Lobe-specific increases in malondialdehyde DNA adduct formation in the livers of mice following infection with Helicobacter hepaticus

Rajinder Singh6,, Chiara Leuratti, Shylaja Josyula2,, Marek A. Sipowicz3,, Bhalchandra A. Diwan4,, Kazimierz S. Kasprzak3,, Herman A. J. Schut2,, Lawrence J. Marnett5,, Lucy M. Anderson3, and David E. G. Shuker1,

Medical Research Council, Toxicology Unit, Hodgkin Building, University of Leicester, PO Box 138, Lancaster Road, Leicester LE1 9HN,
1 Department of Chemistry, The Open University, Walton Hall, Milton Keynes, MK7 6AA, UK,
2 Department of Pathology, Medical College of Ohio, Toledo, OH 43614,
3 Laboratory of Comparative Carcinogenesis, Division of Basic Sciences, National Cancer Institute at Frederick and
4 Intramural Research Support Program, SAIC-Frederick, Building 538, Fort. Detrick, Frederick, MD 21702 and
5 Department of Biochemistry and Chemistry, Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Helicobacter hepaticus infection is associated with chronic hepatitis and the development of liver tumours in mice. The underlying mechanism of this liver carcinogenesis is not clear but the oxidative stress associated with H.hepaticus infection may result in induction of lipid peroxidation and the generation of malondialdehyde. Malondialdehyde can react with deoxyguanosine in DNA resulting in the formation of the cyclic pyrimidopurinone N-1,N2 malondialdehyde–deoxyguanosine (M1dG) adduct. This adduct has the potential to cause mutations that may ultimately lead to liver carcinogenesis. The objective of this study was to determine the control and infection-related levels of M1dG in the liver DNA of mice over time, using an immunoslot-blot procedure. The level of M1dG in control A/J mouse livers at 3, 6, 9 and 12 months averaged 37.5, 36.6, 24.8 and 30.1 adducts per 108 nucleotides, respectively. Higher levels of M1dG were detected in the liver DNA of H.hepaticus infected A/JCr mice, with levels averaging 40.7, 47.0, 42.5 and 52.5 adducts per 108 nucleotides at 3, 6, 9 and 12 months, respectively. There was a significant age dependent increase in the level of M1dG in the caudate and median lobes of the A/JCr mice relative to control mice. A lobe specific distribution of the M1dG adduct in both infected and control mice was noted, with the left lobe showing the lowest level of the adduct compared with the right and median lobes at all time points. In a separate series of mice experimentally infected with H.hepaticus, levels of 8-hydroxy-deoxyguanosine were significantly greater in the median compared with the left lobe at 12 weeks after treatment. In conclusion, these results suggest that M1dG occurs as a result of oxidative stress associated with H.hepaticus infection of mice, and may contribute to liver carcinogenesis in this model.

Abbreviations: MDA, malondialdehyde; M1dG, malondialdehydedeoxyguanosine (3-(2'-deoxy-ß-D-erythro-pentofuranosyl) pyrimido-[1,2{alpha}] purin-10(3H)-one); M1G, malondialdehyde-guanine (pyrimido-[1,2{alpha}]purin-10(3H)-one); 8-oxo-dG, 8-hydroxy-deoxyguanosine.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Helicobacter hepaticus was discovered in 1992 to be the cause of hepatitis and hepatic neoplasia in an untreated control group of A/JCr mice, a strain which normally has a low incidence of hepatic disease (1,2). H.hepaticus is found in the lower intestinal tract of all exposed mice and in susceptible strains colonizes the hepatic bile canaliculi, with male mice showing a greater incidence and severity of hepatitis (3,4). The exact nature of the mechanism whereby H.hepaticus infection can lead to liver carcinogenesis in mice is of considerable interest in view of the association between human gastric cancer and infection by Helicobacter pylori. The possible genotoxic and epigenetic mechanisms which may be involved in H.hepaticus associated liver carcinogenesis have been reviewed (5). The lack of formation of N-7 methylguanine and O6 methylguanine DNA adducts suggests a mechanism that does not involve the nitrosation of endogenous amines by nitric oxide. No mutations have been observed in ras oncogenes or the p53 gene in carcinomas and adenomas from infected mice, indicating that an epigenetic mechanism may be involved (6). Infection with H.hepaticus does enhance the hepatic carcinogenesis induced by the alkylating agent N-nitrosodimethylamine, which implies the involvement of a tumour promotion mechanism (7). However, there is evidence for a role for reactive oxygen species (ROS): 8-hydroxy-deoxyguanosine (8-oxo-dG) levels were significantly higher in infected mice and increased with time (8). Perfusion of livers with nitro blue tetrazolium revealed that increased superoxide was produced within hepatocytes, rather than by inflammatory cells (8).

