Mouse Liver DNA Research Articles

Persistence of N7-(2,3,4-trihydroxybutyl)guanine adducts in the livers of mice and rats exposed to 1,3-butadiene.

Liquid chromatography (LC) in combination with tandem mass spectrometry (MS/MS) and stable isotope methodology was employed for the analysis of the N7-guanine (Gua) adducts derived from 1,2:3, 4-diepoxybutane (BDO2) a reactive metabolite of 1,3-butadiene (BD). Two diastereomeric forms of N7-(2,3,4-trihydroxybutyl)guanine (THBG) were identified in the livers of both mice and rats. One of the diastereomers [(+/-)-THBG] was formed by reaction of DNA with (+/-)-BDO2, and the other diastereomer (meso-THBG) was formed by reaction of DNA with meso-BDO2. There was significantly more (+/-)-THBG and meso-THBG in the liver DNA of the mice when compared with those of the rats during the 10 days of exposure to BD and the 6 days of postexposure that were monitored. There was a 2-fold excess of (+/-)-THBG over meso-THBG in the rat liver at all the time points. In the mouse liver after 10 days of exposure to BD, the (+/-)-THBG (3.9 adducts/10(6) normal bases) was also present in an almost 2-fold excess over meso-THBG (2.2 adducts/10(6) normal bases). However, 6-days after exposure to BD, (+/-)-THBG (1.2 adducts/10(6) normal bases) and meso-THBG (1.0 adduct/10(6) normal bases) were present in almost equal amounts in the mouse liver. Furthermore, there was an almost 5-fold excess of the two THBG diastereomers in the mouse liver DNA 6 days after exposure to BD when compared with rat liver DNA. The half-lives of (+/-)-THBG and meso-THBG appeared to be slightly longer in mouse liver (4.1 and 5.5 days, respectively) than in rat liver (3.6 and 4.0 days, respectively). The apparent persistence of these adducts in the mouse may contribute to the increased susceptibility of this species to BD-induced carcinogenesis. It is possible that (+/-)-THBG and meso-THBG could have also been derived from the reaction of DNA with the hydrolysis product of BDO2, 1,2-dihydroxy-3,4-epoxybutane (DHEB). Surprisingly, a vast majority of the studies in which the mutagenic and carcinogenic potential of BDO2 have been examined have only employed the commercially available (+/-)-BDO2. In light of the present findings, additional studies will be required to determine the potency of meso-BDO2 and the DHEB that is the precursor to meso-THBG as mutagens and carcinogens.

Adeno-Associated Viral Vector-Mediated Gene Transfer of Human Blood Coagulation Factor IX Into Mouse Liver

AbstractRecombinant adeno-associated virus vectors (AAV) were prepared in high titer (1012 to 1013 particles/mL) for the expression of human factor IX after in vivo transduction of murine hepatocytes. Injection of AAV-CMV-F.IX (expression from the human cytomegalovirus IE enhancer/promoter) into the portal vein of adult mice resulted in no detectable human factor IX in plasma, but in mice injected intravenously as newborns with the same vector, expression was initially 55 to 110 ng/mL. The expression in the liver was mostly transient, and plasma levels decreased to undetectable levels within 5 weeks. However, long-term expression of human F.IX was detected by immunofluorescence staining in 0.25% of hepatocytes 8 to 10 months postinjection. The loss of expression was likely caused by suppression of the CMV promoter, because polymerase chain reaction data showed no substantial loss of vector DNA in mouse liver. A second vector in which F.IX expression was controlled by the human EF1α promoter was constructed and injected into the portal vein of adult C57BL/6 mice at a dose of 6.3 × 1010 particles. This resulted in therapeutic plasma levels (200 to 320 ng/mL) for a period of at least 6 months, whereas no human F.IX was detected in plasma of mice injected with AAV-CMV-F.IX. Doses of AAV-EF1α-F.IX of 2.7 × 1011particles resulted in plasma levels of 700 to 3,200 ng/mL. Liver-derived expression of human F.IX from the AAV-EF1α-F.IX vector was confirmed by immunofluorescence staining. We conclude that recombinant AAV can efficiently transduce hepatocytes and direct stable expression of an F.IX transgene in mouse liver, but sustained expression is critically dependent on the choice of promoter.

