CYP1B1 determines susceptibility to low doses of 7,12-dimethylbenz[a]anthracene-induced ovarian cancers in mice: correlation of CYP1B1-mediated DNA adducts with carcinogenicity

Jeroen Buters1,8, Leticia Quintanilla-Martinez2, Wolfgang Schober3, Volker J. Soballa4, Josef Hintermair5, Thomas Wolff5, Frank J. Gonzalez6 and Helmut Greim7

1 Division of Environmental Dermatology and Allergology, GSF/TUM, Centre for Allergy and Environment, Technische Universität München, Biedersteinerstrasse 29, 80802 München, Germany,
2 GSF-Forschungszentrum für Umwelt und Gesundheit, Institut für Pathologie, Ingolstädter Landstrasse 1, 85764 Oberschleißheim, Germany,
3 Institut für Lebensmittelchemie, Technische Universität München, Lichtenbergstrasse 4, 85747 Garching, Germany,
4 Produktsicherheit/Toxikologie, Degussa AG, 83303 Trostberg, Germany,
5 GSF-Forschungszentrum für Umwelt und Gesundheit, Institut für Toxikologie, Ingolstädter Landstrasse 1, 85764 Oberschleißheim, Germany,
6 National Cancer Institute, Building 37, Room 3E24, Bethesda, MD, USA and
7 Institut für Toxikologie und Umwelthygiene, Technische Universität München, Lazarettstrasse 61, 80636 München, Germany


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We showed previously that CYP1B1-null mice developed 10 times less lymphomas than wild-type mice after receiving 7,12-dimethylbenz[a]anthracene (DMBA). In this study a 10-fold lower dose was applied to differentiate between toxicity induced lymphomas (200 µg/mouse/day) and tumor initiation (20 µg/day). DMBA adducts to DNA of organs of mice, or to DNA of V79 cells expressing single mice or human cytochrome P450 isoenzymes were also measured. Mice were dosed three cycles of 5 days/week with DMBA in corn oil orally. Histopathology was determined at intermittent death or 1 year after dosing. DMBA–DNA adducts were assayed by 32P-postlabeling. At 20 µg/day, wild-type mice developed ovary (71%, stromal cells derived), skin (36%), uterus (64%) and lung (14%) hyperplasias. At this dose the CYP1B1-null mice developed no lymphomas, 25% ovary (epithelial cells derived), 8% skin, 58% uterus and 33% lung tumors. Oil control mice (n = 35) developed only eight, mostly different, hyperplasias. Wild-type mice had more DMBA–DNA adducts than the CYP1B1-null mice. The differences were highest in thymus, spleen, ovaries and testes (5–7-fold). Additionally, one specific DMBA–DNA adduct was reduced in CYP1B1-null mice. V79-cells expressed mouse CYP1B1 was 35 times more active than mouse CYP1A1 in forming DMBA–DNA adducts. Human CYP1B1 was 2.5 times less active than mouse CYP1B1 but 2.3-fold more active than human CYP1A1. CYP1B1 is the dominant enzyme in metabolizing DMBA to carcinogenic metabolites at high and low doses in mice, leading to an increased tumor rate of especially the ovaries at low doses of DMBA. Wild-type mice had more DMBA–DNA adducts than CYP1B1-null mice. Additionally, a specific adduct was less present in the CYP1B1-null mice. Human CYP1B1 was less active than mouse CYP1B1, but more active than human CYP1A1 in forming DMBA–DNA adducts. Thus, we expect CYP1B1 to be an important DMBA activating enzyme in humans also.

Abbreviations: CYP, cytochrome P450; DMBA, 7,12-dimethylbenz[a] anthracene.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Some polycyclic aromatic hydrocarbons are potent rodent carcinogens. Ample evidence suggests that they are also human carcinogens (1), but direct evidence is lacking. Alternative evidence could be obtained by explaining the toxicologic mechanism on a molecular level in an animal model. The individual steps leading to the development of cancers in animals could be separately checked in human tissues and compared with the animal activities. Thus, a stepwise prediction of the human risk for chemical carcinogenesis would be possible.

To obtain such a stepwise-based prediction, detailed knowledge on the toxicological mechanism in vivo is needed. Null mice (knockout mice) lacking the expression of genes involved in carcinogenesis provide the in vivo information needed (2).

DMBA is a polycyclic aromatic hydrocarbon that is widely used as a model chemical carcinogen that has been applied to the rat mammary tumor model (3). It is probably the best studied polycyclic aromatic hydrocarbon available, besides benzo[a]pyrene. DMBA, like other polycyclic aromatic hydrocarbons, needs metabolic activation by cytochrome P450 to deploy its carcinogenicity and its 3,4-diol metabolite is thought to be the procarcinogen that is further metabolized by cytochrome P450 to the ultimate carcinogen, 1,2-epoxide-3,4-diol-DMBA, which forms adducts to DNA (46). These adducts lead to mutations (7,8), that are a prerequisite for the development of tumors. The cytochrome P450 responsible for this metabolic activation was for decades assumed to be CYP1A1 (9). Expressed or induced CYP1A1 was capable of activating DMBA to the procarcinogen 3,4-diol DMBA (10,11). That CYP1A1 was crucial in vivo for the development of cancers was then deduced from these, often in vitro, data. However, later on it was discovered that the regulation of CYP1B1 expression is similar to the regulation of CYP1A1 (12), and the observed in vivo effects could have been equally attributed to CYP1B1. The existence of CYP1B1 was not known before 1994 (13). Indeed, recent data revealed that in vivo CYP1B1, and not CYP1A1, is the predominant enzyme in the metabolic activation of DMBA to carcinogenic metabolites (14). CYP1B1 is expressed in human lung and could play an important role in the metabolic activation of components of tobacco smoke like polycyclic aromatic hydrocarbons to carcinogenic metabolites.

Metabolic activation of polycyclic aromatic hydrocarbons and the formation of DNA adducts are a prerequisite of chemical carcinogenesis, but additional factors are needed to transform these adducts into mutations and subsequently tumors. Metabolic activation by CYP1B1 is the first step in the long pathway of chemical carcinogenesis leading to tumors.

