Showing papers on "7,12-Dimethylbenz[a]anthracene published in 1986"

Dietary glucarate as anti-promoter of 7,12-dimethylbenz[a]anthracene-induced mammary tumorigenesis.

Abstract: Using as a criterion the inhibition of serum beta-glucuronidase activity, dietary calcium D-glucarate is shown to serve as an efficient slow-release source in vivo of D-glucaro-1,4-lactone, the potent endogenous inhibitor of this enzyme. Using the 7,12-dimethylbenz[a]anthracene model of mammary tumor induction in rats it is shown for the first time that feeding the rats calcium D-glucarate-supplemented diet after treatment with the carcinogen, inhibits tumor development by over 70%. Supportive evidence is presented for the theory that calcium D-glucarate inhibits or delays the promotion phase of mammary carcinogenesis by lowering endogenous levels of estradiol and precursors of 17-ketosteroids. Therefore, dietary glucarate can be used to lower blood and tissue levels of beta-glucuronidase, and in turn of those carcinogens and promoting agents which are excreted, at least in part, as glucuronide conjugates.

Modification by antioxidants and p,p'-diaminodiphenylmethane of 7,12-dimethylbenz[a]anthracene-induced carcinogenesis of the mammary gland and ear duct in CD rats.

Abstract: The effects of antioxidants on mammary gland carcinogenesis pretreated with 7,12-dimethylbenz[a]anthracene (DMBA) in female Sprague-Dawley rats were examined. The antioxidants used were butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), sodium L-ascorbate, alpha-tocopherol, ethoxyquin and p,p'-diaminodiphenylmethane (DDPM), which is an inhibitor of carcinogenesis in the liver, kidney and urinary bladder. Female Sprague-Dawley rats of 50 days old were treated with 2.5 mg/100 g body weight of DMBA, and from 1 week later were given diet supplemented with 1% BHA, 0.7% BHT, 5% sodium L-ascorbate, 1.5% alpha-tocopherol, 0.5% ethoxyquin or 0.1% DDPM for 33 weeks and then killed. The incidences of mammary tumors, carcinomas and fibroadenomas in DMBA-treated animals were reduced by diet containing BHA or ethoxyquin. Diet containing BHT or DDPM inhibited the induction of only fibroadenomas. The incidence of ear duct tumors in DMBA-treated animals was reduced by diet containing BHT, alpha-tocopherol or ethoxyquin.

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.

Abstract: 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.

Influence of Dietary Retinyl Acetate on Normal Rat Mammary Gland Development and on the Enhancement of 7,12-Dimethylbenz[a]anthracene-Induced Rat Mammary Tumorigenesis by High Levels of Dietary Fat

Abstract: The effect of high levels of dietary fat and retinyl acetate (ROA) on 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary tumor development and growth was examined. Female Sprague-Dawley rats, 51-53 days of age, were treated ig with 5 mg DMBA. At 55-57 days of age, the animals were divided into the following dietary treatment groups: A) 4.5% fat [control fat (CF)]; B) CF + 1.0 mmol ROA/kg diet (CF + ROA); C) 20.0% fat [high fat (HF)]; and D) HF + ROA. HF diets significantly increased mammary tumor multiplicity, with or without ROA, but did not significantly influence mammary tumor growth. ROA treatment reduced mammary tumor multiplicity regardless of the level of dietary fat and inhibited mammary tumor growth in the presence of normal levels of dietary fat. High levels of dietary fat did not significantly influence normal mammary gland growth and development. ROA significantly decreased normal mammary gland growth and development regardless of the level of dietary fat. Blood retinoids in rats fed ROA were primarily in the form of retinyl esters, i.e., retinyl linoleate, retinyl palmitate-oleate, and retinyl stearate. Free retinol levels in blood were not significantly influenced by ROA feeding. Blood retinyl ester levels were lower in rats fed the HF + ROA diet as compared to rats fed the CF + ROA diet.

