Determination of the spectrum of mutations induced by defined-wavelength solar UVB (313-nm) radiation in mammalian cells by use of a shuttle vector.

Mutations induced by UVB (313-nm) radiation, a wavelength in the region of peak effectiveness for sunlight-induced skin cancer in humans, have been analyzed at the sequence level in simian cells by using a plasmid shuttle vector (pZ189). We find that significant differences exist between the types of mutations induced by this solar wavelength and those induced by nonsolar UVC (254-nm) radiation. Compared with 254-nm radiation, 313-nm radiation induces more deletions and insertions in the region sequenced. In addition, although the types of base substitutions induced by the two wavelengths are broadly similar (in both cases, the majority of changes occur at G-C base pairs and the G-C to A-T transition is predominant), an analysis of the distribution of these base changes within the supF gene following irradiation at 313 nm reveals additional hot spots for mutation not seen after irradiation at 254 nm. These hot spots are shown to arise predominantly at sites of mutations involving multiple base changes, a class of mutations which arises more frequently at the longer solar wavelength. Lastly, we observed that most of the sites at which mutational hot spots arise after both UVC and UVB irradiation of the shuttle vector are also sites at which mutations arise spontaneously. Thus, a common mechanism may be involved in determining the site specificity of mutations, in which the DNA structure may be a more important determinant than the positions of DNA photoproducts.     

Chromosomal mutations induced by triplex-forming oligonucleotides in mammalian cells.

Specific recognition of a region of duplex DNA by triplex-forming oligonucleotides (TFOs) provides an attractive strategy for genetic manipulation. Based on this, we have investigated the ability of the triplex-directed approach to induce mutations at a chromosomal locus in living cells. A mouse fibroblast cell line was constructed containing multiple chromosomal copies of the lambdasupFG1 vector carrying the supFG1 mutation-reporter gene. Cells were treated with specific (psoAG30) or control (psoSCR30) psoralen-conjugated TFOs in the presence and absence of UVA irradiation. The results demonstrated a 6- to 10-fold induction of supFG1 mutations in the psoAG30-treated cells as compared with psoSCR30-treated or untreated control cells. Interestingly, UVA irradiation had no effect onthe mutation frequencies induced by the psoralen-conjugated TFOs, suggesting a triplex-mediated but photoproduct-independent process of mutagenesis. Sequencing data were consistent with this finding since the expected T.A-->A.T transversions at the predicted psoralen crosslinking site were not detected. However, insertions and deletions were detected within the triplex binding site, indicating a TFO-specific induction of mutagenesis. This result demonstrates the ability of triplex-forming oligonucleotides to influence mutation frequencies at a specific site in a mammalian chromosome.     

Specificity of mutations induced in transfected DNA by mammalian cells

DNA transfected into mammalian cells is subject to the high mutation frequency of approximately 1% per gene. We present data bearing on the derivation of the two main classes of mutations detected, base substitutions and deletions. The DNA sequence change is reported for nearly 100 independent base substitution mutations that occurred in shuttle vectors as a result of passage in simian cells. All of the mutations occur at G:C base pairs and involve either transition to A:T or transversion to T:A. To identify possible mutational intermediates, various topological forms of the vector DNA were introduced separately. Supercoiled and relaxed DNA are mutated at equal frequencies. However, linearized DNA leads to a greatly elevated frequency of deletions. Nicked and gapped templates stimulate both deletions and base substitutions. We discuss a model involving intracellular degradation of the transfected DNA which explains these observations.     

