Mouse Liver DNA Research Articles

Adduction of DNA with MTBE and TBA in mice studied by accelerator mass spectrometry

Methyl tert-butyl ether (MTBE) is a currently worldwide used octane enhancer substituting for lead alkyls and gasoline oxygenate. Our previous study using doubly (14)C-labeled MTBE [(CH(3))(3) (14)CO(14)CH(3)] has shown that MTBE binds DNA to form DNA adducts at low dose levels in mice. To elucidate the mechanism of the binding reaction, in this study, the DNA adducts with singly (14)C-labeled MTBE, which was synthesized from (14)C-methanol and tert-butyl alcohol (TBA), or (14)C-labeled TBA in mice have been measured by ultra sensitive accelerator mass spectrometry. The results show that the methyl group of MTBE and tert-butyl alcohol definitely form adducts with DNA in mouse liver, lung, and kidney. The methyl group of MTBE is the predominant binding part in liver, while the methyl group and the tert-butyl group give comparable contributions to the adduct formation in lung and kidney.

HPLC−ESI+-MS/MS Analysis of N7-Guanine−N7-Guanine DNA Cross-Links in Tissues of Mice Exposed to 1,3-Butadiene

1,3-butadiene (BD) is a major industrial chemical used in rubber and plastics production and is recognized as an animal and human carcinogen. Although the exact mechanism of BD-induced carcinogenesis is unknown, chemical reactions of epoxide metabolites of BD with DNA to form nucleobase adducts are likely to contribute to multistage carcinogenesis. Among BD-derived epoxy metabolites, 1,2:3,4-diepoxybutane (DEB) appears to be the most genotoxic and carcinogenic, probably because of its bifunctional nature. Initial DNA alkylation by DEB produces N7-(2'-hydroxy-3',4'-epoxybut-1'-yl)guanine monoadducts, which can then be hydrolyzed to N7-(2',3',4'-trihydroxy-1'-yl)guanine or can react with another site in double-stranded DNA to form 1,4-bis(guan-7-yl)-2,3-butanediol (bis-N7G-BD) cross-links. While (2',3',4'-trihydroxy-1'-yl)guanine lesions have been previously quantified in vivo, they cannot be used as a biomarker of DEB because the same lesions are also formed by another, more prevalent BD metabolite, 1,2-epoxy-3,4-butanediol. In contrast, bis-N7G-BD can only be formed from DEB, potentially providing a specific biomarker of DEB formation. We have developed a quantitative HPLC-ESI+-MS/MS method for measuring racemic and meso forms of bis-N7G-BD in DNA extracted from tissues of BD-exposed laboratory animals. In our approach, bis-N7G-BD adducts are released from DNA as free bases by neutral thermal hydrolysis, purified by solid-phase extraction, and subjected to HPLC-ESI+-MS/MS analysis. Selected reaction monitoring is performed by following the loss of a guanine moiety from protonated molecules of bis-N7G-BD and the formation of protonated guanine under collision-induced dissociation. Quantitative analysis of racemic and meso forms of bis-N7G-BD is based on isotope dilution with the corresponding 15N-labeled internal standards. The lower limit of quantification of our current method is 10-20 fmol/0.1 mg of DNA. The accuracy and precision of the new method were determined by spiking control mouse liver DNA with racemic and meso forms of bis-N7G-BD (10 fmol each), followed by sample processing and HPLC-ESI+-MS/MS analysis. Calculated amounts of racemic and meso forms of bis-N7G-BD were within 20% of the theoretical value (9.7 +/- 2 and 9.2 +/- 1.9 fmol, respectively, N = 4). DNA extracted from liver and lung tissues of mice exposed to 625 ppm butadiene for 5 days contained 3.2 +/- 0.4 and 1.8 +/- 0.5 racemic adducts per 10(6) guanines, respectively, while the amounts of meso-bis-N7G-BD were below the detection limits of our method (1 per 10(7) guanines). Control animals did not contain either bis-N7G-BD lesion. Sensitive and specific quantitative methods for bis-N7G-BD analysis developed in this work provide a unique biomarker of DEB-induced DNA alkylation following exposure to BD.