Because the lethality in our previous study was high (14), toxicity induced tumors might have obscured the direct mutagenic effect of DMBA. In this study we prevented premature death by dosing the animals with 10-fold lower concentrations of DMBA. In an attempt to estimate the human capacity for metabolic activation we compared the catalytic activity of cDNA-expressed human and mouse cytochromes P450 that play a role in the metabolism of DMBA.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials
The following chemicals were purchased from suppliers in Germany: 7,12-dimethylbenz[a]anthracene, micrococcal nuclease, potato apyrase (grade VI), RNase A, and RNase T1 from Sigma (Deisenhofen), spleen phosphodiesterase and proteinase K from Boehringer (Mannheim), T4 polynucleotide kinase from Amersham (Frankfurt), [{gamma}-32P]ATP-tetraethylammonium salt (sp. act. 3000 Ci/mmol) from NEN-Dupont (Dreieich), and polyethyleneimine-cellulose TLC sheets, 0.1 mm from Macherey-Nagel (Düren). DMEM, trypsin, L-glutamine, sodium pyruvate and penicillin/streptomycin were from Biochrom (Berlin). Corn oil was obtained from a commercial source (Mazola®, Heilbronn). 7,12-Dimethylbenz[a]anthracene trans 3,4-dihydrodiol was obtained from NCI Chemical Carcinogenesis Reference Repositories, Midwest research Institute, Kansas City, USA.

Carcinogenesis study.
Seven-week-old female wild-type and CYP1B1-null mice, both with a mixed genetic background of C57Bl/6 and 129/Sv, were dosed intragastrically with either 200 µg (adduct study) or 20 µg (carcinogenicity study) DMBA/mouse (6.7 and 0.7 mg/kg, respectively) in 100 µl corn oil or corn oil alone, once daily for 5 days/week for 3 weeks. An additional study for the measurement of adducts was done with animals dosed for 1 week only. The average body weights of the groups were determined and the concentration of DMBA was adjusted so that the animals received the same dose per kilogram body weight, delivered in 100 µl oil. The mice were subsequently observed for 7 months to ~1 year for the appearance of tumors (termination at 7 months for 200 µg/mouse groups, at 12.7 months for wild-type and 13.6 months for CYP1B1-null receiving 20 µg/mouse and 12 months for oil control mice). In the DMBA–DNA adduct studies the mice were killed half a day after the last dose. Mice were killed by carbon dioxide asphyxiation during this period when either a sudden weight loss of >20% or a tumor >1 cm occurred, and were prepared for autopsy immediately. Some mice died without these symptoms. After 1 year the surviving mice were killed by carbon dioxide. All mice were analyzed by histopathology for the presence and identity of tumors.

Pathology.
All mice underwent a complete autopsy. The organs were stored in 10% buffered formalin and embedded in paraffin. In every case, a pathologist reviewed the hematoxylin and eosin stained sections. The organs analyzed included lungs, thymus, lymph nodes, spleen, liver, pancreas, kidneys, adrenal glands, intestine and female or male genital system. When needed, macroscopically detected tumors were embedded separately and also examined.

Cell culture.
The following cell lines were kindly provided by Prof. Dr J.Doehmer: Human CYP1A1 [V79MZh1A1 (15)], human CYP1A2 [V79MZh1A2 (16)], human CYP1B1 [V79MZh1B1 (17)], mouse CYP1A1 [V79MZm1A1 (18)], mouse CYP1B1 (V79MZm1B1) and parental V79 cells [V79MZ (19)]. V79 cells were cultured in DMEM (Dulbecco’s modified eagle medium with 3.7 g/l sodium bicarbonate and 4.5 g/l glucose) supplemented with 1 mM sodium pyruvate, 4 mM L-glutamine, 10% fetal calf serum, 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C, 7.5% CO2 and 90% humidity.

32P-Postlabeling.
Groups of mice other than those used for the carcinogenicity study were treated with 200 µg/mouse either 1 or 3 weeks as described above and killed by carbon dioxide asphyxiation at the fifth day after dosing. The organs were removed, frozen in liquid nitrogen and stored at –80°C until analysis.

All subsequent manipulations were performed under reduced light (20). About 400 mg of a tissue was thawed and DNA was isolated as described by Gupta (21) using RNase A, RNase T1 and proteinase K treatment followed by phenol–chloroform extraction, ethanol precipitation and quantified by UV-spectroscopy. Samples of isolated DNA (2.5 µg) were digested to 3'-nucleotide monophosphates with a mixture of micrococcal nuclease and spleen phosphodiesterase. The butanol enrichment protocol of Gupta was employed with minor modifications (22,23). Briefly, the dried butanol extract of the hydrolysate of 2.0 µg DNA was labeled with [{gamma}-32P]ATP and treated with apyrase to degrade excess ATP. Two-dimensional PEI-cellulose chromatography was performed to separate the adducts. Quantification of the radioactive spots was performed with a Canberra-Packard electronic autoradiography instant imager (Meriden, CT). All values were corrected for the background level. For statistical analysis the values below detection limit were treated as the detection limit of 0.5 adducts/109 bases.

To analyze the DMBA adducts to the DNA of V79 cells expressing the different cytochromes P450, the cells were seeded in three 10 cm plates at 0.5 • 106 cells/plate. The cells were allowed to grow for one day (doubling time 18–24 h), and the medium was then replaced by fresh medium containing DMBA. The cells were incubated for 6 or 24 h, harvested by trypsinization, washed with PBS and the cell pellet was stored at –80°C until analysis. 32P-Postlabeling was done as described above.

To identify the adducts to nucleotides, 0.17 mM of 40mer oligonucleotides of either poly-A, C, T or G or poly-A annealed to poly-T (poly-AT) or poly-G annealed to poly-C (poly-GC) were incubated with 20 µM 3,4-diol-DMBA for 10 or 30 min at 37°C with 0.5 mg/ml TCDD induced mice liver microsomes and 1 mM NADPH in 30 mM Tris buffer pH 7.4. Annealing was achieved by mixing equal amounts of polynucleotides, heating to 95°C, and slowly cooling to room temperature. Analysis of the adducts was as described above. In selected cases the samples were also co-eluted with organ derived DNA adducts.

Statistical analysis.
Differences between groups were determined with a two-sided, heteroscedastic Student’s t-test unless an F-test indicated a normal distribution; then a homoscedastic Student’s t-test was employed. P < 0.10 was considered statistically significant (24).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Previously, we dosed mice at 200 µg/mouse/day (6.7 mg/kg/day) (14). Wild-type mice died predominantly of lymphoblastic lymphoma whereas CYP1B1-null mice were resistant to the toxicity of DMBA. No sex difference was noticed in the frequency of any tumor.