G-to-T transversions and small tandem base deletions are the hallmark of mutations induced by ultraviolet a radiation in mammalian cells

Ultraviolet A (UVA) radiation received from the sun and from the widespread use of tanning beds by populations residing in areas of northern latitude represents a potential risk factor for human health. The genotoxic and cancer-causing effects of UVA have remained controversial. A mutagenic role for UVA based on DNA damage formation by reactive oxygen species as well as by generation of photoproducts such as cyclobutane pyrimidine dimers (CPDs) has been suggested. Here, we investigated the mutagenicity of UVA in relation to its DNA damaging effects in transgenic Big Blue mouse embryonic fibroblasts. We determined the formation of a typical oxidative DNA lesion, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), and of CPDs, as well as quantified the induction of mutations in the cII transgene in cells irradiated with a 2000 W UVA lamp. UVA irradiation at a dose of 18 J/cm(2) produced significant levels of 8-oxo-dG in DNA (P < 0.03) but did not yield detectable CPDs. UVA irradiation also increased the cII mutant frequency almost 5-fold over background (P < 0.01) while showing moderate cytotoxicity (70% cell viability). UVA-induced mutations were characterized by statistically significant increases in G-to-T transversions and small tandem base deletions (P = 0.0075, P = 0.008, respectively) relative to spontaneously derived mutations. This mutational spectrum differs from those previously reported for UVA in other test systems; however, it corresponds well with the known spectrum of mutations established for oxidative base lesions such as 8-oxo-dG. We conclude that UVA has the potential to trigger carcinogenesis owing to its mutagenic effects mediated through oxidative DNA damage.     

Forward mutations and DNA-protein crosslinks induced by ammonium metavanadate in cultured mammalian cells.

Ammonium metavanadate yielded a dose-dependent increase in mutation frequency at the V79 hprt locus following a 24-h exposure period in serum-free F12 medium. Vanadate also increased the mutation frequency of V79 cells by exposure of cells in salts-glucose medium, but these effects were not as striking, or as dose-dependent as they were in serum-free F12 medium. Ammonium metavanadate enhanced the mutation frequency in a V79 variant containing a transfected bacterial gpt gene. These cells are known to be more responsive to oxidative type mutations, and to mutations involving deletions. Although the absolute level of mutations was greater in these cells with ammonium metavanadate, so was the background, and these cells did not exhibit an enhanced mutagenic response to vanadate when compared to the wild-type V79 cells. The vanadate results were compared to a positive control potassium chromate, which exhibited a dose-dependent increase in mutation frequency. Ammonium metavanadate induced DNA-protein crosslinks formation in both Chinese hamster ovary and human MOLT4 cells, and the role of these relatively unrepaired genetic lesions in the mutations produced by vanadate and chromate are discussed.     

Reversion analysis of mutations induced by 5-bromodeoxyuridine mutagenesis in mammalian cells.

Two protocols have been developed, both of which utilize the thymidine analog 5-bromodeoxyuridine (BrdUrd) to induce mutations in mammalian cells in culture (E. R. Kaufman and R. L. Davidson, Proc. Natl. Acad. Sci. USA 75:4982-4986, 1978; E. R. Kaufman, Mol. Cell. Biol. 4:2449-2454, 1984). The first protocol, termed incorporational (INC) mutagenesis, utilizes high concentrations of BrdUrd in the culture medium to generate a high intracellular ratio of BrdUTP/dCTP. The second protocol, termed replicational (REP) mutagenesis, entails the incorporation of BrdUrd into DNA under nonmutagenic conditions, the removal of all BrdUrd from the culture medium, and the subsequent replication of the bromouracil-containing DNA in the presence of high intracellular levels of dTTP and dGTP. Genetic studies using reversion analysis at the hypoxanthine-guanine phosphoribosyltransferase locus were used to determine whether the mechanisms of these two BrdUrd mutagenesis protocols had enough specificity to be distinguishable by their ability to revert various mutants. The results of these studies indicated that (i) mutants induced by INC mutagenesis were induced to revert only by REP mutagenesis and not by INC mutagenesis, (ii) mutants induced by REP mutagenesis were more efficiently reverted by INC mutagenesis than by REP mutagenesis, and (iii) both spontaneous mutants and mutants induced by the chemical mutagen ethyl methanesulfonate showed a high degree of specificity when tested for reversion by the BrdUrd mutagenesis protocols.     

