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.

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.

Benzo(e)pyrene-induced alterations in the binding of benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene to DNA in Sencar mouse epidermis.

Benzo(e)pyrene [B(e)P] cotreatment slightly increases the tumor-initiating activity of benzo(a)pyrene [B(a)P] and greatly decreases the tumor-initiating activity of 7,12-dimethylbenz(a)anthracene (DMBA) in Sencar mice (DiGiovanni et al., Carcinogenesis 3: 371-375, 1982). The effects of B(e)P on the binding of B(a)P and DMBA to Sencar mouse epidermis were investigated using a protocol similar to the mouse skin tumorigenicity studies. After 12 h of exposure to 50 nmol [3H]B(a)P and low or high doses of B(e)P, the level of [3H]B(a)P bound to mouse epidermal DNA increased by 30%. However, after 24 h exposure to 50 nmol [3H]B(a)P and after 12 or 24 h of exposure to 200 nmol [3H]B(a)P, B(e)P had no effect on the amount of [3H]B(a)P bound to DNA. The ration of anti-(the isomer with the epoxide and benzylic hydroxyl on opposite faces of the molecule) B(a)P-7,8-diol-9,10-epoxide [B(a)PDE]-deoxyribonucleoside adducts to syn- (the isomer with the epoxide and benzylic hydroxyl on the same face of the molecule) B(a)PDE-deoxyribonucleoside adducts did not change at either initiating dose of B(a)P or at any time regardless of the dose of B(e)P. After 12 h of exposure to high doses of B(e)P and a 50-nmol initiating dose of B(a)P the level of [3H]B(a)P bound to DNA increased but there was no change in the proportion of particular B(a)PDE-deoxyribonucleoside adducts present. In contrast, B(e)P inhibited the binding of initiating doses of DMBA (5 and 20 nmol) to DNA after 12 and 48 h of exposure to all dose ratios of B(e)P:DMBA tested. The three major adducts, tentatively identified as anti-DMBA-3,4-diol-1,2-epoxide (DMBADE):deoxyguanosine, syn-DMBADE:deoxyadenosine and anti-DMBADE:deoxyadenosine, decreased to the same relative extent as the dose of B(e)P increased. Thus, the effects of B(e)P on the total binding of these hydrocarbons to DNA in epidermis correlate with the cocarcinogenic and anticarcinogenic effects of B(e)P on B(a)P and DMBA, respectively, in a mouse skin initiation-promotion assay. These results indicate that the mechanism of the co- or anticarcinogenic action of hydrocarbons such as B(e)P involves alteration of the binding of carcinogenic hydrocarbons to DNA. They also suggest that measurement of carcinogenic hydrocarbon-DNA adducts formed during cotreatment with other hydrocarbons will provide a rapid method for predicting the co- or anticarcinogenic effect of the other hydrocarbons.

Correlation between formation of a specific hydrocarbon-deoxyribonucleoside adduct and tumor-initiating activity of 7,12-dimethylbenz(a)anthracene and its 9- and 10-monofluoroderivatives in mice.

