect ODC gene repression, as for the stimulating effect it could be related to ODC post-transcriptional modulation, owing to the decrease of proteolytic action.

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.

1-Chloromethylpyrene: a reference skin sensitizer and genotoxin.

1-Chloromethylpyrene (1-CMP) has been evaluated as a model mutagen and toxin related to the ultimate electrophiles derived from benzo[a]pyrene and 1-nitropyrene. It was mutagenic to Salmonella (greater than 100 pg/plate) and exceptionally reactive to DNA when assessed by the 32P-postlabelling technique. 1-CMP was inactive in a mouse bone micronucleus assay when administered by gavage, probably due to hydrolysis, whose kinetics have been studied (t1/2 approximately 23 min at 37 degrees C). However, as expected, it was a potent skin toxin as determined by its activity as a mitogen to mouse skin and its contact allergenicity, as determined using the local lymph node proliferative assay. It is concluded that 1-CMP will probably be a potent human skin carcinogen and contact allergen.

Study of various transforming effects of the anabolic agents trenbolone and testosterone on Syrian hamster embryo cells.

Trenbolone, a synthetic androgen together with testosterone, a natural androgen, were studied comparatively for their transforming effects on Syrian hamster embryo (SHE) cells. The data indicated that both androgens exhibited weak positive complete transforming activities without a dose response relationship. Trenbolone is more toxic than testosterone when the concentrations tested are higher than 10 micrograms/ml, but is less able to transform SHE cells. Medium H21 offers a higher transformation frequency than medium H16. Their transforming effect can be amplified by TPA. However, both products can also reduce the transforming effect of benzo[a]pyrene (B[a]P) either in sequential treatment or when mixed together. The transforming effects of the two androgens including TPA effects can be inhibited completely by dexamethasone, which suggests that such transformation in SHE cells is an epigenetic effect. In conclusion, trenbolone and testosterone themselves exhibit a weak transforming effect on SHE cells, predominantly as promoting potential, especially when associated with 12-O-tetradecanoyl-phorbol-13-acetate, which is related to hormonal action. They also exhibit weak anti-transforming effects when associated with B[a]P.

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.

Epidermal cell proliferation and modulation of the protective potency of dexamethasone against phorbol ester-induced ornithine decarboxylase activity.

Treating mouse skin with dexamethasone (DXME, 1 mumol) after a single TPA (3.25 nmol) application, inhibited both the dermal inflammatory reaction and the induction of epidermal ornithine decarboxylase (ODC) activity. At the hyperplastic stage, DXME was active against inflammation, though inhibited weakly the induction of ODC. In DXME-protected skin, the hyperplastic stage was delayed; unexpectedly, before that stage, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced strongly ODC activity in the epidermal cell layer. Provided that the proliferation process was induced, epidermal cells were increasingly sensitive toward TPA action; they may have been less dependent on inflammatory factors which may modulate the induction of ODC.

Effect of long-term ingestion of asbestos fibres in rats.

The effects of ingested asbestos fibres were studied in Wistar Han rats. Chrysotile and a mixture of chrysotile/crocidolite (75%/25%) in palm oil were given for 24 months to 70 males and 70 females per group (daily doses 10, 60 and 360 mg); one control group was fed with normal diet, a second with normal diet plus palm oil. The animals were observed for a further 6 months after the end of the treatment. The results indicate that ingestion of asbestos fibres at high doses had no toxic effects and did not affect animal survival; in addition, there was no evidence of carcinogenic effects.

Biological effects of asbestos fibres on rat lung maintained in vitro.

The in vitro systems used to determine whether asbestos acts as an initiator or as a promoter have failed to give definitive answers. We studied the effect of chrysotile and crocidolite in an initiation-promotion model on the Fischer rat embryo lung. Two assay systems were used in succession: organ culture of the lung cultured for 24 days and epithelial cell culture derived from treated or untreated explants cultured for 25 passages. Apart from the control groups, three major groups were analysed: (1) fibres with complete carcinogenic potency: explants and/or cells treated with fibres alone; (2) fibres with initiating potency; short treatment with fibres, followed by treatment with the classical promoter TPA; (3) fibres with promoting potency: short benzo[a]pyrene treatment followed by treatment with the fibres. In organ culture, fibres alone induce only cytotoxic lesions; in the 'fibres with promoting potency' group, precancerous lesions were observed. In epithelial cell culture, several transformation criteria are analysed. Our results with the cell system confirm that fibres act as a promoter, but also as a complete carcinogen. However, for equal doses, crocidolite needs a longer treatment time than chrysotile. These different assays failed to demonstrate any initiating activity of the fibres. The use of organ and cell culture in succession makes it possible to demonstrate the in vitro promoting effect of chrysotile and crocidolite.

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.

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.

Sister chromatid exchanges and chromosomal aberrations in lymphocytes of nurses handling cytostatic agents.

A cohort study of 29 nurses who constantly handled cytostatic drugs, and 29 controls matched according to sex and age, was carried out between 1983 and 1986. Cytogenetic damage was assessed by sister chromatid exchanges (SCE) and chromosomal aberrations. No significant increase in mean number of SCE was found for nurses (7.37) as compared to matched controls (7.00), whereas a significant excess of SCE (p less than 0.001) was observed for smokers (8.23) as compared to non-smokers (6.75). The number of SCE was studied in relation to the amount and nature of cytostatics handled as well as to the duration of exposure. A significant association (p less than 0.05) was found between individual mean number of SCE and the total number of drugs handled after adjustment for confounding factors. In contrast, the number of SCE was not significantly related to the nature of drugs handled or to the duration of exposure. With regard to chromosomal damage, no significant difference was observed between nurses and controls in gap, break, dicentric and translocation frequencies.

