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Query: UNIPROT:P00492 (hypoxanthine-guanine phosphoribosyltransferase)
 2,385 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have developed a culture system for detecting and isolating rare hypoxanthine phosphoribosyltransferase-deficient mutants of human epidermal keratinocytes. A thioguanine-resistant variant, 3T3M1, of the Swiss mouse fibroblast line 3T3 was used as a feeder layer to support clonal growth of mutant keratinocytes. A near-diploid, epidermal squamous cell carcinoma line, SCC-13Y, was used as a prototype to determine mutagen treatment conditions, plating density, and phenotypic expression time for maximum mutant recovery. To extend this system to normal keratinocytes, we improved the culture conditions by adding insulin, adenine, and Ham's nutrient mixture F-12, which increased colony-forming efficiencies to 30% in early passage and made feasible the detection of rare mutants in normal epidermal keratinocyte populations. We have quantitated mutation in SCC-13Y and three strains of normal human epidermal keratinocytes after exposure to polycyclic aromatic hydrocarbons, which are activated to their mutagenic forms by cellular mixed-function oxidases. 7,12-Dimethylbenz[a]anthracene and benzo[a]pyrene caused almost no cytotoxicity, but induced thioguanine-resistant mutants at frequencies as much as 50-fold higher than the spontaneous frequency of approximately 10(-6). The mutants were aminopterin-sensitive and possessed no measurable hypoxanthine phosphoribosyltransferase activity; their behavior was indistinguishable from that of keratinocytes cultured from individuals with Lesch-Nyhan syndrome. This mutagenesis assay system should also be applicable to other feeder layer-dependent human epithelial cell types, such as urothelial, mammary, and tracheal epithelial cells.
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PMID:Polycyclic aromatic hydrocarbon mutagenesis of human epidermal keratinocytes in culture. 644 Jan 45

Aryl hydrocarbon (benzo[a]pyrene) hydroxylase inducibility by benzo[a]anthracene was studied in 29 somatic cell hybrid clones, developed by fusing mouse spleen or peritoneal cells from four different inbred strains with hypoxanthine phosphoribosyltransferase-deficient Chinese hamster E36 cells. Karyotype analysis plus 25 markers assigned to 16 autosomes and the X chromosome were examined. In 28 of the 29 clones, the presence or absence of inducibility is associated with the presence or absence, respectively, of mouse chromosome 17. Liver microsomal aryl hydrocarbon hydroxylase induction by 3-methylcholanthrene or benzo[a]anthracene was assessed in appropriate backcrosses with the Mus musculus molossinus, M. m. castaneus, MOR/Cv, PL/J, SM/J and DBA/2J inbred strains and in 13 NX8 recombinant inbred lines. Twenty-seven biochemical genetic markers representing all but four autosomes were tested for possible linkage with the hydroxylase inducibility, and no linkage was found. The hepatic Ah receptor was quantitated in 26 BXD recombinant inbred lines; the Ah phenotype did not match exactly any of the more than 70 genes with established strain distribution patterns representing 12 autosomes and at least five unlinked markers. It is concluded that a major gene controlling aryl hydrocarbon hydroxylase inducibility by benzo[a]anthracene is located on chromosome 17. Because there is no significant linkage with any of three biochemical markers in the upper third of the chromosome, we conclude that the inducibility gene is located in the distal 40% of mouse chromosome 17. Whether this trait represents the Ah locus, i.e., the gene encoding the cytosolic Ah receptor, will require further study.
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PMID:Aryl hydrocarbon hydroxylase induction by benzo[a]anthracene: regulatory gene localized to the distal portion of mouse chromosome 17. 654 99