In the present study only females were dosed at a 10-fold lower dose of 20 µg/mouse/day (0.7 mg/kg, cumulative 10 mg/kg/mouse). Additionally male and female wild-type, and male CYP1B1-null control mice were treated with only the solvent corn oil. To determine the toxicologic mechanism the DNA adducts of DMBA at 200 µg/mouse were measured in the organs of separately treated mice. To extrapolate to humans the DMBA–DNA adduct forming capacity of individual mouse and human cytochromes P450 was determined.

Carcinogenicity of DMBA.
In control mice treated with corn oil only, eight hyperplasias in 35 separate animals were observed: one lung adenoma and one liver adenoma in male wild-type mice (n = 10), one follicular lymphoma and one endometrial cystic hyperplasia in wild-type females (n = 9), and no tumors in CYP1B1-null male mice (n = 8), and one lymphoma, one lung adenoma, and two endometrial cystic hyperplasia in female CYP1B1-null mice (n = 8).

At a dose of 20 µg/mouse/day, wild-type mice developed more malignancies than CYP1B1-null mice. The target organs at 20 µg/mouse/day were however different from those at 200 µg/mouse/day.

At 200 µg/mouse/day ~60%, at 20 µg/mouse/day 29% of the wild-type mice died during the observation period whereas only 8.3% (one out of 12) of the CYP1B1-null mice treated with 20 µg DMBA/day died during the same period (Figure 1Go). Upon pathological analysis, 71% of the wild-type females had ovary tumors at 20 µg/mouse/day, whereas only 25% of the CYP1B1-null mice had ovary tumors. In addition, the ovary tumors in the CYP1B1-null mice were cystadenomas, which originate from ovarian surface epithelium, whereas the tumors in the wild-type mice were granulosa cell tumors, originating from the ovarian stromal cells. Twenty-one percent of the wild-type female mice developed lymphomas and 36% skin hyperplasias whereas the CYP1B1-null female mice developed less of these hyperplasias (none and 8%, respectively). The origin of the lymphomas at 20 µg/mouse/day was mostly follicular (i.e. always B-cell origin), whereas previously at 200 µg/mouse/day the origin was exclusively lymphoblastic (i.e. mostly T-cell origin). The CYP1B1-null female mice developed an equal amount of uterus malignancies as the wild-type mice (64 versus 58%). The CYP1B1-null female mice developed more lung malignancies (33%, n = 4) than the wild-type mice (14%, n = 2) at 20 µg/mouse/day but the difference is small. Although CYP1B1-null mice developed less hyperplasias than the wild-type mice, they developed more hyperplasias than control mice treated with corn oil only.



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Fig. 1. Lethality of wild-type and CYP1B1-null mice after treatment with DMBA. Seven weeks old mice were treated with three cycles of 5 days/week with DMBA dissolved in corn oil at the indicated dose i.p., and subsequently observed for the appearance of tumors (7 months for the 200 µg/mouse/day, and ~12 months for the 20 µg/mouse/day and oil control mice). Animals surviving this observation period were killed by carbon dioxide asphyxiation.

 
One wild-type female mouse died during the first 3 weeks of dosing without any pathological finding and was omitted from analysis. The first death after treatment occurred 74 days into the observation period (wild-type, 95 days after initiation of treatment).

The CYP1B1-null female mice were 9% heavier at the start of dosing and 30% at the end of the observation period than the wild-type mice (growth curve 4.7•X0.375 versus 6.7•X0.252, respectively, r2 > 0.98).

Adducts in the organs
Total adduct level of DMBA metabolites to the DNA of organs of the mice treated with 200 µg/mouse/day was determined by 32P-postlabeling. Levels of adducts after 20 µg/mouse/day dosage were not measured as they are below the detection limit. Mice treated with oil only did not show DNA adducts. Limit of detection was 0.5 adducts/109 nucleotides.

The sum of all measured adducts per mouse was two to three times higher in the wild-type (females 4034 ± 2336, males 4883 ± 2538 adducts/109 bases) than CYP1B1-null mice (females 1287 ± 996, males 2542 ± 729 adducts/109 bases), reaching statistical significance in the female group treated for 3 weeks (female versus female, P < 0.10). Pathways other than CYP1B1 metabolism must however also contribute to the appearance of DMBA–DNA adducts, as all organs of the CYP1B1-null mice showed substantial DMBA–DNA adduct levels. These other pathways must involve factors that are not present in V79 cells but present in tissues, i.e. other enzymes, as viable V79 cells, devoid of cDNA-expressed cytochrome P450 but otherwise functional, showed no DMBA–DNA adducts (Figure 3Go and Table IIIGo).



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Fig. 3. TLC-maps of 32P-labeled DMBA–DNA adducts from V79 cells (expressing different cytochrome P450 isoenzymes) treated with DMBA. V79 cells expressing cytochrome P450 isoenzymes of either human (h) or mouse (m) origin were incubated with 20 µM DMBA for 24 h. Exposure times on an Imaging system (Canberra-Packard ‘Instant Imager’) were 19, 22, 15, 20 and 5 min for hCYP1A1, hCYP1A2, hCYP1B1, mCYP1A1 and mCYP1B1, respectively. Control cells were parental V79 cells devoid of cytochrome P450. The spots from mCYP1A1 appeared closer to the origin.

 

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Table III. Relative amounts of the major adducts of DMBA to the DNA of selected organs after 3 weeks of DMBA dosage: adduct 1 is put at 1 (100%); means ± SD are given (n = 3)
 
Organs with high DMBA–DNA adduct levels, like heart and lung, were not the organs where tumors developed. Although the absolute amounts of adducts were almost always higher in the wild-type than in the CYP1B1-null mice, in some organs the total amount of DMBA–DNA adducts was selectively increased. In those organs (spleen, thymus and ovaries) tumors developed. An exception were testis, where despite a 7-fold difference in adduct level between wild-type and CYP1B1-null mice no tumors were observed (Tables I and IIGoGo).


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Table I. Tumors in wild-type and CYP1B1-null mice after DMBA administration (tumor bearing animals versus total animals in percent)
 

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Table II. Total DMBA–DNA-adduct level in tissues of mice treated with 200 µg/mouse/day (percent of total adducts per animal): the same method as in Figure 2Go was used
 
The difference in adduct levels after 1 or 3 weeks treatment was only quantitative, again after 1 week of treatment the spleen, thymus and ovaries having relatively the most DMBA–DNA adducts of the analyzed organs.

