Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P00492 (hypoxanthine-guanine phosphoribosyltransferase)
 2,385 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Conjugation and detoxification of mixed function oxidase (MFO)-mediated benzo(a)pyrene [B(a)P] metabolites with glucuronic acid and glutathione (GSH) are major pathways of B(a)P elimination and ultimately excretion in vivo. We have studied the effects of uridine diphosphate alpha-D-glucuronic acid (UDPGA) and GSH, a cofactor for the synthesis of glucuronide and GSH conjugates, respectively, on B(a)P-induced cytotoxicity and mutagenicity in mammalian cells. The S9-mix used in the Chinese hamster ovary cell/hypoxanthine-guanine phosphoribosyltransferase (CHO/HPRT) mutational assay was supplemented with either UDPGA, GSH, or GSH plus purified GSH-S-transferases (GSHTs), to study modulation of glucuronide and GSH detoxification mechanisms on B(a)P-induced cytotoxic and mutagenic effects. We found that the addition of UDPGA to S9-mix reduces cytotoxicity induced by either B(a)P or B(a)P 6-OH but not by B(a)P 7,8-diol [B(a)P-diol]. The reduction of B(a)P and B(a)P 6-OH-induced cytotoxicity by glucuronide conjugation is likely due to elimination of cytotoxic phenols and quinones. The addition of GSH to the S9-mix resulted in a reduction of B(a)P- and B(a)P-diol-induced cytotoxicity. GSH plus GSHT reduced B(a)P-induced cytotoxicity and mutagenicity. GSH inhibited the mutagenicity at low concentrations of B(a)P-diol. GSH plus GSHTs inhibited the cytotoxicity and mutagenicity of B(a)P-diol at concentrations not affected by GSH alone. These studies demonstrate that mechanisms of detoxification can affect the biological activity of B(a)P and B(a)P-diol as profoundly as bioactivation by the MFO system. Future research should address studies of mutagenicity modulation by metabolic effectors at both the molecular (DNA sequence) and cellular (quantitative mutagenesis) level.
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PMID:Modulative effects of metabolic effectors on benzo(a)pyrene-induced cytotoxicity and mutagenicity in mammalian cells. 785 67

The molecular basis for putative aberrant splicing of hypoxanthine (guanine) phosphoribosyltransferase (hprt) pre-mRNA in Chinese hamster V-79 cells was determined for 75 independent (+)-7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]-pyrene [(+)-BPDE]-induced and 6 spontaneous 8-azaguanine-resistant mutant clones that had exon deletions in their hprt cDNA. Genomic DNA fragments corresponding to the missing exons and their flanking intron regions were amplified by PCR and sequenced. The results indicated that each of these mutants generated a normal-sized PCR product and resulted from aberrant splicing. For (+)-BPDE-induced aberrant splicing mutants, 81% (61 of 75 clones) had base substitution mutations, 5% (4 of 75 clones) had a single base deletion, and 13% (10 of 75 clones) lacked a detectable mutation in the skipped exon, its flanking intron sequences, or in the upstream donor site of the preceding intron. All mutations at a splice donor site resulted in skipping of the entire upstream neighboring exon, whereas alterations at a splice acceptor site caused skipping of the downstream neighboring exon or activation of a cryptic acceptor site in the downstream exon. Fifty-nine % of the splicing mutants had a mutation occurring at the splice site consensus sequence in the intron, and 28% of the splicing mutants had mutations within exon sequences. Among 21 aberrant splicing mutant clones with a mutation inside an exon sequence, seven were in exon 2, two were in exon 3, and twelve were in exon 4. Evidence is presented that a stemloop structure sequesters the splice donor site of exon 2 in pre-mRNA and plays a role in exon 2 skipping. Mutant clones with mutations stabilizing the proposed stemloop structure inhibited the use of the normal exon 2 splice site which resulted in exon 2 skipping in the hprt mRNA. These mutant clones expressed a mixed population of mRNAs, and both normal-sized and truncated mRNA were formed. Similar to our earlier finding that treatment of V-79 cells with (+)-BPDE resulted in a dose-dependent mutation profile within the coding region of the hprt gene, we also observed the presence of dose-dependence in the profile of (+)-BPDE-induced base substitutions in aberrant splicing mutants. As the dose of (+)-BPDE was decreased, the proportion of base substitution mutations at AT base pairs that affected RNA splicing was increased.
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PMID:Characterization of hprt splicing mutations induced by the ultimate carcinogenic metabolite of benzo[a]pyrene in Chinese hamster V-79 cells. 788 64