Adeno-Associated Viral Vector-Mediated Gene Transfer of Human Blood Coagulation Factor IX Into Mouse Liver

Recombinant adeno-associated virus vectors (AAV) were prepared in high titer (1012 to 1013 particles/mL) for the expression of human factor IX after in vivo transduction of murine hepatocytes. Injection of AAV-CMV-F.IX (expression from the human cytomegalovirus IE enhancer/promoter) into the portal vein of adult mice resulted in no detectable human factor IX in plasma, but in mice injected intravenously as newborns with the same vector, expression was initially 55 to 110 ng/mL. The expression in the liver was mostly transient, and plasma levels decreased to undetectable levels within 5 weeks. However, long-term expression of human F.IX was detected by immunofluorescence staining in 0.25% of hepatocytes 8 to 10 months postinjection. The loss of expression was likely caused by suppression of the CMV promoter, because polymerase chain reaction data showed no substantial loss of vector DNA in mouse liver. A second vector in which F.IX expression was controlled by the human EF1α promoter was constructed and injected into the portal vein of adult C57BL/6 mice at a dose of 6.3 × 1010 particles. This resulted in therapeutic plasma levels (200 to 320 ng/mL) for a period of at least 6 months, whereas no human F.IX was detected in plasma of mice injected with AAV-CMV-F.IX. Doses of AAV-EF1α-F.IX of 2.7 × 1011particles resulted in plasma levels of 700 to 3,200 ng/mL. Liver-derived expression of human F.IX from the AAV-EF1α-F.IX vector was confirmed by immunofluorescence staining. We conclude that recombinant AAV can efficiently transduce hepatocytes and direct stable expression of an F.IX transgene in mouse liver, but sustained expression is critically dependent on the choice of promoter.

Oxidative DNA Damage in Mouse Heart, Liver, and Lung Tissue Due to Acute Side-Stream Tobacco Smoke Exposure

Environmental tobacco smoke (ETS) is a hotly debated social, political, and scientific issue, pitting smokers’ rights against the health and safety of nonsmokers. Striking an acceptable balance between the two depends largely on the potential health hazard assessment of ETS. Studies from this laboratory have shown that exposure to side-stream cigarette smoke (SSS), the major component of ETS, significantly increases oxidative stress in mouse heart, liver, and lung tissues. This study measures the level of oxidative damage to mouse liver, lung, and heart DNA as a result of this oxidative stress. Adult female Balb/c mice were exposed to a regimen consisting of sequences of a 30-min exposure followed by a 90-min nonexposure. This regimen was performed once for the single exposure and repeated three times for the triple exposure. The heart, lung, and liver were excised from the mice, and DNA was extracted and analyzed for the presence of the oxidative product 8-hydroxy-2′-deoxyguanosine (8-OHdG). In all three tissues, the exposure increased the presence of 8-OHdG above the control levels. In some instances, the increased levels returned to normal by the end of the nonexposure period, while other tissues showed a further increase following nonexposure. These studies demonstrate that limited exposure to SSS produces measurable DNA oxidative damage.

Dose-dependent binding of trichloroethylene to hepatic DNA and protein at low doses in mice

Trichloroethylene (TCE) is a widely used industrial chemical and a low level contaminant of surface and ground water in industrialized areas. It is weakly mutagenic in several test systems and carcinogenic in rodents. However, the mechanism for its carcinogenicity is not known. We investigated the binding of [1,2- 14C]TCE ([ 14C]TCE) to liver DNA and proteins in male B6C3F1 mice at doses more relevant to humans than used previously. The time course for the binding was studied in animals dosed with 4.1 μg [ 14C]TCE/kg body weight (b.w.) and sacrificed between 0.5 and 120 h after i.p. injection. A dose response study was carried out in mice given [ 14C]TCE at doses between 2 μg/kg and 200 mg/kg b.w. and sacrificed 2 h post-treatment. [ 14C]TCE associated with the DNA and protein extracts was measured using accelerator mass spectrometry. The highest level of protein binding (2.4 ng/g protein) was observed 1 h after the treatment followed by a rapid decline, indicating pronounced instability of the adducts and/or rapid turnover of liver proteins. DNA binding was biphasic with the first peak (75 pg/g DNA) at 4 h. However, the highest binding (120 pg/g DNA) was found between 24 and 72 h after the treatment. Dose response curves were linear for both protein and DNA binding. The binding of TCE metabolites to DNA was ca. 100-fold lower than to proteins when calculated per unit weight of macromolecules and when measured 2 h post-exposure. This study shows that TCE metabolites bind to DNA and proteins in a dose-dependent manner in liver, one of the target organs for its tumorigenicity. Thus, protein and DNA adduct formation should be considered as a factor in the tumorigenesis of TCE.