Another likely outcome of increased ROS in H.hepaticus infected livers is lipid peroxidation. Peroxidation of the polyunsaturated fatty acids of biological membranes in cells can result in many potential genotoxic products, with malondialdehyde (MDA) being a major example (911). Malondialdehyde can also be formed as a by-product of arachidonic acid metabolism in the biosynthesis of prostaglandins (12). The major adduct formed following the reaction of MDA with DNA is the highly fluorescent, cyclic N-1,N2 malondialdehyde–deoxyguanosine (M1dG). Reaction with adenine and cytosine deoxynucleosides occurs to a much lesser extent (13,14). The mechanism of formation of MDA by lipid peroxidation and subsequent reaction with deoxyguanosine is summarized in Figure 1Go. The M1dG adduct is repaired by both bacterial and mammalian nucleotide excision repair pathways (15).



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Fig. 1. Formation of malondialdehyde by lipid peroxidation and reaction with deoxyguanosine in DNA.

 
Background levels of the endogenously formed M1dG adduct have been detected in a variety of human and animal tissues. Chaudhary et al. (16) have detected levels of M1dG ranging from 50 to 120 adducts per 108 nucleotides in human disease-free liver DNA using gas chromatography/mass spectrometry. Following dosing of rats with carbon tetrachloride, which is an inducer of lipid peroxidation, the same authors reported levels of M1dG at 38 compared with 21 adducts per 108 nucleotides in control rat liver DNA. Malondialdehyde is genotoxic, as indicated by the induction of mutations in bacterial and mammalian systems (1719). The carcinogenic potential of MDA has been shown by the production of thyroid gland and pancreatic tumours in F344/N rats treated chronically with MDA over a period of 2 years (20). It was therefore of interest to determine the levels of M1dG in liver DNA of mice infected with H.hepaticus (A/JCr) compared with non-infected mice (A/J). The results indicate that a significant increase of the M1dG adduct occurred in the infected mice compared with controls as a function of age and that, unexpectedly, this increase was limited to the caudate and median lobes of the liver.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials
Calf thymus DNA, 1,1,3,3-tetramethoxypropane, propidium iodide, Tween-20 and guanine were purchased from Sigma (Poole, UK). All other reagents (analytical grade) and HPLC grade solvents were purchased from Fisher Scientific (Loughborough, UK). Phosphate buffered saline was prepared using Oxoid tablets (Oxoid, Basingstoke, UK). HPLC grade water, 18.2 M output quality was obtained from Maxima purification equipment (Elga, High Wycombe, UK).

Animals and livers
The DNA for this study was the same as that used previously for assessment of 32P-post-labeled indigenous DNA adducts (I compounds) (21). In brief, the controls were male A/J mice obtained at 4 weeks of age from the Jackson Laboratory, Bar Harbor, Maine. They were maintained in a separate room free of H.hepaticus in parallel with male A/JCr mice from a colony, originating from the Frederick Cancer Research Facility Animal Production Area, that was naturally infected with H.hepaticus. Livers were removed at random from 3, 6, 9 and 12 month old H.hepaticus infected A/JCr mice within a 1 week period. Control A/J mice were killed at 6, 9 and 12 months of age within 2 weeks of the infected mice; those 3 months old were killed 4 weeks later. All mice were killed in the morning. The livers were separated into lobes, a portion of each lobe fixed in formalin for histopathological examination, and the remainder stored at –80°C. Representative liver sections from each lobe were evaluated by a board-certified veterinary pathologist (Dr Miriam Anver, SAIC, Frederick, MD) and scored semi-quantitatively with regard to extent and severity of hepatitis and associated inflammatory lesions, as indicated in Table IGo (21). The livers of four mice were selected for DNA adduct determinations at each time point so as to provide a range of disease severity for comparison purposes.