Effects of Dietary Protein, Fat and Energy Intake during an Initiation Phase Study of 7,12-Dimethylbenz[a]anthracene-Induced Breast Cancer in Rats

Abstract: A factorial experiment was conducted to examine the effects of dietary protein (8, 16, 32% of energy from casein) and dietary fat (12, 24, 48% of energy from corn oil) on the initiation of 7,12-dimethylbenz[a]anthracene (DMBA)-induced breast carcinogenesis in rats. Forty weanling female Sprague-Dawley rats were assigned to each of nine diets fed ad libitum. After 4 wk each rat received DMBA (20 mg/kg) via gastric intubation. For an additional 22 wk after carcinogen administration all rats consumed a diet containing 16% of dietary energy from protein and 24% from fat. Dietary fat, protein and ad libitum energy consumption exhibited statistically significant effects on final tumor prevalence, but interactive effects were not found. At necropsy, rats fed corn oil at 12, 24 and 48% of energy prior to DMBA administration showed tumor prevalences of 58, 58 and 85% with 116, 153 and 231 total tumors, respectively. The data indicate a significant nonlinear effect of dietary fat. Corresponding numbers for rats fed casein at 8, 16 and 32% of energy prior to DMBA were prevalences of 79, 65 and 59%, with total tumor counts of 194, 144 and 162. Higher dietary protein during the initiation phase was associated with a significant reduction in tumor prevalence, which was most striking between 8 and 16% of energy from protein. In addition, results of multiple logistic regression showed that tumorigenesis was increased with greater ad libitum energy intake. The odds of a tumor at necropsy were multiplied by 1.19 for each kilocalorie increase in ad libitum energy intake averaged over the post-DMBA phase of the experiment. An additional six weanling rats fed each diet for 4 wk were killed for assay of hepatic carcinogen metabolizing enzymes at the time corresponding to DMBA administration in the initiation experiment. Both protein and fat showed independent effects on the activity of several enzymes. However, enzyme activity did not suggest a unifying mechanism whereby these nutrients influence DMBA-induced mammary carcinogenesis.

Effect of Moderate Vitamin A Supplementation and Lack of Dietary Vitamin A on the Development of Mammary Tumors in Female Rats Treated with Low Carcinogenic Dose Levels of 7,12-Dimethylbenz(a)anthracene

Abstract: We examined the effect of moderately increased and of marginal continued dietary supplementation of vitamin A (retinyl acetate) and the effect of lack of dietary vitamin A on the initiation and promotion stages of mammary tumorigenesis in female Sprague-Dawley rats treated with a single low (0.5 mg/100 g body weight) or very low (0.1 mg/100 g body weight) dose of i.v.-administered 7,12-dimethylbenz( a )anthracene. The number of mammary tumors was significantly ( P < 0.05) reduced if prior to and during initiation with 7,12-dimethylbenz( a )anthracene the rats were fed a moderately increased (30 µg/day) or marginal (3 µg/day) amount of vitamin A, compared to rats fed an adequate (10 µg/day) amount of vitamin A. The number of mammary tumors was also significantly ( P < 0.05) reduced when a moderately increased or marginal amount of vitamin A was provided during the tumor promotion phase. In addition, the number of mammary tumors was significantly ( P < 0.05) reduced by the lack of dietary vitamin A during both the initiation and promotion stages of this tumorigenic process, when compared to vitamin A adequate, ad libitum -fed rats, but not when compared to vitamin A adequate, food-restricted controls. The reduction in numbers of mammary tumors observed in these studies was reflected primarily in significant ( P < 0.05) decreases in mammary fibroadenomas; the number of mammary carcinomas was often reduced, but due to a low frequency of the carcinomatous lesions, this reduction did not reach the 5% level of statistical probability. Plasma and liver vitamin A levels were determined during both the initiation and promotion stages. As the dietary supplementation of vitamin A increased from 0 to 30 µg/day, there was an increase in mean liver and plasma vitamin A levels. No consistent correlation between plasma and liver vitamin A levels and the occurrence of mammary tumors was observed, except with the moderately increased (30 µg/day) intake of vitamin A, that resulted in a small, but statistically significant ( P < 0.05) increase of serum retinol at initiation; this may account for the observed reduction in mammary tumors. These results provide evidence that moderate alterations in vitamin A consumption can modulate low-dose chemically induced mammary gland tumorigenesis. Most importantly, suppression of mammary gland tumorigenesis can be achieved by moderately increased, frequent, and regular consumption of vitamin A; prolonged consumption of vitamin A-deficient diets or diets marginal in vitamin A does not enhance the risk of mammary tumor development.