Electrochemical Detection and Quantification of the Acetylated and Deacetylated C8-Deoxyguanosine DNA Adducts Induced by 2-Acetylaminofluorene

The genotoxic agent 2-acetylaminofluorene induces, upon metabolic activation, two main types of DNA adducts in animal tissue, i.e., (deoxyguanine-8-yl)-aminofluorene (dG-C8-AF) andN-(deoxyguanine-8-yl)-acetylaminofluorene (dG-C8-AAF). Quantification of the frequency of these adducts usually relies on the use of radioactively labeled 2-acetylaminofluorene. Here, we report the development of a sensitive, non-radioactive method for the quantification of dG-C8-AF and dG-C8-AAF. Essentially, the modified DNA bases are separated by high-performance liquid chromatography (HPLC) and quantified by electrochemical detection. We established that both modified bases guanine-C8-aminofluorene and guanine-C8-acetylaminofluorene are electrochemically active. Subsequently, a procedure was developed to quantify dG-C8-AF and dG-C8-AAF in genomic DNA. Following DNA hydrolysis the adducted bases were extracted by ethyl acetate, separated by HPLC, and detected electrochemically. This procedure has been applied in the analysis of dG-C8-AAF inN-acetoxy-2-acetylaminofluorene-modified calf thymus DNA and in the detection of dG-C8-AAF and dG-C8-AF in liver DNA of mice injected intraperitoneally with 150–450 mgN-hydroxy-2-acetylaminofluorene/kg. The quantification of relatively low dG-C8-AF and dG-C8-AAF adduct levels (i.e., 0.1–1 adduct/106nucleotides) in mouse liver DNA demonstrates the sensitivity of this electrochemical detection procedure. The detection limit of the method is 1 adduct per 106nucleotides for both adducts using 20 μg of DNA and 4 adducts per 108nucleotides using 500 μg DNA.

Dietary restriction modulated carcinogen-DNA adduct formation and the carcinogen-induced DNA strand breaks

Dietary restriction (DR) alters the activities of hepatic drug metabolizing enzymes and modulates the formation of carcinogen-DNA adducts in carcinogen treated animals. Our previous results showed that a 40% restriction of diet (60% of ad libitum (AL) food consumption) reduced the hepatic metabolic activation of aflatoxin B 1 (AFB 1) but increased the activation of benzo[a]-pyrene (BaP) in both rats and mice. In this study, the focus was directed toward the levels of carcinogen-DNA adducts formation and the carcinogen-induced DNA strand breaks in mouse kidney and liver DNA. DR significantly inhibited both AFB 1-DNA adduct formation and AFB 1-induced DNA strand breaks in kidney DNA of mice that received a single dose of [ 3H]AFB 1 (5 mg/kg). The levels of AFB 1-DNA adduct formation in mouse kidney DNA correlated well with increased AFB 1-induced DNA strand breaks. The correlation between the levels of AFB 1-DNA-adducts formed and DNA strand breaks in kidney DNA of DR-mice was less linear than between its AL-counterpart suggesting that other factors, such as different rates of DNA repair, may be involved. In addition, DR enhanced hepatic BaP- and 6-nitrochrysene (6-NC)-DNA adduct formation in the mice treated with BaP and 6-NC, respectively. The formation of the specific BaP-adduct, 10-(N 2-deoxyguanosinyl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-BaP (N 2-dG-BaP), in mouse liver was proportional to the dose, and was compatible to the BaP-induced DNA strand breaks affected by DR. The enhancement of the total 6-NC-DNA adduct formation in DR-mouse was also in correlation with the increased 6-NC-induced DNA strand breaks. The activity of mouse liver microsomal nitro-reductase increased by 2-fold in response to DR indicating that the nitroreduction may contribute to the increase of the metabolic activation of 6-NC. Our present results indicate that the effect of DR on the carcinogen activation is dependent upon the DR-modulated carcinogen metabolizing enzyme activities.