Orphan Nuclear Receptor Constitutive Active/Androstane Receptor–Mediated Alterations in DNA Methylation during Phenobarbital Promotion of Liver Tumorigenesis

Altered DNA methylation is an epigenetic mechanism that plays a key role in the carcinogenesis process, and the nongenotoxic rodent hepatocarcinogen phenobarbital (PB) alters the methylation status of DNA in mouse liver. The constitutive active/androstane nuclear receptor (CAR) mediates half of the PB-induced hepatic gene expression changes and it is essential for liver tumor promotion in PB-treated mice. Here, a technique involving methylation-sensitive restriction digestion, arbitrarily primed PCR, and capillary electrophoresis was utilized to detect PB-induced regions of altered DNA methylation (RAMs) in CAR wildtype (WT) mice that are sensitive to promotion by PB and resistant CAR knockout (KO) mice. The CAR WT mice developed preneoplastic lesions after 23 weeks of PB treatment (precancerous) and liver tumors after 32 weeks, while the CAR KO mice did not develop tumors (Y. Yamamoto, et al., 2004, Cancer Res. 64, 7197-7200). Our goal was to discern those RAMs which are playing important roles in tumor formation by comparing the RAMs that form in sensitive and resistant groups of mice. Using this novel approach, 42 unique RAMs were identified in the precancerous as compared to the CAR KO, 23-week PB-treated tissue. Of these 42 RAMs, 14 carried forward to the tumor tissue, and additionally, 104 total unique RAMs were observed in the tumor tissue. These results indicate that there are unique RAMs occurring in the sensitive CAR WT mice and that a portion of these are seen in both the precancerous and tumor tissue. We hypothesize that these unique RAMs may be facilitating the tumorigenesis process, and these data support the view that DNA methylation plays a causative role in PB-induced tumorigenesis.

Detection and Quantitation of Benzo[a]pyrene-Derived DNA Adducts in Mouse Liver by Liquid Chromatography−Tandem Mass Spectrometry: Comparison with 32P-Postlabeling

The polycyclic aromatic hydrocarbon, benzo[a]pyrene (B[a]P) is a proven animal carcinogen that is potentially carcinogenic to humans. B[a]P is an ubiquitous environmental pollutant and is also present in tobacco smoke, coal tar, automobile exhaust emissions, and charred food. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using electrospray ionization and selected reaction monitoring (SRM) has been developed for the detection of 10-(deoxyguanosin-N(2)-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]PDE-N(2)dG) adducts formed in DNA following the metabolic activation of B[a]P to benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (B[a]PDE). The method involves enzymatic digestion of the DNA sample to 2'-deoxynucleosides following the addition of a stable isotope internal standard, [(15)N(5)]B[a]PDE-N(2)dG, and then solid phase extraction to remove unmodified 2'-deoxynucleosides prior to analysis by LC-MS/MS SRM. The limit of detection of the method was 10 fmol (approximately 3 B[a]PDE-N(2)dG adducts per 10(8) 2'-deoxynucleosides) using 100 microg of calf thymus DNA as the matrix. Calf thymus DNA reacted with B[a]PDE in vitro and mouse liver DNA samples at different time points following dosing intraperitoneally with 50, 100, and 200 mg/kg B[a]P was analyzed. Three stereoisomers of the B[a]PDE-N(2)dG adduct were detected following the reaction of calf thymus DNA with B[a]PDE in vitro. The levels of B[a]PDE-N(2)dG DNA adducts in the mice livers were found to increase in a dose-dependent manner with adducts reaching maximal levels at 1-3 days and then gradually decreasing over time but still detectable after 28 days. A very good correlation (r = 0.962, p < 0.001) was observed between the results obtained for the mouse liver DNA samples using LC-MS/MS SRM as compared to those obtained using a (32)P-postlabeling method. However, the levels of adducts observed following (32)P-postlabeling using butanol enrichment were approximately 3.7-fold lower. The LC-MS/MS method allowed the more precise quantitation of DNA adduct levels that were structurally characterized, in addition to a reduction in the time taken to perform the analysis when compared with the (32)P-postlabeling method.