When populations of repair-proficient diploid human fibroblasts were treated with (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) during early S phase, just as the hypoxanthine phosphoribosyltransferase gene (HPRT) was being replicated, 5% of the induced base substitutions were found at nt 212, and 5% of the substitutions were found at nt 229 in exon 3. However, when the population was treated in early G1 phase to allow at least 12 hr for repair before the onset of S phase, 21% of the substitutions were found at nt 212, and 10% were found at nt 229. No such cell-cycle-dependent difference in distribution of base substitutions occurred in excision-repair-deficient cells. To test whether the increase in the relative frequency of mutations resulted from inefficient repair at these sites, we adapted ligation-mediated PCR to measure the rates of removal of BPDE adducts from individual sites in exon 3 of the HPRT gene. Cells were treated with 0.5 microM BPDE in early G1 phase and harvested immediately or after 10, 20, and 30 hr for repair. the nontranscribed strand of exon 3 was analyzed for the original distribution of adducts and those remaining after repair, using Escherichia coli UvrABC excinuclease to excise the adducts and annealing a 5' biotinylated gene-specific primer to the DNA and extending it with Sequenase 2.0 to generate a blunt end at the site of each cut. A linker was ligated to the blunt end, and the desired fragments were isolated from the rest of the genomic DNA by using magnetic beads, amplified by PCR, and analyzed on a sequencing gel. The distribution of fragments of particular lengths indicated the relative number of BPDE adducts initially formed or remaining at specific sites. The rates of repair at individual sites varied widely along exon 3 of the HPRT gene and were very slow at nt 212 and 229, strongly supporting the hypothesis that inefficient DNA repair plays an important role in the formation of mutation hotspots.
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PMID:Site-specific rates of excision repair of benzo[a]pyrene diol epoxide adducts in the hypoxanthine phosphoribosyltransferase gene of human fibroblasts: correlation with mutation spectra. 789 48

In the present study 34 agents, related to carcinogenesis in different ways, were investigated with respect to their recombinogenic activity in mammalian cells. The induction of intrachromosomal recombination was studied using the spontaneous mutant clone SP5 derived from V79 Chinese hamster cells, which exhibits a duplication of exon 2 and its flanking regions in the hprt gene, which was found to be inserted between the two EcoR1 sites of intron 1. Earlier studies on the removal of this insertion fragment in the SP5 clone indicated that such loss involved intrachromosomal recombination and was detectable by using a reversion mutation assay. The categories of agents investigated here included monofunctional alkylating agents, polyaromatic hydrocarbons giving rise to bulky adducts, chlorinated compounds giving small cyclic adducts, intercalating agents, DNA cross-linkers, UV and ionizing radiation, inhibitors of DNA synthesis and topoisomerases, DNA bases and base analogues, radical formers and tumour promotors. Statistically significant enhancements in the frequency of reversion in SP5 cells were observed after treatment with aflatoxin B1, 9-aminoacridine, benzo[a]-pyrene-7,8-dihydrodiol, benzo[a]pyrene-7,8-diol-9,10-epoxide, camptothecin, dimethylbenzanthracene, dimethyl-nitrosamine, ethidium bromide, ethylmethanesulfonate, N-ethyl-N'-nitrosourea, fluorodeoxyuridine, ICR 191, N-methyl-N'-nitrosoguanidine, mitomycin C and UV irradiation. Only slight inducing effects were indicated in the case of methylmethanesulfonate, N-methyl-N'-nitrosourea and gamma irradiation, although not statistically significant. Negative results were found after treatment with 3-amino-benzamide, 5-azacytidine, bleomycin, 5-bromodeoxyuridine, 1,2-dichloroethane, ethylene oxide, etoposide, formaldehyde, hydrogen peroxide, methotrexate, propylene oxide, quercitin, sodium azide, 12-O-tetradecanoylphorbol-13-acetate, thymidine and a complex mixture consisting of a cigarette smoke condensate. Our results on chemically or physically induced recombinogenic effects in the endogenous SP5 hprt gene are in agreement with data obtained in non-endogenous gene systems based on transgenic cell lines containing integrates of tandem mutated tk or hygromycin resistance genes, but not completely consistent with findings on integrates based on the neo genes. This suggests that many factors influence the recombination process, including a difference in the mechanisms underlying inter- and intrachromosomal recombination. Consequently, the endogenous SP5/V79 system is suggested to be more representative than integrated systems for investigating induction of recombination, as well as for mechanistic studies of recombination at the molecular level, e.g. intrachromosomal recombination.
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PMID:Studies on intrachromosomal recombination in SP5/V79 Chinese hamster cells upon exposure to different agents related to carcinogenesis. 795 71