A strain of Chinese hamster ovary cells that is deficient in nucleotide excision repair, strain UV5, was compared with the normal parental CHO cells in terms of cytotoxicity and mutagenesis after exposure to several chemical carcinogens that are known to produce bulky, covalent adducts in DNA. Induced mutations were measured at the hprt locus using thioguanine resistance and at the aprt locus using azaadenine resistance. The compounds tested that required metabolic activation (using rat or hamster microsomal fractions) were 7,12-dimethylbenz(a)anthracene, 3-methylcholanthrene, benzo(a)pyrene, aflatoxin B1, 2-acetylaminofluorene, and 2-naphthylamine. The direct-acting compounds (+/-)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, N-acetoxy-2-acetylaminofluorene, and N-OH-2-naphthylamine were also studied. For all compounds except 2-naphthylamine and its active metabolite, the repair-deficient cells were significantly more sensitive to killing than the normal CHO cells. Mutation induction at both loci was also more efficient in UV5 cells in each instance where enhanced cytotoxicity was observed. By using tritium-labeled N-acetoxy-2-acetylaminofluorene, normal and mutant cells were shown to bind mutagen to their nuclear DNA with similar efficiency, and a greater amount of adduct removal occurred in the normal cells. From this study it is concluded that the use of excision repair-deficient CHO cells provides enhanced sensitivity for detecting mutagenesis and that a positive differential cytotoxicity response gives an indication of repairable, potentially lethal genetic damage.
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PMID:Hypersensitivity to cell killing and mutation induction by chemical carcinogens in an excision repair-deficient mutant of CHO cells. 665 96

A series of 8-azaguanine resistant mutants was induced by treatment of V79 Chinese hamster cells with either r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (antiBPDE) or methylnitrosourea (MNU). Hypoxanthine phosphoribosyltransferase (HPRT) activity in the mutants was determined for both hypoxanthine and azaguanine as substrates. With antiserum to purified brain HPRT, cross-reacting material was also determined and analysed by two dimensional polyacrylamide gel electrophoresis. By these criteria mutants induced by anti-BPDE or MNU did not differ appreciably and the data obtained was consistent with the induction of point mutations by both carcinogens. The relevance of these results to the correlation of carcinogenicity with mutagenicity in V79 cells, but not in bacteria, is discussed.
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PMID:Biochemical and immunological characterisation of mutants induced in V79 Chinese hamster cells by a benzo[a]pyrene diolepoxide. 681 Nov 44

The aim of this study was to screen for potential agents affecting the mutagenicity of tobacco products. The influence of a number of compounds which have been suggested to be antimutagenic some of which are present in tobacco products, was investigated on the mutagenicity of a cigarette smoke condensate (CSC) and, in some cases, an extract of oral Swedish moist snuff (SNUS), using a screening procedure of the Ames Salmonella/microsome assay (STY). For some of the compounds the V79/hprt mutagenicity assay with benzo[a]pyrene metabolites as mutagens was used to obtain complementary and confirmatory information on mammalian cells. The antimutagens used included two selenium compounds, sodium selenite and ebselen; the flavonoids and polyphenols, ellagic acid, (+)-catechin hydrate, scopoletin, chlorogenic acid and rutin trihydrate; the porphyrin derivatives, bovine hemin, biliverdine dihydrochloride, chlorophyllin and a plant extract containing chlorophyll; the terpenoids, beta-carotene, retinol and a mixture of the two epimers (4R) and (4S) of (1S,2E,6R,7E,11E)-cembra-2,7,11-triene-4,6-diols (CBD); and cyclohexanol and ubiquinone. Screening of antimutagenic activities using the STY involves problems with toxicity. In several cases in this study mutagenicity was decreased below the control level without signs of toxicity in the background growth of bacteria. Since the survival of mutants and slight bacteriostatic effects on the background growth cannot be determined accurately in the STY, a reduction in mutagenicity may simply be due to toxicity. Only in cases where a dose-response curve declines to a level at or above the background and then levels off, can toxicity be excluded. An antimutagenic effect determined using this test system is therefore often not sufficient for classifying a compound as antimutagenic until these findings are confirmed in other test systems and, preferably, the mechanism behind this effect is clarified. The results obtained with the selenium compounds were considered to be inconclusive since the reduction in the mutation rate declined below the background level and might only reflect the toxic effects of these compounds. For ellagic acid an almost complete inhibition of the mutagenicity of CSC and SNUS in STY was indicated. This indication of antimutagenicity was confirmed in V79 cells using two metabolites of the CSC constituent benzo[a]pyrene, i.e., trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene and (+)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-oxy-7,8,9,10- tetrahydrobenzo[a]pyrene (BPDE). Chlorogenic acid and (+)--catechin reduced the mutagenicity of CSC and chlorogenic acid also strongly inhibited SNUS mutagenicity. Scopoletin and rutin trihydrate inhibited the mutagenicity of CSC, but showed confounding effects with SNUS.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Screening for agents inhibiting the mutagenicity of extracts and constituents of tobacco products. 751 27