Phosphate Alkylation in Different DNA Substrates: The Role of Local DNA Sequence and Electrophile Character in Determining the Nonrandom Nature of Phosphotriester Adduct Formation

DNA phosphate oxygens are sites for alkylation leading to DNA phosphotriester adduct (PTE) formation. Previously, we have reported that the manifestation of PTEs was nonrandom in mouse liver DNA treated in vivo [Guichard et al. (2000) Cancer Res. 60, 1276-1282], and while further studies revealed possible PTE repair, this was determined not to play a role in the observed nonrandom manifestation in vivo [Le Pla et al. (2004) Chem. Res. Toxicol. 17, 1491-1500]. In the present study, to determine whether the nonrandom manifestation of PTEs in vivo was specifically due to their nonrandom formation, we have compared the in vitro formation of diethylsulfate (DES)-induced PTEs in h2E1/OR human B-lymphoblastoid cells, their isolated nuclei, and their isolated DNA, using the 5' nearest neighbor analysis postlabeling procedure developed by Le Pla et al.. Furthermore, to determine the role of electrophile character in PTE manifestation, prepared oligonucleotides ([dT](20)[dG](20):[dC](20)[dA](20)) were treated with three alkylating agents of differing electrophilic character (DES, methylnitrosourea, and ethylnitrosourea), and PTE manifestation was assessed by postlabeling. The formation of PTEs was determined to be nonrandom in the whole cells, nuclei, and DNA, with PTEs being formed to a greater extent 3' to pyrimidine moieties than 3' to purine moieties. The studies with the oligonucleotides confirm these observations and demonstrate that the nonrandom formation of PTEs is primarily determined by DNA sequence, and not by DNA packaging/chromatin factors, and that the extent of the nonrandom formation of PTEs is also governed by electrophile reactivity, with the more reactive electrophiles yielding a more random formation of PTEs. From our observations, we propose a model for the nonrandom formation of PTEs, which is governed by (i) the phosphate oxygens having to compete with adjacent nucleophilic sites for the alkylating electrophile and (ii) the electrophile's inherent reactivity.

Susceptibility of DNA to oxidative stressors in young and aging mice

The changes that accompany aging may be a result of oxidative damage to DNA that accumulates as a result of aging and age-related illnesses. Furthermore, a higher susceptibility is thought to be more common among elderly than young individuals. In the present study, we examined the severity of DNA damage caused by carbon tetrachloride (CCl 4) and H 2O 2 in cells from young (2 month old) and older (14 month old) mice using both in vivo and in vitro exposures. CCl 4 is known to generate radical oxidative species (ROS) throughout its biotransformation in the liver. Therefore, 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxdGuo) was quantified in liver DNA obtained from young and older mice treated with CCl 4. In addition, DNA single-strand breaks were measured by the Comet assay in primary lung fibroblasts cultured from young and older mice and treated in vitro with H 2O 2. Intracellular ROS production and mitochondrial enzyme activity were determined in parallel. 8-oxodGuo levels were significantly higher in older mouse liver DNA than younger, and increased significantly with CCl 4 treatment. When the basal DNA damage was subtracted, the net damage was almost equal for both. In addition, untreated cells cultured from older mice had significantly greater levels of strand breaks than cells derived from young mice. H 2O 2 increased the level of damage in both cell cultures. Our findings indicate that the DNA damage observed in older animals probably results from the accumulation of endogenous damage with age, perhaps due to insufficient repair, which enhances the injury caused by exposure to the toxic agents.