Introduction, distribution, and removal of 7-methylguanine in different liver chromatin fractions of young and old mice

The distribution and elimination of 7-methylguanine (m 7Gua) from different liver DNA chromatin fractions has been studied after treating young and old mice with N-methyl- N-nitrosourea (MNU). Guanine methylation kinetics was first studied in total liver DNA following intraperitoneal injections of 25 mg/kg and 50 mg/kg MNU doses. MNU-induced DNA alkylation, as measured by m 7Gua levels, was dose-dependent in liver tissues of young (9-11 month) and old mice (28–29 month). However, liver DNA in old mice incurred approximately 50% more damage than young mice for weight-normalized doses of MNU. The kinetics of adduct removal from total DNA was biphasic. A more rapid phase of m 7Gua removal was observed during the first 24 h to 48 h following MNU administration; thereafter, the remaining m 7Gua adducts were hydrolyzed much more slowly. Similar amounts of m 7Gua were removed by 24 h at both the 25 mg/kg and 50 mg/kg MNU doses for a single age-group, but old liver tissue removed significantly more m 7Gua than young liver tissue during this initial phase. Following a single injection of carcinogen (50 mg/kg), liver nuclei were isolated and chromatin was sheared by limited Micrococcal nuclease digestion. Chromatin was separated into nuclease-soluble, low-salt, high-salt and nuclear matrix fractions. All four fractions of young liver chromatin were methylated to the same degree. In contrast, there were differences in m 7Gua levels between old liver chromatin fractions. DNA in the nuclease-sensitive fraction was most heavily alkylated, whereas nuclear matrix sequences were modified the least. Removal of m 7Gua occurred at relatively uniform rates in all chromatin fractions regardless of age, indicating that m 7Gua was not preferentially repaired in different nuclease-susceptible regions of chromatin. These results suggest that the N-methylpurine-DNA glycosylase responsible for eliminating m 7Gua from the mammalian genome is not deficient in senescent liver tissue. However, there may be age-related changes in chromatin composition or structure that make some genomic sequences more accessible to alkylating agents in liver tissue of older animals.

Food Restriction Reduces Aflatoxin B1 (AFB1)-DNA Adduct Formation, AFB1-Glutathione Conjugation, and DNA Damage in AFB1-Treated Male F344 Rats and B6C3F1 Mice ,

The objective of this study was to examine effects of food restriction (FR) on the metabolic activation of aflatoxin B1 (AFB1) in rats and mice, which are AFB1-sensitive and -resistant rodent species, respectively. Forty percent FR [60% of ad libitum (AL) food consumption] reduced the metabolic activation of AFB1 in both rats and mice, causing formation of hepatic AFB1-DNA adducts to be 43% and 31% lower, respectively. The AFB1-DNA adduct 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7-Gua) was predominantly formed in rat liver DNA; the formation of the ring-open analogue of AFB1-N7-Gua, AFB1-formamidopyrimidine (AFB1-FAP), was predominantly found in mouse liver DNA. In contrast to the in vivo results, the in vitro AFB1-DNA adduct formation mediated by the microsomes of liver, kidney or lung from FR-mice was greater than the formation of AFB1-DNA adducts mediated by the tissue microsomes from the AL-mice. Food restriction induced hepatic glutathione S-transferase (GST) activity, as measured by the formation of AFB1-glutathione conjugates (AFB1-SG), in both rats and mice; AFB1-SG was also formed in mouse kidney. Food restriction–induced GST activity assayed in an in vitro system, using [3H]AFB1-8,9-epoxide and glutathione (GSH) as substrates, was also found when mouse kidney and lung cytosolic fractions were used. Food restriction inhibited the AFB1-induced DNA double strand breaks in mouse kidney. The reduction of levels of AFB1-DNA adduct formation in mouse kidney was comparable to the degree of AFB1-induced DNA strand breakages. The results of this study indicate that the metabolic activation of AFB1 can be modulated by FR through the alteration of the formation of AFB1-DNA adducts and AFB1-SG conjugation. However, species and tissue specificities exist regarding the metabolic activation of AFB1.

