Molecular analysis of spontaneous and ethyl methanesulphonate-induced mutations of the hprt gene in hamster cells

Independent spontaneous or ethyl methanesulphonate (EMS)-induced mutants lacking HPRT enzyme activity were analysed for changes in hprt gene structure. Of 21 spontaneous mutants, 6 had total gene deletions, 2 had partial gene deletions, and 13 were indistinguishable from wild-type by Southern analysis. In contrast a sample of 23 EMS-induced mutants, each of which showed potentially interesting characteristics (e.g. high reversion frequency, X-chromosome rearrangement), showed no detectable hprt gene changes. RNA isolated from 59 mutants with presumptive point mutations (13 spontaneous, 46 EMS-induced) was analysed on dot blots for changes in the amount of hprt mRNA. A wide range of mRNA levels was found, from mutants with undetectable amounts to those with more than wild-type amounts. However, Northern blots of all these mutant RNAs revealed only one (EMS-induced) mutation with a change in hprt mRNA size. Taken with our previously-published data on these mutants, it is argued that they represent a broad range of mutational types, and that the hprt gene mutation system provides a sensitive means of distinguishing mutational spectra of different DNA-damaging agents.     

In vivo hprt mutant frequencies in T-cells of normal human newborns

Mutation at the hypoxanthine-guanine phosphoribosyl transferase locus (hprt; HPRT enzyme) in the human fetus was studied by clonal assay of placental cord blood samples from full-term newborns. Conditions for determining hprt mutant frequencies, as defined for adults, were also optimal for studies in newborns. The mean mutant frequency for 45 normal human newborns (37 male, 8 female) was 0.64 X 10(-6) (SD = 0.41 X 10(-6); median value = 0.58 X 10(-6). These values are approx. 10-fold lower than corresponding adult hprt mutant frequency values. Factors such as limiting-dilution cloning efficiencies, delay prior to study of sample, sex, cryopreservation or technician performing the assay did not significantly affect assay results. Maternal smoking did not result in elevated mutant frequency values. Most wild-type and mutant clones studied were CD4 surface antigen positive (helper/inducer). All hprt mutants analyzed lacked HPRT activity.     

The frequency of illegitimate TCRbeta/gamma gene recombination in human lymphocytes: influence of age, environmental exposure and cytostatic treatment, and correlation with frequencies of t(14;18) and hprt mutation.

Chromosome translocations in lymphoid malignancies often involve V(D)J recombinase mediated events giving rise to aberrant T-cell receptor (TCR) and immunoglobulin genes, which have been suggested to be useful as markers of genomic instability, genotoxic exposure and cancer risk. Illegitimate rearrangements involving the TCRbeta/gamma loci on chromosome 7 create TCRbeta/gamma hybrid genes which occur at low frequency in peripheral blood lymphocytes (PBLs) of normal healthy individuals. To evaluate the utility of this marker, we studied the possible effects of age and genotoxic exposures on the TCRbeta/gamma gene variant frequency (VF), and compared the frequencies of hypoxanthine guanine phosphoribosyl transferase (hprt) mutation, hprt exon 2/3 deletion, t(14;18) and TCRbeta/gamma gene rearrangements in cells from the same donors. The TCRbeta/gamma VF ranged five-fold among 16 middle aged blood donors with a mean of 0.74+/-0.29/10(5) PBLs, which is consistent with our previous estimate in healthy subjects. The TCRbeta/gamma VF was found to increase from birth until early adult life, and then to decrease with increasing age. Four testis cancer patients, who 6 years earlier had been treated with etoposide and other cytostatic drugs, showed TCRbeta/gamma VF similar to that in healthy controls. No increase of the TCRbeta/gamma VF was found among non-smoking PAH-exposed aluminum smelter workers compared to non-smoking controls. Smoking smelter workers showed decreased TCRbeta/gamma VF compared to non-smoking workers and controls, but in a follow-up study 2 years later the difference was no longer statistically significant, although the smoking smelter workers still showed a lower TCRbeta/gamma VF than the controls. No correlation was obtained between the TCRbeta/gamma VF and the t(14;18) or hprt mutant frequency (MF) in a group of healthy individuals. However, there was a statistically significant correlation between the TCRbeta/gamma VF and the hprt exon 2/3 deletion frequency in PBL DNA from the same donors. These results show that the TCRbeta/gamma VF in healthy individuals changes with age and correlates with the frequency of hprt exon 2/3 deletion, another marker of aberrant V(D)J recombination in T-cells. However, no effect of smoking or present or previous exposure to genotoxic agents on TCRbeta/gamma VF was observed in this study. Thus, further studies are needed to prove the utility of TCRbeta/gamma gene rearrangement as a marker of genotoxic exposure.     

