Mouse Liver DNA Research Articles

DNA-mediated restoration of phenylalanine hydroxylase gene expression in enzyme-deficient derivatives of enzyme-constitutive mouse cell hybrids.

Phenylalanine hydroxylase (PH) gene expression is not extinguished in hybrids between PH- mouse A9 cells, or its neomycin-resistant derivative A9Neo-3, and PH+ mouse erythroleukemia (MEL) cells, PHC-3A, in contrast to its extinction in hybrids between A9Neo-3 and PH+ rat hepatoma cells, FT-2. Two different types of 6-thioguanine (TG) -resistant derivatives of these A9 X PHC-3A hybrids (LP), are generated in regard to PH gene expression. In regular growth medium supplemented with 10(-4) M TG (Tyr+/TG), TGr derivatives, all of which continue to express PH, occur with high frequency (approximately equal to 10(-3). In contrast, in tyrosine-deficient selective medium, supplemented with 10(-4) M TG (Tyr-/TG), no actively growing colonies are observed. Nevertheless, small colonies containing quiescent cells can be rescued by supplementing the medium with tyrosine. The rescued TGr clones do not express any detectable level of PH. Biochemical, hybridization, and cybridization analyses of one such rescued clone, LPTG-3, showed that these cells lack the regulatory factor capable of activating PH gene in PH- MEL cells. The PH- phenotype of LPTG-3 cells can be converted to the PH+ phenotype by transfection with restriction enzyme-digested or -undigested PHC-3A or mouse liver DNA. Therefore, these cells could be used to clone a fragment of DNA involved in PH gene regulation through DNA-mediated gene transfer methods.

32P-post-labelling analysis of DNA adducts formed in the livers of animals treated with safrole, estragole and other naturally-occurring alkenylbenzenes. I. Adult female CD-1 mice.

The binding of a series of alkenylbenzenes to liver DNA of adult female CD-1 mice, isolated 24 h after i.p. administration of non-radioactive test compound (2 or 10 mg/mouse), was investigated by a modified 32P-post-labelling assay. The known hepatocarcinogens, safrole, estragole and methyleugenol, exhibited the strongest binding to mouse-liver DNA (1 adduct in 10 000 - 15 000 DNA nucleotides or 200 - 300 pmol adduct/mg DNA after administration of a 10 mg dose), while several related compounds, which have not been shown thus far to be carcinogenic in rodent bioassays, bound to mouse-liver DNA at 3 - 200x lower levels. The latter compounds included allylbenzene, anethole, myristicin, parsley apiol, dill apiol and elemicin. Eugenol did not bind. Low binding to mouse-liver DNA was also observed for the weak hepatocarcinogen, isosafrole. Two main 32P-labelled adducts, which appeared to be guanine derivatives, were detected for each of the binding chemicals on thin-layer chromatograms. The loss of safrole adducts from liver DNA was biphasic: a rapid loss during the first week (t 1/2 approximately 3 days) was followed by a much slower decline up to 20 weeks after treatment (t 1/2 approximately 2.5 months). Adducts formed by reaction of 1'-acetoxysafrole, a model ultimate carcinogen, with mouse-liver DNA in vitro were chromatographically identical to safrole-DNA adducts formed in vivo. Pretreatment with pentachlorophenol, a known inhibitor of sulphotransferases, inhibited the binding of safrole to mouse-liver DNA, providing further evidence that the metabolic activation of the allylbenzenes proceeds by the formation of 1'-hydroxy derivatives as proximate carcinogens and 1'-sulphoöxy derivatives as ultimate carcinogens.

32P-post-labelling analysis of DNA adducts formed in the livers of animals treated with safrole, estragole and other naturally-occurring alkenylbenzenes. II. Newborn male B6C3F1 mice.