Differential carcinogenic effects of intraperitoneal initiation with 7,12-dimethylbenz(a)anthracene or urethane and topical promotion with 12-O-tetradecanoylphorbol-13-acetate in skin and internal tissues of female SENCAR and BALB/c mice

Abstract: Groups of female SENCAR or BALB/c mice were initiated once intraperitoneally with 300 micrograms/mouse of 7,12-dimethylbenz(a)anthracene (DMBA) or 20 mg/mouse of urethane at 7 weeks of age. Beginning one week later, mice received topically applied acetone or 12-O-tetradecanoylphorbol-13-acetate (TPA), once weekly, at 2.5 micrograms/mouse for weeks 1 through 6 and 1.25 micrograms/mouse for weeks 7 through 52. The skin lesions were evaluated clinically. A complete necropsy was performed on all mice at week 52. SENCAR mice exposed to DMBA/TPA and urethane/TPA had more skin tumors than SENCAR mice exposed to DMBA or urethane alone and more than BALB/c mice in any treatment group. Of all skin carcinomas diagnosed histologically in DMBA/TPA-exposed mice, less than one-third had been identified clinically while the mice were alive. Most of the carcinomas arose within papillomas. BALB/c mice developed more vascular and uterine tumors than did SENCAR mice injected with DMBA and more lung and vascular tumors than did SENCAR mice injected with urethane. TPA exposure after treatment with either initiator had no significant effect on internal tumor development in either SENCAR or BALB/c mice.

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

Abstract: 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 ∼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.

Effect of dietary polyunsaturated fat and 7,12-dimethylbenz(a)-anthracene on rat splenic natural killer cells and prostaglandin E synthesis.

Abstract: Dietary polyunsaturated fat has been shown to stimulate mammary tumorigenesis induced in rats by 7,12-dimethylbenz(a)anthracene (DMBA). Studies were undertaken to investigate the effect of polyunsaturated fat and DMBA on splenic natural killer (NK) activity and prostaglandin E (PGE) synthesis. In a first experiment, splenic NK activity at 33, 55, 75, and 110 days of age was measured in Sprague-Dawley rats fed 0.5% low fat (LF), 5% normal fat (NF), or 20% high fat (HF) corn oil diets from 23 days of age. At 55 days of age, half of the rats from the 75 and 110 day age groups were given 5 mg DMBA. Ten days after the initiation of the diets splenic NK activity against YAC-1 lymphoma was decreased from 50% cytotoxicity in rats fed NF diet to 21% cytotoxicity in rats fed HF diet, but was not affected by LF feeding. No difference in NK activity was observed among the groups at the later time periods. DMBA had no effect on NK activity at 20 or 55 days after its administration. In a second experiment, where DMBA (15 mg/rat) was given to half of the rats at 50 days of age and NF or HF diets were started 3 days later, NK activity was 35% in rats fed NF diet and 21% in rats fed HF diet, 5 days after the diets were started. No difference in NK activity in rats fed either diet was observed at later time periods. DMBA decreased both NK activity and spleen cellularity transiently. In both experiments, PGE synthesis by spleen cells cultured for 18 h was not affected by dietary fat intake, but was slightly increased 3 days after DMBA administration. Results from these experiments suggest that the stimulation of DMBA-induced mammary tumorigenesis by polyunsaturated fat and by DMBA itself may possibly be mediated by a transient decrease in splenic NK cell activity.

