Immunohistochemical detection of 1,N6-ethenodeoxyadenosine, a promutagenic DNA adduct, in liver of rats exposed to vinyl chloride or an iron overload

Yan Yang, Jagadeesan Nair, Alain Barbin1 and Helmut Bartsch2

Division of Toxicology and Cancer Risk Factors, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany and
1 Unit of Gene–Environment Interactions, International Agency for Research on Cancer, 150 cours Albert-Thomas, F-69172 Lyon, France


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Etheno adducts in DNA bases are formed from exogenous agents such as vinyl chloride and urethane, but also via endogenous lipid peroxidation products like trans-4-hydroxy-2-nonenal. An immunohistochemical method was developed to localize the promutagenic 1,N6-ethenodeoxyadenosine DNA adduct in liver of rats exposed to vinyl chloride or an iron overload with or without carbon tetrachloride. Six monoclonal antibodies, previously produced through collaborative efforts, were screened for their optimal adduct recognition and low background formation. The antibody generated by clone EM-A-4 was found to be most suitable. Semi-quantitative image analysis of relative pixel intensity showed ~1.5 times higher adduct levels (P < 0.05) in the livers of rats treated with vinyl chloride or an iron overload when compared with untreated controls. Significantly elevated adduct levels persisted in vinyl chloride-treated rat liver 14 days after cessation of exposure, suggesting that this adduct is not rapidly eliminated from rat liver DNA. Using the new immunohistochemical method it is possible to visualize this promutagenic etheno-DNA adduct that may play a role in oxidative stress and lipid peroxidation-induced DNA damage in carcinogenesis.

Abbreviations: DAB, 3,3'-diaminobenzidine; {varepsilon}dA, 1,N6-ethenodeoxyadenosine; {varepsilon}dC, 3,N4-ethenodeoxycytidine; LPO, lipid peroxidation; PBS, phosphate-buffered saline; PBST, 0.2% Triton-X100 in PBS; RPI, relative pixel intensity; VC, vinyl chloride.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The etheno-DNA adducts 1,N6-ethenodeoxyadenosine ({varepsilon}dA), 3,N4-ethenodeoxycytidine ({varepsilon}dC) and ethenodeoxyguanosine (1,N2- or N2,3-) are formed from reactive metabolites of the human carcinogen vinyl chloride (VC) and the rodent multi-organ carcinogen urethane. Other structurally diverse carcinogens and mutagens can also form etheno-DNA adducts in vitro and in vivo (1). In recent years, this class of exocyclic DNA adducts has received renewed attention because of the increasing evidence that they are also generated by oxidative stress and lipid peroxidation (LPO)-mediated reactions. Oxidation of arachidonic acid and LPO-derived reactive hydroxyalkenals such as trans-4-hydroxy-2-nonenal, upon epoxidation, has been shown to yield etheno-DNA adducts after reaction with nuclear bases (24). Using a sensitive immunoaffinity/32P-post-labelling method (5), increased {varepsilon}dA and {varepsilon}dC levels were detected in liver DNA of patients with the metal storage diseases Wilson's disease and primary hemochromatosis (6) and also of Long-Evans Cinnamon rats (7), a model for human Wilson's disease. In these cases DNA damage is caused by copper/iron storage that induces oxidative stress and LPO. High dietary intake of linoleic acid (an {omega}-6 polyunsaturated fatty acid that can easily be oxidized to reactive hydroxyalkenals) resulted in markedly increased etheno-DNA adduct levels in female, but not in male, volunteers (8). The postulated pathways of etheno-DNA adduct formation from exogenous agents such as VC and via endogenous processes (LPO) are depicted in Figure 1Go. The two etheno-DNA adducts {varepsilon}dA and {varepsilon}dC have been shown to be useful biomarkers for oxidative stress/LPO-induced DNA damage, which may play a role in the aetiology of human malignant and neurodegenerative diseases (9). For a better understanding of the formation and repair of etheno-DNA adducts at a cellular level, an immunohistochemical method has been developed and standardized for the detection of {varepsilon}dA in tissues. In this paper we describe the new methodology and its application to the analysis of liver tissue from rats exposed to VC or an iron overload with or without CCl4.