Use of T-cell receptor gene probes to quantify the in vivo hprt mutations in human T-lymphocytes

T-cell receptor (Ti) gene restriction fragment patterns (RFPs) were determined by Southern blots of genomic DNA obtained from T-lymphocyte colonies isolated from a single normal individual. 4 wild-type colonies and 11 in vivo derived 6-thioguanine-resistant mutant colonies with previously characterized hprt gene structural alterations were studied. Among the hprt mutants, 10 of the 11 showed unique Ti RFPs indicating their origins in different in vivo progenitors. Unique Ti RFPs were also seen among the wild-type T-cell colonies. One, however, shared its Ti RFP with a mutant. These results suggest that mutation in vivo of the hprt gene in human T-lymphocytes occurred after thymic maturation and that the 11 recovered hprt mutants probably resulted from 11 independent mutational events.     

Induction of mutations by bismuth-212 alpha particles at two genetic loci in human B-lymphoblasts.

The human lymphoblast cell line TK6 was exposed to the alpha-particle-emitting radon daughter 212Bi by adding DTPA-chelated 212Bi directly to the cell suspension. Cytotoxicity and mutagenicity at two genetic loci were measured, and the molecular nature of mutant clones was studied by Southern blot analysis. Induced mutant fractions were 2.5 x 10(-5)/Gy at the hprt locus and 3.75 x 10(-5)/Gy at the tk locus. Molecular analysis of HPRT- mutant DNAs showed a high frequency (69%) of clones with partial or full deletions of the hprt gene among radiation-induced mutants compared with spontaneous mutants (31%). Chi-squared analyses of mutational spectra show a significant difference (P < or = 0.005) between spontaneous mutants and alpha-particle-induced mutants. Comparison with published studies of accelerator-produced heavy-ion exposures of TK6 cells indicates that the induction of mutations at the hprt locus, and perhaps a subset of mutations at the tk locus, is a simple linear function of particle fluence regardless of the ion species or its LET.     

The effect of folate deficiency on the hprt mutational spectrum in Chinese hamster ovary cells treated with monofunctional alkylating agents.

Folic acid deficiency acts synergistically with alkylating agents to increase DNA strand breaks and mutant frequency at the hprt locus in Chinese hamster ovary (CHO) cells. To elucidate the mechanism of this synergy, molecular analyses of hprt mutants were performed. Recently, our laboratory showed that folate deficiency increased the percentage of clones with intragenic deletions after exposure to ethyl methanesulfonate (EMS) but not N-nitroso-N-ethylurea (ENU) compared to clones recovered from folate replete medium. This report describes molecular analyses of the 37 hprt mutant clones obtained that did not contain deletions. Folate deficient cells treated with EMS had a high frequency of G>A transitions at non-CpG sites on the non-transcribed strand, particularly when these bases were flanked on both sides by G:C base pairs. Thirty-three percent of these mutations were in the run of six G's in exon 3. EMS-treated folate replete cells had a slightly (but not significantly) lower percentage of G>A transitions, and the same sequence specificity. Treatment of folate deficient CHO cells with ENU resulted in predominantly T>A transversions and C>T transitions relative to the non-transcribed strand. These findings suggest a model to explain the synergy between folate deficiency and alkylating agents: (1) folate deficiency causes extensive uracil incorporation into DNA; (2) greatly increased utilization of base excision repair to remove uracil and to correct alkylator damage leads to error-prone DNA repair. In the case of EMS, this results in more intragenic deletions and G:C to A:T mutations due to impaired ligation of single-strand breaks generated during base excision repair and a decreased capacity to remove O6-ethylguanine. In the case of ENU additional T>A transversions and C>T transitions are seen, perhaps due to mis-pairing of O2-ethylpyrimidines. Correction of folate deficiency may reduce the frequency of these types of genetic damage during alkylator therapy.     

