Interspecies differences in the major DNA adducts formed from benzo(a)pyrene but not 7,12-dimethylbenz(a)anthracene in rat and human mammary cell cultures

Mammary epithelial cells from rats and humans show both quantitative and qualitative species- and carcinogen-specific differences in their abilities to activate benzo(a)pyrene (B(a)P) and 7,12-dimethylbenz(a)anthracene (DMBA). Previous studies of the DNA binding of these compounds in mammary epithelial cells demonstrated that rat cells bound relatively more DMBA than B(a)P to DNA under identical treatment conditions, while the opposite pattern was exhibited by human mammary epithelial cells. The specific DNA adducts formed in these cells after 24-h incubations with [3H]DMBA and [3H]B(a)P were analyzed to determine if there were qualitative as well as quantitative differences in the amounts of individual adducts. Similar proportions of specific DMBA-DNA adducts were found in both rat and human cells, although the total amount of adducts formed was significantly higher in the rat cells. In contrast, an essentially qualitative species-specific difference was observed in the major B(a)P-DNA adduct present in the rat and human cells. The major B(a)P adduct formed in the human mammary epithelial cells was identified as the (+)-anti-B(a)P-7,8-dihydrodiol-9, 10-epoxide(BPDE)-deoxyguanosine adduct. However, this adduct was formed at very low levels in the rat mammary epithelial cells. The rat cells contained a large proportion of syn-BPDE adducts, and other unidentified B(a)P-DNA adducts. The high level of the (+)-anti-BPDE-deoxyguanosine adduct in the human but not the rat mammary cells is consistent with the potential role of (+)-anti-BPDE in the high mutagenic activity of B(a)P in the cell-mediated mutagenesis assays using the human mammary cells as activators, and the low mutagenic activity of B(a)P when rat cells were used as activators. The quantitative differences in the activation of DMBA by cells from these two species are also consistent with the cell-mediated mutagenic activities of DMBA using these cells as activators. These results suggest that the higher carcinogenic activity of DMBA compared to B(a)P in the rat mammary gland may not be indicative of the relative carcinogenic potencies of these compounds for human mammary cells.     

Comparison of metabolism-mediated binding to DNA of 7-hydroxymethyl-12-methylbenz(a)anthracene and 7, 12-dimethylbenz(a)anthracene.

Comparison of the binding to DNA of 7-hydroxymethyl-12-methylbenza(a)anthracene and 7, 12-dimethylbenz(a)anthracene (DMBA) catalyzed by mouse embryo cells in culture or by rat liver microsomes indicates that the products formed are different for the two hydrocarbons. Thus, the hydroxy compound is not an intermediate in the binding of DMBA to DNA in these systems. Binding of the hydroxy compound to DNA in mouse embryo cells is less efficient than for DMBA and is inhibited by 1,1,1-trichloropropylene 2,3-oxide, an inhibitor of epoxide hydrase. This and the fluorescence spectra of the hydroxy compound-DNA adducts indicate that the hydroxy compound is activated for DNA binding through the formation of a diol-epoxide in the 1,2,3,4-ring. As previously found for DMBA, this is consistent with the activation of this compound through a bay-region diol-epoxide.     

Benzo(e)pyrene-induced alterations in the stereoselectivity of activation of 7,12-dimethylbenz(a)anthracene to DNA-binding metabolites in hamster embryo cell cultures