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Fig. 1. Pathways for the formation of etheno-DNA adducts, e.g. from exogenous agents such as VC or via endogenous processes through LPO of linoleic acid.

 

    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
RNase, DNase and proteinase K were purchased from Boehringer Mannheim (Mannheim, Germany). Biotinylated goat anti-mouse IgG and avidin–biotin–peroxidase (ABC) complex (VECTAstain elite ABC kit) and the 3,3'-diaminobenzidine (DAB) substrate kit were purchased from Linaris (Werthheim-Bettingen, Germany). 3-Triethoxysilyl propylamine and Kaiser's glycerin–gelatin mounting medium were obtained from Merck (Darmstadt, Germany). The monoclonal antibodies EM-A-1–EM-A-6 (cell culture supernatant) raised against {varepsilon}dA were obtained from Dr Petra Lorenz (University of Essen, Essen, Germany). Details of the production of mAbs were reported earlier (10). The ImmunoPure(A/G)IgG purification kit was purchased from Pierce (Bruchsal, Germany) for antibody purification and Microcon microconcentrators were obtained from Millipore (Eschborn, Germany) for antibody concentration. VC (3% in N2, purity 99.9%) was obtained from Airgaz (Sérézin du Rhône, France).

Animal experiments
The animal experiments were performed at IARC (International Agency for Research on Cancer) in Lyon, France.

VC exposure.
Female Sprague–Dawley rats (n = 3 per group), 10 days of age, were exposed together with their foster mothers to 600 p.p.m. VC for 4 h/day for 5 days. They were killed either immediately after treatment (28 15-day-old animals) or 14 days later (16 29-day-old animals). Control groups included the same numbers of rats and were housed at the same time under the same conditions as the treated animals. In this study, liver samples from young animals were analysed.

Iron overload.
Male Sprague–Dawley rats (n = 3) were fed 3.5% iron fumarate (Merck, Darmstadt. Germany) in the diet (pellet diet for breeding obtained from Altromin, France) or 3.5% iron fumarate together with 0.1 ml/kg body wt CCl4 per os 5 days a week for 3 weeks; both regimens induced an iron overload in the liver. Animals were killed after treatment and liver tissues were frozen immediately in liquid nitrogen and stored at –80°C prior to analysis.

Immunohistochemical detection of etheno-DNA adducts
Frozen liver tissue samples were sectioned (6 µm) on a cryostat (Leica Jung CM3000; Germany), placed on 3-triethoxysilyl propylamine/acetone-coated glass microscope slides, fixed with acetone at –20°C for 10 min and air dried. Slides were dipped in phosphate-buffered saline (PBS) and in 0.3% H2O2 in absolute methanol for <20 min to quench endogenous peroxidase activity and then washed with PBS. To remove histone and non-histone proteins from DNA and increase EM-A-4 antibody accessibility, slides with liver sections were treated with proteinase K (10 µg/ml) in double distilled H2O at room temperature for 10 min. After washing with PBS, sections were further treated with RNase (100 µg/ml) in Tris buffer (pH 7.2) at 37°C for 1 h. Non-specific binding sites were blocked with 10% bovine serum albumin in PBST (0.2% Triton-X100 in PBS) at 37°C for 45 min and washed in PBS once. To denature DNA, sections were treated with 4 N HCl for 10 min at room temperature and subsequently rinsed with water and PBS. The pH was neutralized with 50 mM Trisma base by incubation at room temperature for 5 min. The sections were rinsed again with water, PBS and 10% bovine serum albumin in PBST at room temperature for 30 min. The sections were further incubated with each of the purified primary mAbs for {varepsilon}dA (EM-A-1–EM-A-6, dilution 1:10) at 4°C overnight, as indicated by the manufacturer, with minor modifications [ImmunoPure(A/G)IgG purification kit]. After washing with PBS, the sections were incubated with the biotinylated second goat anti-mouse antibody for 30 min and with the avidin–biotin–peroxidase complex (VECTAstain ABC kit), as per the manufacturer's protocol. To visualize the reaction, DAB was used as a chromogen. To increase the sensitivity of the immunoreaction, a nickel/cobalt solution was added to the DAB working solution. After stopping the reaction in PBS, rinsing under tap water and in deionized water for 5 min, slides were mounted with Kaiser's glycerin–gelatin. All sections were subjected to the same procedure under standardized conditions. As negative controls, serial sections of each sample were either treated with DNase (100 µg/ml for 30 min at 37°C) before incubation with the primary mAb or with the primary mAb substituted by PBS.