Chromosome evolution in eukaryotes: a multi-kingdom perspective

In eukaryotes, chromosomal rearrangements, such as inversions, translocations and duplications, are common and range from part of a gene to hundreds of genes. Lineage-specific patterns are also seen: translocations are rare in dipteran flies, and angiosperm genomes seem prone to polyploidization. In most eukaryotes, there is a strong association between rearrangement breakpoints and repeat sequences. Current data suggest that some repeats promoted rearrangements via non-allelic homologous recombination, for others the association might not be causal but reflects the instability of particular genomic regions. Rearrangement polymorphisms in eukaryotes are correlated with phenotypic differences, so are thought to confer varying fitness in different habitats. Some seem to be under positive selection because they either trap favorable allele combinations together or alter the expression of nearby genes. There is little evidence that chromosomal rearrangements cause speciation, but they probably intensify reproductive isolation between species that have formed by another route.     

The molecular basis of mutations induced by deoxyribonucleoside triphosphate pool imbalances in mammalian cells

Alterations of the balanced supply of the precursors of DNA synthesis, the deoxyribonucleoside triphosphates, have dramatic genetic consequences for mammalian cells including the induction of mutations, the sensitization to DNA damaging agents, and the production of gross chromosomal abnormalities. The use of recombinant DNA techniques has allowed the analysis of some of these effects and has revealed further mechanisms by which mammalian cells control the accuracy of DNA replication.     

Electrophoretically detectable mutations induced in CHO cells by varying doses of ultraviolet radiation

Chinese hamster ovary (CHO) cells were subjected to ultraviolet radiation (UV) at doses resulting in 100% (no irradiation), 50–30%, 20–10% and ≈1% survival. 2 divisions after UV exposure surviving cells were cloned and clones expanded for electrophoretic analysis of the products of ≈40 enzyme loci. 4 different classes of variants (electrophoretic shifts, nulls, enzyme re-expression and enzyme modification) were detected in 29 of 1329 clones analyzed and proven mutants by subclone analysis. The frequency of mutants in the irradiated groups (28/38391 loci screened or 7.3 × 10^?4) was significantly higher than controls. The frequency of shift mutants at 10–20% survival was higher than shifts at 30–50% survival and was significantly higher than shifts at ≈1% survival. The frequency of nulls increased with dose. 12 of the 28 mutants obtained in the irradiated groups were at only 3 of the mean 41 loci screened/clone. The results indicated that shift mutants could be detected more efficiently than nulls at lower dose and that loci varied widely with respect to their susceptibility to UV mutagenesis. Multiple null mutants at 2 loci, isocitrate dehydrogenase 2 and hexokinase 2, indicated they may be hemizygous in CHO cells.     

Metformin (dimethyl-biguanide) induced DNA damage in mammalian cells

Metformin (dimethyl-biguanide) is an insulin-sensitizing agent that lowers fasting plasma-insulin concentration, wherefore it's wide use for patients with a variety of insulin-resistant and prediabetic states, including impaired glucose tolerance. During pregnancy it is a further resource for reducing first-trimester pregnancy loss in women with the polycystic ovary syndrome. We tested metformin genotoxicity in cells of Chinese hamster ovary, CHO-K1 (chromosome aberrations; comet assays) and in mice (micronucleus assays). Concentrations of 114.4 μg/mL and 572 μg/mL were used in in vitro tests, and 95.4 mg/kg, 190.8 mg/kg and 333.9 mg/kg in assaying. Although the in vitro tests revealed no chromosome aberrations in metaphase cells, DNA damage was detected by comet assaying after 24 h of incubation at both concentrations. The frequency of DNA damage was higher at concentrations of 114.4 μg/mL. Furthermore, although mortality was not observed in in vitro tests, the highest dose of metformin suppressed bone marrow cells. However, no statistically significant differences were noted in micronuclei frequencies between treatments. In vitro results indicate that chronic metformin exposure may be potentially genotoxic. Thus, pregnant woman undergoing treatment with metformin should be properly evaluated beforehand, as regards vulnerability to DNA damage.     

Frequency and nature of specific-locus mutations induced in female mice by radiations and chemicals: a review.