Benzo(e)pyrene [B(e)P], a weakly carcinogenic polycyclic aromatic hydrocarbon, modifies tumor induction in mouse skin and the induction of mutation in mammalian cells by carcinogenic hydrocarbons. To determine how B(e)P alters the activation of the carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) to DNA-binding metabolites, the hydrocarbon-DNA adducts formed in Syrian hamster embryo cell cultures were analyzed after 24, 48, or 72 h of exposure to 0.1 microgram DMBA/ml medium in the presence of various doses of B(e)P. The total binding of DMBA to DNA was inhibited 3- to 4-fold by high doses of B(e)P, while the binding of DMBA to DNA was increased by low doses of B(e)P at 48 and 72 h of exposure. The amounts of the three major adducts tentatively identified as anti-DMBA-3,4-diol-1,2-epoxide (DMBADE): deoxyguanosine, syn - DMBADE: deoxyadenosine (dAdo), and anti-DMBADE:dAdo decreased in the presence of 1.5 micrograms B(e)P/ml. In contrast, exposure to low doses of B(e)P, 0.1 and 0.3 microgram/ml medium, resulted in an increase in the amount of both anti-DMBADE:deoxyribonucleoside adducts and a decrease in the amount of syn-DMBADE:deoxyribonucleoside adduct present after 48 and 72 h of exposure. Thus, low doses of B(e)P specifically enhanced the formation of anti-DMBA-diol-epoxide:deoxyribonucleoside adducts, and this resulted in an increase in the total amount of DMBA bound to DNA. High doses of B(e)P resulted in a decrease in the formation of all DMBA:DNA adducts and consequently a decrease in the total binding of DMBA to DNA. The amount of DMBA bound to DNA in cultures exposed to a higher dose of DMBA, 0.2 microgram DMBA/ml medium, for 48 h decreased in the presence of both low and high concentrations of B(e)P. This decrease resulted from a reduction in the formation of all three major DMBA-DNA adducts as the dose of B(e)P increased, but the decrease was larger for the syn-DMBADE:dAdo adduct than for the anti-DMBADE:deoxyguanosine and :dAdo adducts. These results demonstrate that the effects of B(e)P on the metabolic activation of DMBA depend upon both the ratio of B(e)P:DMBA and the dose of DMBA. The ability of B(e)P to alter the stereochemical selectivity of activation of DMBA as well as the total amount of activated metabolites also suggests that the ratio of B(e)P:DMBA may be an important factor in B(e)P-induced modifications of the induction of biological effects by DMBA.     

Biochemical basis for cytotoxicity of 7,12-dimethylbenz(a)anthracene in rat liver epithelial cells.

When the effects of 7,12-dimethylbenz(a)anthracene (DMBA) on normal and malignant rat liver epithelial cells were compared in a colony inhibition assay, this carcinogen showed a preferential cytotoxic action on the normal cells. In investigations of the biochemical basis of this selective toxicity, it was found that both cell lines were similarly effective in binding DMBA to DNA and that both cell lines had the capacity to metabolize this carcinogen. However, the hepatoma cells were more efficient than were the normal cells in generating very polar metabolites (not organic solvent extractable). These studies suggest that the basis of the resistance of the hepatoma cells to the toxicity induced by DMBA lies in their ability to detoxify biologically active metabolites. Several phenols were examined as possible toxic metabolites of DMBA, but these were not toxic at dose levels at which DMBA kills most of the normal cells.     

7,12-dimethylbenz[a]anthracene-induced DNA binding and repair synthesis in susceptible and nonsusceptible mammary epithelial cells in culture

The effect of age and parity on the binding of 7,12-dimethybenz[a]anthracene (DMBA) to DNA and the repair of DMBA-damaged DNA have been demonstrated in logarithmic phase and confluent mammary epithelial cell cultures from young virgin (YV), old virgin (OV), and parous (P) noninbred and inbred Sprague-Dawley rats. Over a dose range of 0.1-0.4 micrograms DMBA/ml, DNA binding was 1.5-to 2.0-fold higher in YV cells than in OV or P cells. In addition, a steeper slope of the dose-response curve was obtained with YV cells, suggesting a greater susceptibility of YV cells to DMBA. Excision repair was determined by measuring, in the presence of hydroxyurea and 5-bromodeoxyuridine, tritiated thymidine incorporation into DNA during the repair process. At high doses od DMBA (0.5-2.0 micrograms/ml), excision repair in YV cells was 1.5 times higher than in OV cells and 2 times higher than in P cells. However, with lower DMBA doses (less than 0.5 micrograms/ml) similar levels of repair were obtained in all 3 groups of rats. Since binding to DNA is higher in YV cells at these low DMBA doses, ti is apparent that OV and P cells exhibit a greater DNA repair per unit damage. These results, therefore, suggest that age and parity not only lower the binding of DMBA to mammary epithelial cell DNA but also increase the efficiency of DNA repair processes, which may explain the lower susceptibility of OV and P rats to DMBA-induced mammary carcinogenesis.     