The formation of epidermal DNA adducts from 9-fluoro-7,12-dimethylbenz(a)anthracene (9-F-DMBA) was compared with 7,12-dimethylbenz(a)anthracene (DMBA) and 10-fluoro-7,12-dimethylbenz(a)anthracene (10-F-DMBA) in SENCAR mice. 9-F-DMBA is equipotent, whereas 10-F-DMBA is more potent than DMBA for skin tumor initiation in this mouse stock. The quantity of covalently bound DNA adducts was essentially identical between 9-F-DMBA and DMBA at all doses tested in the range of 10 to 100 nmol/mouse. These results correlated closely with the dose-response relationships for tumor initiation by the two hydrocarbons. A quantitative comparison of the hydrocarbon-DNA adducts formed after topical application of 100 nmol of DMBA, 9-F-DMBA, and 10-F-DMBA yielded interesting results. The total binding for the three hydrocarbons at this dose was 16.2 +/- 2.6, 18.4 +/- 2.4, and 52.3 +/- 6.8 pmol/mg of epidermal DNA, respectively. Analysis of these DNA adduct samples by dihydroboronate chromatography demonstrated marked reductions in the percentage of syn-diol-epoxide-DNA adducts with both 9-F-DMBA (24%) and 10-F-DMBA (18%) compared with DMBA (57%). Analysis of DNA adduct samples from DMBA-, 9-F-DMBA-, and 10-F-DMBA-treated mice (100 nmol/mouse) by high-pressure liquid chromatography revealed qualitatively similar profiles. However, a quantitative comparison of the three major DNA adducts, tentatively identified as anti-diol-epoxide-deoxyguanosine (Peak I), syn-diol-epoxide-deoxyadenosine (Peak II), and anti-diol-epoxide-deoxyadenosine (Peak III), revealed significant differences. With both 9-F-DMBA and 10-F-DMBA there were marked increases (236% and 644%, respectively) in the quantity of Peak I compared to DMBA. On the other hand, Peak II was formed in approximately equal amounts with DMBA and 10-F-DMBA but only 50% of the DMBA value with 9-F-DMBA. Interestingly, Peak III was formed in approximately equal amounts with both DMBA and 9-F-DMBA but was increased to 337% of the DMBA value with 10-F-DMBA. Thus, the actual level of Peak III (tentatively identified as anti-diol-epoxide-deoxyadenosine) correlated closely with the tumor-initiating activity of these three hydrocarbons, whereas the levels of the other two adducts did not. These data suggest that formation of a specific DNA adduct may be important for DMBA skin tumor initiation. These data are discussed in relation to skin tumor initiation by other hydrocarbons.

Kinetics of formation and disappearance of 7,12-dimethylbenz(a)anthracene:DNA adducts in mouse epidermis.

The rates of formation and disappearance of 7,12-dimethylbenz(a)anthracene (DMBA):DNA adducts were analyzed in the epidermis of SENCAR mice over a 21-day time course. Mice were treated topically with 10 nmol of tritium-labeled DMBA per mouse at various times prior to sacrifice. Under these experimental conditions, total covalent binding of DMBA to epidermal DNA reached a peak at 24 h, and thereafter, DMBA:DNA adduct disappearance was biphasic. The early phase of DMBA:DNA adduct disappearance (Phase A) between 24 and 72 h had a half-life of 3.17 +/- 1.1 days, whereas the later phase (Phase B) had a half-life of 6.46 +/- 1.3 days. A comparison of the biphasic disappearance of total DMBA:DNA adducts with total benzo(a)pyrene:DNA adducts at comparable tumor-initiating doses (i.e., doses producing similar papilloma responses in SENCAR mice) revealed that the half-life for Phase A disappearance of benzo(a)pyrene:DNA adducts was approximately 3 times faster than for DMBA:DNA adducts (1.08 +/- 0.3 days versus 3.17 +/- 1.1 days), respectively. Phase B disappearance of DNA adducts was essentially identical for both hydrocarbons and was similar to the rate of loss of label in epidermal DNA due to cell turnover. The rates of formation and disappearance of the three major DNA adducts derived from DMBA were also examined. Peaks II (syn-diol-epoxide deoxyadenosine) and III (anti-diol-epoxide deoxyadenosine) disappeared more rapidly than Peak I (anti-diol-epoxide deoxyguanosine) beyond 24 h. The data support the conclusion that, for a particular hydrocarbon such as DMBA, deoxyadenosine adducts disappear from epidermal DNA faster than the corresponding deoxyguanosine adducts. In addition, the data suggest that, at the doses used, total DMBA:DNA adducts disappear initially more slowly from epidermal DNA than benzo(a)pyrene:DNA adducts.

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 approximately 2-times more potent than 3-MCA. The data suggest that 3-MCA-trans-9,10-diol is a proximate carcinogen for mouse skin.

Formation and disappearance of benzo[a]pyrene DNA-adducts in mouse epidermis.