The DMBA–DNA adduct patterns were similar between the wild-type and CYP1B1-null mice (Figure 2Go, exemplified by heart and spleen). To compare the relative intensities of the adducts, adduct 1 was set at 100% and the relative intensities of the other adducts were calculated (Table IIIGo). In the CYP1B1-null mice one specific DMBA–DNA adduct was especially low, adduct 3, the other adducts were of comparable relative intensity between wild-type and CYP1B1-null mice. Adduct 3 was 2.3 (spleen) and 2.0 times (heart) lower in the male CYP1B1-null mice and 1.6 and 1.9 times lower, respectively, in the females (P < 0.01).



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Fig. 2. TLC-maps of 32P-labeled DMBA adducts to the DNA of organs of mice treated with DMBA. Mice were treated for 3 weeks with 200 µg/mouse (6.7 mg/kg) DMBA/day and killed by carbon dioxide asphyxiation. Representative organs are shown. Exposure time to X-ray film was 7 h for all. The arrow indicates the less abundant adduct 3 in CYP1B1-null mice. WT, wild-type mice; KO1B1, CYP1B1-null mice.

 
Identification of DNA adducts
Because the adduct of interest, adduct 3, was shown to be a C1-3,4-dihydrodiol-1,2-epoxide-DMBA adduct to N6-adenosine (25), DNA preparations were incubated with the 3,4-diol metabolite of DMBA. Single-stranded poly-G reacted strongly with microsomal activated 3,4-dihydrodiol-DMBA, followed by single-stranded poly-A. A low reactivity was observed with single-stranded poly-T and no detectable adducts were seen with single-stranded poly-C. The adducts observed from single-stranded polymers were different from the adducts found after dosing DMBA to whole animals or double-stranded poly-AT or poly-GC. Double-stranded poly-GC showed only one adduct, co-migrating with adduct 1. Because single-stranded poly-C did not react with metabolically activated 3,4-dihydrodiol-DMBA, the adduct from poly-GC, and thus adduct 1 is likely to stem from guanosine. Double-stranded poly-AT gave several adducts, the most intense adduct co-migrating with adduct 2. Adduct 3 was only seen in the double-stranded poly-AT incubations (data not shown).

V79 cells expressing different cytochrome P450 isoenzymes
Parental V79 cells that do not express any cytochrome P450 were devoid of DMBA–DNA adducts (Figure 3Go and Table IVGo). All cDNA-expressed cytochromes P450 investigated were able to form all detectable DMBA adducts to the DNA of the V79 cells. No cytochrome P450 isoenzyme-specific pattern was detected. The difference in adduct pattern between the cytochrome P450 isoenzymes was purely quantitative. Adduct 3 (Figure 2Go), which was formed less in the CYP1B1-null compared with the wild-type mice, was present in all preparations. Interestingly, mouse CYP1B1 predominantly formed adduct 3.


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Table IV. Total levels of DMBA–DNA adducts of V79 cells that express different cytochrome P450 isoenzymes, treated with DMBA (h = human, m = mouse, RAL = radioactive adduct level): 100 000 cells in a 6 cm Petri dish were incubated with 2 or 20 µM DMBA for 6 or 24 h (n = 1)
 
Mouse CYP1B1 formed 20 (2 µM, 24 h) to 37-fold (20 µM, 24 h) more total DMBA–DNA adducts than mouse CYP1A1 (Table IVGo). Of the human cytochrome P450 isoenzymes CYP1B1 was the most active in forming total adducts. The difference between hCYP1B1 and hCYP1A1 was however only ~2.3-fold (at 2 µM, 6 h incubation). Human CYP1A2 played a minor role in the formation of DMBA–DNA adducts. Longer incubations and higher concentrations did result in equal or less DNA adducts. This was not due to cytotoxicity because at 20 µM DMBA, 6 h cytotoxicity was below 10% for all cell lines. At 20 µM DMBA, 24 h toxicity was 28% in V79 parental cells and an additional cytotoxicity due to cytochrome P450 expression was 9% for hCYP1A1, 2% for hCYP1A2, 17% for mCYP1B1 and none for hCYP1B1 or mCYP1A1 (n = 12 for all incubations). An inhibition of hCYP1B1, hCYP1A1 and mCYP1A1 activity with DMBA metabolites could have occurred. This effect was less pronounced with mCYP1B1. Product inhibition by DMBA was described (26) and could occur with some but not all cytochromes, although the effect could also stem from enzyme saturation.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mice treated with corn oil only developed little hyperplasias after 1-year observation. Thus, almost all observed tumors stem from treatment with DMBA, a potent carcinogen. At both doses, 200 and 20 µg/mouse/day, wild-type mice developed more malignancies than CYP1B1-null mice (Table IGo). Thus, the predominant enzyme in the process of activating DMBA to tumor initiating metabolites was clearly CYP1B1. Although the CYP1B1-null mice developed less hyperplasias than wild-type mice, they exhibited more hyperplasias than the oil control treated mice indicating that enzymes other than CYP1B1 must also be capable of activating DMBA to tumorigenic metabolites.

The main target tissues for DMBA administered at 200 µg/mouse/day were bone marrow, skin and lung (14). At 20 µg/mouse/day used in this study, which is one-fiftieth of the daily dose that is usually given to induce tumors in mice, the main target tissues were ovaries, uterus, skin and lung. Qing et al. (27) also reported a difference in organ specificity depending on the dose of DMBA.

Although the CYP1B1-null mice grew faster than wild-type mice (food intake ad libitum) this should not influence the conclusions as mice with a higher body mass, in this case CYP1B1-null mice, would be expected to have a higher tendency to develop malignancies than their leaner counterparts (28), but the opposite was the case.

The 200 µg/mouse/day dose is toxic to cells of the immune system, as less viable cells were isolated from spleen and thymus in mice after 14 days dosing of 90 or 300 µg DMBA/day/mouse as compared with controls (29,30). DMBA concentrations toxic to the lymph cells increase the replacement rate and guarantee that proliferating cells are exposed to DMBA and its carcinogenic metabolites, favoring the rapid development of lymphoblastic lymphomas (T-cell lymphomas). 20 µg/mouse/day are not toxic to the bone marrow or lymph cells (27) resulting in an increased survival of the mice due to the absence of toxicity induced lymphoblastic lymphomas (Figure 1Go and Table IGo). This gave other tissues enough time to proceed from mutated tissue to visible malignancies, resulting in the observed organ specificity.