516. Analysis of AAV Serotype 8 Vector Integration in Normal and DNA-PKcs-Deficient Scid Mice by a Novel Strategy

rAAV vectors integrate into host chromosomal DNA infrequently. However, we still may need to consider the possibility of adverse effects caused by rAAV vector integration because 1) rAAV2 vector integrations in animals accompany host chromosomal deletions or translocations, and occur preferentially in or near gene regulatory sequences; and 2) 3 to 4 % integrations occur in or near cancer-related genes. In order to further address this issue and understand the biology of rAAV vectors in vivo, we investigated rAAV8 vector integration in normal and DNA-PKcs-deficient scid mice. Scid mice were chosen because DNA-PKcs has been shown to be involved in rAAV vector genome processing. Here, we developed a novel strategy for isolation of a large number of rAAV proviruses directly from vector-transduced animal tissues with high efficiency and reliability, and without any selective pressure. The salient features of the strategy are: 1) the use of a shuttle rAAV8 vector carrying an ISceI site and AmpR/Ori (AO) cassette, 2) physical separation of integrated and extrachromosomal vector genomes following ISceI digestion of tissue DNA; 3) removal of unwanted genomic DNA by digestion with a DNA conformation-dependent nuclease; 4) mixing the tissue DNA with yeast genomic DNA at 1:1 ratio at the beginning of the procedure, with yeast DNA serving as a tag sequence to monitor for undesired intermolecular recombination that may arise during the procedure. We injected normal and scid mice with 7.2|[times]|10e12 vg of this rAAV8 shuttle vector via the tail vein, and made rAAV8 integration provirus libraries from transduced mouse liver DNA. Transformation of bacteria with |[sim]|0.3 |[mu]|g liver DNA generated over a thousand colonies in each library. Initial sequencing results of 121 and 69 clones from normal and scid mouse libraries demonstrated that 23 (19%) and 52 (75%) of the sequenced clones carried rAAV8 vector-cellular DNA junctions, respectively. Importantly, we have not seen any intermolecular recombination between rAAV8 vector and yeast genomes, establishing the high reliability of this strategy. A preliminary analysis suggested that integration sites in scid mice are more dispersed than in normal mice. In addition, we found that a majority of rAAV8 genomes in scid mouse liver form extrachromosomal double-stranded |[ldquo]|dog bone|[rdquo]| structures, i.e., with both terminal hairpin loops closed. Such dog bone forms are present at undetectable levels in rAAV2-injected mouse livers, but may represent intermediates toward various vector forms. Thus the isolation of a large number of rAAV proviral genomes from quiescent somatic cells in animals is feasible by our novel strategy, and high-throughput rAAV integration site analyses in normal and scid mouse tissues and detailed characterization of the dog bone forms will provide new insights into the mechanisms of rAAV vector transduction and integration in animal tissues.

Levels of 4‐aminobiphenyl‐induced somatic H‐ras mutation in mouse liver DNA correlate with potential for liver tumor development

The utility of liver H-ras codon 61 CAA to AAA mutant fraction as a biomarker of liver tumor development was investigated using neonatal male mice treated with 4-aminobiphenyl (4-ABP). Treatment with 0.1, 0.3, or 1.0 mumol 4-ABP produced dose-dependent increases in liver DNA adducts in B6C3F(1) and C57BL/6N mice. Eight months after treatment with 0.3 mumol 4-ABP or the DMSO vehicle, H-ras codon 61 CAA to AAA mutant fraction was measured in liver DNA samples (n = 12) by allele-specific competitive blocker-polymerase chain reaction (ACB-PCR). A significant increase in average mutant fraction was found in DNA of 4-ABP-treated mice, with an increase from 1.3 x 10(-5) (control) to 44.9 x 10(-5) (treated) in B6C3F(1) mice and from 1.4 x 10(-5) to 7.0 x 10(-5) in C57BL/6N mice. Compared with C57BL/6N mutant fractions, B6C3F(1) mutant fractions were more variable and included some particularly high mutant fractions, consistent with the more rapid development of liver foci expected in B6C3F(1) mouse liver. Twelve months after treatment, liver tumors developed in 79.2% of 4-ABP-treated and 22.2% of control B6C3F(1) mice; thus measurement of H-ras mutant fraction correlated with subsequent tumor development. This study demonstrates that ACB-PCR can directly measure background levels of somatic oncogene mutation and detect a carcinogen-induced increase in such mutation.

Formation of MTBE-DNA adducts in mice measured with accelerator mass spectrometry

Methyl tert-butyl ether (MTBE) is a gasoline oxygenate and antiknock additive substituting for lead alkyls currently in use worldwide. Previous studies have shown that MTBE at very high doses induces tumors in rodents. The aim of the present study was to examine directly the binding ability of MTBE onto DNA, demonstrating its potential genotoxicity. MTBE-DNA adducts and their decay kinetics in mice have been measured by using doubly 14C-labeled MTBE with an advanced, ultrasensitive technique: accelerator mass spectrometry (AMS). It was found that MTBE definitely formed adducts with DNA in mouse lung, liver, and kidney in a log/log linear dose-response relationship. The distribution sequence of DNA adducts in these tissues is: lung > liver > kidney. The level of MTBE-DNA adducts peaked at 12 h postadministration in the lung and peaked at 6 h postadministration in the liver. Then the adducts declined rapidly until 5 days postadministration and thereafter declined much more slowly. To our knowledge, this is the first report on DNA adduction with MTBE in vivo. The mechanism of the formation of MTBE-DNA adducts also is discussed.