DNA binding and adduct formation of 7,12-dimethylbenz[a]-anthracene by rat mammary epithelial cell aggregates in vitro

Abstract: Freshly isolated mammary epithelial cell aggregates from female Sprague-Dawley rats metabolized 7,12-dimethylbenz-[a]anthracene (DMBA) to bay-region anti- and syn-dihydrodiolepoxides that bound to deoxyguanosine and deoxyadenosine residues in cellular DNA. After 24 h of incubation 68% of the DMBA (0.4 micrograms/ml) was metabolized and 58% of the extracellular metabolites were water-soluble. DMBA-DNA binding increased rapidly during the initial 24 h of incubation. Formation of the bay-region syn-dihydrodiolepoxide:deoxyadenosine adduct increased linearly throughout the 24 h, whereas formation of deoxyadenosine and deoxyguanosine adducts with the bay-region anti-dihydrodiolepoxide increased rapidly following a delay of 12 h.

Comparative DNA binding of 7,12-dimethylbenz[a]anthracene and some of its metabolites in mouse epidermis in vivo as revealed by the 32P-postlabeling technique.

Abstract: The binding of some mouse skin metabolites and related derivatives of the tumor initiator 7,12-dimethylbenz[a]anthracene (DMBA) was investigated by 32P-postlabeling analysis after its topical administration. DMBA and trans-3,4-dihydro-3,4-dihydroxy-DMBA (DMBA-3,4-dihydrodiol) both led to the formation of four DNA adducts, which showed a very similar pattern of spots on thin-layer chromatograms. With trans-8,9-dihydro-8,9-dihydroxy-7,12-dimethylbenz[a]anthracene (DMBA-8,9-dihydrodiol) one major adduct was obtained which was chromatographically indistinguishable from one of the DMBA adducts. In contrast, 7-hydroxymethyl-12-methylbenz[a]anthracene (7-OHM-12-MBA) gave rise to two major adducts which were separable from DMBA adducts. 3-hydroxy-7,12-dimethylbenz[a]anthracene (3-OH-DMBA) and 7,12-dimethylbenz[a]anthracene-7,12-epoxide (DMBA-O2) did not lead to detectable amounts of adducts. Quantitative determination of DNA binding showed that an initiating dose (i = 100 nmol) of DMBA yielded approximately 12 adducts/10(7) normal nucleotides. Adduct formation with the same dose of DMBA-3,4-dihydrodiol was 7-8 times higher. At a 4-fold higher dose level, DMBA-8,9-dihydrodiol exhibited a 3- to 6-times weaker binding and 7-OHM-12-MBA a slightly stronger binding than DMBA. Chromatography of the DMBA and DMBA-3,4-dihydrodiol adducts with a solvent containing borate showed a decreased mobility of two out of four adducts in each case. These adducts were also sensitive to oxidation by periodate. The results suggest that two DMBA adducts carried vicinal cis-hydroxyl groups and thus were probably derived from the anti-3,4-dihydrodiol-1,2-oxide(s) of DMBA. The other two adducts were probably derived from the syn-stereoisomer(s). When the DNA-modifying capabilities and initiating activities of the more prominent mouse-skin metabolites are considered in relation to DMBA, DMBA-3,4-dihydrodiol is postulated to be a proximate and DMBA-3,4-dihydrodiol-1,2-oxide(s) to be ultimate initiators.

Progression-linked properties of specific-lesion cell populations from 7,12-dimethylbenz[a]anthracene-exposed rat tracheal implants.

