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N Battula

Publications and source records attributed to N Battula.

10 recordsLinked to original sources
PubMed1992

Stable expression of human cytochrome P450IIE1 in mammalian cells: metabolic activation of nitrosodimethylamine and formation of adducts with cellular DNA.

To introduce cytochrome P450IIE1 DNA stably into the chromosomal DNA of mammalian cells, we constructed recombinant retroviruses containing the full-length complementary DNA for human cytochrome P450IIE1 and a selectable neo gene. Rat and mouse cells were infected with these viruses, and clones expressing the neo marker gene product were selected in G418-containing medium. Analysis of the DNA of the clones by Southern blotting showed that the viral DNA was integrated into the cellular DNA. Enzymatic analysis of the clones showed that the transduced DNA directed the expression of enzymatically active cytochrome P450IIE1. Treatment of the cells with the carcinogen [14C]-nitrosodimethylamine and analysis of the cellular DNA by CsCl equilibrium density gradients showed incorporation of the label into DNA, indicating the formation of covalent adducts with the cell DNA. Construction of recombinant cell lines constitutively expressing cytochrome P450IIE1 provides a permanent source for this enzyme and can aid in the analysis of its catalytic properties, such as the metabolic activation of chemical mutagens/carcinogens, and the consequent cytotoxic and genotoxic effects of these compounds.

3T3 Cells↗
PubMed1992

Biotransformation of caffeine and theophylline in mammalian cell lines genetically engineered for expression of single cytochrome P450 isoforms.

Primary steps in the metabolism of caffeine and theophylline are cleavage of methyl groups and/or hydroxylation at position 8, mediated by cytochromes P450. V79 Chinese hamster cells genetically engineered for stable expression of single forms of rat cytochromes P450IA1, P450IA2 and P450IIBI and human P450IA2 and rat liver epithelial cells expressing murine P450IA2 were used to overcome problems arising in the proper allocation of metabolic pathways to specific isoforms by conventional techniques. These cell lines were exposed to caffeine and/or theophylline, and concentrations of metabolites formed in the medium were determined by HPLC. Caffeine was metabolized by human, rat and murine P450IA2, resulting in the formation of four primary demethylated and hydroxylated metabolites. However, there were differences in the relative amounts of the metabolites. The human and the mouse P450IA2 isoforms predominantly mediated 3-demethylation of caffeine. The rat cytochrome P450IA2 mediated both 3-demethylation and 1-demethylation of caffeine to a similar extent. Theophylline was metabolized mainly via 8-hydroxylation. All cell lines tested were able to carry out this reaction, with highest activities in cell lines expressing rat or human P450IA2, or rat P450IA1. These results support the hypothesis that caffeine plasma clearance is a specific in vivo probe for determining human P450IA2 activity.

Animals↗
PubMed1991

Cytochrome P4501A2 constitutively expressed from transduced DNA mediates metabolic activation and DNA-adduct formation of aromatic amine carcinogens in NIH 3T3 cells.

We transduced mouse cytochrome P4501A2 DNA into NIH 3T3 cells by retrovirus-mediated gene transfer. The capacity of the transduced cytochrome P4501A2 for metabolic activation and DNA-carcinogen adduct formation of aromatic amine carcinogens was investigated. Clones of NIH 3T3 cells that constitutively express cytochrome P4501A2 and controls were exposed to a prototype food-derived carcinogenic heterocyclic amine, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and an aromatic amine, 2-acetylaminofluorene (AAF), and their genomic DNAs were analyzed for adducts by 32P-postlabeling assays. Kinetic analysis of DNA-carcinogen adducts indicated that adduct formation was dependent on the level of the enzyme, the dose of carcinogen, and the duration of exposure. Addition of 7,8-benzoflavone, an inhibitor of P4501A2, blocked both the enzyme activity and DNA-adduct formation, indicating the specific role of P4501A2 in metabolic activation and adduct formation. Three specific IQ-DNA adducts were detected in cells expressing P4501A2. Fingerprints of the in situ DNA adducts were similar to those of the in vivo adducts in rodent hepatic DNA after the administration of IQ. A single AAF-DNA adduct was observed in cells exposed to AAF, but other minor adducts were also detected in vivo. These results show that cells expressing constitutive levels of single cytochrome P450s provide an excellent in situ model system for analyzing the catalytic specificity, metabolic activation, and genotoxicity of putative toxic, mutagenic, and carcinogenic substances.

3T3 Cells↗
PubMed1991

Rat hypoxanthine phosphoribosyltransferase cDNA cloning and sequence analysis.

We have determined the nucleotide sequence of the rat hprt (hypoxanthine phosphoribosyltransferase; EC 2.4.2.8.) mRNA coding region and of adjacent, untranslated 5' and 3' mRNA, and we have designed an oligonucleotide primer pair for efficient PCR amplification of the rat hprt coding region. These sequence data and rat-specific primer pair will aid workers interested in coupling well-developed rat toxicologic and carcinogenicity bioassays with quantitative and molecular analyses of somatic mutation induction in rat cells in vivo and in vitro.

Animals↗
PubMed1978

Sites of integration of infectious DNA of avian reticuloendotheliosis viruses in different avian cellular DNAs.

The pattern of integration for the infectious DNA of two avian reticuloendotheliosis viruses whose DNA is not inactivated by digestion with the restriction endonuclease, Eco RI was determined. High molecular weight DNA from infected chicken, turkey and pheasant cells was digested with Eco RI, electrophoresed through agarose gels and assayed for infectivity. The same patterns of integration of infectious viral DNA were found for these species of avian cells infected at high or low multiplicities with two reticuloendotheliosis viruses. There were multiple sites of integration in acutely infected cells with concomitant cell death. There was a single site of integration in chronically infected cells with no cell death. There were more integrated infectious viral DNA molecules per cell in acutely infected cells than in chronically infected cells. These results are consistent with the hypotheses that the cell death in the acute phase of infection is a result of the integration of the infectious viral DNA at multiple sites, and that only those cells survive that have the infectious viral DNA integrated exclusively at the single site.