Iron staining
Serial sections of liver were stained to localize iron, using an iron staining kit (Sigma-Aldrich, Deisenhofen, Germany) according to the manufacturer's protocol. The sections were stained for nuclei with pararosaniline.

Imaging and semi-quantitative analysis of {varepsilon}dA
The intensity of staining was evaluated semi-quantitatively with a Leitz Laborlux 11 microscope (Leitz, Germany) equipped with a colour camera (Hitachi HV-C20M, 3CCD). The relative mean pixel intensity of 100 randomly selected nuclei was measured by imaging software (Qwin; Leica, Germany).

Statistical analysis
The statistical analysis was performed by analysis of variance of the relative mean intensity with subsequent Student's t-test for each treatment group, compared with controls.


    Results
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
The mAb EM-A-1 raised against {varepsilon}dA (in a previous collaborative project with Dr Manfred Rajewsky, University of Essen, Essen, Germany; see ref. 10) that is currently being used in the immunoaffinity/32P-post-labelling method (5) was first tested for immunorecognition and background staining. This mAb was found not to be suitable for immunohistochemistry because it produced high background staining. Therefore, the remaining available clones (EM-A-2–EM-A-6) were tested and EM-A-4 was found to be the best. For all subsequent experiments this mAb (EM-A-4) was used.

Figure 2Go shows the immunohistochemical staining of {varepsilon}dA in the livers of rats treated with VC and from untreated controls. The control livers showed a uniform light staining of the adducts in all cells. The VC-treated liver sections showed increased intensity of the staining over the controls and the staining was significantly higher in parenchymal and non-parenchymal cells. Treatment of rats with dietary iron fumarate alone or together with CCl4 for 3 weeks resulted in hepatic iron accumulation localized in the extracellular space around the portal vein (Figure 3Go). Adduct staining in these sections was uniform, but higher than in the controls. However, we did not observe increased immunostaining around the portal vein, where iron accumulation occurred.



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Fig. 2. Photomicrographs of immunohistochemically stained liver sections of an untreated control (A) and a VC-treated rat (B). (C and D) Liver sections without primary mAb and after DNase treatment, respectively.

 


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Fig. 3. Photomicrographs of immunohistochemical and iron staining of livers from untreated control rats (A and B) and from rats after an iron overload with iron fumarate (C and D).

 
In order to compare the intensity of stained nuclei, the pixel intensity was measured in 100 randomly selected nuclei, using imaging software. For each treatment and control group sections from 3–6 animals were used. The mean ± SD relative pixel intensities (RPI) averaged for each slide are given in Figure 4Go. The RPI were elevated ~1.5 times in the groups treated with VC or by an iron overload, when compared with the controls (P < 0.05). The group of rats that received VC for 15 days and were killed 14 days later still had significantly higher adduct intensities than the controls (P < 0.05), suggesting that the adducts are persistent in rat liver. The Fe + CCl4-treated group was investigated in order to assess the formation of etheno-DNA adducts due to enhanced LPO conditions. However, no difference in the RPI values was observed between treatment with Fe alone or together with CCl4.



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Fig. 4. Comparison of relative pixel intensities of immunostained rat liver sections (number of rats). C, control (n = 5); VC-15 (n = 3), 10-day-old rats exposed to VC for 5 days and then killed; VC-29 (n = 6), rats exposed to VC for 5 days and killed 14 days after cessation; Fe, iron overload with iron fumarate alone (n = 3) or with iron fumarate plus carbon tetrachloride (n = 3).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This is the first demonstration of an immunohistochemical detection of {varepsilon}dA in rat liver sections. Out of several mAbs tested, EM-A-4 was found to be the optimal one, yielding the most intense staining of the nuclei combined with the lowest background staining. Although the cross-reactivity of EM-A-4 has not been thoroughly investigated, the low background levels obtained with this antibody suggest that it does not react strongly with normal nucleotides. We obtained an even lower background immunostaining with EM-A-4 as compared with EM-A-1. The cross-reactivity of the latter towards structurally related modifications (etheno-2'-deoxycytidine and ethenoguanine) was >1000 times lower (10). Exclusion of the primary antibody or treatment of the section with DNase prior to the primary mAb treatment resulted in almost no detectable staining of the nuclei. This result and the intensive staining only within the nuclei strongly support the assumption that the staining involves nuclear DNA.