When a series of nine alkenylbenzenes were administered to preweanling male mice, safrole, estragole and methyleugenol induced a significant incidence of hepatic carcinomas, while eugenol, anethole, elemicin, myristicin, dill apiol and parsley apiol did not (Miller et al., Cancer Res., 43, 1124-1134, 1983). Following the protocol used to test seven of these compounds, male C57Bl X C3H/He F1 mice were injected with 0.25, 0.5, 1.0 and 3.0 mumol of a compound on days 1, 8, 15 and 22 after birth, respectively. Groups of mice were killed and their liver DNA isolated on days 23, 29 and 43, and analysed by a modified 32P-post-labelling procedure. Highest levels of adducts were detected with methyleugenol (72.7 pmol/mg DNA), estragole (30.0) and safrole (17.5). After correction for liver growth it was estimated that most of these adducts were still present at 43 days. Significant levels of DNA binding by myristicin (7.8 pmol/mg DNA) and elemicin (3.7) were also found but in the former case the adducts were less persistent. Only low levels of adducts were detected with anethole, dill apiol and parsley apiol (less than 1.4 pmol/mg DNA); no DNA binding was detected with eugenol. Thus, all but one of the alkenylbenzenes studied became bound to newborn mouse-liver DNA, but the levels and the persistence of adducts formed by the carcinogenic compounds were greater.

Enhancement of ribonucleic acid synthesis by chromium(III) in mouse liver

The effect of Cr(III) administration on hepatic RNA synthesis in mice was studied. It was found that Cr accumulated in mouse liver. Forty-eight hours after intraperitoneal injection of CrCl 3 (0.005–5 mg Cr/kg body weight) approximately 10% of the administered dose perg of tissue remained. The accumulated Cr was still retained 64 days after administration (5 mg Cr/kg) with only a slight decrease. Approximately 20% of the hepatic Cr was detected in the nuclei. By administering CrCl 3, RNA synthesis in mouse liver was markedly enhanced without altering the pool size of nucleotides. This enhancement was dose-dependent and statistically significant at doses of 0.05 ( p < 0.05), 0.5 ( p < 0.01), and 5 mg Cr/kg ( p < 0.01), and remained so for at least 16 days after administration of 5 mg Cr/kg. The syntheses of DNA and protein in mouse liver were not significantly changed by CrCl 3 administration. On the other hand, Cr(VI) administration did not enhance but rather inhibited RNA synthesis in mouse liver. These results suggest that Cr(III) specifically enhances RNA synthesis in mouse liver.

CDNA clone for the alpha subunit of the acetylcholine receptor from the mouse muscle cell line BC3H-1.

Sequences from a gene coding for mouse acetylcholine receptor alpha subunit have been inserted into a recombinant plasmid and cloned in Escherichia coli. mRNAs for acetylcholine receptors occur in low abundance in vertebrate muscle. To clone the mouse alpha-subunit cDNA, we made use of (i) a cell line, BC3H-1, that overproduces the alpha-subunit mRNA and (ii) a polysome fractionation procedure that results in enrichment of alpha-subunit mRNA. Polyadenylylated RNA was used to construct a cDNA library of 750 recombinant clones. Acetylcholine receptor-specific sequences were identified by hybrid-selected translation, followed by monoclonal antibody precipitation and peptide mapping of the translation product. One clone (pA59) that fit these criteria was found in the first 80 isolates. It had a 700-base-pair insert that was excised with Pst I. Blot-hybridization experiments with nick-translated pA59 DNA showed that BC3H-1 cells contain 100-1,000 times more alpha-subunit mRNA than does newborn or adult mouse muscle. Blot hybridization of restriction digests of mouse liver DNA revealed that pA59 is homologous to a very small number (probably one) of genomic sequences.