The main pathologic finding at 20 µg/mouse/day was, as compared with 200 µg/mouse/day, the increased tumor incidence of the ovaries in the wild-type mice. Striking was the switching from ovarian granulosa cell tumors in the wild-type mice to cystadenomas in the CYP1B1-null mice. Granulosa cell tumors originate from ovarian stromal cells (fibroblasts). Mouse fibroblasts exclusively express CYP1B1 but no CYP1A1, not even after induction by Ah-receptor ligands like TCDD (31,32). Cystadenomas originate from ovarian epithelial cells. Epithelial cells express CYP1B1 constitutively, but also express CYP1A1 after induction. With the 200 µg DMBA/mouse regimen, CYP1A1 was clearly induced in lung fibroblasts (14), and to a lesser extent with 20 µg/mouse. CYP1A1 is also capable of activating DMBA to the procarcinogen, i.e. 3,4-diol metabolite, although at one-fifth the rate of CYP1B1 (10). In the absence of CYP1B1, as is the case in the CYP1B1-null mice, induced CYP1A1 in ovary epithelium but not in ovary fibroblasts could be responsible for the switching of tumor origin in these mice.

Qing et al. (27) noticed mammary tumors at 20 µg/mouse/day in SENCAR (sensitive to carcinogenesis) mice, whereas none were observed in this study, but ovary tumors instead. Both the mammary gland and the ovary express CYP1B1 (3335). The absence of mammary tumors could be explained by the 3 weeks (this study) instead of Qing’s 6 weeks tumor induction protocol, resulting in half the cumulative dose of DMBA, or by a different genetic sensitivity between SENCAR and the mice used in this study. The absence of ovary tumors in Qing’s study is probably due to the fact that in the present study all organs were analyzed microscopically, thus finding tumors in ovary, uterus and lung that are macroscopically not always visible whereas Qing et al. only investigated macroscopic observed tumors in detail.

At 20 µg/mouse/day the CYP1B1-null mice develop more tumors than the oil control mice. Thus, enzymes other than CYP1B1 must be able to activate DMBA to carcinogenic metabolites. A candidate enzyme is CYP1A1 that is also capable of activating DMBA (see above). Other DMBA activating pathways, like the radical cation pathway (36) or peroxidation (37) are also likely to be relevant. Mice lacking microsomal epoxide hydrolase, required for the hydrolysis of the 3,4-epoxide of DMBA, still developed skin papillomas after DMBA application to the skin followed by TPA, although about four times less than wild-type mice (38). These alternative pathways, perhaps CYP1A1-mediated metabolism, could be especially active in uterus and lung, where tumors in the CYP1B1-null mice appeared. Indeed, a specific localization of DMBA in lung tissue of ß-naphthoflavone or PCB 126 induced, but not in uninduced mice was demonstrated (39). Against this hypothesis speaks the observation that equal amounts of DMBA–DNA adducts were found in the lungs and uteri of CYP1B1-null and wild-type mice.

CYP1B1 is expressed extrahepatically in bone marrow, lung, heart, kidney, ovaries, skin, uterus, breast and many other extrahepatic tissues (33,4044). In all tissues analyzed DNA adducts of DMBA were found. Mutations of these tissues are thus likely. However, to transform a mutation to a hyperplasia or full-blown cancer more factors are needed. Cell proliferation provides these factors and it was in the actively proliferating tissues that the CYP1B1 induced tumors developed, i.e. bone marrow, ovaries, skin and uterus (8). Exceptions were lung and testes. Testes of wild-type mice had a 7-fold higher adduct level than their CYP1B1-null counterparts, but developed no tumors. In testes, the mutated DNA might not be able to form resident transformed cells. If mutated spermatozoids fertilize eggs, the resulting embryos might carry mutations. Indeed, mutations in the offspring of DMBA treated males were found (45). A difference in DNA repair rate among tissues could also explain the higher incidence of tumors in some organs (8). For the higher tumor incidence in the lungs of the CYP1B1-null mice, while having comparable adduct levels with wild-type mice (Table IIGo, n.s.), we have no explanation. Thus, two factors seem to coincide: specific adducts preferentially generated by CYP1B1 and secondly a proliferation rate that fixes these adducts into mutations and drives them to develop into tumors.

The adduct level in the different organs did not correlate well with the site of origin of tumors and hyperplasias. Not those organs with the most DMBA–DNA adducts like heart, liver and lung but those organs with the greatest difference in DMBA–DNA adducts between wild-type and CYP1B1-null mice were those organs where the tumors appeared. In these organs CYP1B1 must be predominantly active. Additionally a DMBA–DNA adduct, adduct 3, was specifically reduced in the CYP1B1-null mice. Thus, two explanations for the lower incidence of tumors in the CYP1B1-null mice are possible:

  1. the tumors stem from a difference of total adducts in sensitive organs, or
  2. the tumors are predominantly initiated by a specific adduct in sensitive organs.

The latter explanation might be more appropriate because in LacZ transgenic mice (Mutamouse®) the mutations as a consequence of DMBA adducts to the DNA are 42% AT to TA mutations (46). In the Big Blue rat a similar number of 48% AT to TA transversions was documented (8). We could not determine the absolute identity of adduct 3. However, because single-stranded poly-A reacted, but poly-T hardly reacted with metabolically activated 3,4-dihydrodiol-DMBA, and poly-AT did, we expect the adducts from the double-stranded poly-AT to stem from adenosine. Thus, adduct 3 stems from a 3,4-dihydrodiol-DMBA metabolite to adenosine. Indeed, using the same method as employed in this study, other authors analyzed adduct 3 to be the syn bay 3,4-dihydrodiol 1,2 epoxide DMBA-C1 adduct to N6-adenosine (6,25).

These lines of evidence point at DMBA–adenosine as the main culprit leading to mutations that cause tumors, which was also supported by evidence presented by Tang et al. (47).