Both 1- and 3-nitrobenzo[a]pyrene (nitro-BaP) are environmental contaminants, potent mutagens in Salmonella, and moderate mutagens in Chinese hamster ovary (CHO) cells. The mutagenicity of their oxidized metabolites,trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-epoxy -7,8,9,10-tetrahydro-1-nitrobenzo[a]pyrene (1-nitro-BaP-DE) and trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydro-3-nitrobenzo[a]- pyrene (3-nitro-BaPDE), together with trans-7,8-dihydroxy-anti-9, 10-ep- oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaP-DE), was determined in CHO-K1 cells, and the resulting mutations at the hprt locus were characterized by polymerase chain reaction (PCR) amplification of reverse-transcribed hprt mRNA, followed by DNA sequence analysis. The mutant frequencies, in mutants/10(6) clonable cells, at 30 and 100 ng/ml, were BaP-DE, 248 and 456; 1-nitro-BaP-DE, 68 and 260; 3-nitro-BaP-DE, 81 and 232, respectively. In general, the three diolepoxides exhibited similar mutational spectra: 1) 64% (23/36 sequenced mutants) of BaP-DE, 53% (19/36) of 1-nitro-BaP-DE, and 64% (23/36) of 3-nitro-BaP-DE mutants resulted from simple base pair substitution, with the predominant mutation being G-->T transversion; 2) 90%, 100%, and 100% of mutations at G:C had the mutated dG on the nontranscribed DNA strand; and 3) about one quarter of the mutants produced by each mutagen had one or more PCR products with partial or complete exon deletions. The mutagens induced few frameshifts or complex mutations. Among the differences in mutational specificity for the three diolepoxides, the proportion of substituted dGs with 3' purines was significant (P < 0.05) for BaP-DE (16/19, 84%) and 3-nitro-BaP-DE (17/20, 85%), but not significant for 1-nitro-BaP-DE-induced mutants (11/17, 65%, P > 0.05). Also, high proportions of BaP-DE and 3-nitro-BaP-DE base pair substitutions at G:C occurred in DNA sequence contexts of 5'-GG-3', 5'-GGA-3', and 5'-TGGA-3', while the proportions of 1-nitro-BaP-DE mutants in these contexts were often lower. The results indicate that nitro substitution at C1 or C3 of BaP-DE reduces mutational potency in CHO cells and appears to have only subtle effects upon the mutational pattern in the hprt gene.
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PMID:Molecular characterization of mutation and comparison of mutation profiles in the hprt gene of Chinese hamster ovary cells treated with benzo[a]pyrene trans-7,8-diol-anti-9,10-epoxide, 1-nitrobenzo[a]pyrene trans-7,8-diol-anti-9,10-epoxide, and 3-nitrobenzo[a]pyrene trans-7,8- diol-anti-9,10-epoxide. 862 44

