Elevation In Mutation Frequency Research Articles

Elevated somatic mutation and evidence of genomic instability in veterans with Gulf War illness

AimsGulf War illness (GWI) is thought to be associated with exposures experienced by soldiers deployed in the 1991 Gulf War. A major question is how these exposures continue to influence the health of these individuals three decades later. One potentially permanent effect of such exposures is the induction of genetic mutations. We investigated whether veterans with GWI exhibited persistently elevated levels of somatic mutation. Materials and methodsWe applied the blood-based glycophorin A (GPA) somatic mutation assay to a cohort of veterans diagnosed with GWI and a set of both concurrent and historic age-matched controls. This assay quantifies red blood cells with a phenotype consistent with loss of one allele at the genetic determinant for the MN blood group, the GPA gene. Key findingsAs a population, those affected with GWI exhibited an uninduced mutation frequency at the GPA locus that was effectively twice that observed in controls, a result that was statistically significant. This result was influenced by an increase in the incidence of individuals with aberrantly high mutation frequencies, seemingly higher than would be expected by dose extrapolation and consistent with the induction of localized genomic instability in the hematopoietic bone marrow stem cells. When these “outliers” were removed from consideration, the remaining affected population retained a significantly higher mean allele loss mutation frequency, suggesting that both dose-dependent bone marrow genotoxicity and induction of genomic instability are contributing to the elevation in mutation frequency in these affected veterans. SignificanceThis study provides evidence that manifestation of GWI is associated with increased cumulative exposure to agents capable of inducing persistent mutations in bone marrow stem cells. Whether these mutations are involved in the clinical aspects of the condition or are simply biomarkers of overall exposure has yet to be determined. The increased incidence of genomic instability suggests that this persistent mutation can have important delayed effects on cellular integrity.

The Murine Model for Hantaan Virus-Induced Lethal Disease Shows Two Distinct Paths in Viral Evolutionary Trajectory with and without Ribavirin Treatment

In vitro, ribavirin acts as a lethal mutagen in Hantaan virus (HTNV)-infected Vero E6 cells, resulting in an increased mutation load and viral population extinction. In this study, we asked whether ribavirin treatment in the lethal, suckling mouse model of HTNV infection would act similarly. The HTNV genomic RNA (vRNA) copy number and infectious virus were measured in lungs of untreated and ribavirin-treated mice. In untreated, HTNV-infected mice, the vRNA copy number increased for 10 days postinfection (dpi) and thereafter remained constant through 26 dpi. Surprisingly, in ribavirin-treated, HTNV-infected mice, vRNA levels were similar to those in untreated mice between 10 and 26 dpi. Infectious virus levels, however, were different: in ribavirin-treated mice, the amount of infectious HTNV was significantly decreased relative to that in untreated mice, suggesting that ribavirin reduced the specific infectivity of the virus (amount of infectious virus produced per vRNA copy). Mutational analysis revealed a ribavirin-associated elevation in mutation frequency in HTNV vRNA similar to that previously reported in vitro. Codon-based analyses of rates of nonsynonymous (dN) and synonymous (dS) substitutions in the S segment revealed a positive selection for codons within the HTNV N protein gene in the ribavirin-treated vRNA population. In contrast, the vRNA population in untreated, HTNV-infected mice showed a lower level of diversity, reflecting purifying selection for the wild-type genome. In summary, these experiments show two different evolutionary paths that Hantavirus may take during infection in a lethal murine model of disease, as well as the importance of the in vivo host environment in the evolution of the virus, which was not apparent in our prior in vitro model system.

Assessment of Biological Effect of Tritiated Water by Using Hypersensitive System

An exposure of human or animals to tritium radiation from nuclear fusion reactor is expected to be a low dose and with low dose-rate. We are focusing on the biological effects of tritiated water (HTO) given at low dose and/or with low dose rate. Hypersensitive assay systems for radiation biological experiments using cultured mammalian cells or transgenic mice have been established and their availability for assessment of biological effects of HTO were tested. A hypersensitive detection system for mutagenesis at Hprt locus was established by using hamster cells carrying a human X-chromosome. The cells present more than 50-fold sensitivity for Hprt-deficient mutation, and this allowed us to detect a significant elevation of mutation frequency by radiations at the dose of less than 0.2 Gy.Because the reverse dose rate effect has been reported for mutation induction by high LET radiation such as neutrons, we first tested whether the reverse dose rate effects could be seen for HTO (tritium beta-rays) or not. No significant change in mutation frequency by HTO was observed in the range of dose rates between 0.05 cGy/h and 2.0 cGy/h, whereas clear reverse dose rate effects was observed in the case of fission neutrons or carbon beam. The result suggests that the reverse dose rate effect does not apply in the case of mutation induction by HTO. In addition, mutation spectrum in the mutants induced by HTO was similar to those in spontaneously induced mutants, suggesting that exposure to a certain level of HTO could enhance the spontaneous mutagenesis.