Abstract: The purpose of this study was to determine whether there are any relationships between the morphological expressions of the progression of neoplasia in tracheal epithelium in vivo, and the increased in vitro growth autonomy expressed by carcinogen-altered cell populations isolated from the same tracheas. Rat tracheal implants were exposed for 2 weeks or 4 weeks to 200 micrograms 7,12-dimethylbenz[a]anthracene (DMBA)-beeswax pellets. In the first phase of this study reported earlier (1), the numbers of carcinogen-altered cell populations, identified by their lack of need for exogenous pyruvate and insulin for survival in cell culture, were quantitated 2, 6 and 9 months after the start of the exposures. Before generating the cell cultures, lesions on the pieces of pre-exposed tracheal implants were identified by placing them in organ culture for 24 h and collecting the exfoliated cells from the medium for diagnostic cytopathology. Although the pieces of 2-week DMBA-exposed tracheas had not developed any markedly atypical lesions by 9 months, there was a progressive increase in the growth autonomy of the small number of subculturable cell populations (approximately 1/trachea) obtained from this exposure group. This was seen in the decreased time needed in culture to expand the population for testing anchorage-independent growth and tumorigenicity in nude mice. Also, anchorage-independent growth was markedly enhanced from 25 to 80% between 2 and 6 months and then to 100% at 9 months. Tumorigenicity did not show an increase. In the 4-week DMBA-exposed group, 74% of the large number of subculturable cell populations (approximately 5.0/trachea) already showed anchorage-independent growth at 2 months. This percentage increased to 94-100% at 6 and 9 months. A progressive increase in tumorigenic cell populations was also clearly seen. At 2 months, 26% of the cell populations inoculated into nude mice formed tumors. This number increased from 45 to 61% at 6 and 9 months, respectively. The pieces of tracheas taken at 2 months after the start of the 4-week DMBA exposure had no markedly atypical lesions. At 6 months one marked atypia was detected, and at 9 months five markedly atypical lesions and two carcinomas in situ were present. The cell populations derived from the explants harboring these lesions showed a high incidence of growth autonomy, e.g. anchorage-independent growth and tumor formation, indicating a close relationship between these properties and the evolving morphological manifestations of tumor development.

Inhibition of the Binding of 7,12-Dimethylbenz[a]anthracene to DNA by Ascorbic Acid, Reduced Glutathione and Cysteine in Chick Embryo Cells Cultured in vitro

Abstract: A study on the capacity of ascorbic acid, reduced glutathione and cysteine to interfere with 3H-7,12-dimethylbenz[a]anthracene (3H-DMBA) binding to DNA in cultured fibroblast-like cells from 11-day-old chick embryos showed that, although the total amount of 3H-DMBA in the treated cells was greater than in the untreated cells, the DNA-bound 3H-DMBA was less. Comparisons between the various experimental groups demonstrated that the greater 3H-DMBA in the ascorbic acid-, reduced glutathione-, and cysteine-treated groups could not be attributed to an initially higher number of cells, nor to a treatment-induced increase in DNA synthesis. It is proposed that the three substances examined inhibit the oxidative degradation of 3H-DMBA, thereby favoring its accumulation within the cell and reducing the formation of DNA-binding metabolites.

Immediate Ascites Conversion of Mammary Tumors Induced in NYLR/Nya Mice by 7,12-Dimethylbenz-[a] Anthracene and Urethane Feeding and by Forced Breeding

Abstract: Intragastric feeding of dimethylbenz-[a]anthracene in corn oil together with urethane in the drinking water and forced breeding were successful in rapidly inducing (2-4 months) mammary tumors (adenoacanthomas) in inbred NYLR/Nya mice, which have a low incidence of spontaneous breast tumors and of other tumors. The tumors could be quickly and permanently transformed to an ascites form by intraperitoneal inoculation of enzyme-dissociated cells and subsequent serial passage of free cells. Only 10 (3 in some cases) serial passages were required, thus conveniently providing multiple isogeneic carcinoma cell lines in this strain of mice. Some tumor cell lines proliferated strongly in the abdominal cavity even on the first passage.