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.     

A comparison of covalent DNA binding of benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene in respiratory tissues from human, rat and mouse

In vivo and in vitro covalent DNA binding was investigated in an attempt to explain the higher susceptibility of A/J mouse lung and Fischer-344 rat trachea to 7,12-dimethylbenz[a]anthracene (DMBA) as compared to benzo[a]pyrene (BP), and to evaluate the relative susceptibility of the human respiratory tract to these compounds. After in vivo administration of either BP or DMBA to A/J mice covalent DNA binding was higher in the liver than in the lungs. Forty-eight hours after administration, but not before, binding of DMBA was higher than that of BP in both organs. In vitro studies using cultured explants of both human and A/J mouse peripheral lung, as well as human bronchus and Fischer-344 rat trachea, revealed that covalent DNA binding of DMBA to mouse lung and rat trachea were similar and that both were significantly higher than that of BP to these organs. Binding of BP and DMBA was similar in both human tissues and did not differ from BP binding in the animal tissues. Enzymatic hydrolysis and HPLC separation of the DNA-hydrocarbon adducts revealed that patterns of adducts in human and mouse peripheral lung were similar and qualitatively resembled known patterns in other target and non-target tissues. It is concluded that the higher susceptibility of the mouse lung and rat trachea to DMBA as compared to BP may be related to the higher covalent DNA binding of the former and that the relative carcinogenic risk of the human respiratory tract after exposure to DMBA may be the same as that after BP exposure.     

Effect of naturally occurring coumarins on the formation of epidermal DNA adducts and skin tumors induced by benzo[a]pyrene and 7,12- dimethylbenz[a]anthracene in SENCAR mice

Several naturally occurring coumarins previously found to be potent inhibitors of mouse hepatic ethoxyresorufin-O-deethylase (EROD) and/or pentoxyresorufin-O-dealkylase (PROD) were examined for their effects on formation of benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA) DNA adducts in mouse epidermis, as well as, their effects on skin tumor initiation by these polycyclic aromatic hydrocarbons (PAH). Bergamottin, a potent inhibitor of hepatic EROD, given topically 5 min prior to an initiating dose of B[a]P, significantly decreased total covalent binding of B[a]P to DNA in a dose-dependent manner 24 h after treatment. A dose of 400 nmol bergamottin reduced covalent binding of B[a]P by 72%. Coriandrin, at a dose of 400 nmol also significantly reduced total covalent binding of B[a]P by 59%. In addition, formation of the major (+)anti-B[a]P-diol epoxide-N2-dGuo adduct was selectively reduced by both of these coumarins. In contrast, bergamottin and coriandrin did not significantly decrease covalent binding of DMBA to epidermal DNA at doses of either 400 nmol or 800 nmol. Imperatorin and isopimpinellin, which are more potent inhibitors of hepatic PROD activity, significantly reduced overall binding of DMBA to epidermal DNA by 67% and 52%, respectively, when applied at doses of 400 nmol. These two coumarins also inhibited B[a]P-DNA adduct formation at similar doses but to a lesser extent. Imperatorin at a dose of 400 nmol dramatically decreased formation of covalent DNA adducts derived from both the anti and syn diol epoxides of DMBA. Bergamottin was a potent inhibitor of tumor initiation by B[a]P while coriandrin was less effective in this regard. Imperatorin was an effective inhibitor of skin tumor initiation by DMBA and also inhibited complete carcinogenesis by this PAH. At dose levels higher than those effective against DMBA, imperatorin also inhibited tumor initiation by B[a]P. The results demonstrate that several naturally occurring coumarins possess the ability to block DNA adduct formation and tumor initiation by PAHs such as B[a]P and DMBA. The mechanism for reduced DNA adduct formation and tumor initiation appears to involve inhibition of the P450s involved in the metabolic activation of these hydrocarbons. Finally, the differential effects of certain coumarins on B[a]P vs DMBA DNA adduct formation and tumor initiation may be useful for dissecting the role of specific cytochromes P450 in their metabolic activation.      