Sulfur dioxide, a ubiquitous air pollutant, is a co-carcinogen for benzo[a]pyrene (BP). We have demonstrated previously that the interaction between sulfite, the physiological form of sulfur dioxide, and (+/-) -7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE), the ultimate carcinogenic form of BP, results in an enhanced mutagenic effect in Salmonella typhimurium strains TA98 and TA100. We report here that this same co-mutagenic effect of sulfite occurs in a mammalian cell line. Treatment of Chinese hamster V79 cells with 50 nM anti-BPDE, a concentration on the linear portion of the dose-response, resulted in a four-fold increase in mutations at the hprt locus relative to the spontaneous rate. When V79 cells were exposed to 1 or 10 mM sulfite immediately prior to the addition of anti-BPDE, the mutation rate increased by 73% and 210%, respectively, over that elicited by anti-BPDE alone. Sulfite itself was moderately cytotoxic, but caused no increase in mutation over the spontaneous rate. Characterization of the dose- and time-dependance of this enhancement of diol epoxide mutagenicity by sulfite closely resembled the effects seen previously in the bacterial system. In particular, enhancement by sulfite was evident when sulfite was added to the cells between 60 min and 1 min prior to the addition of the diol epoxide. Concurrent addition of sulfite and the diol epoxide attenuated the enhancement, and the effect was lost altogether when sulfite was added 10 min after the diol epoxide. The specificity of this effect of sulfite was shown by comparison with sulfate, which at concentrations of either 1 or 10 mM exhibited modest cytotoxicity, but neither was directly mutagenic nor able to enhance the mutagenic effect of anti-BPDE. Binding studies with labeled anti-BPDE showed that the addition of 10 mM sulfite increased binding of anti-BPDE to DNA by over 43%, corresponding to the observed increase in mutant frequency. Interestingly, this difference in level of DNA modification was not apparent after 30 min to 2 h exposures, but only emerged at the 4 h time point. The 4 h point was routinely used for all mutagenicity studies. Binding of anti-BPDE-derived materials to cellular RNA was not altered by 10 mM sulfite. The emergence of increased DNA modification at the latest time point suggests either a more prolonged period of active DNA binding than would occur with diol epoxide, or a difference in the ability to recognize and clear specific DNA adducts. Both possibilities are discussed in regard to the observed formation of 7r,8t,9t-trihydroxy-7,8,9,10-tetrahydrobenzo[a] pyrene-10c-sulfonate (BPT-10-sulfonate) in those incubations. BPT-10-sulfonate is a relatively stable BP derivative which retains the ability to covalently modify DNA. The role of this derivative in the enhancement of diol epoxide mutagenicity by sulfite is strongly suggested by these data.
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PMID:Enhancement of benzo[a]pyrene diol epoxide mutagenicity by sulfite in a mammalian test system. 864 Sep 14

Studies showing that different types of DNA adducts are repaired in human cells at different rates suggest that DNA adduct conformation is the major determinant of the rate of nucleotide excision repair. However, recent studies of repair of cyclobutane pyrimidine dimers or benzo[a]pyrene diol epoxide (BPDE)-induced adducts at the nucleotide level in DNA of normal human fibroblasts indicate that the rate of repair of the same adduct at different nucleotide positions can vary up to 10-fold, suggesting an important role for local DNA conformation. To see if site-specific DNA repair is a common phenomenon for bulky DNA adducts, we determined the rate of repair of 1-nitrosopyrene (1-NOP)-induced adducts in exon 3 of the hypoxanthine phosphoribosyltransferase gene at the nucleotide level using ligation-mediated PCR. To distinguish between the contributions of adduct conformation and local DNA conformation to the rate of repair, we compared the results obtained with 1-NOP with those we obtained previously using BPDE. The principal DNA adduct formed by either agent involves guanine. We found that rates of repair of 1-NOP-induced adducts also varied significantly at the nucleotide level, but the pattern of site-specific repair differed from that of BPDE-induced adducts at the same guanine positions in the same region of DNA. The average rate of excision repair of 1-NOP adducts in exon 3 was two to three times faster than that of BPDE adducts, but at particular nucleotides the rate was slower or faster than that of BPDE adducts or, in some cases, equal to that of BPDE adducts. These results indicate that the contribution of the local DNA conformation to the rate of repair at a particular nucleotide position depends upon the specific DNA adduct involved. However, the data also indicate that the conformation of the DNA adduct is not the only factor contributing to the rate of repair at different nucleotide positions. Instead, the rate of repair at a particular nucleotide position depends on the interaction between the specific adduct conformation and the local DNA conformation at that nucleotide.
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PMID:Site-specific excision repair of 1-nitrosopyrene-induced DNA adducts at the nucleotide level in the HPRT gene of human fibroblasts: effect of adduct conformation on the pattern of site-specific repair. 866 88