DNA repair-deficient (xeroderma pigmentosum group A (XPA)) and DNA repair-proficient (normal) human skin fibroblasts were genetically engineered by transformation with a controllable human cytochrome P450 (CYP)1A1 expression vector. Induction of CYP1A1 enabled these cells to metabolize (+/-)-benzo[a]pyrene-trans-7,8-dihydrodiol (BPD) into a potent cytotoxicant and mutagen. The XPA cells were more susceptible than the normal cells to the cytotoxic effects of both CYP1A1-metabolized BPD and exogenously supplied (+/-)-anti-benzo[a]pyrene-trans-7,8-dihydrodiol-9,10- epoxide (BPDE). Furthermore, the differential cytotoxicity between XPA and normal cells induced by CYP1A1-metabolized BPD was 8.4-fold greater than that induced by exogenously supplied BPDE. The two cell lines had similar CYP1A1 activities, suggesting that a difference in metabolic potential was not the cause of the differential response to BPD. At comparable cytotoxicity in both XPA and normal cells, BPD treatment induced more mutants and more DNA adducts than BPDE treatment did. At similar levels of DNA adducts in XPA cells, the levels of cytotoxicity induced by CYP1A1-metabolized BPD and exogenously supplied BPDE were similar, but CYP1A1-metabolized BPD induced a threefold higher hypoxanthine phosphoribosyltransferase mutation frequency. In contrast, at similar levels of adducts in CYP1A1-expressing normal cells, BPD induced less cytotoxicity and a lower mutation frequency. DNA adducts were identified and quantified by 32P-postlabeling analyses. The principal adduct formed by both CYP1A1-metabolized BPD and exogenously supplied BPDE was 10-beta-(deoxyguanosin-N2-yl)-7 beta,8 alpha,9 alpha-trihydroxy-7,8,9,10- tetrahydrobenzo[a]pyrene, indicating that the differential effects of BPD- and BPDE-induced adducts were not due to a difference in the types of adducts formed. The results of these studies suggest that CYP1A1-metabolized BPD may form adducts preferentially in transcriptionally active genes or that the intracellular concentration of BPDE may influence the balance between cytotoxicity and mutagenicity (or both).
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PMID:Differential mutagenicity and cytotoxicity of (+/-)-benzo[a]pyrene-trans-7,8-dihydrodiol and (+/-)-anti-benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide in genetically engineered human fibroblasts. 766 21

Trans-stilbene oxide, trans-beta-methylstyrene 7,8-oxide, trans-beta-ethylstyrene 7,8-oxide, trans-beta-propylstyrene 7,8-oxide and 4-fluorochalcone oxide were investigated for genotoxic activity in bacterial and mammalian cells, in the absence of external xenobiotic-metabolising systems. All compounds strongly enhanced the frequency of sister-chromatid exchanges (SCE) in cultured human lymphocytes. None of them was mutagenic in Salmonella typhimurium (reversion of the his- strains TA98, TA100 and TA104). The limit of detection was 1/20,000 to 1/10(6) of the activity of the positive control, benzo[a]pyrene 4,5-oxide, depending on the compound and the bacterial strain. Trans-beta-methylstyrene 7,8-oxide and 4-fluorochalcone oxide were additionally tested for induction of SCE and gene mutations in the same target cells, namely Chinese hamster V79 cells. Their influence on the level of SCE was similar to that observed in human lymphocytes, whilst gene mutations (at the hprt locus) were not induced. The four investigated styrene oxide derivatives are known to be excellent substrates for a mammalian enzyme, cytosolic epoxide hydrolase (cEH). 4-Fluorochalcone oxide is a potent selective inhibitor of this enzyme and is structurally similar to the investigated styrene oxide derivatives. These properties of the test compounds however cannot explain the observed discrepancies in the results, since the genetic end point (SCE versus gene mutations) was decisive, and SCE were induced in cEH-proficient human lymphocytes as well as in cEH-deficient V79 cells.
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PMID:Some substrates and inhibitors of cytosolic epoxide hydrolase induce sister-chromatid exchanges in mammalian cells, but do not induce gene mutations in Salmonella typhimurium and V79 cells. 769 7