To extrapolate the findings in mice to the human situation, DMBA was incubated with V79 cells expressing mouse and human cytochromes P450. CYP1A1 and CYP1B1 are the main DMBA-metabolizing enzymes in humans (10,48). The levels of expression between the different cytochrome P450 cell lines is similar (Krebsfaenger et al., manuscript in preparation). Mouse CYP1B1 is clearly the dominant enzyme in forming metabolites that form adducts to DNA, more than CYP1A1. For the human enzymes this is also true, but the contribution of CYP1A1 in activating DMBA to form DNA adducts is six times greater than that of mouse CYP1A1. Because adduct 3 might be the main DNA-mutating adduct, it could be the difference in adduct 3, irrespective of its origin from CYP1A1 or CYP1B1, that determines the difference in susceptibility between man and mouse. The absolute in vivo amounts of both cytochromes in man and mouse are not known. In man, CYP1A1 is constitutively not detectable (49), unlike rodents that show a constitutive level of CYP1A1 expression (50). CYP1A1 is despite its presence in mice not the main DMBA activating enzyme. If several assumptions are made: (i) that the V79 cDNA-expression system is representative for the in vivo catalytic activity of CYP1A1 and CYP1B1 and (ii) that the level of expression of CYP1B1 is identical between the species, then human CYP1B1 is about two times less active than mouse CYP1B1, but more active than human CYP1A1 in activating DMBA to DNA binding metabolites.

Because of the multi-step nature of carcinogenesis it cannot be concluded from our data that humans, like mice, are sensitive to the carcinogenic potential of DMBA.

DMBA has not been found in the environment, perhaps because of its light sensitivity (20). The results with DMBA might however be applicable to other environmentally present polycyclic aromatic hydrocarbons such as benzo[a]pyrene and dibenzo[a,l]pyrene.

The main enzyme in mice that activates DMBA to carcinogenic metabolites is CYP1B1. An additional role of another enzyme, perhaps CYP1A1, is likely.

DMBA targets all tissues but tumors develop only in rapidly turned-over tissues that express CYP1B1, like bone marrow and ovaries. The classic theory that polycyclic aromatic hydrocarbons are activated by cytochrome P450 to form adducts to DNA that lead to mutations is fully supported by our data. For DMBA and perhaps other polycyclic aromatic hydrocarbons that are, unlike DMBA, environmentally present, the main cytochrome P450 correlated with carcinogenicity is CYP1B1.