Detection of 1,N6-ethenodeoxyadenosine and 3,N4-ethenodeoxycytidine by immunoaffinity/32P-postlabelling in liver and lung DNA of mice treated with ethyl carbamate (urethane) or its metabolites.

The capacity of the chemical carcinogen ethyl carbamate (EC, urethane) and its metabolites vinyl carbamate (VC) and vinyl carbamate epoxide (VCO) to form ethenobases was studied in liver and lung DNA of 12-day-old and adult CD-1, B6C3F1, C3H/HeJ and C57BL/6J mice. Following single and multiple doses of EC, VC or VCO, the formation of 1,N6-ethenodeoxyadenosine (epsilon dA) and 3,N4-ethenodeoxycytidine (epsilon dC) was quantified by an immunoaffinity chromatography/32P-postlabelling technique. Both etheno adducts were detected in untreated control DNA samples from liver and lung in the range of 2-15 adducts/10(9) parent nucleotides. Following five repeated injections of 250 or 280 nmol/g body wt VC to adult mice, 51, 57 and 78 epsilon dA/10(9) dA and 28, 42 and 42 epsilon dC/10(9) dC (means of duplicate analyses) were detected in liver DNA of CD-1, C3H/HeJ and C57BL/6J mice respectively. In lung DNA of these VC-treated mice, the levels were 87, 49 and 58 (epsilon dA/10(9) dA) and 64, 39 and 43 (epsilon dC/10(9) dC) respectively. Under similar dose regimens, lower levels of etheno adducts were detected in B6C3F1 mice. Etheno-DNA adducts were also formed in liver and lung upon treatment with EC in adult mice, but at 3-fold lower levels as compared with VC. In 12-day-old C3H/HeJ and C57BL/6J mice, 2- to 3-fold higher etheno adduct levels were detected in liver DNA, when compared with adults, upon a single treatment with 250 nmol/g body wt VC, suggesting that young animals are more susceptible to adduct formation. Combined analysis of adduct formation in adult CD-1, C3H/HeJ and C57BL/6J mice at the higher dose showed a statistically significant increase in etheno adduct formation in the order EC > VC. The results demonstrate that EC and its activated intermediates bind to liver and lung DNA to form epsilon dA and epsilon dC, and the differences in DNA binding further support the hypothesis that metabolic activation of EC to VC is involved. Preliminary data also suggest that background levels of epsilon dA and epsilon dC in DNA are affected by the type of diet given to the animals.

Construction of libraries for methylation sites by in-gel competitive reassociation (IGCR).

The in-gel competitive reassociation (IGCR) procedure was successfully applied to construct a comprehensive library enriched in DNA fragments containing C5mCGG sequences from mouse liver and brain genomic DNA. For IGCR, methylation-insensitive restriction enzyme (Msp I) digests were used as target DNA and methylation-sensitive restriction enzyme (Hpa II) digests as competitor DNA. Southern blot analysis indicated that 60 to 70% of the clones in the library were derived from the methylated sites and overall enrichment was 200- to 1000-fold. IGCR was further applied to construct a library for the sites differentially methylated between brain and liver DNA. In the library, approximately 20% of the Hpa II sites exhibited different degrees of methylation between these tissues.

Mechanistic data indicate that 1,3-butadiene is a human carcinogen.

A review of the epidemiological and mechanistic data on 1,3-butadiene indicates that this chemical is a human carcinogen for which the mouse is an appropriate model for assessing human cancer risk. Butadiene is carcinogenic at multiple organ sites in laboratory animals, including the induction of lymphomas in mice, while epidemiological studies have consistently found associations between occupational exposure to butadiene and increased mortality from lymphatic and hematopoietic cancers. Activated oncogenes and inactivated tumor suppressor genes in butadiene-induced tumors in mice are analogous to genetic alterations frequently observed in human cancers. Butadiene is metabolized to mutagenic and carcinogenic epoxides in all mammalian species studied, including humans. These metabolites form N7-alkylguanine adducts which have been detected in liver DNA of mice exposed to butadiene and in urine of exposed workers. Increases in hprt mutations were observed in lymphocytes from mice exposed to butadiene and in occupationally exposed humans. The mutational spectra for butadiene and its epoxide metabolites at the hprt locus in mouse lymphocytes are similar to the mutational spectrum of ethylene oxide; all of these chemicals exhibit a high percentage of frameshift mutations. Ethylene oxide, an alkylating agent that also forms an N7-alkylguanine adduct, was recently classified by the International Agency for Research on Cancer as a human carcinogen. Based on these data, we suggest that cancer induction by ethylene oxide and butadiene involve similar molecular mechanisms.