Protection by indole-3-carbinol against covalent binding of benzo[ a]pyrene metabolites to mouse liver DNA and protein

Indole-3-carbinol (I-3-C) is a compound present in many cruciferous vegetables that has been shown to reduce aryl hydrocarbon-induced neoplasia in experimental animals. We examined the relationship between the ability of I-3-C to alter the activity of hepatic aryl hydrocarbon hydroxylase (AHH), and its ability to inhibit the covalent binding of benzo[ a]pyrene (B aP) metabolites to DNA and protein. Using an in vitro system and a hepatic postmitochondrial fraction from mice that had been treated by gavage with I-3-C, we found that up to 90% of the covalent binding of B aP metabolites to macromolecules was eliminated, while AHH activity was unchanged. In experiments in vivo, treatment of mice by gavage with I-3-C before [ 14C]B aP resulted in up to an 80% decrease in covalent binding of 14C to DNA or protein with no concomitant decrease in hepatic AHH activity. These results suggest that I-3-C administered in vivo confers protection against the binding of B aP oxidation products to hepatic cellular macromolecules.

Nucleotide sequence of a region downstream of the mouse CK immunoglobulin gene.

2318 bp downstream of the CK (1) gene segment were sequenced in a clone (L1-D) derived from mouse liver DNA. The 966 bp at the 5' side of this stretch were found to be identical to a sequence which had been determined previously in a myeloma T derived clone, i.e. no somatic mutations had occurred in the transition from the germline to the rearranged configuration. The remaining 1352 bp had not been known and extend the sequenced part of the mouse JK-CK region to about 7.5 kb. Within the newly sequenced area three BspRI sites have been located which were used in chromatin studies (Weischet et al., accompanying publications). In L1-D sequences have been found which are possible targets of aberrant recombination events.

Cloning of endogenous murine leukemia virus-related sequences from chromosomal DNA of BALB/c and AKR/J mice: identification of an env progenitor of AKR-247 mink cell focus-forming proviral DNA.

Recombinant phages containing murine leukemia virus (MuLV)-reactive DNA sequences were isolated after screening of a BALB/c mouse embryo DNA library and from shotgun cloning of EcoRI-restricted AKR/J mouse liver DNA. Twelve different clones were isolated which contained incomplete MuLV proviral DNA sequences extending various distances from either the 5' or 3' long terminal repeat (LTR) into the viral genome. Restriction maps indicated that the endogenous MuLV DNAs were related to xenotropic MuLVs, but they shared several unique restriction sites among themselves which were not present in known MuLV proviral DNAs. Analyses of internal restriction fragments of the endogenous LTRs suggested the existence of at least two size classes, both of which were larger than the LTRs of known ecotropic, xenotropic, or mink cell focus-forming (MCF) MuLV proviruses. Five of the six cloned endogenous MuLV proviral DNAs which contained envelope (env) DNA sequences annealed to a xenotropic MuLV env-specific DNA probe; in addition, four of these five also hybridized to an ecotropic MuLV-specific env DNA probe. Cloned MCF 247 proviral DNA also contained such dual-reactive env sequences. One of the dual-reactive cloned endogenous MuLV DNAs contained an env region that was indistinguishable by AluI and HpaII digestion from the analogous segment in MCF 247 proviral DNA and may therefore represent a progenitor for the env gene of this recombinant MuLV. In addition, the endogenous MuLV DNAs were highly related by AluI cleavage to the Moloney MuLV provirus in the gag and pol regions.

Metabolism of [14C]trichloroethylene to 14CO2 and interaction of a metabolite with liver DNA in rats and mice.

Male Sprague-Dawley rats and male B6C3F1 mice excreted 5-15% of a tracer dose of [14C]trichloroethylene as 14CO2 within 24 h after ip injection of a single dose in a corn-oil vehicle. The proportion of the dose excreted as CO2 was greater in mice than in rats, but increased in the rats after starvation or pretreatment with phenobarbital. As the dose was increased toward the LD50 level, the proportion excreted as 14CO2 decreased slightly, but this was largely due to increased loss of unchanged trichloroethylene. The excretion of 14CO2 was thus correlated with the expected level of microsomal metabolism of trichloroethylene to an electrophilic intermediate capable of binding to glutathione or macromolecules. Liver protein labeling was observed to be relatively high (10,000-23,000 cpm/mg in the mouse), while DNA labeling was consistently observed to be very low, not allowing identification of any adducts by high-performance liquid chromatography (HPLC). Also, no effect on DNA fragmentation was seen by alkaline sucrose gradient centrifugation after injection of an LD50 dose of trichloroethylene. The ability of trichloroethylene to interact with DNA in vivo was thus observed to be very slight.

