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Carcinogenesis Advance Access originally published online on November 17, 2006
Carcinogenesis 2007 28(5):1117-1121; doi:10.1093/carcin/bgl219
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Formation and persistence of DNA adducts formed by the carcinogenic air pollutant 3-nitrobenzanthrone in target and non-target organs after intratracheal instillation in rats

Christian A. Bieler, Michael G. Cornelius, Marie Stiborova1, Volker M. Arlt2, Manfred Wiessler, David H. Phillips2 and Heinz H. Schmeiser*

Division of Molecular Toxicology, German Cancer Research Center, INF 280, 69120 Heidelberg, Germany
1 Department of Biochemistry, Faculty of Science, Charles University, 128 40 Prague 2, Czech Republic
2 Section of Molecular Carcinogenesis, Institute of Cancer Research, Brookes Lawley Building, Sutton, Surrey SM2 5NG, UK

* To whom correspondence should be addressed. Tel: +49 6221 423348; Fax: +49 6221 423375; Email: h.schmeiser{at}dkfz.de


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary data
 References
 
Sprague–Dawley rats were treated by intratracheal instillation with a single dose of 0.2 mg/kg body wt of 3-nitrobenzanthrone (3-NBA), and whole blood, lungs, pancreases, kidneys, urinary bladders, hearts, small intestines and livers were removed at various times after administration. At five posttreatment times (2 days, 2, 10, 20 and 36 weeks), DNA adducts were analysed in each tissue by 32P-postlabelling to study their long-term persistence. 3-NBA-derived DNA adducts consisting of the same adduct pattern were observed in all tissues from animals killed between 2 days and 36 weeks and between 2 days and 20 weeks in blood. DNA isolated from whole blood contained the same 3-NBA-specific adduct pattern as that found in tissues. Although total adduct levels in the blood were much lower than those found in the lung, the target organ of 3-NBA tumourigenicity, they were related (20–25%, R2 = 0.98) to the levels found in lung. In all organs, total adduct levels decreased over time to 20–30% of the initial levels till the latest time point (36 weeks) and showed a biphasic profile, with a rapid loss during the first 2 weeks followed by a much slower decline that reached a stable plateau at 20 weeks after treatment. These results show that uptake of 3-NBA by the lung induces high levels of specific DNA adducts in target and non-target organs of the rat. The correlation between DNA adducts in lung and blood suggests that persistent 3-NBA–DNA adducts in the blood may be useful biomarkers for human respiratory exposure to 3-NBA.

Abbreviations: 3-NBA, 3-nitrobenzanthrone; dG-N2-ABA, 2-(2'-deoxyguanosin-N2-yl)-3-aminobenzanthrone; nitro-PAH, nitropolycyclic aromatic hydrocarbon; TLC, thin-layer chromatography; HPLC, high performance liquid chromatography


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary data
 References
 
Epidemiological studies have shown that occupational exposure to diesel exhaust is associated with an increased risk of lung cancer and environmental exposure to diesel exhaust may also pose a significant cancer risk to the general population (1–4).

Diesel exhaust induces lung tumours in experimental animals, an effect attributed to the particulate phase that contains many absorbed chemicals such as nitropolycyclic aromatic hydrocarbons (nitro-PAHs) (4). 3-Nitrobenzanthrone (3-NBA, 3-nitro-7H-benz[de]anthracen-7-one), a member of this class of compounds, has been detected in the particulate fraction of diesel exhaust and in urban air samples (5, for review). 3-NBA is a potent mutagen (6,7) and is carcinogenic to rodents inducing pulmonary tumours in rats after intratracheal instillation (8). Both in vitro and in vivo studies showed that 3-NBA reacts with DNA through initial reduction of the nitro group primarily catalysed by cytosolic nitroreductases and subsequently esterification catalysed by actetyltransferases and/or sulfotransferases (9–14). DNA adduct formation in rats treated orally, i.p. or intratracheally with 3-NBA has been demonstrated using thin-layer chromatography (TLC) 32P-postlabelling (9,11,15). In these studies, the same adduct pattern consisting of multiple 3-NBA-specific DNA adducts was detected in various organs of rats. Since inhalation is the major route by which airborne materials gain access to the body, primary exposure of the lungs of rats to 3-NBA would constitute a better model system. However, experimental inhalation exposure systems, with appropriate generation and characterization of exposure atmospheres, are expensive to acquire and maintain (16). Alternatively, direct instillation of a test material into the lungs via the trachea has been employed in many studies as an alternative exposure procedure to inhalation. Instillation has also certain advantages over inhalation, the foremost being that the actual dose delivered to the lungs of each animal can be defined accurately.