The pentachlorophenol metabolite tetrachloro- p-hydroquinone induces the formation of 8-hydroxy-2-deoxyguanosine in liver DNA of male B6C3F1 mice

Tetrachloro- p-hydroquinone (TCHQ), the major metabolite of pentachlorophenol (PCP) in mammalian systems, is known to autoxidize to its semiquinone radical under physiological conditions. In this way, PCP could present a potent source of reactive oxygen species (ROS) during metabolization. ROS contribute to numerous modifications of DNA. Formation of 8-hydroxy-2′-deoxyguanosine (8-OH-dG), a product of hydroxyl radical attack on DNA, is monitored as a marker of a major genetic lesion induced by agents which produce oxygen radicals. We studied the properties of TCHQ for the induction of oxidative DNA damage in vivo. Male B6C3F1 mice were fed a diet containing TCHQ for 2 and 4 weeks. These experiments resulted in an enhancement of about 50% of the 8-OH-dG portion in liver DNA after administration of 300 mg TCHQ/kg body /day for 2 weeks. Control levels did not change over the periods of 2 and 4 weeks, respectively. In contrast to these results, a single i.p. injection of 20 or 50 mg/kg body wt. did not affect the 8-OH-dG content after 6 and 24 h, respectively. These data may support a possible contribution of ROS to the carcinogenicity of PCP.

Bromopropylate: Induction of Hepatic Cytochromes P450 and Absence of Covalent Binding to DNA in Mouse Liver

Oral administration of benzilic acid ester-based acaricide bromopropylate at daily doses of 3, 15, 100, and 300 mg/kg body wt to young adult male Tif:MAGf mice for 14 days caused slightly increased liver weights in the high-dose group. A dose-dependent increase of the microsomal cytochrome P450 content was accompanied by elevated ethoxycoumarin O-deethylase, ethoxyresorufin O-deethylase, pentoxyresorufin O-depentylase, and total testosterone hydroxylase activities. When compared with mice treated in parallel with the model compounds for hepatic xenobiotic metabolizing enzyme induction, phenobarbitone, and 3-methylcholanthrene, the enzyme activity changes observed with bromopropylate largely equalled those expressed in phenobarbitone-treated mice. Immunochemical studies with monoclonal antibodies against rat liver cytochrome P450 isoenzymes of the gene families 1A, 2B, 3A, and 4A confirmed that bromopropylate is a phenobarbitone-type inducer in the mouse liver. Titration of liver microsomal suspensions with bromopropylate yielded Type I substrate binding spectra. The specific amplitude was increased 1.5-fold when microsomes from bromopropylate-treated mice (300 mg/kg body wt) were used instead of control microsomes, indicating the induction of cytochromes P450 catalyzing the oxidative metabolism of the test compound. Single oral administration of 300 mg/kg body wt [14C]- bromopropylate to male mice, without or following pretreatment for 14 days with 300 mg/kg body wt unlabeled bromopropylate, gave no indication for DNA binding of the test compound in the liver. This excludes a genotoxic potential via covalent DNA modification The results suggest that, in analogy to phenobarbitone, bromopropylate acts as a tumor promotor rather than a tumor initiator in the mouse liver.

Detection of exocyclic 1,N2-propanodeoxyguanosine adducts as common DNA lesions in rodents and humans.