Cyclopenta[cd]pyrene (CPP) is a widely distributed polycyclic aromatic hydrocarbon with potent mutagenic and carcinogenic activity. In order to acquire an understanding of the mutagenic pathways of CPP, we studied mutations induced by this chemical in human cells. Four independent cultures of a human cell line expressing cytochrome P450 CYP1A1 (cell line MCL-5) were treated with CPP, and mutants at the hypoxanthine phosphoribosyltransferase (HPRT) locus were selected en masse by 6-thioguanine (6TG) resistance. The kinds and positions of the mutations were analyzed using the combination of high-fidelity polymerase chain reaction (hifi-PCR) and denaturing gradient gel electrophoresis (DGGE). The third exon of the HPRT gene was amplified from the 6TG-resistant cells using the hifi-PCR and the amplified fragment was subsequently analyzed by DGGE to separate mutant sequences from the wild-type sequence. Mutant bands were excised from the gel, amplified using PCR and sequenced. Sixteen different mutations were identified and consisted mostly of the G to T and A to T transversions. Other mutations identified included G to A and A to G transitions, a G to C transversion, and a single G deletion. Of these mutations, six occurred within a run of six guanines. The predominance of transversions involving a guanine or an adenine observed with CPP is similar to the data previously reported for the racemic mixtures of benzo[a]pyrene (B[a]P), suggesting that the mechanisms of mutation induced by CPP may be similar to those induced by B[a]P.
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PMID:In vitro mutational spectrum of cyclopenta[cd]pyrene in the human HPRT gene. 772 67

Provisional mutational spectra at the hypoxanthine phosphoribosyl transferase (HPRT) locus in vitro have been worked out for acetaldehyde (AA) and benzo[a]pyrene diolepoxide (BPDE) in human (T)-lymphocytes and for ethylene oxide (EtO) in human diploid fibroblasts using Southern blotting and polymerase chain reaction (PCR)-based DNA sequencing techniques. The results indicate that large genomic deletions are the predominating hprt mutations caused by AA and EO, whereas BPDE induces point mutations that are mainly GC > TA transversions. The mutational spectra induced by the three agents are clearly different from the background spectrum in human T-cells. Thus, the hprt locus is a useful target for the study of chemical-specific mutational events that may help identify causes of background mutation in human cells in vivo.
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PMID:Mutations induced in the hypoxanthine phosphoribosyl transferase gene by three urban air pollutants: acetaldehyde, benzo[a]pyrene diolepoxide, and ethylene oxide. 782 Dec 87

A large number of hprt-mutants were obtained by treating human lymphoblast cells (TK6) with 5 microM K2Cr2O7 for 5 hr and selecting by growth in 6-thioguanine. A combination of high fidelity polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) allowed us to measure mutant frequencies as a function of DNA sequence. Chromium(VI) induced four hotspots in a 104 bp domain of hprt exon 3. Substitutions at G:C base pairs were the predominant mutations. One of the chromium-induced hotspots was located at the same position as previously determined hydrogen peroxide and benzo(a)pyrene diol epoxide hotspots.
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PMID:Use of denaturing-gradient gel electrophoresis to study chromium-induced point mutations in human cells. 784 3


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