The AHH-1 human lymphoblastoid line was exposed to benzo[a]pyrene under markedly different conditions: a single toxic exposure of 30 microM for 28 h, a nontoxic exposure of 0.5 microM for 6 days and an exposure approximating estimates of BP concentration in the human lung of 20 nM for 20 days. Duplicate cultures containing 2 x 10(9) cells each were used to assure the statistical quality of the mutational spectra. Point mutational hotspots were observed in bp 215 to 318 of the third exon of the hprt gene after mutants were selected en masse with 6-thioguanine, using a combination of denaturing gradient gel electrophoresis and high fidelity polymerase chain reaction. The spectra were highly reproducible in replicate experiments but varied dramatically among treatment conditions. These data demonstrate that mutational spectra were critically dependent upon conditions of exposure. The results significantly extend prior reports on this subject and clarify an important issue for the use of mutational spectra obtained in vitro to create hypotheses about what spectra may be expected in humans in vivo. We conclude that commonly used protocols of short-term exposure to mutagenic chemicals at high concentrations should not be used to define such expectations. Rather, the more difficult protocols of long-term and low-concentration mutation studies are justified as conditions necessary, although perhaps not sufficient, to approximate human in vivo mutational pathways.
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PMID:Mutational spectra vary with exposure conditions: benzo[a]pyrene in human cells. 887 96

Benzo[a]pyrenediol-epoxide (BPDE), a metabolite of the ubiquitous environmental carcinogen benzo[a]pyrene (B[a]P), has been implicated as a point mutagen. However, as mutational events other than point mutations are also often associated with cancer, we have investigated whether BPDE can induce other classes of mutation. This was done by analyzing mutation at the aprt and hprt loci, both in hemizygous (D422) and heterozygous (D423) Chinese hamster ovary (CHO) cell strains. Southern blotting analysis indicated that BPDE is not an effective producer of either deletions or insertions in the hemizygous environment. The analysis of mutation in the aprt heterozygote was done to investigate the frequency of loss of heterozygosity (LOH) events following BPDE treatment. Using PCR to produce an artificial restriction fragment length polymorphism in the functional aprt allele, BPDE was found to induce LOH in about one-quarter of the mutants recovered. While the precise mechanism of this phenomenon remains obscure, it is likely to have important implications, since similar events involving homologous recombination in somatic cells may have an impact in tumorigenesis.
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PMID:Benzo[a]pyrenediol-epoxide induces loss of heterozygosity in Chinese hamster ovary cells heterozygous at the aprt locus. 892 79

Big Blue (BB) and generic B6C3F1 mice were given one to three i.p. injections of 50 mg/kg benzo[a]pyrene (B[a]P) in DMSO every other day to achieve cumulative doses of 50 to 150 mg/kg. Three weeks after treatment, the mutation frequency at the endogenous hprt gene and lacI transgene was measured in splenic T cells. Generic mice given 50, 100, and 150 mg/kg B[a]P displayed induced hprt frequencies (observed hprt frequency minus control frequency) of 5.5 +/- 1.0, 11 +/- 2.0, and 19 +/- 2.6 x 10(-6), respectively (average +/- SEM). In contrast, BB mice given 50 and 150 mg/kg B[a]P displayed induced hprt frequencies of 0.9 +/- 0.6 and 9.1 +/- 1.5 x 10(-6). 32P postlabelling revealed that the lower hprt response in BB mice correlated with lower amounts of BP-DNA adducts in spleen, liver, and lung 24 hours after B[a]P exposure. Western blot analysis of liver samples from B[a]P-treated mice suggests that the reduced adduct load in turn may be due to lower P450 1A1 levels in BB mice. The frequency of induced, nonsectored blue plaques (observed blue plaque frequency minus control frequency) in BB mice receiving 50 and 150 mg/kg B[a]P was 41 +/- 9 and 134 +/- 10 x 10(-6) (15- to 40-fold higher than the induced hprt frequency in the same treated animals). Sectored plaques were observed in both control and B[a]P groups but their frequency showed no relationship to dose (sectored frequency in all groups was approximately 20 x 10(-6)). To test whether persistent DNA adducts in the packaged lambda vector were contributing to the observed blue plaque frequency, purified lambda-LIZ DNA was treated in vitro with B[a]P diol epoxide (BPDE), packaged, and plated on E. coli lawn cells. Treatment with BPDE did not produce significant increases in homogeneous blue plaques, suggesting that the majority of mutants obtained from B[a]P-treated BB mice occurred in vivo. These results indicate that B[a]P exposure produces many more mutations at the lacI transgene than at the endogenous hprt locus.
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PMID:Mutagenic response of the endogenous hprt gene and lacI transgene in benzo[a]pyrene-treated Big Blue B6C3F1 mice. 899 Oct 66


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