Exocyclic adducts are unique DNA modifications resulting from binding at two sites of bases that normally are involved in hydrogen-bonding for maintaining the double-helical structure of DNA. These adducts have been shown to be formed in rodents upon exposure to carcinogens. Using a sensitive 32P-postlabeling method combined with high performance liquid chromatography, we obtained evidence that 1,N2-propanodeoxyguanosine adducts of acrolein (AdG) and crotonaldehyde (CdG) are present in the liver DNA of humans and rodents without carcinogen treatment. The identities of these adducts were verified by cochromatography with the synthetic adduct standards. Further proof of identities was obtained by conversion mediated by nuclease P1 of the labeled AdG and CdG 3',5'-bisphosphates to their corresponding 5'-monophosphates. This treatment converted the in vivo adducts into products that again cochromatographed in a characteristic pattern with the synthetic 5'-monophosphates of AdG and CdG. Using this assay, we also demonstrated the in vivo stereoselective formation of one of the AdG isomers. The estimated total levels of modification were 1.0-1.7, 0.2-1.0, and 0.3-2.0 adducts in 10(6) guanine bases in the liver DNA of mice, rats, and humans, respectively. The detection of these adducts in relatively high levels without carcinogen treatment suggests that the endogenous factors such as lipid peroxidation may be important for their formation. This study provides evidence for the presence of acrolein- and crotonaldehyde-derived exocyclic adducts as common lesions in the liver DNA of rodents and humans.

High-performance liquid chromatographic analysis of methylation changes of CCGG sequence in brain and liver DNA of mice during pre- and postnatal development

The change of the methylation of CpG in the CCGG sequence of brain and liver DNAs of mice during late fetal and suckling periods was determined by high-performance liquid chromatography using a reversed-phase column and 0.1 M phosphate buffer (pH 6.0) as the mobile phase. The tissue DNA was digested with the restriction enzyme, MspI, and was labeled at the 5′-end with [γ- 32P]ATP. The cpm% of deoxycytidine 5′-monophosphate (5mdCMP) in total CpG dinucleotides was calculated from the equation 5mdCMP/total CCGG (cpm%) = (5mdCMP) MspI,cpm /{(5mdCMP) MspI,cpm + (dCMP) MspI,cpm } × 100. The brain DNA exhibited a significant decrease in CpG methylation at prenatal day 18 but little change after birth. This marked decline of 5mdCMP in the CCGG sequence may be associated with the increase of enzymes before birth. The liver DNA showed considerable change during the late prenatal period. The observed changes of CpG methylation in liver DNA are indicative of the corresponding alterations of enzymes, multinucleate cells and hepatocytes. The results obtained indicate that both brain and liver cells have the development-associated changes in the conformation and transition of DNA around the time of birth.

Tamoxifen: evidence by 32P-postlabeling and use of metabolic inhibitors for two distinct pathways leading to mouse hepatic DNA adduct formation and identification of 4-hydroxytamoxifen as a proximate metabolite.

Exposure to pentachlorophenol (PCP) strongly intensifies the formation of mouse hepatic DNA adducts elicited by oral administration of tamoxifen (TAM), as previously shown by 32P-postlabeling. To explain this effect, PCP was proposed to interfere with the detoxication by sulfate conjugation of an as yet unidentified hydroxylated proximate TAM metabolite. A comparison of the present and earlier results shows that the hepatic TAM adduct pattern in female ICR mice depended on the route of administration of TAM (120 mumol/kg), with oral administration primarily eliciting formation of more polar adducts (termed group I adducts), while after i.p. administration less polar adducts (group II) predominated over group I adducts by a factor of 17.5. All these adducts were also formed in female Sprague-Dawley rats after i.p. dosing with TAM, but total adduct levels were 3.5- to 5-fold higher than in mice. After four daily i.p. treatments, TAM adducts accumulated in mouse liver DNA in a non-linear fashion. Adduct levels were 30-50 times lower in mouse kidney and lung than in liver. The phenolic metabolite 4-hydroxy TAM (120 mumol/kg) exclusively led to formation of polar (group I) hepatic adducts, and this process was stimulated 8-fold by co-administration of PCP (75 mumol/kg). Co-administration of PCP with the parent compound led to an 11-fold enhancement of group I adduct formation; simultaneously, levels of group II adducts were suppressed 6-fold. Another inhibitor of sulfate conjugation, 2,6-dichloro-4-nitrophenol, unlike PCP, had no effect on group I adducts, but it reduced group II adduct formation 2.2-fold. The PCP metabolite 2,3,5,6-tetrachlorohydroquinone (75 mumol/kg) did not significantly affect any major TAM adduct, suggesting that PCP itself was the active compound. Similar to group II TAM adducts, the formation of hepatic safrole-DNA adducts was inhibited in female ICR mice by both sulfotransferase inhibitors, consistent with the proposal that metabolic alpha-hydroxylation of the ethyl group of TAM followed by sulfate conjugation represented a mechanism of TAM activation. On the other hand, the strong intensification of group I adducts by PCP and the lack of this effect by 2,6-dichloro-4-nitrophenol suggested that inhibition of sulfate conjugation may not have been the primary mechanism underlying the intensification of group I adducts formed from TAM or 4-hydroxy TAM. The results presented herein demonstrate conclusively that TAM was activated to DNA-reactive compounds along two distinct pathways which contrasted in their responses to metabolic inhibitors.