Histologic origin of rat ovarian cancer induced by direct application of 7,12-dimethylbenz(a)anthracene.

Abstract: A direct application of 7,12-dimethylbenz(a)anthracene (DMBA) to the ovary successfully produces an ovarian epithelial cancer in rat. To identify the histogenic process, the DMBA-treated rats were serially examined from the 15th to the 40th week following DMBA application. Furthermore, from the anticipated effect of estrogen on surface epithelial proliferation, the rats were put into an hyperestrogenic condition for immunohistochemical characterization of the tumor tissue. At the 20th week, the surface epithelial proliferations were seen to show remarkable multistratification with cellular pleomorphism. The proliferated surface cells began to infiltrate into the stroma to then form irregular gland structures. In immunoperoxidase stainings, both the adenocarcinoma cell and the surface cell has the same character in the immunohistochemical reaction for estradiol. On the base of these results, the induced cancer in rat was believed to have a similar histogenic process to the common epithelial tumors in human ovary.

Food restriction inhibits [3H] 7,12-dimethylbenz(a)anthracene binding to mouse skin DNA and tetradecanoylphorbol-13-acetate stimulation of epidermal [3H] thymidine incorporation.

Abstract: It has been known for many years that reducing the food intake of laboratory mice and rats inhibits the development of a broad spectrum of chemically induced and spontaneous tumors, but the mechanism of this effect is poorly understood Food restriction of A/J mice for two weeks is now shown to inhibit the binding of topically applied [3H]7,12-dimethylbenz(a)anthracene (DMBA) to skin DNA by 50% and to abolish the stimulation of [3H]-thymidine incorporation in the epidermis produced by topical application of the tumor promoter tetradecanoylphorbol-13-acetate (TPA) Similar effects on the actions of DMBA and TPA are observed following topical application of the adrenal steroid, dehydroepiandrosterone (DHEA), a potent glucose-6-phosphate dehydrogenase (G6PDH) inhibitor, while food restriction for two weeks depresses epidermal G6PDH activity by 60% It is suggested that both the inhibition of [3H]DMBA binding to skin DNA and the TPA stimulation in epidermal [3H]thymidine incorporation result from a reduction in the NADPH cellular pool as a result of G6PDH inhibition

Selective interactions of cytochromes P-450 with the hydroxymethyl derivatives of 7, 12-dimethylbenz[a]anthracene

Abstract: Competition between a hydroxylated metabolite and the parent polycyclic aromatic hydrocarbon (PAH) for metabolism at cytochromes P-450 may result in the generation of hydroxylated dihydrodiol epoxides. The effectiveness of the competition between 7-hydroxymethyl-12-methylbenz[a]anthracene (7HOMMBA) or 12-hydroxymethyl-7-methylbenz[a]anthracene (12HOMMBA) and 7,12-dimethylbenz[a]anthracene (DMBA) is highly dependent on the form(s) of cytochrome P-450 in the microsomes. The inhibitory effects of exogenously added 7HOMMBA or 12HOMMBA on DMBA metabolism were 30- to 50-fold greater in 3-methylcholanthrene (MC)-induced rat liver microsomes (Ki = 0.4 microM) compared to either uninduced or phenobarbital (PB)-induced liver microsomes (Ki = 14 and 11 microM, respectively). Similarly, product inhibition of total DMBA metabolism by metabolites generated in situ was significant only in MC-induced liver microsomes (Ki' = 2.5 microM). Metabolism of 7HOMMBA in these microsomes was strongly restricted by an unusual substrate inhibition derived from the inhibitory binding of a second molecule of 7HOMMBA. This same phenomenon was observed with reconstituted cytochrome P-450c but not with PB-induced or uninduced microsomes. Complex formation by binding of DMBA, 7HOMMBA, and 12HOMMBA to purified P-450c reconstituted in phospholipid micelles was determined by optical spectroscopy and fluorescence quenching. Binding affinities of both the 7HOMMBA and 12HOMMBA (Kd = 95 and 110 nM, respectively), were 2.5-fold higher compared to that of DMBA (Kd = 265 nM). These results provide a first demonstration that hydroxylation of a PAH can lead to preferential metabolism through an increased affinity for cytochrome P-450.