[Epigenetic mechanism of tumor promotion. Interrelationship between inflammation and ornithine decarboxylase activity induced in vitro by the carcinogenic 12-O-tetradecanoyl-phorbol-13-acetate]. / Mécanisme épigénétique de la promotion tumorale. Interrelation entre l'inflammation et l'activité d'ornithine décarboxylase induites in vitro par le cancérogène 12-O-tetradecanoyl-phorbol-13-acetate.

Topical application of 2 micrograms 12-O-tetradecanoyl-phorbol-13-acetate (TPA) regularly induced two early events in mouse skin: inflammatory reaction localized in dermal compartment and stimulation of ornithine decarboxylase activity in epidermal cells, in relation to polyamine synthesis and cell division. These reactions were followed, after 48 hrs. by an epidermal hyperplasia. At this stage, another TPA treatment induced a strong ODC activity concurrent with severe inflammation of the dermis. Inhibition of the synthesis of inflammatory factors may antagonize TPA-induced ODC, but the protective potencies differs according to the evolutive stages of the cell. After the first TPA treatment the anti-inflammatory compounds dexamethasone and indomethacin effectively inhibited ODC activity. In contrast during the proliferative stage epidermal cell function may be less dependent on inflammatory factors; thus only partial protection was observed with the inflammatory inhibitors.

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.

Initiating, promoting and carcinogenic activities of three naphthofurans in a mouse skin long-term study.

The initiating, promoting and carcinogenic activities of three naphthofurans, 2-nitro-7-methoxynaphto[2,1-b]furan (A), 2-nitronaphtho[2,1-b]furan (C) and 7-methoxynaphtho[2,1-b]-furan (E), were determined in a long-term assay using CD1 mice, by means of a two-step skin painting regimen. Compound A was a strong initiator and a weak carcinogen, and compound C was a strong promotor and a moderate carcinogen, whereas compound E did not have any effect. The NO2 group at the 2 position on the molecule was concluded to be responsible for the carcinogenic activity of the former two naphthofurans. The addition of a CH3O group at position 7 enhanced the initiating ability while it diminished the promoting and carcinogenic potentials. These findings indicate that the initiating activity is closely linked to the mutagenic potential previously detected in in vitro and in vivo mammalian cell systems, while the promoting and carcinogenic activities are related to the effects detected in mouse short-term skin tests.

Cytotoxic potential of ketonucleosides.

The relationship between the structure of ketonucleosides and cytotoxicity was investigated in Friend leukemia cells (FLC). When cells were grown in the continuous presence of ketonucleosides, it was shown that the addition of an electrophilic agent (Br-) to the sugar moiety (compound KN-35) increased the cytotoxic potential by ten fold as compared to the unsubstitute compound (KN-43). In contrast, addition of 0-acetyl group (compound KN-3) reduced this effect by three fold. When cells were pre-treated with KN-35, followed by growth in drug-free medium, cell survival was inhibited by 50% (ID50) after 3 min, whereas the same effect was reached after 240 min pre-treatment with KN-43. When drugs were pre-incubated in serum-free medium prior to cell exposure, reduced cytotoxicity was observed. We therefore conclude that the activity of these ketonucleosides may be related to the rate of in fact drug incorporation.

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.

Absence of genotoxic effects in cells exposed to four ketonucleoside derivatives.

The relationship between the structure and genotoxic potential of four new cytostatic compounds--the ketonucleosides KN-35, KN-43, KN-44 and KN-3--was investigated in short-term in vitro assays of hypoxanthine-guanine phosphoribosyl transferase locus mutation in V79 cells, induction of chromosomal aberrations and sister chromatid exchanges on human lymphocytes, induction of chromosomal aberrations and micronuclei in V79 cells, and transformation of Syrian hamster embryo cells. None of the ketonucleosides induced mutagenic effects in any of the assays. Their failure to exhibit significantly genotoxic activity may be ascribed to the probable absence of any reaction between these drugs and the cellular DNA, and indicates that they act by some other mechanism which probably differs from the one observed with alkylating or intercalating antitumoural agents. This suggests that the cytotoxic activity of ketonucleosides cannot be related to genotoxicity.

In vitro and in vivo activity of 12-O-3-N-dansylamino TPA.

Dansyl-TPA, a fluorescent TPA analogue, which is a label with a high affinity for C3H/10T1/2 cells and induces 3H-choline release from these cells (Tran et al. Nouv. J. Chim 1984, 8, 751-757), has been studied for in vitro promoting activity in the same cell line initiated by a carcinogen MNNG and in vivo short-term mouse skin tests. In vitro, dansyl-TPA expresses transforming effect in its own (without MNNG pretreatment) as well as increases the production of transformed foci in MNNG-treated cells. In in vivo skin tests, dansyl-TPA displays lower effects than TPA on mouse skin. These results indicate a low promoting potential of dansyl-TPA.