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Table I. M1dG adducts in individual mice livers as related to severity of hepatitis
 
DNA extraction procedure
DNA was isolated from 0.5 g portions of each lobe of the liver using a direct salt precipitation method as previously described (21). The phenol extraction step following digestion with proteinase K was omitted and salt-aided precipitation was used for the remaining proteins (21). The concentration of DNA was measured by determining the absorbance at 260 nm using a GeneQuant spectrophotometer (Pharmacia Biotech, Cambridge, UK) assuming that one absorbance unit at 260 nm is equal to 50 µg/ml DNA. DNA was stored at –80°C until utilized. No DNA was available for the following samples: for the infected mice, three caudate lobes at 9 months; for the control mice, two caudate lobes at 3 months, one median lobe at 6 months and one caudate lobe at 9 and 12 months.

Preparation of M1 guanine standard
The procedure was adapted from the one described by Hadley and Draper (22). Guanine (100 mg) dissolved in 2.5 ml 1.0 M HCl was incubated with a 1.0 M MDA solution at 40°C for 1 h. The MDA solution was prepared by incubating 1,1,3,3-tetramethoxypropane with 1.0 M HCl at 40°C for 40 min. The reaction mixture was centrifuged at 1000 r.p.m. for 10 min and the supernatant removed. The remaining pellet was extracted a further two times with HPLC grade water at 60°C (2 ml) and centrifuged as before. All three supernatants were combined and evaporated to dryness. The resulting yellow solid was redissolved in 3.0 ml 10 mM formic acid and then centrifuged at 1000 r.p.m. for 10 min. Aliquots of the supernatant were subjected to semi-preparative HPLC using a Gilson HPLC connected to a Hypersil C18 BDS (250x10 mm, 5µ) column. The column was eluted with solvent A (50 mM ammonium formate, pH 5.4) and solvent B (methanol using the following gradient: 0 min–0%B, 15 min–25%B, 25 min–35%B, 30 min–0%B) with a flow rate of 3.5 ml/min. UV absorbance was monitored at 254 nm. HPLC fractions corresponding to peaks were collected, lyophilized and subjected to analysis by electrospray-mass spectrometry. The fraction corresponding to M1G yielded a molecular ion m/z 188 (M+H)+ and was further purified by re-injection onto the Gilson HPLC system as described above but using HPLC grade water as solvent A. Further characterization of the product was performed by determining the UV spectrum, which gave a {lambda}max of 214 nm, {lambda}min of 235 nm and {lambda}max of 253 nm at neutral pH (23).

Preparation of standard DNA treated with malondialdehyde for the immunoslot-blot procedure
Calf thymus DNA (20 mg) dissolved in 0.1 M KH2PO4, pH 4.5 (1 mg/ml) was incubated at 37°C for 3 days with a 2 mM MDA solution (24). The MDA solution was prepared by incubating 1,1,3,3-tetramethoxypropane with 0.1 M HCl at 40°C for 40 min followed by neutralization with 0.1 M KOH. The DNA was precipitated with ice-cold isopropanol and washed with ethanol followed by 70% (v/v) ethanol in water and redissolved in HPLC grade water prior to storage at –80°C. An aliquot of DNA (2 µg) was subjected to acid hydrolysis using 0.1 M formic acid at 70°C for 1 h. The level of M1guanine following hydrolysis was determined using a Waters 600E pump and system controller HPLC coupled to a Waters 484 UV detector in series with a Waters 470 fluorescence detector. A Hypersil C18 BDS (100x2.1 mm, 3µ) column was used which was eluted isocratically with 99:1 (v/v) 0.1 M triethylamine acetate (pH 5.0):methanol at a flow rate of 0.2 ml/min. UV absorbance was monitored at 260 nm and fluorescence was monitored at 360 nm for excitation and at 500 nm for emission.