Dose-dependency of 2-acetylaminofluorene binding to liver DNA and hemoglobin in mice and rats

The carcinogenic activity of 2-acetylaminofluorene (2-AAF) in mice and rats has been accurately described so that it is a suitable archetypical chemical for investigations of parameters of cross-species variation. The binding of orally administered [9- 14C]2-AAF to liver DNA in mice and rats was dose related in a linear manner between 1 to 100 μmol/kg body wt. By Sephadex LH-20 chromatography, it was determined that the formation of 2-AAF adducts over the dose range studied was both qualitatively and quantitatively similar. Hepatic DNA binding was greater in rats compared to mice. No sex difference was observed in the DNA binding in rats so that the higher incidence of liver cancer in male compared to female rats could not be explained by a greater amount of DNA binding in males. The binding of orally administerd [9- 14C]2-AAF to hemoglobin in mice and rats was dose related in a linear manner between 0.1 to 100 μmol/kg. The results of 2-AAF binding to DNA and hemoglobin would indicate that for an oral dose of 2-AAF, the rate of formation of reactive metabolites was similar between 0.1 and 100 μmol/kg body wt. However, the sevenfold greater DNA binding and 2.5-fold greater hemoglobin binding in rats compared to mice were more than an order of magnitude less than 246-fold greater carcinogenicity of 2-AAF in rat liver. The results of the study supported a pharmacokinetic model that related the metabolism of 2-AAF to reactive metabolites to the binding of the metabolites to DNA and hemoglobin. The results did not support a relationship of macromolecular binding to carcinogenic potency.

The determination of O 6-methyl- and 7-methylguanine in mouse liver DNA from dimethylnitrosamine-treated mice by high-performance liquid chromatography with UV absorbance detection

The methylated purines O 6-methyl- and 7-methylguanine were isolated from mouse liver DNA hydrolysates by means of a column cleanup employing a Sep Pak C-18 reverse-phase cartridge. The purine bases were eluted from the cartridge with methanol, evaporated to dryness, and then dissolved in mobile phase for liquid chromatographic analysis by normalphase chromatography. The system consisted of a LiChrosorb Si 60 column with a watersaturated mobile phase of 20% methanol in chloroform containing 0.001% H 3PO 4. The two methylated bases eluted before adenine or guanine. For extremely low-level (<300 pmol) quantitation, the peaks corresponding to O 6-methyl- and 7-methylguanine were collected and then analyzed by reverse-phase chromotography with a LiChrosorb RP-18 column and a mobile phase of 5% methanol in pH 7 phosphate buffer (for 7-methylguanine) or 9.5% methanol/buffer (for O 6-methylguanine). Comparisons were made with fluorescence detection and with scintillation counting (in animal studies where [ 14C]dimethylnitrosamine was used). Minimum detectable levels at 254 nm were about 3 ng (3:1 signal to noise ratio) for each of the title compounds. As low as 10 pmol/mg of each could be detected in DNA hydrolysates. Recoveries of O 6-methyl- and 7-methylguanine from DNA spiked at 750 pmol/mg were greater than 80%.

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Treatment of inbred CBA mice with hydrazine sulfate, a liver carcinogen, plus [14C]formate or L-[methyl-14C]-methionine as a source of "C1" units, produced [14C]7-methylguanine (7-MG) in liver DNA and RNA. This methylated guanine was identified by its chromatographic characteristics in three different systems: on Sephadex G10, on Dowex 50, and by paper co-chromatography with authentic 7-MG. Mice that received [14C]formate alone showed no 7-MG in their DNA or RNA. In addition, mice treated with L-[methyl-14C]methionine alone showed no 7-MG in their DNA. These results suggest that "indirect" alkylation of nucleic acids is a mechanism of cancer initiation by hydrazine.