Using HPLC/RIA and immunoaffinity/32P-post-labelling methods, increased etheno-DNA adduct formation was unequivocally demonstrated in whole livers of rats exposed to VC (10,11). Using our immunohistochemical method we observed that the intensity of staining for {varepsilon}dA was higher in both parenchymal and non-parenchymal cells. VC induces predominantly angiosarcomas of the liver in both humans and rodents, which originate from endothelial cells (12,13). The current immunostaining did not allow us to establish preferential etheno-DNA adduct formation in endothelial cells. It should be possible to address this query by staining endothelial cells with specific antibodies together with mAb for {varepsilon}dA. Detection of significantly higher RPI in rat livers 14 days after cessation of VC exposure indicates that the repair of {varepsilon}dA is slow in liver DNA, as observed earlier using other adduct detection methods (14). Although increased {varepsilon}dA staining was detected in both the Fe and the Fe + CCl4 groups compared with the control, no difference in immunostaining was detected between the treated groups. This could be due to saturation of the steady-state adduct levels or the RPI or both. A major DNA repair pathway of {varepsilon}dA is 3-methyladenine-DNA glycosylase (15,16). Increased etheno-DNA adduct levels were detected in the livers of rats that had received an iron overload and of patients suffering from primary hemochromatosis (6,17). A 90- to 240-fold increase in the relative risk for primary liver cancer has been reported in these patients (18). Our immunohistochemical analysis of rat livers after iron overload revealed the generation of uniform DNA damage, although the iron was found to accumulate in small extracellular foci in rat liver (Figure 3Go).

Etheno-DNA adducts were previously shown to be promutagenic DNA lesions. Their efficiency in inducing base pair substitution mutations in bacterial and mammalian cells has been demonstrated (1,19,20). {varepsilon}dA can induce A->C, A->G and A->T base pair substitutions and {varepsilon}dC leads to C->T and C->A mutations (21). The GC->AT and CG->AT mutations observed in activated Ki-ras genes in angiosarcomas of the liver obtained from VC-exposed workers correspond to the expected mutation pattern from {varepsilon}dC and N2,3-ethenodeoxyguanosine (1,22), whereas the rarely occurring AT->TA transversion observed in the p53 gene in two out of five human angiosarcomas (23) may originate from {varepsilon}dA. Thus, mutations in Ki-ras and in the p53 tumor suppressor gene revealed that mutational changes are consistent with the miscoding properties of {varepsilon}dA, {varepsilon}dC and ethenodeoxyguanosine (21,23). In a recent study on the p53 gene mutation patterns in VC-induced rat liver tumours, nine of 13 point mutations involved A:T base pairs (24).

There has been increasing evidence for a role of reactive oxygen species and LPO in the aetiology of human cancer development and neurodegenerative diseases. A series of studies in humans and rodents have now demonstrated that etheno-DNA adducts are one of the most important classes of DNA damage produced via endogenous pathways as a result of oxidative stress and lipid peroxidation (summarized in ref. 9). Development and application of immunohistochemical methods for all three etheno adducts will elucidate their role at the cellular level in human disease pathogenesis. Such methods will be particularly helpful when the amount of human tissue samples in molecular epidemiological studies is limited.


    Acknowledgments
 
Dr P.Lorenz, University of Essen, is thanked for providing the monoclonal antibodies and Mrs G.Bielefeldt for secretarial assistance. This work was carried out during the tenure of a visiting scientist fellowship awarded to Dr Yan Yang by the DKFZ, Heidelberg, and was in part supported by EU contract ENV4-CT97-0505.


    Notes
 
2 To whom correspondence should be addressed Email: h.bartsch{at}dkfz-heidelberg.de Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received September 22, 1999; revised November 15, 1999; accepted November 25, 1999.