Inhibition of the binding of 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene to DNA in mouse skin epidermis by 1-ethynylpyrene

The effects of 1-ethynylpyrene (EP), 1-vinylpyrene (VP) and 2-ethynlnaphthalene (EN) on the covalent binding of 7,12-dimethylbenz[a]anthracene (DMBA) and of benzo[a]-pyrene (B[a]P) to the epidermal DNA in mouse skin were investigated. When applied topically, 5 min before an initiating dose of 10 nmol DMBA or of 200 nmol B[a]P, EP was an effective inhibitor of the formation of the covalent complexes of these procarcinogenic polycyclic aromatic hydrocarbons (PAHs) with the epidermal DNA. VP, applied under the same conditions, was a significantly less effective inhibitor of the binding of DMBA to DNA and showed even weaker inhibition of the binding of B[a]P. EN was ineffective as an inhibitor of the binding of either DMBA or B[a]P. These results establish that both the pyrene nucleus and the ethynyl substituent of EP contribute to the effective inhibition of the binding of DMBA and B[a]P to the epidermal DNA of mouse skin. No significant changes in the ratios of the anti- to the syndiol epoxide-DNA adducts of DMBA or of B[a]P were produced by doses of EP that produced inhibitions of the binding to DNA. At doses of VP that inhibited covalent binding of both DMBA and B[a]P, no changes in DMBA-DNA adduct distributions were observed but changes in the relative proportions of several B[a]P-DNA adducts were noted. These data are discussed in terms of the potential of aryl acetylenes to act as suicide inhibitors (mechanism-based inactivators) of cytochrome P450-dependent monooxygenase isozymes.     

Induction by 7,12-dimethylbenz(a)anthracene of molecular and biochemical alterations in transformed human mammary epithelial stem cells, and protection by N-acetylcysteine

Several lines of evidence suggest that stem cells are major targets for carcinogens. A normal human breast epithelial cell type was previously shown to possess stem cell characteristics. Further cell lines were derived following sequential transfection with SV40 large T-antigen (immortal, non-tumorigenic M13SV1 cells), exposure to X-rays (weakly tumorigenic M13SV1R2 cells), and ectopic expression of c-erbB2/neu (highly tumorigenic M13SV1R2N1 cells). We evaluated some characteristics of these cells and their susceptibility to the breast carcinogen 7,12-dimethylbenz(a)anthracene (DMBA). Compared to M13SV1 cells, the two untreated tumorigenic cell lines displayed higher levels of connexin 43 expression and NF-kappaB nuclear translocation, and a higher frequency of fhit loss. The baseline nuclear translocation of AP-1 and pCREB was particularly evident in M13SV1R2N1 cells and was further enhanced by DMBA treatment, indicating an interaction between c-erbB2/neu and DMBA-induced signalling. Treatment with DMBA did neither affect the baseline fhit loss nor p53 mutation, whereas it increased NF-kappaB nuclear translocation, the proportion of apoptotic cells, and the levels of connexin 43, common 4977-bp mitochondrial DNA deletion, and bulky adducts to nuclear DNA. DMBA-treated M13SV1 cells underwent significant oxidative DNA damage and exhibited the highest DNA adduct levels, while they had the lowest apoptotic rate. Co-treatment of cells with N-acetylcysteine (NAC) attenuated DMBA-induced toxicity and DNA alterations, particularly in M13SV1 cells. Thus, the immortal cell line derived from the normal human adult breast stem cell without further tumorigenic progression is the most susceptible both to DMBA-related alterations and to the protective effects of NAC.     

