Abstract
7,12-Dimethylbenz[a]anthracene (DMBA) and N-methyl-N-nitrosourea (MNU) are important environmental carcinogens. Their different biological effects were examined in CBA/Ca H-2K haplotype inbred mice on the gene expression of c-myc, Ha-ras and p53 through a 24 hour period. Elevated expression of c-myc and Ha-ras genes was found in the spleen, lung, thymus and lymph nodes 6 and 12 hours after DMBA treatment and in the lung and thymus 3 hours after MNU treatment. In the liver, DMBA induced strong onco/suppressor gene expression as early as 6 hours after the treatment, but MNU increased the p53 gene expression 12 hours after the treatment. The gene expression patterns reflected the different mechanism of the direct acting MNU and metabolically activated DMBA. This phenomenon provides evidence as to the usefulness of detection of onco/supressor key gene expression as early molecular epidemiological biomarkers of carcinogenesis and carcinogenic exposure in animal model, useful in human cancer prevention practice as well.
7,12-Dimethylbenz[a]anthracene (DMBA) and N-methyl-N-nitrosourea (MNU) are important and proven pluripotent carcinogens (Figure 1). Their environmental and possible in vivo formation is also well demonstrated in the literature (1, 2).
In our earlier studies, we elucidated the early effect of DMBA and MNU on the expression of c-myc, Ha-ras and p53 onco/suppressor genes as molecular epidemiological biomarkers of carcinogenic exposure or carcinogenesis (3, 4). Both carcinogenic agents caused an elevation in the expression of these onco/supressor genes within 12-48 hours after in vivo treatment on CBA/Ca H-2K haplotype inbred mice (5, 6). Moreover the mechanism of action of both alkylating agents (MNU and DMBA) involves conversion of the parent molecule into an active carbonium cation and subsequent alkylation of cellular macromolecules, such as DNA (7-10). The molecular mechanisms of oncogenesis induced by the two agents are partially different: DMBA is activated by metabolic enzymes (7, 11), however MNU is a well-known pluripotent direct acting carcinogen (10). Thus, the effect of DMBA is generally delayed in comparison to the effect of MNU (10, 12, 13).
Okamura et al. in rasH2 transgenic mice (14) and El-Sohemy et al. in female Sprague-Dawley rats (15) induced tumours with a single dose of MNU or DMBA. Mutations in codon 12 in the case of MNU or in 61 in the case of DMBA of Ha-ras gene were detected (14-15). We can suppose it is the point mutation that is responsible for overexpression of the Ha-ras gene (as well as p53 and c-myc genes) which was detected after DMBA treatment in our previous in vivo studies (7, 16, 17). It cannot be excluded, however, that mechanisms, other than the DNA adduct-forming effect also take part in the DMBA-induced overexpression of the Ha-ras gene. Earlier it was demonstrated that O6-alkylguanine-DNA alkyltransferase (AGT) may be chemopreventive in MNU toxicity, but such a protective effect of AGT in DMBA-induced carcinogenity has not been reported (18). The difference in features of these two carcinogenic agents may be the result of mechanism(s) independent of adduct formation, e.g. through early elevation of expression of activated oncogenes or depression (of either expression or activity) of tumour suppressor genes, e.g. p53.
The structure of DMBA and MNU.
The metabolic background of DMBA-induced Ha-ras, c-myc and p53 gene expression has been elucidated by in vitro and in vivo experiments. It was found that simultaneous administration of E-2-(4′-methoxybenzylidene)-1-benzosuberone (MBB), a synthetic cyclic chalcone analogue, with DMBA inhibited DMBA-induced Ha-ras, c-myc and p53 gene overexpression in CBA/Ca mice (16, 17). Based on this observation, MBB was believed to inhibit metabolic activation of DMBA, an assumption that was supported by in vitro enzyme kinetic experiments: MBB was found to be a strong inhibitor of CYP1A, the CYP isoenzyme that is mainly responsible for metabolic activation of DMBA (19). These studies provided experimental evidence for the usefulness of the afore mentioned gene expressions as an early molecular epidemiological biomarker of carcinogenesis or carcinogen exposure.
The long-term effects of the two agents are well documented in the literature: both compounds have been reported to cause lung cancer, lymphoma, leukaemia and spleen hemangiosarcoma (6). In addition, MNU induced formation of tumour in the stomach and in the nervous system, and caused overgrowth of thymic, splenic, mandibular and mesenterial lymphatic nodes in different animal species (1, 8, 9, 20). Further target organs of DMBA toxicity were the stomach, lung and skin tissue in wild-type mice (21).