The predominant DNA adducts formed by 3-NBA in vivo have been identified recently as 2-(2'-deoxyguanosin-N2-yl)-3-aminobenzanthrone (dG-N2-ABA) and N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (8,17,18) and are most probably responsible for the induction of GC->TA transversion mutations observed in vivo (7).

DNA damage such as DNA adduct formation is an important first step in the process of chemical carcinogenesis (19). Several human biomonitoring studies have reported higher levels of bulky DNA adducts among subjects heavily exposed to diesel exhaust and urban air pollution (20–22). This correlates with increased cancer risk (23,24). The main metabolite of 3-NBA, 3-aminobenzanthrone, has been found in urine samples of workers occupationally exposed to diesel emissions (25), suggesting that exposure to 3-NBA in diesel emissions may represent a health hazard for large sections of the population. Thus, 3-NBA–DNA adducts may be used as biomarkers for genotoxic risk. It was suggested that DNA adducts formed by 3-NBA not only represent premutagenic lesions (5) but that both higher initial levels of specific adducts and longer persistence in the target tissue such as the lung probably contribute to its mutagenicity and subsequently to tumour development (8,15).

DNA adducts have different stabilities owing to factors such as DNA repair and chemical instability. It was postulated that persistent DNA adducts represent dormant lesions that may become fixed as mutations long after exposure to the carcinogen and contribute to the malignant transformation as a result of cell division (26–28). Recently, we showed that short-term intratracheal treatment of 3-NBA in rats leads to a dose-dependent formation of DNA adducts in target (lung) and non-target organs (e.g. liver, kidney, urinary bladder, pancreas, heart and small intestine) (15). In the present study, 3-NBA–DNA adduct formation and persistence were characterized in target and non-target organs of rats after a single intratracheal instillation of 3-NBA to explore possible relationships between tumour formation by 3-NBA and DNA adduct pharmacokinetics.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary data
 References
 
Chemical
3-NBA was synthesized and identified as described (6,10). The compound was checked by high performance liquid chromatography (HPLC) analysis and no impurities could be detected.

Animal experiments
Adult female Sprague–Dawley rats, so-called ‘ex-breeders’ (4–6 months old, 300–380 g) were treated with 0.2 mg/kg body wt of 3-NBA by intratracheal instillation under ether anaesthesia. 3-NBA was dissolved in tricaprylin at a concentration of 0.4 mg/ml, resulting in a volume of ~180 µl applied per rat. 3-NBA dissolved in tricaprylin was administered to rats at the bronchial bifurcation by injection through a tracheal cannula. Control rats were treated with vehicle only (180 µl of tricaprylin). Animals were killed 2 days (n = 3), 2 (n = 3), 10 (n = 3), 20 (n = 4) and 36 weeks (n = 6) after treatment and blood was collected immediately by puncture of the vena cava inferior and transferred to PAXgene blood collection tubes (PreAnalytiX, Hilden, Germany) for subsequent isolation of DNA. Seven organs (lung, liver, kidney, urinary bladder, pancreas, heart and small intestine) were removed, immediately frozen in liquid nitrogen and stored at -80°C until DNA isolation.

DNA isolation
Isolation of total DNA from whole blood was performed with the PAXgene blood DNA kit (PreAnalytiX) according to the instructions of the manufacturer. Briefly, 8.5 ml of whole blood collected in PAXgene blood DNA tubes was transferred to the processing tube. The solution was mixed to lyse red and white blood cells, centrifuged and the resulting pellet of nuclei and mitochondria was washed and resuspended. After digestion with protease, DNA was precipitated by addition of isopropanol and dissolved in water. Typical yields from 8.5 ml rat blood were 100–200 µg DNA.