The rates of formation and disappearance of benzo[a]pyrene (B[a]P) DNA-adducts were analyzed in the epidermis of SENCAR mice over a 21-day time course. Mice were treated topically with 200 nmol/mouse of [3H]B[a]P at various times prior to sacrifice. The formation and disappearance of total adducts as well as individual adducts was determined and in addition, the rate of DNA turnover was monitored concurrently so that adduct disappearance could be expressed as a function of epidermal cell turnover. Under these experimental conditions, covalent binding of B[a]P to epidermal DNA reached a peak 24 h after treatment. Interestingly, between 24-48 h after application of the hydrocarbon there was a very rapid drop in the level of bound B[a]P to value approximately 50% of the maximum level at 24 h. Thereafter, the level of bound B[a]P disappeared at a much slower rate. In dual-label experiments, where the epidermal DNA was pre-labeled with [14C]thymidine, [3H]B[a]P DNA-adduct disappearance between 24-48 h was clearly more rapid than could be explained on the basis of epidermal DNA turnover. By 72 h and beyond, however, [3H]B[a]P DNA-adduct disappearance approximately paralleled DNA turnover. Examination of the rate of formation and disappearance of individual B[a]P DNA-adducts (nine individual adducts) suggested that some deoxyadenosine adducts were removed more rapidly than deoxyguanosine adducts. The results indicate that at least some epidermal cells have the capacity to repair B[a]P DNA-adducts. The data are discussed in relation to the process of tumor initiation in mouse skin.

Covalent binding of 7,12-dimethylbenz(a)anthracene and 10-fluoro-7,12-dimethylbenz(a)anthracene to mouse epidermal DNA and its relationship to tumor-initiating activity.

10-Fluoro-7,12-dimethylbenz(a)anthracene (10-F-DMBA) is a more potent skin tumor initiator in SENCAR mice when compared with the parent hydrocarbon 7,12-dimethylbenz(a)anthracene (DMBA). To elucidate the mechanism for this difference, the covalent binding of these two hydrocarbons to the DNA of mouse epidermal cells in vivo and in vitro was compared. The quantity of 10-F-DMBA covalently bound to mouse epidermal DNA in vivo was greater than that of DMBA at all doses tested over the range of 4 to 200 nmol/mouse. The magnitude of this binding difference between 10-F-DMBA and DMBA was greater at the higher doses (e.g., 1.5-fold at 4 nmol/mouse versus 3.4-fold at 200 nmol/mouse). These results correlated closely with the dose-response relationships for tumor initiation by the two hydrocarbons. Analysis of the isolated DNA samples by Servacel DHB chromatography revealed the relative proportion of syn-diol-epoxide:DNA adducts derived from DMBA increased dramatically as a function of dose (approximately 30% at 4 nmol/mouse versus approximately 55% at 200 nmol/mouse). Conversely, the relative proportion of syn-diol-epoxide adducts derived from 10-F-DMBA was low and remained essentially constant over the same dose range. High-pressure liquid chromatographic analyses of the DNA adducts derived from DMBA- and 10-F-DMBA-treated mice revealed qualitatively similar profiles. However, as expected, there was a marked reduction in the relative proportion of syn-diol-epoxide:DNA adducts in the profiles of epidermal samples from 10-F-DMBA-treated mice. The major syn-diol-epoxide:deoxy-adenosine adduct was present at a level only 30% that found in high-pressure liquid chromatographic profiles of DMBA samples. Similar results were obtained when primary cultures of mouse epidermal cells were treated with the hydrocarbons. The results suggest that the increased total binding and possibly the decreased proportion of syn-diol-epoxide:DNA adducts confer greater tumor-initiating potency on 10-F-DMBA.

Parents of disturbed enuretic and non-enuretic children.

The values and child-rearing attitudes of parents of three groups of children (enuretics, clinically symptomatic non-enuretics, normals) were evaluated with the Study of Values and Parental Attitude Research Instrument. The parents of the clinically symptomatic enuretics and non-enuretics did not differ. However, the patterns of the two clinical groups differed significantly from the parents of the normals on a number of PARI variables.