The aim of this study was to investigate the early effect of DMBA and MNU within 24 hours to demonstrate the different effect of the two carcinogens based on the expression patterns of the aforementioned key genes.
Materials and Methods
Animals and treatments. Six- to eight-week-old (20±4 g) conventionally maintained, CBA/Ca inbred H-2K haplotype mice (3 males and 3 females in each group) were used for this experiment. Four groups of animals were treated intraperitonealy (i.p.) with a single 30 mg/kg body weight dose of MNU (Sigma Aldrich Budapest, Hungary) dissolved in 0.1 ml Salsol A (Teva Pharmaceutical Industries Ltd. Debrecen, Hungary). Another four groups of mice treated i.p. with 0.1 ml Salsol A served as controls for the MNU-treated groups. Four groups of mice were treated intraperitonealy (i.p.) with a single 20 mg/kg body weight dose of DMBA (Sigma Aldrich Budapest, Hungary) dissolved in 0.1 ml corn oil (Teva Pharmaceutical Industries Ltd. Debrecen, Hungary). Four groups of mice treated i.p. with corn oil served as controls for the DMBA-treated group.
Gene expression investigations. Three, six, twelve and twenty-four hours after the MNU or DMBA treatment, the mice were autopsied by cervical dislocation, their liver, lungs, kidneys, thymus, spleen, lymph nodes and bone marrow were removed and 100 mg samples of each tissue from the respective groups were pooled. After homogenization of the organs, total cellular RNA was isolated using TRIZOL reagent (Invitrogen, Paisley, UK). The RNA quality was checked by denaturing gel-electrophoresis, and absorption measurement was performed at 260/280 nm (A260/A280 was over 1.8). After the necessary dilution, 10 μg RNA were dot-blotted onto Hybond N+ nitrocellulose membrane (ECL kit, Amersham, Little Chalfont, UK) and hybridized with chemiluminescently labelled specific probes for c-myc, p53 and Ha-ras (Professor J. Szeberényi, University of Pécs, Hungary) genes. Isolation of RNA, hybridization and detection were performed according to the manufacturer's instructions. The membranes were rehybridized with constitutively expressed beta-actin gene as a positive control. The chemiluminescent signals were detected on X-ray films, scanned into a computer and evaluated by Quantiscan software (Biosoft, Cambridge, UK). The results were expressed as the percentage of the positive controls.
Results
The effect of DMBA (20 mg/kg, i.p.) and MNU (30 mg/kg, i.p.) was investigated on the expression of the c-myc, Ha- ras and the p-53 genes in isolated RNA from liver, spleen, lung, kidney, thymus, lymph nodes and bone marrow of the female and the male CBA/Ca (sensitive H-2K haplotype) inbred mice. Expressions of the three genes were examined 3, 6, 12 and 24 hours after the treatment of the experimental animals. The results are summarized in Figures 2, 3, 4, 5, 6, 7 and 8.
As shown in Figure 2, 6 hours after the treatment, DMBA caused a strong elevation of expression of all the three examined genes in the liver compared to the control. On the other hand, elevated expression of p53 at the 12 hour time point after treatment of animals with MNU was detected (Figure 2).
In the spleen, treatment with MNU resulted in an increased expression of the c-myc gene at the 3-hour time point. After the DMBA treatment, elevated expression of c-myc gene was observed 6 and 12 hours after the treatment of animals (Figure 3).
c-myc, Ha- ras and p53 gene expression pattern from liver of CBA/CA mice at 3, 6, 12 and 24 hours after treatment (the arbitrary unit is gene expression as % of β-actin).
c-myc, Ha- ras and p53 gene expression pattern from spleen of CBA/CA mice at 3, 6, 12 and 24 hours after treatment (the arbitrary unit is gene expression as % of β-actin).
In the lung, DMBA caused a strong elevation in expression of the c-myc and Ha-ras genes 3, 6 and 12 hours after the treatment. Exposure to MNU also increased expression of c-myc at the 3-hour time point. Twelve hours after the treatment, overexpression of both c-myc and p53 genes was observed (Figure 4).