DNA from organs was isolated by the Qiagen Genomic DNA Purification Procedure (Qiagen, Hilden, Germany). Briefly, 120 mg of tissue was minced and digested by proteinase K for 3 h at 50°C. The digest was applied to a Qiagen Tips 100 column and DNA was isolated following the standard column procedure (Blood and Cell Culture DNA Kit, Qiagen). DNA was precipitated by addition of isopropanol (yield: 80–100 µg) and dissolved in water.

DNA adduct analysis by 32P-postlabelling
The butanol enrichment procedure of the 32P-postlabelling assay was performed as described (29,30). Briefly, 12.5 µg of DNA were digested using micrococcal endonuclease (750 mU per sample, Sigma, Taufkirchen, Germany) and spleen phosphodiesterase (62.5 mU per sample, Calbiochem, Darmstadt, Germany) for 3 h at 37°C. An aliquot (2.5 µg) of the digest was removed and diluted for determination of normal nucleotides. Adducts were enriched by butanol extraction and labelled with [{gamma}-32P]ATP (100 µCi per sample; MP Biomedicals, Heidelberg, Germany) by incubation with T4-polynucleotide kinase (USB, Germany) for 30 min at room temperature. Efficiency of enrichment and excess of ATP were checked with an aliquot of the radiolabelled sample by one-dimensional TLC (PEI-cellulose, 50 mM sodium phosphate, 0.28 M ammonium sulfate; pH 6.5). 32P-labelled adduct nucleoside bisphosphates were separated by chromatography on PEI-cellulose sheets (Macherey and Nagel, Düren, Germany). The following solvents were used: D1, 1 M sodium phosphate, pH 6.5; D3, 3.5 M lithium formate, 8.5 M urea, pH 3.5; D4, 0.8 M lithium chloride, 0.5 M Tris, 8.5 M urea, pH 8.0 and D5, 1.7 M sodium phosphate, pH 6.0. Electronic autoradiography was performed using an Instant Imager (Canberra Packard, Dowers Grove, IL). DNA adduct levels (relative adduct labelling) were calculated as c.p.m. adducts per c.p.m. normal nucleotides.

HPLC analysis of 32P-labelled 3',5'-deoxyribonucleoside bisphosphates
Adduct identification by HPLC analysis was essentially performed as described (11). Briefly, individual adduct spots or origins after D1 detected by the TLC 32P-postlabelling assay were excised from plates, extracted, dried and redissolved in methanol–phosphate buffer (pH 3.5). Aliquots were run on a phenyl-modified reverse-phase column (Luna 5 µm phenyl–hexyl, 4.6 x 150 mm, Phenomenex, Aschaffenburg, Germany) with a linear gradient (from 30 to 55% in 45 min) of methanol in aqueous sodium phosphate (pH 3.5) at a flow rate of 1 ml/min on a Waters 2690 HPLC system. Radioactivity eluting from the column was detected by monitoring Cerenkov radiation with a Flow Scintillation Analyzer (Packard, Dowers Grove, IL).

Characterization of DNA adducts formed in vivo
Standard adduct samples of N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone-3'-phosphate, 2-(2'-deoxyguanosin-N2-yl)-3-aminobenzanthrone-3'-phosphate and 2-(2'-deoxyadenosin-N6-yl)-3-aminobenzanthrone-3'-phosphate were prepared and analysed as described recently (17).

Statistical analysis
Correlation coefficient between the level of total 3-NBA–DNA adducts formed in lung tissue and blood were determined by linear regression using Statistical Analysis System software version 6.12.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary data
 References
 
DNA adduct analysis in rat tissue
Using the butanol enrichment version of the 32P-postlabelling method, DNA adducts were measured in lung, pancreas, kidney, liver, small intestine, urinary bladder and heart of female Sprague–Dawley rats at 2 days, 2, 10, 20 and 36 weeks after administration of a single intratracheal dose of 0.2 mg 3-NBA/kg body wt. Enrichment by butanol extraction was used in preference to nuclease P1 digestion as it has been shown to yield more adduct spots and a better recovery (9). Figure 1 shows representatively DNA adduct patterns obtained with lung. The same specific DNA adduct pattern at all time points was also obtained with the organs investigated (data not shown). This characteristic adduct pattern observed using TLC essentially consists of up to five adduct spots, with spots 1, 3 and 4 being the major adducts and is the same as that found in organs of rats treated with 3-NBA orally or i.p. (9,11). No adduct spots were detected in DNA isolated from organs of rats treated with the vehicle (tricaprylin) alone. At the last time point (36 weeks), the major adduct spot 3 (dG-N2-ABA) was still clearly detectable and accounted for ~75% of the total DNA adduct level (Figure 1E).