The inducibility of heritable mutations in female mammals has been measured in the mouse specific-locus test (SLT). For radiation-induced mutations, a large body of data has been accumulated that includes information about biological and physical factors that influence mutation yields. However, relatively few SLT studies in females have been conducted with chemicals to date. A single estimate of the spontaneous mutation rate in oocytes, 6/536,207, has been derived as the most appropriate one to subtract from experimental rates. This rate is highly significantly below the spontaneous mutation rate in males. Mutations recovered from females mutagenized at any time after about the 12th day post-conception are induced in non-dividing cells. In adult females, most oocytes are arrested in small follicles; maturation from this stage to ovulation takes several weeks. High-dose-rate radiations are more mutagenic in mature and maturing oocytes than in spermatogonia of the male; on the other hand, no clearly induced mutations have been recovered from irradiated arrested oocytes. Efficient repair processes have been invoked to explain the latter finding as well as the upward-curving dose-effect relation for acute irradiation, and the fact that dose protraction drastically reduces mutation yield from mature and maturing oocytes. The dose-protraction effect is much greater than that found in spermatogonia. Radiation-induced mutation rates in embryonic, fetal, and newborn females are overall lower than those in the mature and maturing oocytes of adults. A dose-protraction effect has also been demonstrated at an early developmental stage when the nuclear morphology of mouse oocytes most resembles that of the human. Of only 5 chemicals so far explored for their effect in oocytes, 2 (ethylnitrosourea, ENU, and triethylenemelamine, TEM), and possibly a third (procarbazine hydrochloride, PRC), are mutagenic--with at least one of these (ENU) mutagenic in arrested as well as maturing oocytes. However, the mutation rate is, in each case, lower than for treated male germ cells. By contrast, ENU-induced mutation yield for the maternal genome of the zygote is an order of magnitude higher than that for the zygote's paternal genome or for spermatogonia. A high proportion of mutants derived from chemical treatment of oocytes (including the oocyte genome in zygotes) are mosaics, probably owing to lesions affecting only 1 strand of the DNA. A characteristic of specific-locus mutations induced in oocytes is that they include a considerably higher percentage of large (multi-locus) lesions (LLs) than do mutations induced in spermatogonia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Precise excision of transposons and point mutations induced by chemicals.

The ability of 23 chemicals (carcinogens and non-carcinogens) to induce precise excision of Tn10 and point mutations was studied in experiments with a single strain. The mutation assay was shown to detect a wider spectrum of genotoxic agents than the assay of Tn10 precise excision. The latter was induced only by potent SOS mutagens, which is in accordance with data on the SOS dependence of the induction of precise excision of Tn10. The precise excision assay as an additional test contributing to the knowledge of particular features of the action of a tested mutagen is discussed. The induction of precise excision of Tn10 by pyrene (and its failure to induce point mutations in this strain) demonstrates the value of using the transposon excision assay in cases of 'problem' mutagens.     

High yields of lethal mutations in somatic mammalian cells that survive ionizing radiation

When mammalian cells are irradiated in vitro, the component cells of a normal-appearing survivor colony or clone are commonly thought to have proliferative capacity equivalent to that of the unirradiated cells. We have found, however, that cells appearing in survivor colonies may carry heritable lethal defects which come to light, perhaps only after numerous successful divisions, in the form of plating efficiencies that are reduced below those of unirradiated cells in a dose-dependent manner. We regard these heritable defects as signs of the induction of lethal mutations, which, like non-lethal mutations, may require many generations before they are expressed. This effect has been noted in two very dissimilar mammalian cell lines, one a primary culture from adult tissue, the other an immortal cell line. We suggest that induction of lethal mutations may occur also in somatic cells in vivo; this would account for the well-known observation that previously irradiated but apparently healed tissue is subsequently proved to be extraordinarily sensitive to subsequent exposure to irradiation or cytotoxic drugs. The results of our experiments in vitro suggest that current methods of estimating mutation or transformation yields may yield underestimates. If lethal mutations are induced also in vivo, interpretations of the results of fractionation experiments on normal tissues may have to be reconsidered.     