Immunoslot-blot procedure for the determination of M1 dG DNA adduct levels
This method has been described by Leuratti et al. (25) but several modifications have been made. Liver DNA samples or DNA standards for the calibration line were pipetted (3.5 µg) and the final volume adjusted to 100 µl with 10 mM K2HPO4, pH 7.0. DNA was sonicated for 20 min using an ultrasonic bath (Decon Laboratories, Hove, UK). Following the addition of 150 µl phosphate buffered saline (PBS), the samples were heat denatured at 100°C for 5 min in a boiling water bath and then cooled on ice for at least 10 min before the addition of 250 µl 2 M ammonium acetate. The resulting single-stranded DNA was pipetted (1 µg) in triplicate onto a nitrocellulose filter (Protran® BA 79, 0.1 µm; Schleicher & Schuell, Dassel, Germany) using the Minifold II blotting apparatus (Schleicher and Schuell). Prior to usage the nitrocellulose filter was pre-soaked in HPLC grade water followed by 1 M ammonium acetate. Following the application of DNA, the slots of the blotting apparatus were washed with 200 µl 1 M ammonium acetate. The filter was heated at 80°C for 1.5 h and blocked for non-specific binding for 1 h with 100 ml 5% non-fat milk powder (Marvel, Premier Brands, UK) dissolved in phosphate buffered saline containing 0.1% (v/v) Tween-20 (PBS-T). Following blocking the filter was washed for 5 min twice with 50 ml PBS-T. The filter was incubated at room temperature for 2 h then at 4°C overnight with the primary antibody specific for M1dG (0.3 mg/ml) (26), diluted 1:48 000 with 40 ml 0.5% non-fat milk powder PBS-T. Following washing with 50 ml PBS-T for 1 min and then twice for 5 min, the filter was incubated for 2 h at room temperature with horseradish peroxidase-conjugated secondary antibody (goat anti-mouse; Dako A/S, Denmark), diluted 1:4000 with 32 ml 0.5% non-fat milk powder PBS-T. The filter was washed with 50 ml PBS-T for 15 min followed by a further two 5 min washes. Finally the filter was incubated with the chemiluminescent reagent consisting of 4 ml luminol/enhancer solution plus 4 ml stable peroxide buffer (Supersignal® West Dura extended duration substrate; Pierce, Rockford, IL) for 5 min and exposed to chemiluminescent sensitive hyperfilm prior to an image of the filter being acquired using a Fluor-S MultiImager (Bio-Rad, Hercules, CA). The intensity of chemiluminescent signal for each band was determined using the image analysis software. The level of the adduct in the liver DNA samples was determined from the calibration line generated by the dilution of standard DNA (with control DNA) containing known amounts of the M1dG adduct.

Determination of the amount of DNA bound to the immunoslot-blot filter
The nitrocellulose filter was washed with PBS for 10 min and incubated with a solution of propidium iodide (250 µg) dissolved in PBS (50 ml) for 3 h in the dark. Following incubation, the filter was washed with PBS for 1 h and an image of the filter was captured using the Fluor-S MultiImager. The level of DNA bound to the filter was determined by integrating the intensity of each band on the filter using the image analysis software.