In the kidney, DMBA treatment caused a definite overexpression of the three genes at all the examined time points. MNU treatment also caused overexpression of the c-myc and Ha-ras genes as early as 3 hours after the treatment. In this case, however, overexpression of the genes ceased at the later time points (Figure 5).
c-myc, Ha- ras and p53 gene expression pattern from lung of CBA/CA mice at 3, 6, 12 and 24 hours after treatment (the arbitrary unit is gene expression as % of β-actin).
c-myc, Ha- ras and p53 gene expression pattern from kidney of CBA/CA mice at 3, 6, 12 and 24 hours after treatment (the arbitrary unit is gene expression as % of β-actin).
In the thymus, an elevated expression of all three genes was observed 3, 6 and 12 hours after the DMBA treatment. On the contrary, MNU increased expression of the three genes only at the 3-hour time point. By the 6-hour time point, however, overexpression of the genes ceased (Figure 6).
In the lymph nodes, DMBA caused a definite overexpression of all the three genes 3 hours after the treatment. At the later time points (6, 12 and 24 hours), however, the expression level of most of the examined genes was only slightly increased compared to the control. Exposure to MNU resulted in a slighter overexpression of most of the examined genes at the 3- and the 12- hour time points. Twenty-four hours after the MNU treatment, expression of all the three examined genes was found to decrease below that of the control (Figure 7).
c-myc, Ha- ras and p53 gene expression pattern from thymus of CBA/CA mice at 3, 6, 12 and 24 hours after treatment (the arbitrary unit is gene expression as % of β-actin).
c-myc, Ha- ras and p53 gene expression pattern from lymph nodes of CBA/CA mice at 3, 6, 12 and 24 hours after treatment (the arbitrary unit is gene expression as % of β-actin).
In the bone marrow, DMBA treatment caused an increased expression of all the three genes at the early 3-and 6-hour time points. On the contrary, exposure to MNU resulted in a similar effect only 12 hours after the treatment (Figure 8).
Discussion
We previously demonstrated that significant overexpression of the Ha-ras gene in the liver, lung and kidney as well as that of the c-myc and p53 genes in the kidney of female CBA/Ca (sensitive H-2K haplotype) mice can be detected 24 hours after administration of DMBA (16). These investigations have demonstrated that DMBA resulted in elevated expresssion of all the three investigated genes in the liver as early as 6 hours after the treatment. The rationale for this observation may be the well-documented metabolic activation of DMBA by the CYP enzymes that are expressed dominantly in the liver (7, 16, 19). Such an enzyme is e.g. the mouse CYP1B1, which was originally identified as the enzyme responsible for oxidative metabolism of DMBA (22). Besides CYP1B1, the CYP1A1 enzyme also metabolises DMBA in the liver (19, 23) and is also induced by DMBA treatment (24). These enzymes may cause the early activation of DMBA resulting in the elevated gene expressions detected in our present and earlier (7, 16, 17) studies. The early time point when onco/suppressor gene overexpression was detected can be rationalized by the lipophilic nature of DMBA, which results in its fast distribution in body tissues after exposure (25). Our results provide further experimental evidence to demonstrate the usefulness of detection of expression of these molecular epidemiological biomarker genes to indicate early detection of carcinogenesis as well as of carcinogenic exposure.
c-myc, Ha- ras and p53 gene expression pattern from bone marrow of CBA/CA mice at 3, 6, 12 and 24 hours after treatment (the arbitrary unit is gene expression as % of β-actin).
Dickson et al. reported that the amplification and overexpression of c-myc gene could be an initiator in human and mouse breast carcinogenesis (26). Our results indicate a similar (possible initiator) effect of early elevated c-myc gene expression in the liver caused by DMBA treatment. MNU elevated expression of the p53 gene in the liver 12 hours after the treatment, which was demonstrated in our previous study (6) as an early sign of carcinogenic effect of the compound.
In the spleen, elevated expression of the c-myc gene was observed 6 and 12 hours after DMBA treatment. Our results are in accordance with the results of Doi et al. who reported development of splenic hemangiosarcomas in rasH2 transgenic mice accompanied by elevation of c-myc gene expression after DMBA treatment (20). Exposure to MNU (as early activator) resulted in elevated the gene expression of c-myc at the early 3-hour time point. This result corresponds to the fact that MNU is a direct-acting carcinogen and, in (long-term) experiments, induces spleen hemangiomas in susceptible Wistar rats (27).