Inhibitory Effects of Heterocyclic Amine-Induced DNA Adduct Formation in Mouse Liver and Lungs by Beer

An evaluation of the in vivo antigenotoxic potential of beer components on heterocyclic amines including 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 3-amino-1-methyl-5H-pyrido[4.3-b]indole (Trp-P-2) was determined with particular focus on the target organs of tumorigenesis, and the protective mechanisms involved were investigated. Beer-solution, consisting of a freeze-dried and dissolved sample, given as drinking water, reduced the formation of MeIQx-DNA adducts in mouse liver and lungs. Beer-solution added in the diet as a mimic of food additives also significantly reduced the amount of DNA adducts present in the liver, lung, and kidney DNA of mice fed with MeIQx compared to control mice fed with MeIQx in the absence of beer-solution. The amount of adducts present in the liver of mice with single or continuous administration of Trp-P-2 was significantly reduced when beer-solution was given as part of the diet compared to control mice given Trp-P-2 without beer-solution. Protective effects were observed both with lager- and stout-type samples. In an effort to investigate the mechanisms responsible for the observed protective effects, the effects of beer-solution on metabolizing enzymes for heterocyclic amines were examined. Beer-solutions inhibited the metabolic activation of Trp-P-2 to Trp-P-2(NHOH), as demonstrated by HPLC analysis. Considering the overall suppression of the genotoxicity of MeIQx and Trp-P-2 by beer, we have shown that beer components can inhibit the metabolic activation of heterocyclic amines and subsequently suppress the observed genotoxicity. The results of this study show that beer components are protective against the genotoxic effects of heterocyclic amines on target organs associated with tumorigenesis in vivo.

Further Development of 32P-Postlabeling for the Detection of Alkylphosphotriesters: Evidence for the Long-Term Nonrandom Persistence of Ethyl-Phosphotriester Adducts in Vivo

DNA phosphate oxygens are sites for alkylation leading to phosphotriester adducts (PTEs). PTEs are reported to be both abundant and persistent and so may serve as long-term markers of genotoxicity. Previously, we reported a 32P-postlabeling assay for the specific detection of PTEs plus identification of nucleosides located 5' to PTEs. Using this, we demonstrated the nonrandom nature of ethyl-PTEs (Et-PTEs) in vivo, these results being suggestive of either the nonrandom formation of Et-PTEs in vivo or sequence specific Et-PTE repair. Presently, we report the further development and validation of the 32P-postlabeling assay, to permit the more straightforward determination of nucleosides 5' to PTEs and, using this, have investigated the long-term persistence of PTEs in vivo. Analysis of liver DNA of mice treated in vivo with N-nitrosodiethylamine reveals an initial decline in the level of Et-PTEs (t1/2<24 h) as well as their nonrandom persistence for the duration of the time course, with approximately 37 and approximately 15% of the initial Et-PTEs remaining 4 and 56 days after treatment, respectively. From this, we conclude that Et-PTEs are suitable as long-term markers of genotoxic exposure and that putative PTE repair is not responsible for their nonrandom manifestation. However, the possibility of active repair contributing to the initial decline of Et-PTEs is considered.

DNA adducts in rats and mice following exposure to [4- [formula omitted]]-1,2-epoxy-3-butene and to [2,3- [formula omitted]]-1,3-butadiene

1,3-Butadiene (BD) is a major industrial chemical and a rodent carcinogen, with mice being much more susceptible than rats. Oxidative metabolism of BD, leading to the DNA-reactive epoxides 1,2-epoxy-3-butene (BMO), 1,2-epoxy-3,4-butanediol (EBD) and 1,2:3,4-diepoxybutane (DEB), is greater in mice than rats. In the present study the DNA adduct profiles in liver and lungs of rats and mice were determined following exposure to BMO and to BD since these profiles may provide qualitative and quantitative information on the DNA-reactive metabolites in target tissues. Adducts detected in vivo were identified by comparison with the products formed from the reaction of the individual epoxides with 2′-deoxyguanosine (dG). In rats and mice exposed to [4- 14 C ]-BMO (1–50 mg/kg, i.p.), DNA adduct profiles were similar in liver and lung with N 7-(2-hydroxy-3-butenyl)guanine ( G1) and N 7-(1-(hydroxymethyl)-2-propenyl)guanine ( G2) as major adducts and N 7-2,3,4-trihydroxybutylguanine ( G4) as minor adduct. In rats and mice exposed to 200 ppm [2,3- 14 C ]-BD by nose-only inhalation for 6 h, G4 was the major adduct in liver, lung and testes while G1 and G2 were only minor adducts. Another N 7-trihydroxybutylguanine adduct ( G3), which could not unambiguously be identified but is either another isomer of N 7-2,3,4-trihydroxybutylguanine or, more likely, N 7-(1-hydroxymethyl-2,3-dihydroxypropyl)guanine, was present at low concentrations in liver and lung DNA of mice, but absent in rats. The evidence indicates that the major DNA adduct formed in liver, lung and testes following in vivo exposure to BD is G4, which is formed from EBD, and not from DEB.