Data and statistical analysis
The average adduct level for the left, caudate, median and right liver lobes of each mouse was calculated, and this value was used for comparisons between infected and control mice at each time point, and for examination of time trends. Significances of differences were tested by use of Instat from GraphPad Software Inc., San Diego, CA, and included parametric and non-parametric tests as appropriate.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Determination of M1 dG using the immunoslot-blot procedure
The M1G standard was used to construct calibration lines for HPLC which were then used to determine the level of modification in calf thymus DNA that had been incubated with 1,1,3,3-tetramethoxypropane. Figure 2A and BGo shows the typical UV and fluorescence HPLC chromatograms, respectively, that were obtained following acid hydrolysis of calf thymus DNA. Under the conditions used for acid hydrolysis the maximum yield of M1G was at 1 h (data not shown). The level of modification was determined to be 12.69 pmol M1G/µg DNA. The level of modification was in close agreement with the value (12.88 pmol/µg) obtained for the same DNA sample analysed by using an enzymatic digestion in conjunction with liquid chromatography–mass spectrometry method (J.P.Plastaras, personal communication). The DNA was subsequently used to construct the calibration lines (ranging from 0 to 5.0 fmol M1dG/µg DNA) for the immunoslot-blot procedure by dilution with control (unmodified) DNA. Figure 3AGo shows a typical image of the immunoslot-blot filter with the calibration line on the left and samples pipetted on the right. An aliquot of commercially available human blood (buffy coat) genomic DNA purchased from Boehringer Mannheim was also pipetted onto the filter to ensure the immunoslot-blot gave consistent results over time from one blot to another. Figure 3BGo shows the typical image obtained using the Bio-Rad multimager following staining with propidium iodide. The intensity of the signal was directly proportional to the amount of DNA (up to 3.0 µg) pipetted onto the filter (data not shown). The level of adduct in each sample was corrected for the amount of DNA bound to the filter as determined by propidium iodide staining. The limit of detection attained for the immunoslot-blot method for determining M1dG was 2.5 adducts per 108 unmodified nucleotides, which was comparable with that obtained by published methodology (27). The sensitivity of immunoslot-blot method is greatly enhanced since only 1 µg DNA is required per adduct determination, with the added advantage that each sample is analysed in triplicate. Furthermore, since quantitation of adduct levels is based on a calibration line derived from a DNA standard with known amounts of modification blotted onto the same filter as the liver DNA samples, the results can be standardized for different analyses.



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Fig. 2. Typical HPLC chromatograms obtained after acid hydrolysis for the determination of the level of M1G formed following the incubation of calf thymus DNA with 2 mM MDA by (A) UV and (B) fluorescence detection (1 µg of hydrolysed DNA was injected onto the HPLC column).

 


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Fig. 3. Typical images obtained of the immunoslot-blot filter using the Fluor-S MultiImager following incubation with (A) chemiluminescent reagent and (B) propidium iodide. Each DNA sample (1 µg per slot) was blotted onto the filter in triplicate.

 
Comparison of M1 dG adduct levels between A/J and A/JCr livers
The level of the M1dG adduct in each mouse liver was calculated as the average of the values for the four lobes (Table IGo). The level of the adduct at each time point was derived from the average of the values from the livers of four mice. The level of M1dG in control A/J mice livers at 3, 6, 9 and 12 months averaged 37.5, 36.6, 24.8 and 30.1 adducts per 108 nucleotides, respectively. Higher levels of M1dG were detected in the liver DNA of H.hepaticus infected A/JCr mice, with levels averaging 40.7, 47.0, 42.5 and 52.5 adducts per 108 nucleotides at 3, 6, 9 and 12 months, respectively.

Lower M1 dG adduct levels in left and caudate lobes than in right and median lobes of the liver
At all time points, in both infected and control mice, the level of M1dG adduct was lower in the left liver lobe than in the median and right lobes (Figure 4Go). At most time points, the adduct was also lower in the caudate lobe. The differences among lobes were of statistical significance for control mice at 3 and 12 months and for infected mice at 6 and 12 months by ANOVA, with differences close to significance for controls at 9 months and for infected mice at 3 months.



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Fig. 4. Comparison of M1dG adduct levels ± SD in DNA from different liver lobes in control mice (A–D) and infected mice (E–H) at 3 (A, E), 6 (B, F), 9 (C, G) and 12 months (D, H). P values for ANOVA of all data in each set are shown on the graphs. P values for individual comparisons from the ANOVA are as follows: for control mice at 3 months (A), left and caudate lobe values each significantly lower than that for right lobe, P < 0.05. For control mice at 12 months (D), left lobe values less than those for median and for right lobe, P < 0.01; caudate lobe values less than those for median and right lobe, P < 0.05. For infected mice at 6 months (F), left lobe values significantly less than those for median and for right lobe, P < 0.05; at 12 months (H), left lobe values less than those for median lobe, P < 0.01.