Environmental pollutant 5-chlorouracil is incorporated in mouse liver and testes DNA

Our aquatic environment contains 5-chlorouracil and 5-chlorouridine among other chloro-organics as a result of chlorination of sewage. Mice ingesting 5-chlorouracil in their drinking water incorporate the base heavily in the liver and testes DNA. Another related metabolite, 5-chlorodeoxyuridine, is 5 times more potent than 5-bromodeoxyuridine in inducing sister-chromatid exchanges in Chinese hamster ovary cells.

Chain length heterogeneity of nucleosomal DNA in mouse liver after dimethylnitrosamine administration.

The effect of dimethylnitrosamine on the nucleosomal structure of mouse liver chromatin was studied. After a single oral dose of dimethylnitrosamine (2-75 mg/kg body weight 45 min before sacrifice) liver nuclei were isolated and incubated with micrococcus nuclease. Nucleosomes were separated on sucrose density gradients. There were no differences in nucleosomal sedimentation velocities between preparations from control and dimethylnitrosamine treated animals. The supernatant obtained after centrifugation of the lysed nuclei (2 min at 4,000 gav) and nucleosomal peak fractions were used for isolation of DNA. DNA was heat denatured in 7 M urea or formamide. After electrophoresis on polyacrylamide gels areas under mononucleosomal DNA and smaller fragments were measured and compared with the total DNA area. The increase in DNA fragmentation was dimethylnitrosamine dose response dependent. When expressed as per cent of controls it amounted to 106% for 2 mg; 115% for 10 mg; 127% for 25 mg; 164% for 75 mg dimethylnitrosamine/kg body weight. A good correlation between mobility and log of chain length of phi chi 174 RF DNA-Hae III digest was obtained in nondenaturing 5% polyacrylamide gels and denaturing non-aqueous formamide polyacrylamide gels but not in 12% polyacrylamide gels containing 7 M urea. DNA of mononucleosomal peak fractions contained 200 and that of dinucleosomal peak fractions 400 nucleotides. Fragmentation of DNA was closely related to in vivo dimethylnitrosamine treatment but was not detected in measurements of protein-DNA complexes in the chromatin. It was disclosed on denaturation of DNA followed by polyacrylamide gel electrophoresis.

Alkaline DNA fragmentation in vivo: borderline or negative results obtained respectively with 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene.

Using the in vivo DNA damage alkaline elution assay, a satisfactory correlation with carcinogenicity in the same target organ has been previously shown for a variety of chemical agents. This work was intended to enlarge the exploration of the predictivity of this test. Benzo[a]pyrene (BP) was found negative for damage to liver DNA of mice and rats, and 7,12-dimethylbenz[a]anthracene (DMBA) negative for damage to liver and bone marrow DNA of mice and slightly positive for damage to mammary gland DNA of young female rats. The results were found to be correlated with the extension of DNA arlyation in target organs in similar experimental conditions. From carcinogenicity data reported in the Survey of Compounds Which Have Been Tested for Carcinogenic Activity (vols. 1961-1973) BP and DMBA were both found to be essentially negative as liver carcinogens; however, DMBA was a potent carcinogen in inducing mammary tumors.

Hormone-responsive expression of an endogenous proviral gene of mouse mammary tumor virus after molecular cloning and gene transfer into cultured cells.