Cell Line↗
PubMed1977

Infectious DNA of spleen necrosis virus is integrated at a single site in the DNA of chronically infected chicken fibroblasts.

The infectious DNAs of a number of avian leukosis-sarcoma and reticuloendotheliosis viruses were digested with six nucleotide-specific restriction endonucleases, and the digests were tested for infectivity. All of the enzymes inactivated the viral infectivities except for EcoRI, which did not inactivate the infectivity of the DNA of two of the reticuloendotheliosis viruses, spleen necrosis and chick syncytial viruses. The infectious DNA of spleen necrosis virus after digestion with EcoRI had a buoyant density in CsCl solution greater than the density of the high-molecular-weight infectious viral DNA. The infectious EcoRI-digested spleen necrosis virus DNA from chronically infected chicken cells was uniform in size, 10 megadaltons, which indicated a single site of integration. The infectious EcoRI-digested spleen necrosis virus DNA from acutely infected cells was heterogeneous in size, ranging from 8-14 megadaltons, which indicated multiple sites of integration. These results are consistent with the hypothesis that cells that integrate infectious spleen necrosis virus DNA at a single site survive and multiply, whereas cells that integrate infectious viral DNA at additional sites either die or selectively lose or inactivate the DNA in the additional sites.

Acute Disease↗
PubMed1976

On the fidelity of DNA replication. Lack of exodeoxyribonuclease activity and error-correcting function in avian myeloblastosis virus DNA polymerase.

Homogeneous DNA polymerase ("reverse transcriptase") from avian myeoblastosis virus was assayed for exodeoxyribonuclease activity. The substrates were defined template-initiator complexes in which different radioactive nucleotides were present at the 3'-OH termini of the initiator. Even when the number of molecules of enzyme was equal to the number of initiator termini there was no significant release of radioactivity with any of the template-initiator combinations tested. Under similar conditions, the nuclease activity associated with either Escherichia coli or T4DNA polymerases rendered more than 90% of the initiator termini acid-soluble. The ratio of exodeoxyribonuclease activity to protein with avian myeoblastosis DNA polymerase is less than 0.003% of that obtained with E. coli DNA polymerase I. Furthermore, avian myeloblastosis virus DNA polymerase failed to excise mispaired terminal nucleotides in both the presence and absence of polymerization.

Avian Leukosis Virus↗
PubMed1975

Avian myeloblastosis virus DNA polymerase. Kinetic studies on the incorporation of noncomplementary nucleotides.

The high error rate characteristic of DNA polymerases from RNA tumor viruses has permitted measurements on the simultaneous incorporation of complementary and noncomplementary nucleotides during DNA synthesis. For example, avian myeloblastosis virus DNA polymerase incorporates 1 molecule of dCMP for approximately 500 molecules of dTMP polymerized using polyriboadenylic acid as a template. The parallel incorporation of complementary and noncomplementary nucleotides afer gel filtration of avian myeloblastosis virus DNA polymerase indicates that the observed fidelity is catalyzed by the polymerase itself. Nearest neighbor analysis of the product indicates that noncomplementary nucleotides are incorporated as single base substitutions. The incorporation of the noncomplementary dCMP is not reduced by a 20-fold greater amount of the complementary nucleotide, dTTP. Conversely, the concentration of the noncomplementary nucleotides does not effect the rate of incorporation of the complementary nucleotide. A similar lack of competition between complementary dGTP and noncomplementary dATP is exhibited using poly(rC)-oligo(dG) as a template-primer. Furthermore, there was no detectable competition between the different noncomplementary nucleotides. Possible explanations for this lack of competition are considered.

Avian Leukosis Virus↗
PubMed1975

On the fidelity of DNA replication. Characterization of polynucleotides with errors in base-pairing synthesized by avian myeloblastosis virus deoxyribonucleic acid polymerase.

Polynucleotide templates were copied by avian myeloblastosis virus DNA polymerase ("reverse transcriptase") and the frequency and distribution of errors were determined. The error rate with [r(pA)2500-d(pT)12-18] template-initiator under a variety of conditions was approximately 1/600, i.e. one incorrect dCMP incorporated for 600 correct dTMP polymerized. Addition of the metal chelator o-phenanthroline to the reaction inhibited the incorporation of correct and incorrect nucleotides proportionately. The enzyme inhibited a pH optimum of 8.5 and the error rate remained constant over a range of pH (6.0 to 10.0). The rate of polymerization was greater at higher temperatures and approximately doubled for every 10 degrees increase. The error rate was constant at all temperatures. These results indicate that the purified avian myeloblastosis virus DNA polymerase synthesizes polydeoxynucleotides with an unusually large number of errors in base-pairing. Velocity sedimentation of the products of the reaction obtained at various times during the course of synthesis indicate that: (a) the entire length of the 14 S template was copied, and (b) the incorporation of the incorrect nucleotide did not terminate chain propagation. Isopyknic banding in neutral and alkaline cesium sulfate gradients showed that the noncomplementary nucleotides are incorporated into the polydeoxynucleotide product. Stepwise degradation and nearest neighbor analysis of the reaction product indicated that (a) the correct and incorrect nucleotides are present in phosphodiester linkages, (b) the errors are not concentrated at either termini; and (c) the errors are uniformly distributed throughout the newly synthesized polydeoxynucleotide.

Avian Leukosis Virus↗