Dose responses for DNA adduct formation in tissues of rats and mice exposed by inhalation to low concentrations of 1,3-[2,3- [formula omitted]]-butadiene

Male Sprague–Dawley rats and B6C3F1 mice were exposed to either a single 6 h or a multiple (5) daily (6 h) nose-only dose of 1,3-[2,3- 14 C ]-butadiene at exposure concentrations of nominally 1, 5 or 20 ppm. The aim was to compare the results with those from a similar previous study at 200 ppm. DNA isolated from liver, lung and testis of exposed rats and mice was analysed for the presence of butadiene related adducts, especially the N7-guanine adducts. Total radioactivity present in the DNA from liver, lung and testis was quantified and indicated more covalent binding of radioactivity for mouse tissue DNA than rat tissue DNA. Following release of the depurinating DNA adducts by neutral thermal hydrolysis, the liberated depurinated DNA adducts were measured by reverse phase HPLC coupled with liquid scintillation counting. The guanine adduct G4, assigned as N7-(2,3,4-trihydroxybutyl)- guanine, was the major adduct measured in liver, lung and testis DNA in both rats and mice. Higher levels of G4 were detected in all mouse tissues compared with rat tissue. The dose–response relationship for the formation of adduct G4 was approximately linear for all tissues studied for both rats and mice exposed in the 1–20 ppm range. The formation of G4 in liver tissue was about three times more effective for mouse than rat in this exposure range. Average levels of adduct G4 measured in liver DNA of rats and mice exposed to 5×6 h 1, 5 and 20 ppm 1,3-[2,3- 14 C ]-butadiene were, respectively, for rats: 0.79±0.30, 2.90±1.19, 16.35±4.8 adducts/10 8 nucleotides and for mice: 2.23±0.71, 12.24±2.15, 48.63±12.61 adducts/10 8 nucleotides. For lung DNA the corresponding values were for rats: 1.02±0.44, 3.12±1.06, 17.02±4.07 adducts/10 8 nucleotides, and for mice: 3.28±0.32, 14.04±1.55, 42.47±13.12 adducts/10 8 nucleotides. Limited comparative data showed that the levels of adduct G4 formed in liver and lung DNA of mice exposed to a single exposure to butadiene in the present 20 ppm study and earlier 200 ppm study were approximately directly proportional across dose, but this was not observed in the case of rats. From the available evidence it is most likely that adduct G4 was formed from a specific isomer of the diol-epoxide metabolite, 3,4-epoxy-1,2-butanediol rather than the diepoxide, 1,2,3,4-diepoxybutane. Another adduct G3, possibly a diastereomer of N7-(2,3,4-trihydroxybutyl)-guanine or most likely the regioisomer N7-(1-hydroxymethyl-2,3-dihydroxypropyl)-guanine, was also detected in DNA of mouse tissues but was essentially absent in DNA from rat tissue. Qualitatively similar profiles of adducts were observed following exposures to butadiene in the present 20 ppm study and the previous 200 ppm study. Overall the DNA adduct levels measured in tissues of both rats and mice were very low. The differences in the profiles and quantity of adducts seen between mice and rats were considered insufficient to explain the large difference in carcinogenic potency of butadiene to mice compared with rats.

Inhibition of hepatitis B virus in mice by RNA interference.