Effects of prostaglandins and some anti-inflammatory drugs on the binding of 7,12-dimethylbenz[alpha]anthracene to the DNA of murine epidermal cells in culture.

The effects of prostaglandins (PG) E1, E2, F1 alpha and F2 alpha and some anti-inflammatory drugs such as acetylsalicyclic acid, flufenamic acid, indomethacin, and fluocinolone acetonide (FA) on the binding of [3H]7,12-demthylbenz[alpha]anthracene (DMBA) to DNA of murine epidermal cells have been investigated. PG E1 and E2 significantly inhibit the binding of DMBA to murine epidermal cells (MEC) DNA while PG F1 alpha and F2 alpha do not affec the binding. Salicyclic acid and flufenamic acid also do not alter the binding; whereas, indomethacin and FA lowered the binding of DMBA to DNA.     

Inhibitory effects of naturally occurring coumarins on the metabolic activation of benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene in cultured mouse keratinocytes

Several naturally occurring coumarins to which humans are routinely exposed have been previously found to be potent inhibitors and inactivators of cytochrome P450 (P450) 1A1-mediated monooxygenase in both murine hepatic microsomes and in a reconstituted system using purified human P450 1A1 [Cai et al. (1993) Chem. Res. Toxicol., 6, 872- 879 and Cai et al. (1996) Chem. Res. Toxicol., 9, 729-736]. In the present study, several of these coumarins were investigated for their inhibitory effects on the metabolism and metabolic activation of benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA) in cultured mouse keratinocytes. Initial analysis of B[a]P metabolism in cultured keratinocytes showed that imperatorin, isoimperatorin, coriandrin, and bergamottin, at concentrations of 2 nM equal with B[a]P, reduced the formation of water-soluble metabolites of B[a]P by 33% to 57%. Bergamottin and coriandrin were the most potent inhibitors of the compounds examined. HPLC analysis of organic solvent-soluble metabolites of B[a]P indicated that all the coumarins tested significantly reduced the formation of individual B[a]P metabolites (including phenols, diols and tetraols). However, the greatest effect was on the formation of B[a]P tetraols. Additional experiments determined the ability of selected coumarins to block covalent binding of B[a]P and DMBA to DNA in keratinocytes. Bergamottin preferentially inhibited the binding of B[a]P to DNA by 56%, while coriandrin preferentially inhibited the binding of DMBA to DNA by 48%. Notably, analysis of individual DNA adducts formed from B[a]P and DMBA indicated that both bergamottin and coriandrin specifically inhibited the formation of anti diol-epoxide DNA adducts derived from both hydrocarbons. The preferential inhibitory effect of bergamottin and coriandrin on the formation of anti diol-epoxide adducts derived from DMBA was further confirmed by separation of anti- and syn-diol-epoxide- DNA adducts using immobilized boronate chromatography. The current study demonstrates that certain naturally occurring coumarins inhibited metabolic activation of B[a]P and DMBA in cultured mouse keratinocytes and specifically inhibited the formation of DNA adducts derived from the anti diol-epoxide diastereomers from either hydrocarbon. The current data also suggest that certain naturally occurring coumarins may possess anticarcinogenic activity toward polycyclic aromatic 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.     

