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
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Abstract |
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Abbreviations: CYP, cytochrome P450; DMBA, 7,12-dimethylbenz[a] anthracene.
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Introduction |
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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.
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Materials and methods |
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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 DMBADNA 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 (Dulbeccos 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 phenolchloroform 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 [-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 1824 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 Students t-test unless an F-test indicated a normal distribution; then a homoscedastic Students t-test was employed. P < 0.10 was considered statistically significant (24).
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Results |
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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 DMBADNA 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 1
). 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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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 4.7X0.375 versus 6.7X0.252, 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 DMBADNA adducts, as all organs of the CYP1B1-null mice showed substantial DMBADNA 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 DMBADNA adducts (Figure 3 and Table III
).
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The DMBADNA adduct patterns were similar between the wild-type and CYP1B1-null mice (Figure 2, 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 III
). In the CYP1B1-null mice one specific DMBADNA 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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V79 cells expressing different cytochrome P450 isoenzymes
Parental V79 cells that do not express any cytochrome P450 were devoid of DMBADNA adducts (Figure 3 and Table IV
). 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 2
), 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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Discussion |
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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 1 and Table I
). 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 Qings 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 Qings 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 DMBADNA 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 II, 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 DMBADNA adducts like heart, liver and lung but those organs with the greatest difference in DMBADNA 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 DMBADNA 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:
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 DMBAadenosine 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.
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Notes |
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Acknowledgments |
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References |
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