Mutation frequency and specificity with age in liver, bladder and brain of lacI transgenic mice

Mutation frequency and specificity were determined as a function of age in nuclear DNA from liver, bladder, and brain of Big Blue lacI transgenic mice aged 1.5-25 months. Mutations accumulated with age in liver and accumulated more rapidly in bladder. In the brain a small initial increase in mutation frequency was observed in young animals; however, no further increase was observed in adult mice. To investigate the origin of mutations, the mutational spectra for each tissue and age were determined. DNA sequence analysis of mutant lacI transgenes revealed no significant changes in mutational specificity in any tissue at any age. The spectra of mutations found in aging animals were identical to those in younger animals, suggesting that they originated from a common set of DNA lesions manifested during DNA replication. The data also indicated that there were no significant age-related mutational changes due to oxidative damage, or errors resulting from either changes in the fidelity of DNA polymerase or the efficiency of DNA repair. Hence, no evidence was found to support hypotheses that predict that oxidative damage or accumulation of errors in nuclear DNA contributes significantly to the aging process, at least in these three somatic tissues.     

Cancer chemotherapy and somatic cell mutation

The occurrence of a second neoplasm is one of the major obstacles in cancer chemotherapy. The elucidation of the genotoxic effects induced by anti-cancer drugs is considered to be helpful in identifying the degree of cancer risk. Numerous investigations on cancer patients after chemotherapy have demonstrated: (i) an increase in the in vivo somatic cell mutant frequency (Mf) at three genetic loci, including hypoxanthine-guanine phosphoribosyl-transferase (hprt), glycophorin A (GPA), and the T-cell receptor (TCR), and (ii) alterations in the mutational spectra of hprt mutants. However, the time required for and the degree of such changes are quite variable among patients even if they have received the same chemotherapy, suggesting the existence of underlying genetic factor(s). Accordingly, some cancer patients prior to chemotherapy as well as patients with cancer-prone syndrome have been found to show an elevated Mf. Based on the information obtained from somatic cell mutation assays, an individualized chemotherapy should be considered in order to minimize the risk of a second neoplasm.     

Thioguanine-resistant mutant frequency in T-lymphocytes from a healthy human population.

Resistance to 6-thioguanine in T-lymphocytes was used to study in vivo somatic mutations in normal healthy adults. Donor age had a significant effect on mutant frequency at the hprt locus, showing an increase of 0.09/10(6) cells per year of age. No significant increase was associated with sex of donor, smoking habits, alcohol or coffee/tea intake, or X-ray exposure. The lower mutant frequency seen with contraceptive pill usage was probably due to the age difference between the groups of users and non-users.     

Molecular characterization of hprt mutants induced by low- and hgh-LET radiations in human cells

Southern blotting techniques were employed to examine the spectrum of molecular alterations in DNA induced by internally emitting iodine isotopes and X-rays at and around the hprt locus in a human lymphoblastoid cell line. We analyzed 165 mutant clones using a cDNA probe for the human hprt locus, and 3 anonymous sequenceprobes for regions of the X-chromosome which are linked to hrpt. The results were compared with those for 35 spontaneously arising mutant clones. The majority of ionizing radiation-induced mutants showed changes in the normal restriction patterns at the hprt locus, whereas very few alterations were seen at linked markers along the X chromosome. Total hprt coding sequence deletions comprised 30–48% of the changes observed at this locus, while partial deletions and rearrangements comprised 14–54% of the observed changes. In the case of mutants induced by [^125I]dUrd, a densely ionizing radiation, the spectrum of alterations was dose-dependent; at low doses it was not significantly different from that seen after sparsely ionizing X-ray exposure, whereas a higher proportion of gene deletions and rearrangements occurred after high doses of this incorporated isotope. Changes were rarely observed in the 3 linked markers examined. Overall, these results indicate that the distribution of mutational events at the hprt locus in irradiated human cells may not only be LET-dependent but dose-dependent, and that deletions involving large regions of the X chromosome surrounding the hprt locus are rae events.     

Age-related accumulation of autosomal mutations in solid tissues of the mouse is gender and cell type specific

Most cancers in solid tissues increase with age and invariably contain causal mutations eliminating expression of one or more autosomal tumor suppressor genes. However, very little is known about the effect of age on autosomal mutations, often large in size, in cells of solid tissues. In this study, the frequency and spectrum of autosomal mutations were examined as a function of age for kidney epithelial cells and ear mesenchymal cells in B6D2F1 mice heterozygous for the selectable Aprt locus. Aprt mutant frequencies were found to increase with age in the kidneys of both male and female mice, but at all ages the mutant frequencies were approximately twice as high in the females, which in this strain have shorter lifespans than the males. An age-related increase in Aprt mutant frequencies was also observed for ear cells from female mice, but no significant increases in mutant frequencies were observed for the ear cells of male mice. A molecular analysis showed that the kidney and ear mutational spectra were distinct and that the age-related increases in mutant frequencies did not involve significant shifts in the mutational spectra. In total, the results demonstrate both gender and cell-type-specific patterns of autosomal mutational accumulation as a function of age in two solid tissues of the mouse.     

In vivo mutant frequency of thioguanine-resistant T-cells in the peripheral blood and lymph nodes of melanoma patients

T-cell activation by malignant melanoma would be anticipated to stimulate T-cell proliferation, which in turn has been associated with increasing the likelihood of somatic gene mutation. The purpose of this study was to test the hypothesis that in vivo hypoxanthine guanine phosphoribosyltransferase (hprt) mutant frequencies (MFs) are increased in peripheral blood T-cells from melanoma patients compared to normal controls. Assays were made of 48 peripheral blood samples from melanoma patients with stage 3 (13 patients) and stage 4 (35 patients) disease, 38 normal controls, and of nine tumor bearing lymph nodes. The mean hprt log(10)(MF) in patient peripheral blood was -4.77 (geometric mean hprt MF=17.0x10(-6)) compared to a mean hprt log(10)(MF) of -4.87 (geometric mean hprt MF=13.5x10(-6)) in controls. Although modest, this difference is statistically significant both by t-test (P=0.049) and after adjustment for covariates of age, gender, and cigarette smoking by regression analysis (P=0.001). Among the melanoma patients, the mean log(10)(MF) for the 17 patients who had received potentially genotoxic therapies was not significantly different from the mean log(10)(MF) for the 31 patients not receiving such therapies. The hprt MFs in the nine tumor bearing nodes were compared with MFs in peripheral blood from the same patients and revealed a non-significant (P=0.07) trend for increasing MFs in blood. Furthermore, analyses of T-cell receptor gene rearrangement patterns revealed hprt mutants originating from the same in vivo clone in both peripheral blood and a tumor-bearing node. The finding of elevated hprt MFs not entirely explained by genotoxic therapies in patients compared to controls can be explained either by hypermutability or in vivo T-cell activation. The similar MFs in peripheral blood and tumor bearing lymph nodes, as well as the finding of mutant representatives of the same in vivo T-cell clone in both locations, support monitoring peripheral blood to detect events in the nodes. If in vivo proliferation accounts for the current findings, the hprt deficient (hprt-) mutant fraction in blood may be enriched for T-cells that mediate the host immune response against malignant melanoma. Further studies will characterize the functional reactivity of hprt mutant isolates against melanoma-related antigens.     

DNA repair and mutant frequency in schizophrenia.

The in vivo frequency of mutants resulting from mutation at the hprt locus in human T-lymphocytes was determined with a cloning assay. T-lymphocytes were obtained from 14 individuals diagnosed with schizophrenia and 5 controls. No significant difference in mutant frequency was observed between the 2 groups. In addition, DNA-repair capacity was measured with the unscheduled DNA synthesis technique in lymphocytes from 7 individuals diagnosed with schizophrenia and 7 controls. Repair capacity was determined following treatment with MMS, MNNG, and 20 J/m2 ultraviolet light. No significant differences in DNA repair were observed between the patient and control groups in response to any of the 3 DNA-damaging agents. These results argue against differences between normal and schizophrenic individuals with respect to in vivo mutant frequency or their capacity to repair DNA lesions induced by MMS, MNNG, or ultraviolet radiation.     

Lung-specific mutagenicity and mutational spectrum in B6C3F1 lacI transgenic mice following inhalation exposure to 1,2-epoxybutene

1,3-Butadiene (BD) is carcinogenic and mutagenic in B6C3F1 mice. BD inhalation induces an increased frequency of specific base substitution mutations in the bone marrow and spleen of B6C3F1 lacI transgenic mice. BD is bioactivated to at least three mutagenic metabolites: 1,2-epoxybutene (EB), 1,2-epoxy-3,4-butanediol (EBD), and 1,2,3,4-diepoxybutane (DEB), however, the contribution of these individual metabolites to the in vivo mutational spectrum of BD is uncertain. In the present study, lacI transgenic mice were exposed by inhalation (6h per day, 5 days per week for 2 weeks) to 0 or 29.9ppm of the BD metabolite, EB to assess its contribution to the in vivo mutational spectrum of BD. No increase in lacI mutant frequency was observed in the bone marrow or spleen of EB-exposed mice. The lack of mutagenicity in the bone marrow or spleen likely relate to insufficient levels of EB reaching these tissues. The lacI mutant frequency was increased 2.7-fold in the lungs of EB-exposed mice (mean+/-S.D., 9.9+/-3.0x10(-5)) compared to air control mice (3.6+/-0.7x10(-5)). DNA sequence analysis of 65 and 66 mutants from the lungs of air control and EB-exposed mice, respectively, revealed an increase in the frequency of two categories of base substitution mutation and deletions. Like mice exposed to BD, EB-exposed mice had an increased frequency of A:T-->T:A transversions. However, in contrast to the BD mutational spectra, G:C-->A:T transitions at 5'-CpG-3' sequences, occurred with increased frequency in the EB-exposed mice. The increased frequency of deletions as well as the induction of two tandem mutations and a tandem deletion in the lungs of EB-exposed mice are also inconsistent with previous mutational spectra from BD-exposed mice or EB-exposed cells in culture. We hypothesize that the direct in vivo mutagenicity and further in situ metabolism of EB in the lungs of EB-exposed mice played a prominent role in the generation of the current mutational spectrum.     

Influence of smoking and donor age on the spectrum of in vivo mutation at the HPRT-locus in T lymphocytes of healthy adults

Types and frequencies of in vivo mutation in the hypoxanthine-guanine phosphoribosyl-transferase (HPRT) gene was studied in 142 T cell mutants from 78 healthy nonsmoking and smoking adults with a mean of 65 years. The HPRT mutant frequency in the nonsmokers was 18.7±12.0×10⁻⁶, and in the smokers 26.6±18.5×10⁻⁶ (mean±S.D., P<0.01). Among 107 single base pair substitutions (SBS) in the coding region of the HPRT gene, one new mutable site, one novel nonsense mutation and three not previously reported SBS were identified. Transitions accounted for 59% of the SBS and transversions for 41%. GC>AT transitions were the predominant type of mutation, with 50% of all SBS. The mutations showed a nonrandom distribution along the coding sequence, with three significant hotspots at positions 143, 197 and 617 (13, 14 and 7 mutations, respectively). There was no difference between smokers and nonsmokers with regard to the distribution of mutations at these hotspot positions. However, 85% of the mutations at GC base pairs and 88% of the mutations at AT base pairs in smokers occurred at sites with guanine or thymine, respectively, in the nontranscribed DNA strand. Moreover, smokers had a higher frequency of transversions and lower frequency of transitions than nonsmokers did. Particularly, GC>TA transversions were increased in smokers (11%) compared to nonsmokers (2%), which suggests that tobacco-smoke induced adducts at guanine bases in the nontranscribed DNA strand contributes to the increase of HPRT mutation in smokers. Overall, these results were very similar to the mutational spectra in two younger study populations reported previously [K.J. Burkhart-Schultz, C.L. Thompson, I.M. Jones, Spectrum of somatic mutation at the hypoxanthine phosphoribosyltransferase (HPRT) gene of healthy people, Carcinogenesis 17 (1996) 1871–1883; A. Podlutsky, A.-M. Österholm, S.-M. Hou, A. Hofmaier, B. Lambert, Spectrum of point mutations in the coding region of the hypoxanthine-guanine phosphoribosyltransferase, Carcinogenesis 19 (1998) 557–566]. With the possible exception of an increase of mutations at hotspot position 143, and a decrease of 5-methylcytosine deamination mediated transitions at CpG-sites in the older individuals, there were no differences between the mutational spectra of old and young adults. In conclusion, both smoking and ageing seem to have minor influences on the spectrum of HPRT mutation in T cells.     

Mutations induced by benzo[a]pyrene diolepoxide at the hprt locus in human T-lymphocytes in vitro

Human T-lymphocytes have been treated with benzo[a]pyrene diolepoxide (BPDE) in vitro and T-cell clones mutated in the hprt gene have been isolated. The mutant frequencies in BPDE-treated T-cell cultures were on average 24-fold higher than those of untreated cultures. Thus, BPDE is a potent inducer of gene mutation in this system. In order to examine which types of mutations are induced by BPDE in human cells, 41 spontaneous and 44 BPDE-induced mutant clones have been characterized using the Southern blot technique. In addition, rearrangements of the T-cell-receptor beta and gamma loci have been used to determine the proportion of isolated clones that are unique, and thus likely to represent independent mutational events. Out of 23 independent spontaneous mutants 4 had large hprt alterations that could be detected on Southern blots. Two of these alterations, deletions of exons 2-6, have been confirmed using PCR of hprt cDNA and direct sequencing of the PCR product. All 33 independent BPDE-induced mutants had normal hprt restriction patterns which indicates that BPDE is mainly a point mutagen in this system.     

Molecular analysis of hprt mutants induced by 2-cyanoethylene oxide in human lymphoblastoid cells

The mutagenic epoxide metabolite of acrylonitrile, 2-cyanoethylene oxide (ANO), was used to treat human TK6 lymphoblasts (150 microM x 2 h ANO). A collection of hypoxanthine-phosphoribosyltransferase (hprt) mutants was isolated and characterized by dideoxy sequencing of cloned hprt cDNA. Base-pair substitution mutations in the hprt coding region were observed in 19/39 of hprt mutants: 11 occurred at AT base pairs and 8 at GC base pairs. Two -1 frameshift mutations involving GC bases were also observed. Approximately half (17/39) of the hprt mutants displayed the complete loss of single and multiple exons from hprt cDNA, as well as small deletions, some extending from exon/exon junctions. Southern blot analysis of 5 mutants with single exon losses revealed no visible alterations. Analysis of 1 mutant missing exons 3-6 in its hprt mRNA revealed a visible deletion in the corresponding region in its genomic DNA. The missing exon regions of 4 mutants (one each with exons 6, 7 and 8 loss and one mutant with a 17-base deletion of the 5' region of exon 9) were PCR amplified from genomic DNA and analyzed by Southern blot using exon-specific probes. The exons missing from the hprt mRNA were present in the genomic hprt sequence. DNA sequencing of the appropriate intron/exon regions of hprt genomic DNA from a mutant with exon 8 loss and a mutant exhibiting aberrant splicing in exon 9 revealed point mutations in the splice acceptor site of exon 8 (T----A) and exon 9 (A----G), respectively.     

DNA sequence analysis of 1-nitropyrene-4,5-oxide and 1-nitropyrene-9,10-oxide induced mutations in the hprt gene of Chinese hamster ovary cells

Nitropyrene, the predominant nitropolycyclic hydrocarbon found in diesel exhaust, is a mutagenic and tumorigenic environmental pollutant that requires metabolic activation via nitroreduction and ring oxidation. In order to determine the role of ring oxidation in the mutagenicity of 1-nitropyrene, its oxidative metabolites, 1-nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide, were synthesized and their mutation spectra were determined in the coding region of hprt gene of CHO cells by a PCR amplification of reverse-transcribed hprt mRNA, followed by a DNA sequence analysis. A comparison of the two metabolites for mutation frequencies showed that 1-nitropyrene 9,10-oxide was 2-times higher than 1-nitropyrene 4,5-oxide. The mutation spectrum for 1-nitropyrene 4,5-oxide was base substitutions (33/49), one base deletions (11/49) and exon deletions (5/49). In the case of 1-nitropyrene 9,10-oxide, base substitutions (27/50), one base deletions (15/50), and exon deletions (8/50) were observed. Base substitutions were distributed randomly throughout the hprt gene. The majority of the base substitutions in mutant from 1-nitropyrene 4,5-oxide treated cells were A-->G transition (15/33) and G-->A transition (8/33). The predominant base substitution, A-->G transition (11/27) and G-->A transition (8/27), were also observed in mutant from 1-nitropyrene 9,10-oxide treated cells. The mutation at the site of adenine and guanine was consistent with the previous results, where the sites of DNA adduct formed by these compounds were predominant at the sites of purines. A comparison of the mutational patterns between 1-nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide showed that there were no significant differences in the overall mutational spectrum. These results indicate that each oxidative metabolite exhibits an equal contribution to the mutagenicity of 1-nitropyrene, and ring oxidation of 1-nitropyrene is an important metabolic pathway to the formation of significant lethal DNA lesions.     

Databases and software for the analysis of mutations in the human p53 gene, human hprt gene and both the lacI and lacZ gene in transgenic rodents.

We have created databases and software applications for the analysis of DNA mutations at the human p53 gene, the human hprt gene and both the rodent transgenic lacI and lacZ loci. The databases themselves are stand-alone dBASE files and the software for analysis of the databases runs on IBM-compatible computers with Microsoft Windows. Each database has a separate software analysis program. The software created for these databases permit the filtering, ordering, report generation and display of information in the database. In addition, a significant number of routines have been developed for the analysis of single base substitutions. One method of obtaining the databases and software is via the World Wide Web. Open the following home page with a Web Browser: http://sunsite.unc.edu/dnam/mainpage. html . Alternatively, the databases and programs are available via public FTP from: anonymous@sunsite.unc.edu. There is no password required to enter the system. The databases and software are found beneath the subdirectory: pub/academic/biology/dna-mutations. Two other programs are available at the site, a program for comparison of mutational spectra and a program for entry of mutational data into a relational database.