 
Changes in M1 dG adduct levels in liver lobes with time
In control livers, the adduct levels tended to decrease slightly with age (Figure 5A–DGo), with none of these differences being of statistical significance. The largest apparent decrease was in the left lobe (P = 0.17; Figure 5AGo). In contrast, in the infected livers, the adduct levels in the left and right lobes did not change significantly with age (Figure 5E and HGo), but those in the caudate and median lobes increased significantly (Figure 5F and GGo). This was particularly consistent and striking for the caudate lobe (Figure 5FGo; P < 0.0001).



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Fig. 5. Regression analysis of the changes in M1dG adduct levels with age in control mice (A–D) and infected mice (E–H), in the left lobe (A, E), the caudate lobe (B, F), the median lobe (C, G) and the right lobe (D, H). Significance of the line slopes as different from zero are shown on the graphs.

 
Higher M1 dG adduct levels in caudate plus median and in left lobes of infected mice
In comparisons utilizing combined data from the caudate and median lobes, which showed an age-related increase in the M1dG adduct in infected but not control mice (Figure 5Go), the average values were higher for the infected mice, compared with control mice, starting at 6 months, and of statistical significance at 9 and 12 months (Figure 6A–DGo). Values for the left lobes were also compared, since these decreased in the controls but not the infected mice. The latter had a higher average of the M1dG adduct at all time points, with differences that were significant or close to significant at 9 and 12 months (Figure 6E–HGo).



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Fig. 6. Comparison of M1dG adduct levels ± SD in caudate plus median lobes, control versus infected, at (A) 3, (B) 6, (C) 9 and (D) 12 months. Comparisons for these time points for the left liver lobe are shown in (EH). P values for possibly significant differences are shown on the graphs. In addition, values for the lobes analysed separately were significantly different in control versus infected mice for the caudate lobe at 12 months (P = 0.0001) and for the median lobe close to significance at 6 (P = 0.054), 9 (P = 0.061) and 12 months (P = 0.11).

 
Higher 8-oxo-dG in median versus left lobe in mice experimentally infected with H.hepaticus
Earlier we reported an increase in average levels of 8-oxo-dG in livers of male mice experimentally infected with H.hepaticus, compared with controls, up to 12 weeks after introduction of the bacteria (8). In that study, left and median lobes of each liver were analysed separately, and these per lobe data have now been analysed. As shown in Table IIGo, at 12 weeks, when minimal hepatocellular necrosis was observed in the infected livers, the median lobe had a marked, significant higher level of 8-oxo-dG compared with the left lobe. No consistent or significant differences between these lobes were seen at earlier times or in the control mice at any time. It may be noted, however, that overall average levels of 8-oxo-dG were significantly higher in the infected mice by 2 weeks after treatment (8).


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Table II. Levels of 8-oxo-dG in left and median lobes of livers of mice experimentally infected with Helicobacter hepaticus and non-infected mice
 
Relationship of M1 dG adduct levels to liver disease and 32P-post-labeled adducts
Hepatitis associated with H.hepaticus infection is typically focal but has not been reported to have a lobe specificity, although this possibility has not been systematically studied. Sections from the various lobes in the present study showed no consistent differences among lobes with regard to severity or extent of hepatitis. There were no clear correlations between extent of hepatitis in each liver, as scored semi-quantitatively, and the M1dG adduct in caudate or median lobes, or averaged among all lobes (Table IGo). In the median lobe, the lowest level of the M1dG adduct was found in the livers with most extensive disease at 3 and 6 months, but the opposite was true at 9 months, and at 12 months no pattern was evident. There was also no correlation with total 32P-post-labeled adducts, reported previously (21). Both sets of data were closely inspected for possible relationships between the M1dG adduct and the individual 32P-post-labeled adducts; none were found (data not shown).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The results of this study show that the natural infection of livers of mice by H.hepaticus, previously noted to involve a progressive increase in levels of 8-oxo-dG in DNA with age (8), also led to age-related elevations in M1dG. Strikingly, this increase was limited to the caudate and median lobes of the liver. An apparent increase in the left lobes relative to controls was due to a decrease of the M1dG adduct in this lobe in controls over time.