Figure 1
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  		Fig. 1. Autoradiographic profiles of DNA adducts obtained from lung after intratracheal treatment of Sprague–Dawley rats with a single dose of 0.2 mg/kg body wt 3-NBA using the butanol enrichment version of the 32P-postlabelling assay. (A) Two days after treatment, (B) 2 weeks after treatment, (C) 10 weeks after treatment, (D) 20 weeks after treatment and (E) 36 weeks after treatment. Electronic autoradiography was performed for 15 min for (A), (B) and (C) and for 30 min for (D) and (E). Origins in the bottom left corner were cut off before exposure.

 
To confirm the identity of adducts detected in DNA from organs of rats treated with 3-NBA by intratracheal instillation, HPLC analysis was performed as a second, independent chromatographic system. As demonstrated previously (9,11), adduct spots 1 (retention time 40.5 min) and 2 (retention time 32.5 min) are derived from deoxyadenosine, whereas adduct spots 3 (retention time 23.0 min), 4 (retention time 27.0 min) and 5 (retention time 38.5 min) are formed from reaction of activated 3-NBA with deoxyguanosine (data not shown). Using authentic DNA adduct standards, the two most abundant guanine adducts formed were identified as dG-N2-ABA (adduct 3) and N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (adduct 4/5), whereas adduct 1 was identified as 2-(2'-deoxyadenosin-N6-yl)-3-aminobenzanthrone.

Maximum adduct levels in tissues were found 2 days after treatment, the earliest time point at which DNA samples were collected. As shown in Table I, highest total DNA binding was found in pancreatic tissue, kidney and lung at all time points followed by urinary bladder, heart, small intestine and liver that always showed the lowest level. In all organs examined, dG-N2-ABA was the predominant lesion found, accounting for ~40% of total DNA binding after 2 days. After 2 weeks, dG-N2-ABA made up ~50%, after 10 weeks ~60%, after 20 weeks ~70% and after 36 weeks almost 100% of the total DNA adduct level in most tissues (see Supplementary data).


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  		Table I. Total DNA binding (relative adduct labelling) in various organs at various time points of rats treated with 3-NBA

 
As shown in Figure 2A, for lung total adduct levels decreased over time (39 ± 18 after 2 days) to 30% (11 ± 5 adducts in 108 nucleotides) of the initial level after 36 weeks. Similar adduct kinetics were observed in the other tissues and showed a biphasic profile with a steep decline during the first 2 weeks, then gradually till 20 weeks reaching a plateau thereafter in most organs (Figure 2A and B). In all organs, dG-N2-ABA was still detectable after 36 weeks. Highest total adduct levels after 36 weeks were found in pancreas, lung and kidney (11 and 12 adducts in 108 nucleotides), organs that showed the highest initial levels. DNA adduct removal occurred at similar rates in target and non-target organs.


Figure 2
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  		Fig. 2. Time course of 3-NBA–DNA adduct levels in organs of rats treated intratracheally with a single dose of 0.2 mg/kg body wt 3-NBA. Relative adduct labelling (RAL) values are expressed as adducts per 108 nucleotides. Results represent mean ± SD from three treated rats for time points 2 days, 2 and 10 weeks and four animals for time point 20 weeks and six animals for time point 36 weeks, all analysed in duplicate.