The role of DNA mismatch repair in generating genetic diversity and drug resistance in malaria parasites

Although the mechanisms by which malaria parasites develop resistance to drugs are unclear, current knowledge suggests a main mechanism of resistance is the alteration of target enzymes by point mutation. In other organisms, defects in DNA mismatch repair have been linked to increased mutation rates and drug resistance. We have identified an unusual complement of mismatch repair genes in the Plasmodium genome. An initial functional test of two of these genes (PfMSH2-1 and PfMSH2-2) using a dominant mutator assay showed an elevation in mutation frequency with the PfMSH2-2 homolog, indirectly demonstrating a role for this gene in mismatch repair. We successfully disrupted PbMSH2-2 in the Plasmodium berghei laboratory isolate NK65, and showed that this gene is not essential for parasite growth in either the asexual (rodent) or sexual (mosquito) stages of the lifecycle. Although we observed some differences in levels of drug resistance between wild type and mutant parasites, no uniform trend emerged and preliminary evidence does not support a strong link between PbMSH2-2 disruption and dramatically increased drug resistance. We found microsatellite polymorphism in the PbMSH2-2 disrupted parasites in less than 40 life cycles post-transfection, but not in PbMap2K disrupted controls or mosquito-passaged wild type parasites, which suggests a possible role for PbMSH2-2 in preventing microsatellite slippage, similar to MSH2 in other organisms. Our studies suggest that Plasmodium species may have evolved a unique variation on the highly conserved system of DNA repair compared to the mismatch repair systems in other eukaryotes.

Mitochondrial DNA mutation frequencies in experimentally irradiated compost worms, Eisenia fetida

The compost worm Eisenia fetida is routinely used in ecotoxicological studies. A standard assay to assess genetic damage in this species would be extremely valuable. Since mitochondrial DNA (mtDNA) is known to exhibit an increased mutation rate following exposure to ionising radiation we assessed the validity of a mtDNA-based assay for measuring increases in mutation rate in laboratory-irradiated compost worms. To this end the mutation frequency in the mtDNA of the compost worm E. fetida was quantified following in vivo γ-irradiation of adult worms in three dose groups. Five adult worms exposed to 1.4 mGy/h for 55 days (total dose 1.85 Gy), five adult worms exposed to 8.5 mGy/h for 55 days (total dose 11.22 Gy) and five adult control worms were used to assess the effect of irradiation on mtDNA mutation induction. DNA samples extracted from irradiated adult worms were used in high-fidelity PCR of a 486 bp region of mtDNA spanning the ATPase 8 gene, chosen for its high spontaneous mutation rate. PCR products were cloned and sequenced to identify mutations, with 89–102 clones successfully sequenced per individual. A significant elevation in mtDNA mutation frequency ( p = 0.032) was seen in worms exposed at the higher dose rate (8.5 mGy/h, total dose 11.22 Gy; mutation frequency 27.98 ± 4.85 × 10 −5 mutations/bp) in comparison to controls (mutation frequency 12.68 ± 3.06 × 10 −5 mutations/bp), but no elevation in mutation frequency ( p = 0.764) was seen for the lower dose rate (1.4 mGy/h, total dose 1.85 Gy; mutation frequency 13.74 ± 1.29 × 10 −5 mutations/bp) compared with controls. This indicates that although the technique has the potential to detect an elevation in mutation frequency, it does not have sufficient sensitivity at the doses likely to be encountered in environmental monitoring scenarios.

HPV-16 E6 oncoprotein induces mutations via p53-dependent and -independent pathways.

The E6 oncoprotein of <high risk> human papillomaviruses (HPV) promotes oncogenesis by inactivating tumor suppressor protein p53 i. e. it binds to and enhances the degradation of p53. To study whether inactivation of p53 is solely responsible for E6-induced oncogenesis, we constructed several plasmid vectors expressing wild-type (wt) or mutant (mt) E6 proteins. RKO cells that express wt p53 were stably transfected with these plasmids and challenged with DNA damaging agents. The level of p53 was significantly increased by DNA damaging agents in control cells and cells transfected with plasmids expressing mt E6 that do not bind to p53. As expected, p53 did not increase in cells transfected with plasmids expressing mt E6 that do bind to p53. To investigate the oncogenic effect of these various E6 proteins, we determined the mutation frequency of the hprt locus in control cells and cells expressing different E6 proteins. We found that cells expressing wt E6 and mt E6 (capable or incapable of binding to p53) showed notable increases in the mutation frequency at hprt locus compared with that of control cells. The elevation of mutation frequency in cells expressing mt E6 was similar to that in cells expressing wt E6. These data indicate that E6-induced mutagenicity is induced not only via p53 inactivation, but also via p53-independent pathways.