In a separate group of mice experimentally infected with H.hepaticus, analysis of 8-oxo-dG had been carried out only on left and median lobes. At 12 weeks after the start of the infection, levels of 8-oxo-dG were significantly higher in the median compared with the left lobes, confirming elevation of ROS in this lobe. The reason for this lobe restriction in elevation of both M1dG and 8-oxo-dG is unclear. The caudate and median lobes are centrally located in proximity to the gall bladder. This may be relevant, since H.hepaticus bacteria inhabit the bile canaliculi.

As in the case of 32P-post-labeled adducts (21), the levels of MldG adducts did not correlate with extent of hepatitis in a representative section of each liver lobe. We obtained a similar result for 8-oxo-dG (unpublished data). It is possible that, since the disease is very focal within the liver, the representative sections do not provide an accurate indication of overall extent of inflammatory disease within the lobe. Alternatively, the various DNA adducts may reflect events other than an inflammatory response. Perfusion of H.hepaticus infected livers with nitro blue tetrazolium revealed that superoxide was formed within the cytoplasm of hepatocytes, in association with induction of cytochrome P450 2A5 and, surprisingly, not in association with inflammatory cells (8). Whatever the source of the ROS, it is likely that lipid peroxidation within such cells results in generation of malondialdehyde and M1dG. Intracellular generation of ROS within the hepatocytes, followed by 8-oxo-dG, M1dG and other forms of DNA damage, may have potential for initiation of neoplasia. The chronic gastritis observed with H.pylori infection has been associated with the accumulation of oxidative DNA damage in humans (28).

A further interesting observation from the current study was the liver lobe specific distribution of the M1dG adduct, in controls as well as infected mice (Figure 4Go). At all time points, the level of M1dG in the left lobe was lower than in other lobes. This is in contrast to 32P-post-labeled adducts in the same livers, which were slightly, but consistently, higher in the left lobe (21). At several time points M1dG was also lower in the caudate lobe. Lawson and Pound (29) have observed greater liver damage in the right compared with the left lobe of rats dosed with carbon tetrachloride, implying that the right lobe may be more sensitive to lipid peroxidation derived damage. The reason for this is not known, but might be worth further study. Richardson et al. (30) have shown that there is a lobe-specific distribution of the O4 ethylthymidine adduct formed following dosing of rats with N-nitrosodiethylamine. The highest levels of the adduct were observed in the left and right median lobes of the liver which corresponded to the highest distribution of radiolabelled N-nitrosodiethylamine in the left lobe (30). Differential circulation to the left lobe could influence steady state levels of MDA.

It is of interest that in the control livers the average levels of M1dG did not increase with age, and may have experienced a slight decrease, particularly in the left lobe. This is in contrast to the steady increase in 8-oxo-dG with age that has been repeatedly demonstrated in rodents (8,31). This result suggests that, in spite of the considerable between-lobe and between-animal variability, M1dG levels are physiologically regulated through control of MDA production, repair of M1dG or other processes. In another study of A/J mice, none of several antioxidant parameters measured in liver changed significantly between 3 and 6 months of age; however, total cytochrome P450 increased by 30% over this period (32).

In conclusion, formation of M1dG in mice is a liver lobe-specific phenomenon, and in mice infected by H.hepaticus this adduct increases significantly over time in the caudate and median lobes. It is therefore possible that this DNA adduct contributes to initiation of hepatocellular tumours in infected animals. Lobe distribution of hepatic neoplasms associated with H.hepaticus infection has not been reported thus far but should be investigated.


    Notes
 
6 To whom correspondence should be addressedEmail: rs25{at}le.ac.uk Back


    Acknowledgments
 
R.Singh acknowledges the UK Foods Standards Agency for financial support. Prof. L.J.Marnett acknowledges the NIH for financial support (research grant CA77839).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received February 20, 2001; revised April 23, 2001; accepted April 24, 2001.