Genotoxic effects of the carbamate insecticide, methomyl. II. In vivo studies with pure compound and the technical formulation, “lannate 25”

The carbamate insecticide, methomyl, and the methomyl-containing technical formulation, "Lannate 25", were tested for the induction of DNA damage in vivo. Swiss CD1 mice were treated intraperitoneally with test substances and the following tests were performed: alkaline elution of liver and kidney DNA, 8-hydroxyguanosine detection in liver DNA, and 32P-postlabelling analysis of DNA adducts in liver DNA. The clastogenic activity of the two pesticide preparations was also evaluated as micronucleus frequency in bone marrow. No DNA adducts were detected in liver DNA of mice treated with pure methomyl, while a dose-related increase in DNA adducts was found in Lannate 25-treated animals. All other tests were positive with both methomyl and Lannate 25. A summary of genotoxic activity of methomyl is also presented. The hypothesis that the observed genotoxic effects of methomyl are induced indirectly, through formation of active oxygen species, is discussed.

Origin and maintenance of germ-line V genes.

The distribution of nucleotide variability within the upstream of germ-line VH186.2-related variable genes was studied. The data in this report of work in progress indicate non-random selection for variability in the second antigen-contact or complementarity-determining region (CDR2) for 12 such genes isolated using the polymerase chain reaction (PCR) technique from genomic C57BL/6 mouse liver DNA. The translated protein sequences of these and three additional previously published genes also display a pronounced Wu-Kabat peak of amino acid variability in CDR2. In the CDR1 and CDR2 regions of this set of related germ-line genes, there are few [corrected] silent nucleotide changes, and most amino acid replacements (or non-synonymous changes) are non-conservative. In contrast, there is selection against amino acid replacement in the framework regions (FW), as indicated by the significant number of silent (or synonymous) mutational changes from the VH186.2 reference sequence. This is surprisingly similar to the Wu-Kabat variability patterns observed in somatically mutated immune response antibodies. These data could imply similar diversification mechanisms acting on B cell-expressed V genes in the soma (i.e. in a germinal centre) and in the germ-line DNA of male and/or female germ cells. While always possible, we consider this unlikely. Similarly, we consider as unlikely an explanation based on a classical Darwinian model involving simple stepwise whole animal selection prior to reproduction for each VH and VL gene now phylogenetically stored in the V segment arrays of the genomic DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutations in liver DNA of lacI transgenic mice (Big Blue) following subchronic exposure to 2-acetylaminofluorene

2-Acetylaminofluorene (2-AAF) was administered at levels of 0, 300 and 600 ppm in the diet for 28 days to female transgenic mice bearing the lacI gene in a lambda vector (Big Blue® mice). The lambda vector was excised from liver DNA and packaged in vitro into bacteriophage particles which were allowed to infect E. coli bacteria, forming plaques on agar plates. Approximately 10 5 plaques were screened per animal for the appearance of a blue colour, indicative of mutations in the lacI gene which had resulted in an inactive gene product. Background mutation rate was 2.7 × 10 −5 (pooled results of two animals, 8 mutant plaques/289530 plaques). At 300 ppm in the diet, the rate of 3.5 × 10 −5 (8/236300) was not significantly increased over background. At 600 ppm in the diet, the rate increased approximately 3 fold to 7.7 × 10 −5 (17/221240). In comparison to the usual single or 5-day carcinogen exposure regimes, the 4-week exposure protocol allowed the use of much lower dose levels (10–1000 fold lower). Overt toxicity could thus be avoided. The daily doses used were somewhat higher than those required in 2-year carcinogenicity studies with 2-AAF.