Hepatitis B virus (HBV) infection substantially increases the risk of chronic liver disease and hepatocellular carcinoma in humans. RNA interference (RNAi) of virus-specific genes has emerged as a potential antiviral mechanism. Here we show that RNAi can be applied to inhibit production of HBV replicative intermediates in cell culture and in immunocompetent and immunodeficient mice transfected with an HBV plasmid. Cotransfection with plasmids expressing short hairpin RNAs (shRNAs) homologous to HBV mRNAs induced an RNAi response. Northern and Southern analyses of mouse liver RNA and DNA showed substantially reduced levels of HBV RNAs and replicated HBV genomes upon RNAi treatment. Secreted HBV surface antigen (HBsAg) was reduced by 94.2% in cell culture and 84.5% in mouse serum, whereas immunohistochemical detection of HBV core antigen (HBcAg) revealed >99% reduction in stained hepatocytes upon RNAi treatment. Thus, RNAi effectively inhibited replication initiation in cultured cells and mammalian liver, showing that such an approach could be useful in the treatment of viral diseases.

Novel nuclear and mitochondrial glycosylases revealed by disruption of the mouse Nth1 gene encoding an endonuclease III homolog for repair of thymine glycols

Endonuclease III, encoded by nth in Escherichia coli, removes thymine glycols (Tg), a toxic oxidative DNA lesion. To determine the biological significance of this repair in mammals, we established a mouse model with mutated mNth1, a homolog of nth, by gene targeting. The homozygous mNth1 mutant mice showed no detectable phenotypical abnormality. Embryonic cells with or without wild-type mNth1 showed no difference in sensitivity to menadione or hydrogen peroxide. Tg produced in the mutant mouse liver DNA by X-ray irradiation disappeared with time, though more slowly than in the wild-type mouse. In extracts from mutant mouse liver, we found, instead of mNTH1 activity, at least two novel DNA glycosylase activities against Tg. One activity is significantly higher in the mutant than in wild-type mouse in mitochondria, while the other is another nuclear glycosylase for Tg. These results underscore the importance of base excision repair of Tg both in the nuclei and mitochondria in mammals.

Involvement of mammalian OGG1(MMH) in excision of the 8-hydroxyguanine residue in DNA 1,2

8-Hydroxyguanine (7,8-dihydro-8-oxoguanine, abbreviated as 8-OH-G or 8-oxoG) is the site of a frequent mutagenic DNA lesion produced by oxidative damage. MutM of E. coli and OGG1 of Saccharomyces cervisiae are known to possess 8-OH-G glycosylase and apurinic (AP) site lyase activity. cDNA clones of four isoforms (types 1a, 1b, 1c, and 2) of human OGG1 homologs (hMMH) were isolated. In order to examine whether expression of hMMH (hOGG1) protein actually occurs in human cells, we prepared type 1a specific antibody, and by using this antibody, we showed that type 1a protein isolated from HeLaS3 has 8-OH-G glycosylase/lyase activity. Furthermore, we showed that type 1a protein is a major enzyme for repair of the 8-OH-G lesion in human cells. In our second study, we generated a mouse line carrying an inactivated mutant Mmh allele by targeted gene disruption. Liver extracts of Mmh homozygous mutant mice were found to have loss of the nicking activity for the 8-OH-G site. In addition, the amount of endogenous 8-OH-G in liver DNA of the homozygous mice increased linearly with age, reaching 7-fold increase in 14 week old mice, over that of wild-type or heterozygous mice. Furthermore, when homozygous mice were fed the oxygen radical-forming agent KBrO 3, to provide oxidative stress, the level of 8-OH-G in kidney DNA was tremendously increased: more than 200-fold as that of control mice without oxidative stress after 12 weeks of age. These results indicate that Ogg1/ Mmh plays an essential role in the repair of the 8-OH-G residue in DNA produced by oxidative stress.

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

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,N(2) 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 10(8) 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 10(8) 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.

Mechanisms of hormonal carcinogenesis in the p53+/- hemizygous knockout mouse: studies with diethylstilbestrol.