 
DNA adduct analysis of blood from rats treated intratracheally with 3-NBA
In DNA, isolated from total blood of rats treated intratracheally with a single dose of 0.2 mg/kg body wt 3-NBA, a similar adduct pattern as found in tissues was observed (Figure 4). However, adduct spot 5, which has the lowest abundance in other tissues, was not detected. Blood DNA isolated from rats treated with vehicle showed no adduct spots in the region of interest. Generally, maps of 32P-postlabelling analyses from blood DNA were not as clear and total adduct levels were lower than in DNA from tissues. As observed in the tissues, total adduct levels decreased over time also in blood (Figure 3), however, only the most abundant DNA adduct, dG-N2-ABA (adduct 3) was detectable 20 weeks after treatment (Figure 4C) representing 38% (3.6 ± 0.21 adducts in 108 nucleotides) of the initial level. At the last time point (36 weeks), no 3-NBA-derived spots were found (Figure 4D).


Figure 3
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  		Fig. 3. Total DNA adduct levels in whole blood at various time points after treatment with a single dose of 0.2 mg/kg body wt 3-NBA. Relative adduct labelling (RAL) values are expressed as adducts per 108 nucleotides. Results represent mean ± SD from three treated rats for time points 2 days and 2 weeks and four animals for time point 20 weeks and six animals for time point 36 weeks, all analysed in duplicate; ND, not detected.

 


Figure 4
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  		Fig. 4. Autoradiographic profiles of DNA adducts obtained from whole blood of rats treated intratracheally with a single dose of 0.2 mg/kg body wt 3-NBA using the butanol enrichment version of the 32P-postlabelling assay. (A) Two days after treatment, (B) 2 weeks after treatment, (C) 20 weeks after treatment and (D) 36 weeks after treatment.

 
At 2 days, 2 and 20 weeks, adduct levels in blood were ~20% of the levels in lung (R2 = 0.98).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary data
 References
 
Although no definite correlation has been established between exposure to nitro-PAHs and human cancer, some evidence suggests that engine exhausts, which contain significant amounts of these compounds, are carcinogenic to humans (1,4). The aromatic nitroketone 3-NBA, an extremely potent mutagen and lung carcinogen, has been identified as an air pollutant, diesel emissions being the predominant source (6). As a consequence, 3-NBA like other nitro-PAHs is widely distributed in the environment and human exposure to 3-NBA is thought to occur primarily via the respiratory tract (31,32). Thus, it is important to study the effect of 3-NBA following administration into the lungs.

Previously, we and others demonstrated by the TLC 32P-postlabelling technique that 3-NBA forms multiple DNA adducts after metabolic activation in vitro in cells (9,33–37) and in vivo in rats and mice after oral and i.p. administration (7,9). In our laboratory, the observed adduct pattern was essentially the same consisting of up to five spots when analysed by the butanol enrichment version.

In the present study, we demonstrated the formation and persistence of 3-NBA–DNA adducts in several organs of rats treated intratracheally with a single dose of 3-NBA (0.2 mg/kg body wt). The observed adduct pattern was the same in all organs and qualitatively similar to those obtained from in vitro reactions indicating that no adducts other than those already detected in vitro were detectable in vivo. Initially, high adduct levels were detected in the lung (Table I), but similarly high initial levels were also found in the pancreas and kidney followed by lower levels in heart, urinary bladder, small intestine and liver, suggesting that 3-NBA or its metabolites are distributed via the blood stream to other organs.

The high DNA binding found in the pancreas of rats after intratracheal or i.p. (11) treatment with 3-NBA is consistent with the high nitroreductase activity in this organ and has led to the suggestion by Anderson et al. (38) that exposure to nitro-PAHs might be a risk factor for pancreatic cancer in humans.

Using two independent chromatographic systems (TLC and HPLC), we showed clearly that adduct 1 represents 2-(2'-deoxyadenosin-N6-yl)-3-aminobenzanthrone (Figure 1) and adduct 2 is also derived from reaction with deoxyadenosine but has not been identified yet. Moreover, adducts 3, 4 and 5 were shown to be reaction products with deoxyguanosine and identified as dG-N2-ABA (adduct 3) and N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (adduct 4/5). According to these DNA adduct structures, N-hydroxy-3-aminobenzanthrone, which is formed through initial reduction of the nitro group primarily catalysed by cytosolic nitroreductases and subsequent esterification, is the critical intermediate in DNA adduct formation in vivo and in vitro (9,11,13).