Tulipa gesneriana bulb extracts activate promutagenic 7,12-dimethylbenz[a]anthracene in the Salmonella/Ames assay

Abstract: Crude extracts from Tulipa gesneriana bulbs have been tested for their ability to activate 7,12-dimethylbenz[a]anthracene (DMBA) in the Salmonella mutagenicity assay. Bacteria of strain TA98 were incubated for 30 min at 37 degrees C with the mixture of the promutagen and bulb extracts prior to plating. The frequency of his+ revertants increased in relation to both the promutagenic dose and the amount of bulb extract in the mixture, and under optimal conditions, was more than 50 times higher than the value found after the action of the promutagen alone. Addition of NADP and glucose 6-phosphate to the incubation mixture did not seem to be obligatory.

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.

Abstract: 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.

Epidermal Changes following Application of 7,12-Dimethylbenz(a)anthracene and 12-O-Tetradecanoylphorbol-13-acetate to Human Skin Transplanted to Nude Mice Studied with Histological Species Markers

Abstract: Effects of the tumor initiator 7,12-dimethylbenz( a )anthracene (DMBA) and of the tumor promoter 12- O -tetradecanoylphorbol-13-acetate (TPA) on epidermis of human fetal and adult skin were studied in the nude mouse/human skin model. Human skin grafts on NC nude mice were exposed to two topical applications of 1 mg of DMBA in 50 µl of acetone with an interval of 3 days and/or to applications of 10 µg of TPA in 50 µl of acetone twice weekly. In some animals, it was attempted to augment the susceptibility of the grafts to the tumor-initiating effect of DMBA by pretreatment with TPA or ultraviolet light. The mice were sacrificed 8–32 wk after the initial treatment. Tumors did not appear in the central portions of any of the grafts, but epidermal tumors were seen at the graft border in 34.9% of the DMBA-treated animals. To identify human epidermis on the grafts and to determine the species origin of the induced tumors, two independently working histological marker methods were applied. ( a ) The first is detection of a human Blood Group B-like antigen present in mouse epidermis and in chemically induced murine epidermal tumors. This antigen cannot be demonstrated in human epidermis and in epidermal tumors of human patients. ( b ) The second is histological staining with the DNA-specific fluorochrome, bisbenzimide, displaying a characteristic pattern of 5–10 intranuclear fluorescent bodies in murine nonneoplastic epidermal cells and in murine epidermal tumor cells. Such a pattern is not seen in human epidermis and in epidermal tumors of human patients. The studies showed that TPA treatment resulted in epidermal hyperplasia in both the human epidermis and the adjacent mouse epidermis and that the induced tumors were derived from murine tissue. The mechanisms behind the DMBA action in the nude mouse/human skin model are discussed, and suggestions for future carcinogenesis studies on the model are given.

Cell-mediated immune response in mice during the two-step carcinogenesis experiment by 7,12-dimethylbenz(a)anthracene and 12-O-tetradecanoylphorbol-13-acetate.

Abstract: Cell mediated immune response in ICR mice during the two step carcinogenesis experiment induced by DMBA plus TPA was followed by determining the delayed type hypersensitivity to two antigens, ie DNFB applied to skin and SRBC injected intraperitoneally In mice developing tumors from 3 to 15 weeks after DMBA initiation, delayed type hypersensitivity to DNFB was suppressed to as low as 30% that of normal mice This suppression was not recovered by treatment of indomethacin No difference in delayed type hypersensitivity to SRBC was observed on between the mice on the two-step carcinogenesis experiment and normal mice