Human cell-mediated benzo(a)pyrene cytotoxicity and mutagenicity in human diploid fibroblasts

A human epithelial cell-mediated cytotoxicity and mutagenicity assay system for benzo(a)pyrene [B(a)P] has been developed with human fibroblasts as the target cells. Lethally X-irradiated human kidney carcinoma-derived epithelial cells, which had constant levels of B(a)P-metabolizing activity, were cocultivated with target human skin fibroblasts (XP12BE), which lack excision repair capability for B(a)P-DNA adducts. The optimal conditions determined for the cell-mediated cytotoxicity assay were a 48-hr exposure to B(a)P concentrations ranging from 0.1 to 1 microM at a metabolizing cell:target cell ratio of at least 1:1. Under these conditions, the frequency of mutations to 6-thioguanine resistance induced in the target XP12BE cells by B(a)P as well as the binding of tritium-labeled B(a)P to DNA was also shown to be concentration dependent. High-pressure liquid chromatography analysis of enzymatically degraded B(a)P-DNA adducts revealed two peaks: a major peak (82%) which cochromatographed with the guanosine adduct-standard synthesized from anti-isomeric-7,8-dihydrodiol-9,10-epoxide of B(a)P; and a minor peak (18%) which cochromatographed with the guanosine adduct-standard synthesized from syn-isomeric-7,8-diol-9,10-epoxide of B(a)P. Human liver carcinoma- and lung carcinoma-derived cell lines, capable of metabolizing B(a)P, proved equal to or better than the kidney carcinoma-derived cell line in producing cytotoxic B(a)P metabolites in the cell-mediated cytotoxicity assay with target XP12BE cells.     

Distribution, covalent binding, and DNA adduct formation of 7,12-dimethylbenz(a)anthracene in SENCAR and BALB/c mice following topical and oral administration

SENCAR mice are much more susceptible to tumor initiation by 7,12-dimethylbenz(a)anthracene (DMBA) administered topically than p.o. and are also more susceptible to initiation by topically applied DMBA than are BALB/c mice. To determine how the distribution and metabolic activation of DMBA differed in these strains and with route of administration, BALB/c and SENCAR mice were exposed to [3H]DMBA topically and p.o., and the distribution and DNA binding of DMBA were analyzed. Both the amount of DMBA in skin and the covalent binding of DMBA to epidermal DNA were greater following topical administration than after p.o. administration in both strains. Differences in DMBA distribution and macromolecular binding were found between SENCAR and BALB/c mice, with the binding of DMBA to DNA in epidermis tending to be greater in BALB/c mice than in SENCAR mice when differences were observed. The formation of individual DMBA:DNA adducts in epidermis was also examined in SENCAR and BALB/c mice following topical administration of DMBA. No substantial qualitative or quantitative differences in DMBA:DNA adducts were found between SENCAR and BALB/c mice. The anti/syn-DMBA-diol-epoxide-DNA adduct ratios calculated from the three major DMBA:deoxyribonucleoside adducts increased with time in both SENCAR and BALB/c mice. The data suggest that differences in the distribution and macromolecular binding of DMBA are responsible for the much greater skin tumor initiating activity of DMBA applied topically than p.o. but do not account for the greater sensitivity of the SENCAR mouse to DMBA-induced epidermal tumorigenesis.     

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.     

Effect of dehydroepiandrosterone (DHEA) on the metabolism of 7,12-dimethylbenz[a]anthracene (DMBA) in rats

The influence of dehydroepiandrosterone (DHEA), an adrenal steroid, on the biotransformation of the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) in rats has been investigated. Male Sprague-Dawley rats (2-3 months old) were fed DHEA for 14 days at a dietary level of 0.8%. There was an increase in liver weights with increases per whole liver, in total protein, microsomal and cytosolic protein and cytochrome P-450, and cytosolic glutathione transferase activity in DHEA fed rats. DNA content of the liver, however, remained constant. Forty-eight hours after a single i.p. dose of [3H]DMBA (133 mumol/kg body weight, 102 muCi/rat) binding of DMBA derived metabolites to DNA decreased significantly both per unit of DNA (605 versus 194 pmol/mg DNA) as well as per whole liver DNA (25.4 versus 8.5 nmol) in DHEA fed rats. However, a significantly higher amount of DMBA-derived metabolites were bound to total hepatic protein (455 versus 288 nmol) in the steroid fed rats. Microsome mediated binding of DMBA to DNA was 3-fold higher in DHEA fed rats. Excretion of DMBA-derived metabolites in urine was 2-fold higher in DHEA fed rats. The results of this study demonstrate that DHEA inhibits binding of DMBA to hepatic DNA in vivo in spite of the increased metabolic activation of the carcinogen perhaps due to increased detoxification and competitive binding of its active species to proteins.     