    Notes
 
8 To whom correspondence should be addressed Email: buters{at}lrz.tum.de Back


    Acknowledgments
 
This work was funded in part by Deutsche Forschungsgemeinschaft grant DO 242/9-1 and 242/9-3 (J.B.).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. IARC (1983) Polynuclear Aromatic Hydrocarbons, Part 1: Chemical, Environmental and Experimental Data. IARC Scientific Publications, IARC, Lyon.
  2. Gonzalez,F.J. (2001) The use of gene knockout mice to unravel the mechanisms of toxicity and chemical carcinogenesis. Toxicol. Lett., 120, 199–208.[CrossRef][ISI][Medline]
  3. Russo,J. and Russo,I.H. (1996) Experimentally induced mammary tumors in rats. Breast Cancer Res. Treat., 39, 7–20.[ISI][Medline]
  4. Dipple,A., Moschel,R.C. and Bigger,C.A.H. (1984) Polynuclear aromatic hydrocarbons. In Searle,C.E. (ed.), Chemical Carcinogens. American Chemical Society, Washington, DC, Vol. 1, pp. 245–314.
  5. Cheng,S.C., Prakash,A.S., Pigott,M.A., Hilton,B.D., Roman,J.M., Lee,H.M., Harvey,R.G. and Dipple,A. (1988) Characterization of 7,12-dimethylbenz[a]anthracene-adenine nucleoside adducts. Chem. Res. Toxicol., 1, 216–221.[ISI][Medline]
  6. Cheng,S.C., Prakash,A.S., Pigott,M.A., Hilton,B.D., Lee,H., Harvey,R.G. and Dipple,A. (1988) A metabolite of the carcinogen 7,12-dimethylbenz[a]anthracene that reacts predominantly with adenine residues in DNA. Carcinogenesis, 9, 1721–1723.[Abstract]
  7. Dipple,A., Khan,Q.A., Page,J.E., Ponten,I. and Szeliga,J. (1999) DNA reactions, mutagenic action and stealth properties of polycyclic aromatic hydrocarbon carcinogens (review). Int. J. Oncol., 14, 103–111.[ISI][Medline]
  8. Shelton,S.D., Cherry,V. and Manjanatha,M.G. (2000) Mutant frequency and molecular analysis of in vivo lacI mutations in the bone marrow of Big Blue rats treated with 7, 12-dimethylbenz[a]anthracene. Environ. Mol. Mutagen., 36, 235–242.[CrossRef][ISI][Medline]
  9. Kawajiri,K. (1999) CYP1A1. IARC Scientific Publications, Chapter 15, IARC, Lyon, pp. 159–172.
  10. Savas,U., Carstens,C.P. and Jefcoate,C.R. (1997) Biological oxidations and P450 reactions. Recombinant mouse CYP1B1 expressed in Escherichia coli exhibits selective binding by polycyclic hydrocarbons and metabolism which parallels C3H10T1/2 cell microsomes, but differs from human recombinant CYP1B1. Arch. Biochem. Biophys., 347, 181–192.[CrossRef][ISI][Medline]
  11. Sato,S. and Tomita,I. (1998) Response differences among mouse strains in DNA damage and skin carcinogenicity of 7,12-dimethylbenz[a]anthracene are due to inducible aryl hydrocarbon hydroxylase activity. Biol. Pharm. Bull., 21, 90–92.[ISI][Medline]
  12. Angus,W.G., Larsen,M.C. and Jefcoate,C.R. (1999) Expression of CYP1A1 and CYP1B1 depends on cell-specific factors in human breast cancer cell lines: role of estrogen receptor status. Carcinogenesis, 20, 947–955.[Abstract/Free Full Text]
  13. Savas,Ü., Bhattacharyya,K.K., Christou,M., Alexander,D.L. and Jefcoate,C.R. (1994) Mouse cytochrome P-450EF, representative of a new 1B subfamily of cytochrome P-450s. Cloning, sequence determination and tissue expression. J. Biol. Chem., 269, 14905–14911.[Abstract/Free Full Text]
  14. Buters,J.T.M., Sakai,S., Richter,T., Pineau,T., Alexander,D.L., Savas,U., Doehmer,J., Ward,J., Jefcoate,C.R. and Gonzalez,F.J. (1999) Cytochrome P450 CYP1B1 determines susceptibility to 7,12-dimethylbenz[a]anthracene-induced lymphomas. Proc. Natl Acad. Sci. USA, 96, 1977–1982.[Abstract/Free Full Text]
  15. Schmalix,W.A., Maser,H., Kiefer,F., Reen,R., Wiebel,F.J., Gonzalez,F., Seidel,A., Glatt,H., Greim,H. and Doehmer,J. (1993) Stable expression of human cytochrome P450 1A1 cDNA in V79 Chinese hamster cells and metabolic activation of benzo[a]pyrene. Eur. J. Pharmacol., 248, 251–261.[Medline]
  16. Wölfel,C., Heinrich-Hirsch,B., Schulz-Schalge,T., Seidel,A., Frank,H., Ramp,U., Wächter,F., Wiebel,F., Gonzalez,F., Greim,H. and Doehmer,J. (1992) Genetically engineered V79 chinese hamster cells for stable expression of human cytochrome P4501A2. Eur. J. Pharmacol. Environ. Toxicol. Pharmacol. Sec., 228, 95.
  17. Luch,A., Coffing,S.L., Tang,Y.M., Schneider,A., Soballa,V., Greim,H., Jefcoate,C.R., Seidel,A., Greenlee,W.F., Baird,W.M. and Doehmer,J. (1998) Stable expression of human cytochrome P450 1B1 in V79 chinese hamster cells and metabolically catalyzed DNA adduct formation of dibenzo[a,l]pyrene. Chem. Res. Toxicol., 11, 686–695.[CrossRef][ISI][Medline]
  18. Schober,W. (2000) Metabolische Aktivierung von Bay- und Fjord-Region enthaltenden polyzyklische aromatische Kohlenwasserstoffe durch Cytochrome P450 von Maus, Ratte und Mensch. Dissertation: Institut für Toxikologie und Umwelthygiene, Fakultät für Chemie. Technische Universität München, München, pp. 117.
  19. Glatt,H., Gemperlein,I., Turchi,G., Heinritz,H., Doehmer,J. and Oesch,F. (1987) Search for cell culture systems with diverse xenobiotic-metabolizing activities and their use in toxicological studies. Mol. Toxicol., 1, 313–334.[Medline]
  20. Baird,W.M. and Dipple,A. (1977) Photosensitivity of DNA-bound 7,12-dimethylbenz[a]anthracene. Int. J. Cancer, 20, 427–431.[ISI][Medline]
  21. Gupta,R.C. (1984) Nonrandom binding of the carcinogen N-hydroxy-2-acetylaminofluorene to repetitive sequences of rat liver DNA in vivo. Proc. Natl Acad. Sci. USA, 81, 6943–6947.[Abstract]
  22. Gupta,R.C. (1985) Enhanced sensitivity of 32P-postlabeling analysis of aromatic carcinogen: DNA adducts. Cancer Res., 45, 5656–5662.[Abstract]
  23. Werner,S., Topinka,J., Wolff,T. and Schwarz,L.R. (1995) Accumulation and persistence of DNA adducts of the synthetic steroid cyproterone acetate in rat liver. Carcinogenesis, 16, 2369–2372.[Abstract]
  24. Lorenz,R.J. (1989) Biometrie. Grundbegriffe der Biometrie. Gustav Fischer Verlag, Stuttgart.
  25. Schmeiser,H., Dipple,A., Schurdak,M.E., Randerath,E. and Randerath,K. (1988) Comparison of 32P-postlabeling and high pressure liquid chromatographic analyses for 7,12-dimethylbenz[a]anthracene-DNA adducts. Carcinogenesis, 9, 633–638.[Abstract]
  26. Christou,M., Wilson,N.M. and Jefcoate,C.R. (1984) The role of secondary metabolism in the metabolic activation of 7,12-dimethylbenz[a]anthracene by rat liver microsomes. Carcinogenesis, 5, 1239–1247.[Abstract]
  27. Qing,W.G., Conti,C.J., LaBate,M., Johnston,D., Slaga,T.J. and Macleod,M.C. (1997) Induction of mammary cancer and lymphoma by multiple, low oral doses of 7,12-dimethylbenz[a]anthracene in SENCAR mice. Carcinogenesis, 18, 553–559.[Abstract]