Mitochondrial DNA point mutations and a novel deletion induced by direct low-LET radiation and by medium from irradiated cells

Radiation damage incurred by nuclear DNA is well documented and interest is increasing in the properties of 'bystander' factor(s) and their ability to induce radiation-like damage in cells never exposed to radiation. 'Bystander' and direct low-LET radiation effects on the mitochondria, and more particularly the mitochondrial genome are less well understood. In this study HPV-G cells (a human keratinocyte cell line derived from human neonatal foreskin transfected with the HPV-16 virus) were exposed to either gamma-radiation doses as low as 5 mGy and up to 5 Gy from a 60Co teletherapy unit, or to growth medium taken from similarly irradiated cells, i.e. irradiated cell conditioned medium (ICCM). Mutation and deletion analysis was performed on mitochondrial DNA (mtDNA) 4-96 h after exposure. Primers flanking the so-called mitochondrial 'common deletion' were employed to assess its possible induction. Single-strand conformation polymorphism (SSCP) analysis was conducted to identify induced point mutations. The relative mitochondrial number per cell was analysed by semi-quantitative PCR (sqPCR). Results indicate the induction of a relatively novel deletion in the mitochondrial genome as early as 12 h after direct exposure to doses as low as 0.5 Gy and 24 h after exposure to 0.5-Gy ICCM. SSCP analysis identified the induction of point mutations, in a non-consistent manner, in only the D-loop region of the mitochondrial genome and only in cells exposed to 5 Gy, and neither in cells exposed to lower doses of direct radiation nor in those exposed to ICCM. SqPCR also identified an increase in the number of mitochondria per cell after both exposure to low level gamma-radiation and ICCM, indicative of a possible mechanism to respond to mitochondrial stress by increasing the number of mitochondria per cell.     

Frequency of spontaneous and induced "point" mutations in higher eukaryotes

Data are presented on comparative mutation rates in Drosophila, mice, and humans, as derived from the study of both biochemical and morphological traits. There is a notable similarity in these rates, despite the large differences in average generation time, mean body temperature, and number of cell divisions intervening between fertilization of the egg and production of functional gametes. This suggests the evolution of superior genetic (DNA) repair strategies in (long-lived) humans. Evidence is reviewed that raises the possibility of higher mutation rates in mostly tropical-dwelling, tribal human populations than in temperate-dwelling civilized groups. The data suggesting that humans may be somewhat less sensitive to the genetic effects of radiation than mice are presented, and the question of whether this apparent difference is real or spurious is discussed. Finally, some possible new directions in the study of the mutation-selection balance are presented.     

Long-lived mutagenic radicals induced in mammalian cells by ionizing radiation are mainly localized to proteins

We have provided evidence that long-lived radicals, produced by ionizing radiation, are highly mutagenic and transforming in mammalian cells. Long-lived radicals are scavenged effectively by vitamin C or by epigallocatechin-3-O-gallate (EGCG). Long-lived radicals are not involved in lethality or in the induction of chromosome aberrations. We now report the results of experiments that define the relative amounts of long-lived radicals in DNA and proteins and identify the major protein radicals as sulfinyl radicals (R-CH2-S-O*). To make these assignments, yields of long-lived radicals in gamma-irradiated salmon sperm DNA and albumin were compared by ESR. ESR spectra of long-lived radicals produced in irradiated Syrian hamster embryo (SHE) cells were analyzed precisely and compared with ESR parameters obtained by density functional theory calculations. Long-lived radicals yields of 99.8% were produced in proteins. We also identified a new type of long-lived radical as H-added phenylalanine radicals. While our evidence does not rule out the possibility of important biological consequences of the low-level long-lived radicals created by radiation, it implicates radicals in proteins as playing a key role in genetic effects of ionizing radiation. We suggest that these novel radicals, wherever they reside, need to be considered in explanations of biological sequela of radiation.