The work reported here has explored the long-term persistence (36 weeks) of DNA adducts in organs and blood of adult female Sprague–Dawley rats after a single instillation of 3-NBA. Nagy et al. (8) have studied adduct formation and persistence after single instillation of 3-NBA in young female Fischer 344 rats using HPLC 32P-postlabelling although they used a much higher dose (10 mg/kg body wt). Consistent with our results, maximal adduct levels were observed after 2 days in the lung and the kidney, whereas much lower levels were found in the liver. In addition, dG-N2-ABA was the most prominent adduct found in each organ examined at all time points. However, in contrast to our results the authors did not detect adducts above background 16 days after treatment. In the light of our detection of adducts up to 36 weeks after treatment, we would have expected Nagy et al. (8) to have detected adducts at 16 days, given their detection limit by HPLC 32P-postlabelling of one adduct in 108 nucleotides and their use of a 50-fold higher dose of compound than in the present study. It should be noted that in the two studies different strains of rats were used, adducts were analysed in different ways (TLC versus HPLC 32P-postlabelling), but whether these or other factors account for the difference in the detection of long-term persistent adducts between the two studies is unclear, and will require further investigation

In all organs, target and non-target, adduct levels decreased over time and showed a similar profile reaching a plateau at 20 weeks after treatment. In fact, such pharmacokinetics for DNA adduct persistence have already been reported for several other carcinogens (26,27,39–42). However, adduct removal studies for times >20 weeks are rare. The plant carcinogen aristolochic acid I, a nitro-PAH-like 3-NBA showed comparable initial total adduct levels in several organs after single oral treatment (5 mg/kg body wt) and similar removal curves till 36 weeks after treatment (27). Likewise, Randerath et al. (26) reported triphasic adduct removal profiles from mouse skin after topical application of 7,12-dimethylbenz[a]anthracene. In this case, 7,12-dimethylbenz[a]anthracene–DNA adducts attained virtually constant levels up to 42 weeks after treatment even in a proliferating tissue. Several studies have found a positive correlation between carcinogen–DNA adduct levels and persistence in relation to target organ specificity for tumour formation (43–45), suggesting the critical importance of persistent carcinogen–DNA adducts. However, it is not clear why a fraction of the damage persists, even in a proliferating tissue such as skin.

DNA adduct patterns and DNA adduct removal in blood of rats instilled with a single dose of 3-NBA were similar to those observed in the tissues. The most abundant DNA adduct, dG-N2-ABA (adduct 3), was still detectable 20 weeks after treatment, to our knowledge, the longest period of persistence for an adduct in blood. The collateral pharmacokinetics for DNA adduct persistence in lung and total blood suggest that blood adduct-based dosimetry may reflect adduct patterns and levels in other tissues remote from the site of administration. Thus, blood DNA adducts may prove to be useful dosimeter for the biologically effectively delivered dose of DNA adduct-forming carcinogens at their target tissue.

In summary, a single intratracheal administration of 3-NBA to rats resulted in the formation of a specific adduct pattern in each of the seven organs investigated and in blood. These TLC 32P-fingerprints consisted of multiple adducts chromatographically identical to authentic DNA adduct standards clearly indicating the importance of the nitroreduction pathway in the bioactivation of 3-NBA. More importantly, the results of the present study support the conclusion that some or all of the detected 3-NBA–DNA adducts represent premutagenic lesions involved in mutagenesis and/or carcinogenesis. Recent data indicate that G:C to T:A transversion mutations induced by 3-NBA in the transgenic MutaMouse assay are caused by misreplication of adducted guanine residues, the adduct type most persistent in the target organ.


   Supplementary data
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary data
References
 
Supplementary data are available at Carcinogenesis Online.


   Acknowledgments
 
This work was supported financially by Baden-Württemberg (BWPLUS, BWR 22009), GACR (303/05/2195) and Cancer Research UK. V.M.A. and D.H.P. are members of the European Environmental Cancer Risk, Nutrition and Individual Susceptibility Network of Excellence. We thank Reinhold Klein for the animal treatment.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary data
 References
 

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Received September 8, 2006; revised November 1, 2006; accepted November 4, 2006.


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