A recombinant lambda phage containing mouse mammary tumor virus (MMTV) proviral DNA was isolated from a gene library constructed from GR mouse liver DNA. Restriction enzyme analyses reveal that the cloned molecule contains a copy of one of the GR endogenous MMTV proviruses flanked on both sides by 2--3 kb of mouse genomic DNA. In this report we have examined the expression of the cloned MMTV provirus after cotransfection with the herpes thymidine kinase (TK; ATP:thymidine 5'-phosphotransferase,, EC 2.7.1.21) gene and integration into mouse LTK- cells. Nine individual TK+ transformants were selected, and all were found to contain MMTV-transfected DNA. One of the TK+ transformants was chosen for further study. Total poly(A)-containing RNA was isolated from the cells, and liquid hybridization analyses with MMTV cDNA showed that it contained 0.02% MMTV-specific RNA. The sizes of the MMTV-specific species were determined and found to correspond to the 35S and 24S mRNAs synthesized in MMTV-infected cells. Glucocorticoid hormones have been shown to increase the concentration of MMTV RNA in virus-infected cultured cells. Therefore, we tested the effect of dexamethasone on the concentration of MMTV-specific RNA in cells transfected with the MMTV proviral DNA. The amount of MMTV-specific poly(A)-containing RNA found in the cells grown in the presence of hormone was 0.17%. Therefore, dexamethasone causes an 8-fold increase in the amount of MMTV-specific RNA in mouse cells containing several copies of a cloned and transfected MMTV proviral gene.

In vitro assay of erythropoietin in fetal mouse liver cultures. II. Effects of human transferrin bound iron and of serum on the stimulation of incorporation of 3H-thymidine into DNA.

Iron bound to human transferrin but not apotransferrin, increases the effect of erythropoietin in stimulating incorporation of 3H-thymidine into DNA in fetal mouse liver cells in vitro. The effect of erythropoietin, with or without transferrin-iron is blocked by pre-incubation of the erythropoietin with rabbit anti erythropoietin serum. Human sera contain factors in addition to erythropoietin and transferin-iron which may modify the stimulation of incorporation of 3H-thymidine into fetal mouse liver DNA induced by erythropoietin. Heat treatment of sera at 56 degrees C for 30 min does not necessarily destroy these factors. Acid heat treatment of sera (pH 5.5 and 100 degrees C for 5 min) may destroy inhibitory factors and can result in an apparent increase in serum erythropoietin activity assessed in this fetal mouse liver system.

Mapping of heavy chain genes for mouse immunoglobulins M and D.

A single DNA fragment containing both mu and delta immunoglobulin heavy chain genes has been cloned from normal BALB/c mouse liver DNA with a new lambda phage vector Charon 28. The physical distance between the membrane terminal exon of mu and the first domain of delta is 2466 base pairs, with delta on the 3' side of mu. A single transcript could contain a variable region and both mu and delta constant regions. The dual expression of immunoglobulins M and D on spleen B cells may be due to alternate splicing of this transcript.

J genes for heavy chain immunoglobulins of mouse.

A 15,8-kilobase pair fragment of BALB/c mouse liver DNA, cloned in the Charon 4A lambda phage vector system, was shown to contain the mu heavy chain constant region (CHmu) gene for the mouse immunoglobulin M. In addition, this fragment of DNA contains at least two J genes, used to code for the carboxyl terminal portion of heavy chain variable regions. These genes are located in genomic DNA about eight kilobase pairs to the 5' side of the CHmu gene. The complete nucleotide sequence of a 1120-base pair stretch of DNA that includes the two J genes has been determined.

Sequence organization of cloned intracisternal A particle genes.

Seven recombinant DNA clones containing mouse intracisternal A particle genes were isolated and analyzed by restriction enzyme digestion, Southern blot analysis and heteroduplex mapping. The sequence organization of the individual genes was found to differ, with one end of the gene region being most variable, while a central segment of 1.8 kb was missing from two of the clones. A third region, common to all the clones and containing the 3' end of the gene, is present in about 1800 copies per haploid genome, but the central portion is found in only 650 copies. The same reiteration frequency is found in both myeloma tumor and mouse liver DNA. The most abundant intracisternal A particle RNA in two different myeloma lines was found to be 3.5 kb, and RNA/DNA hybrids show that the RNA is homologous to all but a small internal segment of one of the clones.