The early effect of DMBA in the lung causing increased expression of the c-myc and the Ha-ras genes 3, 6 and 12 hours after treatment correlates well to the previous literature data reporting development of lung adenocarcinomas caused by single DMBA treatment of rasH2 mice (20). Earlier we reported an overexpression of c-myc and p53 genes in the lung 12 hours after MNU treatment of experimental animals (6). This finding is in accordance with the results demonstrating that MNU initiated development of lung adenoma in long-term studies (28). The finding that expression of the c-myc oncogene increased 3 hours after MNU treatment supports the possible initiator activity of elevated c-myc gene expression in MNU-initiated lung adenocarcinomas (28).
In the kidney, DMBA treatment resulted in an overexpression of all three genes at each examined time point. MNU increased expression of the c-myc and Ha-ras genes at the 3-hour time point but its effect ceased at the further time points (Figure 5). These latter findings are in accordance with our previous results (6). According to the literature, exposure of p53+/- mice to MNU caused pale-coloured kidneys due to lymphoma of the hematopoietic system in (long-term) experiments (8). In addition, other organs such as the thymus, spleen, liver, lungs and kidneys were also affected (8). Our results are in accordance with a rapid effect of MNU as a direct-acting carcinogen and suggest an initiator role of the early c-myc and Ha-ras overexpression in the MNU-induced kidney neoplasms followed by malignant transformation in (long-term) experiments. While MNU is a water soluble compound it becomes concentrated in the kidney through filtration in the nephrons, but due to its rapid spontaneous degradation (T1/2=15 min), its early biological effect on gene expression ceases in a short time (29). Contrary to the effect of DMBA, exposure to MNU did not cause elevated expression of the p53 gene (Figure 5). This finding suggests that the protective role of p53 is one of the key points in respect to the divergence of the tumour-inducing mechanisms of the two carcinogenic agents (30). Moreover, DMBA seems to act both as a putative initiator and as a promoter in tumourogenesis, contrary to MNU, of which only an initiator effect is expected (30). This could explain why O6-alkylguanine-DNA alkyltransferase (AGT) could be chemopreventive in some cases against MNU toxicity contrary to DMBA-induced carcinogenity (18).
The thymus is a target organ of both DMBA and MNU toxicity. DMBA treatment caused an increased expression all of the examined genes from the 3-hour to the 12-hour time point. On the contrary, exposure to MNU elevated the investigated onco/suppressor gene expressions only 3 hours after the treatment. These data are in accordance with the initiating and promoting effect of DMBA and the initiating role of MNU in thymus carcinomas (due to the direct-acting effect of MNU) as above (28).
DMBA treatment induced overexpression of all the three investigated genes at the 3-, 6- and 12-hour time points, which is in accordance with the well-documented lymphogenic effect of the compound. Expression of the p53 gene decreased beyond the control after 12 hours and that of c-myc completely ceased after 24 hours of the treatment. The latter observation suggests a putative negative feedback mechanism in regulation of expression of these two genes by each other (31). Exposure to MNU, which is reported to cause thymic lymphomas in long-term studies (28), resulted in overexpression of all the three investigated genes at the 3- and the 12-hour time points. In accordance with the results of our previous study (6), the expression level of all the three examined genes decreased beyond the control level, according to the spontaneous degradation of MNU.
In the bone marrow, exposure to DMBA induced overexpression of the investigated genes earlier (at 3 and 6 hours) than that of MNU (12 hours) (Figure 8). This observation is in agreement with the results of our previous study (6). Although a leukemogenic effect was observed for both carcinogenic agents (28), our findings indicate that the two compounds affect the expression pattern of the investigated molecular epidemiological biomarker genes differently.
In summary, we can conclude that results of the present study provide further experimental evidence on the usefulness of the detection of expression of these key epidemiological biomarker genes to indicate early detection of carcinogenesis as well as of carcinogenic exposure, both in animal models and in human cancer prevention practice as well (32, 33). In the future, we are planning to elucidate the effect of MNU and DMBA exposure on the expression of the AGT gene in our experimental model (32) to investigate whether point mutations on onco/supressor genes and epigenetic molecular biological alterations (such as DNA adduct formation and gene expression changes) together can lead to divergence in (long-term) carcinogenic biological effects of MNU and DMBA.
Acknowledgements
The authors express their special thanks to Zsuzsanna Bayer and Mónika Herczeg for valuable technical assistance.
- Received January 6, 2009.
- Revision received March 13, 2009.
- Accepted April 2, 2009.
- Copyright © 2009 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved