Mutagenicity and clastogenicity of proflavin in L5178Y/TK +/- -3.7.2.C cells

We evaluated the ability of proflavin to induce specific-locus mutations at the heterozygous thymidine kinase (tk) locus of L5178Y/TK +/- -3.7.2C mouse lymphoma cells, which appears to permit the recovery of mutants due to single-gene and chromosomal mutations. Proflavin was highly mutagenic at the tk locus, producing 724-965 TK mutants/10(6) survivors (background = 56-85/10(6); survival = 29-32%). Most of the mutants were small colonies, which suggested that proflavin may induce chromosomal mutations. The potent clastogenicity of proflavin was confirmed by cytogenetic analysis for chromosomal aberrations. At the highest dose analyzed (1.5 micrograms/ml), proflavin produced 82 aberrations/100 metaphaes (background = 2/100). The large-colony TK mutant frequency produced by proflavin (48-109/10(6) survivors; background = 23/10(6); survival = 57-61%) was similar to published HPRT mutant frequencies produces by proflavin in L5178Y and CHO cells (50-100/10(6) survivors; background = 2-50/10(6); survival = 50-62%). These results lead to the conclusion that proflavin is a potent clastogen and induces a high frequency of small-colony TK mutants; however, it induces a low frequency of HPRT mutants and a low frequency of large-colony TK mutants.     

Molecular changes in UV-induced and γ-ray-induced mutations in human lymphoblastoid cells

We have characterized the structural changes in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene of 14 UV-induced, 15 γ-ray-induced and 17 spontaneous mutants of human lymphoblastoid cells selected for 6-thioguanine (6TG) resistance. Southern blot analysis using the full-length HPRT cDNA as a probe revealed that 29% (5/17) of the spontaneous mutants contained detectable alterations in their restriction fragment patterns. Among the 15 mutants induced by γ rays, 7 (47%) had such alterations indicative of large deletions in the HPRT gene. In contrast, all 14 UV-induced mutants exhibited hybridization patterns indistinguishable from those of the wild-type cells. These results suggest that UV is likely to induce point mutations at the HPRT locus on the human chromosome and that the molecular mechanism of UV-induced mutation is quite different from that of ionizing radiation-induced mutation or spontaneous mutation in human cells.     

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.     

Radiation-induced HPRT mutations resulting from misrejoined DNA double-strand breaks

DNA double-strand breaks (DSBs) are the most severe lesions induced by ionizing radiation, and unrejoined or misrejoined DSBs can lead to cell lethality, mutations and the initiation of tumorigenesis. We have investigated X-ray- and alpha-particle-induced mutations that inactivate the hypoxanthine guanine phosphoribosyltransferase (HPRT) gene in human bladder carcinoma cells and in hTERT-immortalized human fibroblasts. Fifty to 80% of the mutants analyzed exhibited partial or total deletions of the 9 exons of the HPRT locus. The remaining mutants retained unaltered PCR products of all 9 exons but often displayed a failure to amplify the HPRT cDNA. Hybridization analysis of a 2-Mbp NotI fragment spanning the HPRT gene with a probe 200 kbp distal to the HPRT locus indicated altered fragment sizes in most of the mutants with a wild-type PCR pattern. These mutants likely contain breakpoints for genomic rearrangements in the intronic sequences of the HPRT gene that allow the amplification of the exons but prevent HPRT cDNA amplification. Additionally, mutants exhibiting partial and total deletions of the HPRT exons also frequently displayed altered NotI fragments. Interestingly, all mutations were very rarely associated with interchromosomal exchanges analyzed by FISH. Collectively, our data suggest that intrachromosomal genomic rearrangements on the Mbp scale represent the prevailing type of radiation-induced HPRT mutations.     

Genotoxicity of 3'-azido-3'-deoxythymidine in the human lymphoblastoid cell line, TK6: relationships between DNA incorporation, mutant frequency, and spectrum of deletion mutations in HPRT.

Perinatal treatment with 3'-azido-3'-deoxythymidine (AZT) has been found to reduce the rate of maternal-infant transmission of HIV; however, AZT is genotoxic in mammalian cells in vitro and induces tumors in the offspring of mice treated in utero. The purpose of the present study was to investigate the relationships between incorporation of AZT into DNA, and the frequency and spectrum of mutations at the HPRT locus of the human lymphoblastoid cell line, TK6, following in vitro exposures to AZT. Cells were cultured in medium containing 0 or 300 microM AZT for 1, 3, or 6 day(s) (n = 5/group). The effects of exposure duration on incorporation of AZT into DNA and HPRT mutant frequency were determined using an AZT radioimmunoassay and a cell cloning assay, respectively. AZT accumulated in DNA in a supralinear manner, approaching a plateau at 6 days of treatment (101.9 +/- 14.7 molecules AZT/10(6) nucleotides). After 3 days of AZT exposure, HPRT mutant frequency was significantly increased (1.8-fold, p = 0.016) compared to background (mutant frequency = 3.78 x 10(-6)). Multiplex PCR amplification of genomic DNA was used to determine the frequency of exon deletions in HPRT mutant clones from untreated cells versus AZT-treated cells. Molecular analyses of AZT-induced mutations revealed a significant difference in the frequency of total gene deletions (44/120 vs. 18/114 in controls, p = 0.004 by the Mann-Whitney U-statistic). In fact, the Chi-square test of homogeneity demonstrate that the differences between the control and AZT-treatment groups is attributed mainly to this increase in total gene deletion mutations (p = 0.00001). These data indicate that the primary mechanism of AZT mutagenicity in human TK6 cells is through the production of large deletions which occur as a result of AZT incorporation into DNA and subsequent chain termination. The data imply that perinatal chemoprophylaxis with AZT may put children of HIV-infected women at potential risk for genetic damage.     

Dose-dependent changes in the spectrum of mutations induced by ionizing radiation

We examined the influence of dose on the spectrum of mutations induced at the hypoxanthine guanine phosphoribosyltransferase (Hprt) locus in Chinese hamster ovary (CHO) cells. Independent CHO-K1 cell mutants at the Hprt locus were isolated from cells exposed to 0, 0.5, 1.5, 3.0 and 6.0 Gy (137)Cs gamma rays, and the genetic changes responsible for the mutations were determined by multiplex polymerase chain reaction (PCR)-based exon deletion analysis. We observed dose-dependent changes in mutation spectra. At low doses, the principal radiation-induced mutations were point mutations. With increasing dose, multibase deletion mutations became the predominant mutation type such that by 6.0 Gy, there were almost three times more deletion mutations than point mutations. The dose response for induction of point mutations was linear while that for multibase deletions fit a linear-quadratic response. There was a biphasic distribution of deletion sizes, and different dose responses for small compared to large deletions. The frequency of large (>36 kb) total gene deletions increased exponentially, implying that they develop from the interaction between two independent events. In contrast, the dose response for deletion mutations of less than 10 kb was nearly linear, suggesting that these types of mutations develop mostly from single events and not the interactions between two independently produced lesions. The observation of dose-dependent changes in radiation-induced mutation spectra suggests that the types of alterations and therefore the risks from low-dose radiation exposure cannot be easily extrapolated from high-dose effects.     

Illegitimate recombination induced by DNA double-strand breaks in a mammalian chromosome.

We examined DNA double-strand-break-induced mutations in the endogenous adenine phosphoribosyl-transferase (APRT) gene in cultured Chinese hamster ovary cells after exposure to restriction endonucleases. PvuII, EcoRV, and StuI, all of which produce blunt-end DNA double-strand breaks, were electroporated into CHO-AT3-2 cells hemizygous at the APRT locus. Colonies of viable cells containing mutations at APRT were expanded, and the mutations that occurred during break repair were analyzed at the DNA sequence level. Restriction enzyme-induced mutations consisted of small deletions of 1 to 36 bp, insertions, and combinations of insertions and deletions at the cleavage sites. Most of the small deletions involved overlaps of one to four complementary bases at the recombination junctions. Southern blot analysis revealed more complex mutations, suggesting translocation, inversion, or insertion of larger chromosomal fragments. These results indicate that blunt-end DNA double-strand breaks can induce illegitimate (nonhomologous) recombination in mammalian chromosomes and that they play an important role in mutagenesis.     

Molecular analysis of spontaneous mutations at the gpt locus in Chinese hamster ovary (AS52) cells

AS52 cells are Chinese hamster ovary (CHO) cells that carry a single functional copy of the bacterial gpt gene and allow the isolation of 6-thioguanine-resistant (6TGr)mutants arising from mutation at the chromosally integrated gpt locus. The gpt locus in AS52 cells is extremely stable, giving rise to 6TGr mutants at frequencies comparable to the endogenous CHO hprt locus. In this study, we describe the spectrum of spontaneous mutations observed in AS52 cells by Southern blot and DNA sequence analyses. Using the polymerase chain reaction (PCR) and the Thermus aquaticus (Taq) polymerase, we have enzymatically amplified 6TGr mutant gpt sequences in vitro. The PCR product was then sequenced without further cloning manipulations to directly identify gpt structural gene mutations. Deletions predominant among the 62 spontaneous 6TGr-AS52 mutant clones analyzed in this study. Of these, 79% (49/62) of the mutations were identified as deletions either by Southern blotting, PCR amplification or DNA sequence analysis. Among these deletions is a predominant 3-base deletion that was observed in 31% (19/62) of the mutants. These data provide a basis for future comparisons of induced point mutational spectra derived in the AS52 cell line, and demonstrate the utility of PCR in the generation of DNA sequence spectra derived from chromosomally integrated mammalian loci.     

Transformation of the gene for hypoxanthine phosphoribosyltransferase.

Purified DNA from wild-type Chinese ovary (CHO) cells has been used to transform three hypoxanthine phosphoribosyltransferase (HPRT) deficient murine cell mutants to the enzyme positive state. Transformants appeared at an overall frequency of 5 x 10(-8) colonies/treated cell and expressed CHO HPRT activity as determined by electrophoresis. One gene recipient, B21, was a newly isolated mutant of LMTK- deficient in both HPRT and thymidine kinase (TK) activities. Transformation of B21 to HPRT+ occurred at 1/5 the frequency of transformation to TK+; the latter was, in turn, an order of magnitude lower than that found in the parental LMTK- cells, 3 x 10(-6). Thus both clonal and marker-specific factors play a role in determining transformability. The specific activity of HPRT in transformant extracts ranged from 0.5 to 5 times the CHO level. The rate of loss of the transformant HPRT+ phenotype, as measured by fluctuation analysis, was 10(-4)/cell/generation. While this value indicates stability compared to many gene transferents, it is much greater than the spontaneous mutation rate at the indigenous locus. The ability to transfer the gene for HPRT into cultured mammalian cells may prove useful for mutational and genetic mapping studies in this well-studied system.     

Intervention of somatic mutational events in vivo by a germline defect at the adenine phosphoribosyltransferase locus

Both germline and somatic mutations are known to affect phenotypes of human cells in vivo. In previous studies, we cloned mutant peripheral blood T cells from germline heterozygous humans for adenine phosphoribosyltransferase (APRT) (EC 2.4.2.7) deficiency and found that approximately 1.3 × 10^–4 peripheral T cells had undergone in vivo somatic mutations. Loss of heterozygosity (LOH) was the major cause of the mutations at the APRT locus since approximately 80% of the mutant T cell clones exhibited loss of normal alleles. In the present study, we identified three heterozygous individuals for APRT deficiency (representing two separate families), in whom none of the somatic mutant cells exhibited LOH at the APRT locus. The germline mutant APRT alleles of these heterozygotes from two unrelated families had the same gross DNA abnormalities detectable by Southern blotting. None of the germline mutant APRT alleles so far reported had such gross DNA abnormalities. The data suggest that the germline mutation unique to these heterozygous individuals is associated with the abrogation of LOH in somatic cells. The absence of LOH at a different locus has already been reported in vitro in an established cell line but the present study describes the first such event in vivo in human individuals. Received: 10 May 1996     

The effect of folate deficiency on the cytotoxic and mutagenic responses to ethyl methanesulfonate in human lymphoblastoid cell lines that differ in p53 status

Folic acid deficiency acts synergistically with alkylating agents to increase genetic damage at the HPRT locus in Chinese hamster ovary cells in vitro and in rat splenocytes in vivo. The present studies extend these observations to human cells and, in addition, investigate the role of p53 activity on mutation induction. The human lymphoblastoid cell lines TK6 and WTK1 are derived from the same parental cell line (WI-L2), but WTK1 expresses mutant p53. Treatment of folate-replete or deficient WTK1 and TK6 cells with increasing concentrations (0-50microg/ml) of ethyl methanesulfonate (EMS) resulted in significantly different HPRT mutation dose-response relationships (P<0.01), indicating that folate deficiency increased the EMS-induced mutant frequency in both cell lines, but with a greater effect in TK6 cells. Molecular analyses of 152 mutations showed that the predominant mutation (65%) in both cell types grown in the presence or absence of folic acid was a G>A transition on the non-transcribed strand. These transitions were mainly at non-CpG sites, particularly when these bases were flanked 3' by a purine or on both sides by G:C base pairs. A smaller number of G>A transitions occurred on the transcribed strand (C>T=14%), resulting in 79% total G:C>A:T transitions. There were more genomic deletions in folate-deficient (15%) as compared to replete cells (4%) of both cell types. Mutations that altered RNA splicing were common in both cell types and under both folate conditions, representing 33% of the total mutations. These studies indicate that cells expressing p53 activity exhibit a higher rate of mutation induction but are more sensitive to the toxic effects of alkylating agents than those lacking p53 activity. Folate deficiency tends to reduce toxicity but increase mutation induction after EMS treatment. The p53 gene product did not have a major influence on the molecular spectrum after treatment with EMS, while folate deficiency increased the frequency of deletions in both cell types.     

Analysis of repair processes by the determination of the induction of cell killing and mutations in two repair-deficient Chinese hamster ovary cell lines

Two UV sensitive DNA-repair-deficient mutants of Chinese hamster ovary cells (43-3B and 27-1) have been characterized. The sensitivity of these mutants to a broad spectrum of DNA-damaging agents: UV254nm, 4-nitroquinoline-1-oxide (4NQO), X-rays, bleomycin, ethylnitrosourea (ENU), ethyl methanesulphonate (EMS), methyl methanesulphonate (MMS) and mitomycin C (MMC) has been determined. Both mutants were not sensitive to X-rays and bleomycin. 43-3B was found to be sensitive to 4NQO, MMC and slightly sensitive to alkylating agents. 27-1 was sensitive only to alkylating agents. The results suggest the existence of two repair pathways for UV-induced cytotoxicity: one pathway which is also used for the removal of 4NQO and MMC adducts and a second pathway which is also used for the removal of alkyl adducts. Parallel to the toxicity, the induction of mutations at the HPRT and Na+/K+-ATPase loci was determined. The increased cytotoxicity to UV, MMC and 4NQO in 43-3B cells and the increased cytotoxicity to UV in 27-1 cells correlated with increased mutability. It was observed that the increase in mutation induction at the HPRT locus was higher than that at the Na+/K+-ATPase locus. As only point mutations give rise to viable mutants at the Na+/K+-ATPase locus the lower mutability at this locus suggests that defective excision repair increases the chance for deletions. Despite an increased cytotoxicity to ENU in 27-1 cells the mutation induction by ENU was the same in 27-1 and wild-type cells at both loci, which suggests that the mutations are mainly induced by directly miscoding adducts (e.g. O-6 alkylguanine), which cannot be removed by CHO cells. As EMS and MMS treatment of 27-1 cells caused an increase in mutation induction at the HPRT locus and a decrease at the Na+/K+-ATPase locus it indicates that these agents induce a substantial fraction of other mutagenic lesions, which can be repaired by wild-type cells. This suggests that O-6 alkylation is not the only mutagenic lesion after treatment with alkylating agents.     

Induction of TK mutations in human lymphoblastoid TK6 cells by the rat carcinogen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX)

3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a chlorine disinfection by-product in drinking water, is carcinogenic in rats and genotoxic in mammalian cells in vitro. In the current study, the mechanism of genotoxicity of MX in human lymphoblastoid TK6 cells was investigated by use of the Comet assay, the micronucleus test, and the thymidine kinase (TK) gene-mutation assay. MX induced a concentration-dependent increase in micronuclei and TK mutations. The lowest effective concentrations in the MN test and the TK gene-mutation assay were 37.5μM and 25μM, respectively. In the Comet assay, a slight although not statistically significant increase was observed in the level of DNA damage induced by MX in the concentration range of 25-62.5μM. Molecular analysis of the TK mutants revealed that MX induced primarily point mutations or other small intragenic mutations (61%), while most of the remaining TK mutants (32%) were large deletions at the TK locus, leading to the hemizygous-type loss-of-heterozygosity (LOH) mutations. These findings show that aside from inducing point mutations, MX also generates LOH at the TK locus in human cells and may thus cause the inactivation of tumour-suppressor genes by LOH.     

In vivo human somatic mutation: frequency and spectrum with age.

The number and molecular nature of in vivo mutations in relation to age was studied at the autosomal HLA-A locus in human lymphocytes. Mutant lymphocytes were isolated by immunoselection, cloned at limiting dilution and enumerated, and the HLA-A gene and other polymorphic gene loci on chromosome 6 were studied by Southern blotting to determine gene dosage and loss of heterozygosity. Results of 167 assays in 73 individuals showed that the total number of mutant lymphocytes increased significantly with age from a geometric mean frequency of 0.71 x 10(-5) in neonates to 6.53 x 10(-5) in elderly individuals. Analysis of rearrangement of T lymphocyte receptor beta or gamma chain genes gave a best estimate of 3.3% for the proportion of mutant lymphocytes detected which are clonally related. Molecular study of 434 mutants from 31 individuals showed no change on Southern blotting in 64.7%, gene deletion in 2.8% and mitotic recombination in 32.5%. Two mutants due to gene conversion but no mutants due to non-disjunction were detected. The number of 'no change' and recombination mutants increased significantly with age. There was a significant difference between individuals in the proportion of mutants which resulted from mitotic recombination and the data suggested that the proportion was bimodally distributed. The point of crossing-over in recombination mutants was predominantly randomly distributed between the HLA-A locus and the centromere.     

Elevated frequency of microsatellite mutations in TK6 human lymphoblast clones selected for mutations at the thymidine kinase locus.

A major question in carcinogenesis is, How can a normal cell accumulate multiple mutations in different genes on different chromosomes, when the mutation rate of each gene is in the range of 10(-8) to 10(-5) per cell division? We hypothesize that many mutations may not be isolated events but rather are accompanied by concomitant mutations elsewhere in the genome. To test this hypothesis, 331 independent clones selected for new mutations at the thymidine kinase (TK) locus on chromosome 17q, and 243 nonselected control clones were examined for mutations in 12 random microsatellite loci dispersed throughout the genome. A total of 24 second-site mutations were identified in the TK mutant clones, compared with 3 in the control clones not selected for mutations at TK. The mutations include small deletions, insertions, and loss of heterozygosity. These results provide evidence that a global trans-acting mutagenic process exists in human cells. The activation of this process could be responsible for causing multiple essential mutations in tumor cells.     

HPRT mutations in humans: biomarkers for mechanistic studies.

The X-chromosomal gene for hypoxanthine-guanine phosphoribosyltransferase (HPRT), first recognized through its human germinal mutations, quickly became a useful target for studies of somatic mutations in vitro and in vivo in humans and animals. In this role, HPRT serves as a simple reporter gene. The in vivo mutational studies have concentrated on peripheral blood lymphocytes, for obvious reasons. In vivo mutations in T cells are now used to monitor humans exposed to environmental mutagens with analyses of molecular mutational spectra serving as adjuncts for determining causation. Studies of the distributions of HPRT mutants among T cell receptor (TCR) gene-defined T cell clones in vivo have revealed an unexpected clonality, suggesting that HPRT mutations may be probes for fundamental cellular and biological processes. Use of HPRT in this way has allowed the analyses of V(D)J recombinase mediated mutations as markers of a mutational process with carcinogenic potential, the use of somatic mutations as surrogate markers for the in vivo T cell proliferation that underlies immunological processes, and the discovery and study of mutator phenotypes in non-malignant T cells. In this last application, the role of HPRT is related to its function, as well as to its utility as a reporter of mutation. Most recently, HPRT is finding use in studies of in vivo selection for in vivo mutations arising in either somatic or germinal cells.     

Analysis of point mutations induced by ultraviolet light in human cells

Mutations induced in cultured human cells by 254-nm UV light were analyzed within exon 3 of the hypoxanthine guanine phosphoribosyl transferase (HPRT) gene. Five large independent cultures of human lymphoblastoid cells, line TK6, were exposed to 4 J/m2 of 254-nm UV light and mutants at the HPRT locus were selected en masse by 6-thioguanine (6TG) resistance. Exon 3 of the HPRT gene was amplified from the mutant cells by polymerase chain reaction (PCR) using modified T7 DNA polymerase. Denaturing gradient gel electrophoresis (DGGE) was used to separate the mutant sequences from the wild type as mutant/wild-type heteroduplexes. Individual mutant bands were isolated from the gel and the nature of the mutations was determined by direct sequencing. Eight predominant mutations were detected in the 184-bp exon 3 sequence. Of these, 3 transition, including 2 G-C to A-T and 1 A-T to G-C and 2 A-T to C-G transversions, appeared in all 5 UV-treated cultures but not in untreated cultures and were thus considered to be mutational hotspots. These observations are similar in nature to those previously reported in bacterial and rodent cells. A single G deletion, a tandem substitution of CpT for TpA, and a tandem triple substitution of GpGpA for ApApG were also observed but in only 2, 2 and 3 of the 5 UV-treated cultures, respectively. Numerical analysis of the mutant fractions of these 8 mutations indicated that each of them was distributed as a set of non-random and independent events, i.e., a mutational hotspot.     

Molecular structural analysis of HPRT mutations induced by thermal and epithermal neutrons in Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells were exposed to thermal and epithermal neutrons, and the occurrence of mutations at the HPRT locus was investigated. The Kyoto University Research Reactor (KUR), which has been improved for use in neutron capture therapy, was the neutron source. Neutron energy spectra ranging from nearly pure thermal to epithermal can be chosen using the spectrum shifters and thermal neutron filters. To determine mutant frequency and cell survival, cells were irradiated with thermal and epithermal neutrons under three conditions: thermal neutron mode, mixed mode with thermal and epithermal neutrons, and epithermal neutron mode. The mutagenicity was different among the three irradiation modes, with the epithermal neutrons showing a mutation frequency about 5-fold that of the thermal neutrons and about 1.5-fold that of the mixed mode. In the thermal neutron and mixed mode, boron did not significantly increase the frequency of the mutants at the same dose. Therefore, the effect of boron as used in boron neutron capture therapy (BNCT) is quantitatively minimal in terms of mutation induction. Over 300 independent neutron-induced mutant clones were isolated from 12 experiments. The molecular structure of HPRT mutations was determined by analysis of all nine exons by multiplex polymerase chain reaction. In the thermal neutron and mixed modes, total and partial deletions were dominant and the fraction of total deletions was increased in the presence of boron. In the epithermal neutron mode, more than half of the mutations observed were total deletions. Our results suggest that there are clear differences between thermal and epithermal neutron beams in their mutagenicity and in the structural pattern of the mutants that they induce. Mapping of deletion breakpoints of 173 partial-deletion mutants showed that regions of introns 3-4, 7/8-9 and 9-0 are sensitive to the induction of mutants by neutron irradiation.     

Relationship between specific alkylated bases and mutations at two gene loci induced by ethylnitrosourea and diethyl sulfate in CHO cells

DNA adduct formation and induction of mutations at 2 gene loci, hypoxanthine-guanine-phosphoribosyltransferase (HPRT) and Na,K-ATPase, were determined simultaneously in Chinese hamster ovary (CHO) cells after treatment with 2 ethylating agents, ethylnitrosourea (ENU) or diethyl sulfate (DES). Doses of DES and ENU, which resulted in equal levels of O6-ethylguanine (O6-EtGua) and O4-ethylthymine (O4-EtThy) in the DNA, were found to induce very similar frequencies of 6-thioguanine-resistant (6-TGr) mutants. Formation of these DNA adducts might therefore be correlated with mutations induced at the HPRT locus. When, however, the same analysis was applied to ouabain-resistant (ouar) mutants, it was found that, at similar levels of O6-EtGua and O4-EtThy, DES induced many more ouar mutants than ENU. This result supports the notion that primary DNA lesions other than O6-EtGua and O4-EtThy are involved in the fixation of ENU- and DES-induced mutations at the Na,K-ATPase gene locus.     

The relationship between DNA damage and mutation frequency in mammalian cell lines treated with N-nitroso-N-2-fluorenylacetamide

The induction of DNA single-strand breaks in C3H10T1/2 mouse fibroblasts and Chinese hamster ovary (CHO) cells by N-nitroso-N-2-fluorenylacetamide (N-NO-2-FAA) was demonstrated by the alkaline elution technique. Without metabolic activating system (i.e., rat liver S9 fraction), N-NO-2-FAA exhibits more direct and strong damaging effects on DNA than its parent compound, 2-FAA, at equal concentration in both cell lines. To compare the DNA-damaging potency of N-NO-2-FAA with other well-known carcinogens, such as benzo[a]pyrene, 2-nitrofluorene, and N-methyl-N'-nitrosoguanidine (MNNG), the order of potency is as follows: MNNG (5 microM) greater than N-NO-2-FAA (150 microM) greater than benzo[a]pyrene (20 microM) at equitoxic concentrations, LD37, in the same cell system. Another parallel experiment indicated that N-NO-2-FAA could disrupt the superhelicity of circular plasmid DNA (pBR 322) at a dose range of 0.1-50 mM; however, a complete conversion to form III linear DNA was found at the highest concentration (50 mM). After treatment with various concentrations of N-NO-2-FAA, ouabain resistance (ouar) was induced in C3H10T1/2 cells, while both ouar and 6-thioguanine resistance (6-TGr) were induced in CHO cells. The mutation frequency in the Na+/K+-ATPase locus in CHO cells (1.5 X 10(-6) mutants/microM) is higher than that in C3H10T1/2 cells (1.0 X 10(-6) mutants/microM). The maximal mutation frequency at the Na+/K+-ATPase gene locus was attained with 30 min of exposure in C3H10T1/2 cells, whereas the mutation frequency in CHO cells continued to increase up to 80 min of treatment. Similarly, the maximal mutation frequency at the HPRT locus also continued to increase up to 80 min of treatment. Finally, a linear plot of alkali-labile lesions versus 6-TGr mutations was obtained; but the same relationship was not observed in the case of ouar mutation.