The 2-year rodent bioassay has long had a central role in determining whether a compound is carcinogenic. It has recently been suggested that the use of 6-month studies in transgenic mice could reduce costs and animal numbers, without impairing the validity of cancer risk assessment. The p53+/- hemizygous knockout mouse model is phenotypically stable and develops tumors during the 6-month study period only in response to chemical and physical stimuli, showing a high concordance with genotoxic rodent carcinogens. We treated p53+/- mice and wild-type parent strain (C57BL/6J) animals with diethylstilbestrol (DES). 500 micromol/kg i.p. for 4 days. Following sacrifice, DNA was extracted from various tissues and adducts measured by a modified monophosphate version of the 32P-postlabelling assay. Major DES adducts were detected in the liver DNA of DES-treated wild-type mice at a level of 118.7+/-17.0 (mean +/- SD relative adduct level [RAL]/10(10) nucleotides) compared with 207.7+/-36.4 in p53+/- mice. No such adducts were detected in vehicle-treated animals. Total adduct levels, including endogenous I-compound adducts, in wild-type mice were 192.4+/-17.5 and 311.5+/-58.6 in p53+/- animals. These data support the hypothesis that deficient p53-dependent global genomic repair of DES adducts in p53+/- mice may result in the persistence of exogenous and endogenous DNA adducts that could contribute to earlier carcinogenicity in this model. We also prepared hepatic microsomes from male and female p53+/- and wild-type mice exposed to DES or vehicle. Western blot analysis demonstrated modestly higher basal levels of various cytochrome P450 (CYP) enzymes in the untreated p53+/- mice compared to the wild-type mice. Furthermore, P450 levels were higher in female DES-treated p53+/- mice compared to treated wild-type mice. For the p53+/- knockout mice to be used with contidence in drug safety studies, a further understanding of the metabolic capacity of these animals is needed.

Mutation frequency in the lacI gene of liver DNA from lambda/lacI transgenic mice following the interaction of PCBs with iron causing hepatic cancer and porphyria.

The synergistic interaction of iron overload, AHR: genotype and exposure to a mixture of polychlorinated biphenyls (PCBs) (Aroclor 1254) in mice leads to hepatic porphyria, oxidative DNA damage and cancer. In humans, hepatocellular cancer is associated with iron overload and hepatic porphyria. Neither the mechanism of hepatic carcinogenesis induced by PCBs in rodents nor hepatocellular cancer induced by iron and porphyria in humans are understood. To test the hypothesis that chronic interaction of iron and PCBs may induce mutagenesis in liver DNA, lambda /lacI transgenic C57BL/6 mice were given iron dextran (600 mg iron/kg) and then administered Aroclor 1254 in the diet (0.01%) for 7 weeks. Hepatic iron, CYP1A activity and CYP1A1/1A2 protein were elevated >20-fold as a result of iron or Aroclor treatments, respectively, but porphyria with associated histological changes only developed in the combined iron/Aroclor treatment group. lambda/lacI shuttle vectors were isolated from liver genomic DNA and the mutational frequency (MF) in the lacI gene determined. Both iron and Aroclor treatments alone caused significant small increases in MF (1.5- and 1.4-fold, respectively), however, the MF following the combined iron and Aroclor treatment (1. 6-fold) was not greater than the additive effects. In contrast, the MF was significantly elevated (4.7-fold) in liver DNA of mice 2 weeks following five daily doses of N-nitrosodimethylamine (4 mg/kg). These studies demonstrate that neither PCBs nor iron overload caused marked point mutations even in a combination regime that leads to oxidative damage and cancer. There was also no strong evidence either that porphyrins or chronic CYP1A1 expression induced by the PCBs after this period caused marked point mutagens or simple deletions. Hence, to understand the PCBs-iron synergism more complex scenarios than point mutations or simple deletions must be invoked.

Expression of Hepatitis B Virus X Protein Does Not Alter the Accumulation of Spontaneous Mutations in Transgenic Mice

Chronic infection with hepatitis B virus (HBV) is one of the major etiological factors in the development of human hepatocellular carcinoma. Transgenic mice that express the HBV X protein (HBx) have previously been shown to be more sensitive to the effects of hepatocarcinogens, although the mechanism for this cofactor role remains unknown. The ability of HBx to inhibit DNA repair in transiently transfected cell lines suggests one possible pathway. In the present study, primary hepatocytes isolated from transgenic mice that possess the HBV X gene under the control of the human alpha-1-antitrypsin regulatory region (ATX mice) were found to be deficient in their ability to conduct unscheduled DNA synthesis in response to UV-induced DNA damage. In order to measure the impact of HBx expression on DNA repair in vivo, double-transgenic mice that express HBx and possess a bacteriophage lambda transgene were sacrificed at 30, 90, and 240 days of age. Mutation frequency was determined for high-molecular-weight liver DNA of ATX and control mice by functional analysis of the lambda transgene. Expression of HBx did not significantly increase the accumulation of spontaneous mutations. These results are consistent with previous studies of HBx transgenic mice in which no effect of HBx on liver histology was apparent. This new animal model provides a powerful system in which to investigate the in vivo cooperation between HBx expression and environmental carcinogens.