  28. Hart,R.W., Turturro,A., Leakey,J., Allaben,W.T., Bucher,J.R., Rao,G.N. and Abdo,K. (1995) Diet and test animals. Science, 270, 1419–1420.
  29. Falzon,M., Vu,V.T., Roller,P.P. and Thorgeirsson,S.S. (1987) Relationship between 7,12-dimethyl- and 7,8,12-trimethylbenz[a]anthracene DNA adduct formation in hematopoietic organs and leukemogenic effects. Cancer Lett., 37, 41–49.[CrossRef][ISI][Medline]
  30. Burchiel,S.W., Davis,D.A., Ray,S.D., Archuleta,M.M., Thilsted,J.P. and Corcoran,G.B. (1992) DMBA-induced cytotoxicity in lymphoid and nonlymphoid organs of B6C3F1 mice: relation of cell death to target cell intracellular calcium and DNA damage. Toxicol. Appl. Pharmacol., 113, 126–132.[ISI][Medline]
  31. Eltom,S.E., Larsen,M.C. and Jefcoate,C.R. (1998) Expression of CYP1B1 but not CYP1A1 by primary cultured human mammary stromal fibroblasts constitutively and in response to dioxin exposure: role of the Ah receptor. Carcinogenesis, 19, 1437–1444.[Abstract]
  32. Savas,Ü., Christou,M. and Jefcoate,C.R. (1993) Mouse endometrium stromal cells express a polycyclic aromatic hydrocarbon-inducible cytochrome P450 that closely resembles the novel P450 in mouse embryo fibroblasts (P450EF). Carcinogenesis, 14, 2013–2018.[Abstract]
  33. Shimada,T., Hayes,C.L., Yamazaki,H., Amin,S., Hecht,S.S., Guengerich,F.P. and Sutter,T.R. (1996) Activation of chemically diverse procarcinogens by human cytochrome P-450 1B1. Cancer Res., 56, 2979–2984.[Abstract]
  34. Hushka,L.J., Williams,J.S. and Greenlee,W.F. (1998) Characterization of 2,3,7,8-tetrachlorodibenzofuran-dependent suppression and AH receptor pathway gene expression in the developing mouse mammary gland. Toxicol. Appl. Pharmacol., 152, 200–210.[CrossRef][ISI][Medline]
  35. Bhattacharyya,K.K., Brake,P.B., Eltom,S.E., Otto,S.A. and Jefcoate,C.R. (1995) Identification of a rat adrenal cytochrome P450 active in polycyclic hydrocarbon metabolism as rat CYP1B1. Demonstration of a unique tissue-specific pattern of hormonal and aryl hydrocarbon receptor-linked regulation. J. Biol. Chem., 270, 11595–11602.[Abstract/Free Full Text]
  36. Cavalieri,E.L. and Rogan,E.G. (1995) Central role of radical cations in metabolic activation of polycyclic aromatic hydrocarbons. Xenobiotica, 25, 677–688.[ISI][Medline]
  37. Yamazoe,Y., Zenser,T.V., Miller,D.W. and Kadlubar,F.F. (1988) Mechanism of formation and structural characterization of DNA adducts derived from peroxidative activation of benzidine. Carcinogenesis, 9, 1635–1641.[Abstract]
  38. Miyata,M., Kudo,G., Lee,Y.H., Yang,T.J., Gelboin,H.V., Fernandez-Salguero,P., Kimura,S. and Gonzalez,F.J. (1999) Targeted disruption of the microsomal epoxide hydrolase gene. Microsomal epoxide hydrolase is required for the carcinogenic activity of 7,12-dimethylbenz[a]anthracene. J. Biol. Chem., 274, 23963–23968.[Abstract/Free Full Text]
  39. Granberg,A.L., Brunstrom,B. and Brandt,I. (2000) Cytochrome P450-dependent binding of 7,12-dimethylbenz[a]anthracene (DMBA) and benz[a]pyrene. Arch. Toxicol., 74, 593–601.[CrossRef][ISI][Medline]
  40. Heidel,S.M., Holston,K., Buters,J.T., Gonzalez,F.J., Jefcoate,C.R. and Czupyrynski,C.J. (1999) Bone marrow stromal cell cytochrome P4501B1 is required for pre-B cell apoptosis induced by 7,12-dimethylbenz[a]anthracene. Mol. Pharmacol., 56, 1317–1323.[Abstract/Free Full Text]
  41. Sutter,T.R., Tang,Y.M., Hayes,C.L., Wo,Y.-Y.P., Jabs,E.W., Li,X., Yin,H., Cody,C.W. and Greenlee,W.F. (1994) Complete cDNA sequence of human dioxin-inducible mRNA identifies a new gene subfamily of cytochrome P450 that maps to chromosome 2. J. Biol. Chem., 269, 13092–13099.[Abstract/Free Full Text]
  42. Baron,J.M., Holler,D., Schiffer,R., Frankenberg,S., Neis,M., Merk,H.F. and Jugert,F.K. (2001) Expression of multiple cytochrome p450 enzymes and multidrug resistance-associated transport proteins in human skin keratinocytes. J. Invest. Dermatol., 116, 541–548.[Abstract/Free Full Text]
  43. Spink,D.C., Spink,B.C., Cao,J.Q., Depasquale,J.A., Pentecost,B.T., Fasco,M.J., Li,Y. and Sutter,T.S. (1998) Differential expression of CYP1A1 and CYP1B1 in human breast epithelial cells and breast tumors. Carcinogenesis, 19, 291–298.[Abstract]
  44. Marston,C.P., Pereira,C., Ferguson,J., Fischer,K., Hedstrom,O., Dashwood,W.M. and Baird,W.M. (2001) Effect of a complex environmental mixture from coal tar containing polycyclic aromatic hydrocarbons (PAH) on the tumor initiation, PAH-DNA binding and metabolic activation of carcinogenic PAH in mouse epidermis. Carcinogenesis, 22, 1077–1086.[Abstract/Free Full Text]
  45. Murota,T. and Shibuya,T. (1983) Modifying effects of the enzyme inducers, phenobarbital and 3-methylcholanthrene, on dominant lethal events induced by 7,12-dimethylbenz[a]anthracene in mice. Mutat. Res., 107, 329–336.[CrossRef][ISI][Medline]
  46. Gorelick,N.J., Andrews,J.L., Gu,M. and Glickman,B.W. (1995) Mutational spectra in the lacl gene in skin from 7,12-dimethylbenz[a]anthracene-treated and untreated transgenic mice. Mol. Carcinogen., 14, 53–62.[ISI][Medline]
  47. Tang,M.S., Vulimiri,S.V., Viaje,A., Chen,J.X., Bilolikar,D.S., Morris,R.J., Harvey,R.G., Slaga,T.J. and DiGiovanni,J. (2000) Both (+/–)syn- and (+/–)anti-7,12-dimethylbenz[a]anthracene-3,4-diol-1,2-epoxides initiate tumors in mouse skin that possess -CAA- to -CTA-mutations at Codon 61 of c-H-ras. Cancer Res., 60, 5688–5695.[Abstract/Free Full Text]
  48. Shou,M., Korzekwa,K.R., Krausz,K.W., Buters,J.T.M., Grogan,J., Goldfarb,I., Hardwick,J.P., Gonzalez,F.J. and Gelboin,H.V. (1996) Specificity of cDNA-expressed human and rodent cytochrome P450s in the oxidative metabolism of the potent carcinogen 7,12-dimethylbenz (a)anthracene. Mol. Carcinogen., 17, 241–249.[CrossRef][ISI][Medline]
  49. Larsen,M.C., Angus,W.G.R., Brake,P.B., Eltrom,S.E., Sukow,K.A. and Jefcoate,C.R. (1998) Characterization of CYP1B1 and CYP1A1 expression in human mammary epithelial cells: role of the aryl hydrocarbon receptor in polycyclic aromatic hydrocarbon metabolism. Cancer Res., 58, 2366–2374.[Abstract]
  50. Reiners,J.J., Jones,C.L., Hong,N. and Myrand,S.P. (1998) Differential induction of Cyp1a1, Cyp1b1, Ahd4 and Nmo1 in murine skin tumors and adjacent normal epidermis by ligands of the aryl hydrocarbon receptor. Mol. Carcinogen., 21, 135–146[CrossRef][ISI][Medline]
Received October 1, 2001; revised October 25, 2002; accepted October 29, 2002.