Differences in mediated mutagenesis and poly cyclic aromatic hydrocarbon metabolism in mammary cells from pregnant and virgin rats

The physiological state of pregnancy confers significant resistanceto poly cyclic aromatic hydrocarbon (PAH)-induced mammary tumorigenesis.We have tested the abilities of primary mammary cells from pregnantand virgin rats cultured on collagen-coated plates to metabolizePAHs and convert these carcinogens to mutagenic derivatives.Using a cell-mediated mutagenesis assay, we found that mammaryepithelial cells from pregnant rats produced half the levelsof mutagenic 7,12-dimethylbenz[a]anthracene (DMBA) metabolitesformed by cells from virgin rats. Pregnant-derived mammary cellsalso showed consistently lower levels of metabolism of DMBAand benzo[a]pyrene (B[a]P) than cells from virgin rats. H.p.l.c.analysis of B[a]P metabolism by these cell populations indicatedno significant qualitative differences in the extracellularand intracellular metabolites formed. We have previously shownthat mammary cells from rat strains exhibiting significant differencesin susceptibility to PAH-induced tumors have equivalent qualitativeand quantitative abilities to metabolize PAH carcinogens. Ourcurrent data suggest that modifications in mammary tumor susceptibilityfound in various physiological states, unlike genetically determineddifferences, may be related in part to an altered ability toactivate chemical carcinogens within the mammary gland.     

Human and rat kidney cell metabolism of 2-acetylaminofluorene and benzo(a)pyrene

The metabolism and mutagenic activation of the model carcinogens benzo(a)pyrene [B(a)P] and 2-acetylaminofluorene (AAF) by human and rat kidney cells were measured. A slicing technique followed by enzyme digestion was utilized to obtain the kidney cells. Although levels of total metabolism of B(a)P by rat and human kidney cells were similar, analysis of specific metabolites of B(a)P indicated that species differences existed. Human kidney cells produced the organic-soluble metabolites B(a)P-9,10-diol, B(a)P-4,5-diol, B(a)P-7,8-diol, B(a)P-3,6-quinone, and B(a)P-9-phenol. Rat kidney cells produced organic-soluble B(a)P-pre-9,10-diols, B(a)P-9,10-diol, B(a)P-4,5-diol, and B(a)P-6,12-quinone. Both species produced sulfate and glucuronide conjugates of all products. For AAF, kidney cells from some human tissues produced up to four times the level of total metabolites compared to rat kidney cells. Organic-soluble metabolites were qualitatively similar between the species and consisted of 2-aminofluorene (AF), N-hydroxy-AAF and ring-hydroxylated products at the 1, 3, 5/9, 7, and 8 positions. Sulfate and glucuronide conjugates of these metabolites were also detected. Human interindividual variation with kidney cells was about 2.5-fold for total AAF metabolism and up to 6-fold for individual AAF metabolites. For B(a)P metabolism, human interindividual variation in total metabolism was low while for specific metabolites there was up to a 4-fold variation. Levels of AAF and AF cell-mediated Salmonella typhimurium mutagenesis were significantly higher with human cells as compared to rat kidney cells. It appears that the differences between human and rodent kidney cell metabolism of chemical carcinogens vary with the chemical class and understanding these differences will be necessary in the extrapolation of rodent carcinogenesis data to humans.