Elevated levels of mutation in multiple tissues of mice deficient in the DNA mismatch repair gene Pms2.

The Pms2 gene has been implicated in hereditary colon cancer and is one of several mammalian homologs of the Escherichia coli mutL DNA mismatch repair gene. To determine the effect of Pms2 inactivation on genomic integrity in vivo, hybrid transgenic mice were constructed that carry targeted disruptions at the Pms2 loci along with a chromosomally integrated mutation reporter gene. In the absence of any mutagenic treatment, mice nullizygous for Pms2 showed a 100-fold elevation in mutation frequency in all tissues examined compared with both wild-type and heterozygous litter mates. The mutation pattern in the nullizygotes was notable for frequent 1-bp deletions and insertions within mononucleotide repeat sequences, consistent with an essential role for PMS2 in the repair of replication slippage errors. Further, the results demonstrate that high rates of mutagenesis in multiple tissues are compatible with normal development and life and are not necessarily associated with accelerated aging. Also, the finding of genetic instability in all tissues tested contrasts with the limited tissue distribution of cancers in the animals, raising important questions regarding the role of mutagenesis in carcinogenesis.

Oxygen radical induced mutagenesis is DNA polymerase specific

Oxygen free radicals are produced in large amounts by normal cellular processes. Damage to DNA by these reactive species has been implicated in mutagenesis and may be important in the etiology of a variety of human diseases. In this study we investigate the types of mutations produced in vitro as a result of DNA damage by oxygen free radicals. We used a lacZ alpha forward mutation assay in which M13 viral DNA is damaged in vitro, replicated with purified DNA polymerase alpha or beta, transfected into E. coli, and screened for mutations by reduced alpha-complementation of beta-galactosidase activity. By determining the effects of damaged templates on the fidelity of individual DNA polymerases involved in replication and repair, we address the role of specific DNA polymerases in mutagenesis induced by reactive oxygen species. Aerobic incubation of DNA with 100 microM CuCl, 10 microM H2O2 and 100 microM ascorbic acid results in a 3.3-fold and a 3.6-fold elevation in mutation frequency for polymerases alpha and beta, respectively. The specificity and location of the induced mutations, however, are entirely different. For polymerase alpha, A to C, and C to A transversions and deletions of C are each elevated more than 10-fold over their frequencies on undamaged template. For polymerase beta, A to T, C to T, C to A, G to C, and G to T substitutions, and deletions of G are elevated by damage. The frequency of mutants containing two or more closely spaced substitutions is also markedly increased by template damage although the types of mutations and their positions are again specific to each DNA polymerase. We conclude that, for oxidative lesions, the frequency and the types of mutations are determined in part by the DNA polymerase that encounters the site of damage.

Somatic mutation frequencies in the stamen hairs of Tradescantia grown in soil samples from the Bikini Island.

Somatic pink mutation frequencies in the stamen hairs of Tradescantia BNL 02 clone grown for 76 days in two soil samples taken from the Bikini Island (where a hydrogen bomb explosion test had been conducted in 1954) were investigated. A significantly high mutation frequency (2.58 +/- 0.17 pink mutant events per 10(3) hairs or 1.34 +/- 0.09 pink mutant events per 10(4) hair-cell divisions) was observed for the plant grown in one of the two Bikini soil samples, as compared to the control plants (1.70 +/- 0.14 or 0.88 +/- 0.07, respectively) grown in the field soil of Saitama University. The soil sample which caused the significant increase in mutation frequency contained 6,880 +/- 330 mBq/g 137Cs, 62.5 +/- 4.4 mBq/g 60Co, and some other nuclides; a 150 microR/hr exposure rate being measured on the surface of the soil sample. The effective cumulative external exposures measured for the inflorescences of the plant grown in this soil sample averaged at most 60.8 mR, being too small to explain the significant elevation in mutation frequency observed. On the other hand, internal exposure due to uptake of radioactive nuclides was estimated to be 125 mrad (1.25 mGy) as an accumulated effective dose, mainly based on a gamma-spectrometrical analysis. However, it seemed highly likely that this value of internal exposure was a considerable underestimate, and the internal exposure was considered to be more significant than the external exposure.