ATM-dependent hyper-radiosensitivity in mammalian cells irradiated by heavy ions.

PURPOSE: Low-dose hyper-radiosensitivity (HRS) and the later appearing radioresistance (termed induced radioresistance [IRR]) was mainly studied in low linear energy transfer (LET) radiation with survival observation. The aim of this study was to find out whether equivalent hypersensitivity occurred in high LET radiation, and the roles of ataxia telangiectasia mutated (ATM) kinase. METHODS AND MATERIALS: Survival and mutation were measured by clonogenic assay and HPRT mutation assay. ATM Ser1981 activation was detected by Western blotting and immunofluorescent staining. Pretreatment of specific ATM inhibitor (10 microM KU55933) and activator (20 microg/mL chloroquine) before carbon radiation were adopted to explore the involvement of ATM. The roles of ATM were also investigated in its G2/M checkpoint function with histone H3 phosphorylation analysis and flow cytometric assay, and DNA double strand break (DSB) repair function measured using gamma-H2AX foci assay. RESULTS: HRS/IRR was observed with survival and mutation in normal human skin fibroblast cells by carbon ions, while impaired in cells with intrinsic ATM deficiency or normal cells modified with specific ATM activator or inhibitor before irradiation. The dose-response pattern of ATM kinase activation was concordant with the transition from HRS to IRR. The ATM-dependent "early" G2 checkpoint arrest and DNA DSB repair efficiency could explain the difference between HRS and IRR. CONCLUSIONS: These data demonstrate that the HRS/IRR by carbon ion radiation is an ATM-dependent phenomenon in the cellular response to DNA damage.

A bystander effect observed in boron neutron capture therapy: a study of the induction of mutations in the HPRT locus.

PURPOSE: To investigate bystander mutagenic effects induced by alpha-particles during boron neutron capture therapy, we mixed cells that were electroporated with borocaptate sodium (BSH), which led to the accumulation of (10)B inside the cells, and cells that did not contain the boron compound. The BSH-containing cells were irradiated with alpha-particles produced by the 10B(n,alpha)7Li reaction, whereas cells without boron were affected only by the 1H(n,gamma)2H and 14N(n,rho)14C reactions. METHODS AND MATERIALS: The lethality and mutagenicity measured by the frequency of mutations induced in the hypoxanthine-guanine phosphoribosyltransferase locus were examined in Chinese hamster ovary cells irradiated with neutrons (Kyoto University Research Reactor: 5 MW). Neutron irradiation of 1:1 mixtures of cells with and without BSH resulted in a survival fraction of 0.1, and the cells that did not contain BSH made up 99.4% of the resulting cell population. The molecular structures of the mutations were determined using multiplex polymerase chain reactions. RESULTS: Because of the bystander effect, the frequency of mutations increased in the cells located nearby the BSH-containing cells compared with control cells. Molecular structural analysis indicated that most of the mutations induced by the bystander effect were point mutations and that the frequencies of total and partial deletions induced by the bystander effect were less than those induced by the original neutron irradiation. CONCLUSION: These results suggested that in boron neutron capture therapy, the mutations caused by the bystander effect and those caused by the original neutron irradiation are induced by different mechanisms.

DNA lesion and Hprt mutant frequency in rat lymphocytes and V79 Chinese hamster lung cells exposed to cadmium.

Cadmium is a potential carcinogenic environmental and occupational pollutant. A wide variety of mutagens have been shown to cause DNA damage, but it is not yet clear whether the DNA damage is relative to inducement of mutations. DNA damage and the formation of mutations at the hypoxanthine guanine phosphoribosyl trans ferase (HPRT) induced by cadmium chloride (CdCl(2)) were investigated with rat lymphocytes and V79 Chinese hamster lung cells. The hprt mutant frequency (MF) assay was used as the method to measure gene mutation in the rat lymphocytes and V79 cells exposed to CdCl(2), and comet assay analysis was performed to detect DNA lesion and repair in CdCl(2)-induced V79 cells. The results showed that CdCl(2) treatment caused a strong genotoxic effect and a marginal effect on the frequency of gene mutations. The hprt mutant frequencies in the rat lymphocytes and V79 cells exposed to CdCl(2) were statistically higher than those of the negative control. There was statistical significance in TL, TD and percentage of comet cell with tails. CdCl(2) treatment can induce DNA single-strand breaks. There was a dose-dependent increase between CdCl(2) and DNA lesion. After cells were treated with CdCl(2) and hydrogen peroxide (H(2)O(2)), the TL and TD declined with repair time increasing, which indicated that DNA damages were repaired gradually. However, DNA repair with treatment of CdCl(2) was slower than that of H(2)O(2) in V79 cells, which suggests that CdCl(2) affected DNA repair of damaged cells. The study also showed that the hprt MF and comet assay can be used for genotoxicity testing of heavy metals. DNA damage detected with the comet assay may be relative to mutagenesis.

Telomerase-immortalized human fibroblasts retain UV-induced mutagenesis and p53-mediated DNA damage responses.

Immortalized cells frequently have disruptions of p53 activity and lack p53-dependent nucleotide excision repair (NER). We hypothesized that telomerase immortalization would not alter p53-mediated ultraviolet light (UV)-induced DNA damage responses. DNA repair proficient primary diploid human fibroblasts (GM00024) were immortalized by transduction with a telomerase expressing retrovirus. Empty retrovirus transduced cells senesced after a few doublings. Telomerase transduced GM00024 cells (tGM24) were cultured continuously for 6 months (>60 doublings). Colony forming ability after UV irradiation was dose-dependent between 0 and 20J/m2 UVC (LD50=5.6J/m2). p53 accumulation was UV dose- and time-dependent as was induction of p48(XPE/DDB2), p21(CIP1/WAF1), and phosphorylation on p53-S15. UV dose-dependent apoptosis was measured by nuclear condensation. UV exposure induced UV-damaged DNA binding as monitored by electrophoretic mobility shift assays using UV irradiated radiolabeled DNA probe was inhibited by p53-specific siRNA transfection. p53-Specific siRNA transfection also prevented UV induction of p48 and improved UV survival measured by colony forming ability. Strand-specific NER of cyclobutane pyrimidine dimers (CPD) within DHFR was identical in tGM24 and GM00024 cells. CPD removal from the transcribed strand was nearly complete in 6h and from the non-transcribed strand was 73% complete in 24h. UV-induced HPRT mutagenesis in tGM24 was indistinguishable from primary human fibroblasts. These wide-ranging findings indicate that the UV-induced DNA damage response remains intact in telomerase-immortalized cells. Furthermore, telomerase immortalization provides permanent cell lines for testing the immediate impact on NER and mutagenesis of selective genetic manipulation without propagation to establish mutant lines.

In vitro studies of the genotoxicity of ionizing radiation in human G(0) T lymphocytes.

In an effort to mimic human in vivo exposures to ionizing irradiation, G(0) phase T lymphocytes from human peripheral blood samples were utilized for in vitro studies of the genotoxic effects of (137)Cs low-LET irradiation and (222)Rn high-LET irradiation. Both types of radiation induced mutations in the HPRT gene in a dose-dependent manner, with a mutant frequency (MF) = 4.28 + 1.34x + 7.51x(2) for (137)Cs (R(2) = 0.95) and MF = 4.81 + 0.67x for (222)Rn (R(2) = 0.51). Post (137)Cs irradiation incubation in the presence of cytosine arabinoside, a reversible inhibitor of DNA repair, caused an increase in the MF over irradiation alone, consistent with a misrepair mechanism being involved in the mutagenicity of low-LET irradiation. The spectrum of (137)Cs irradiation-induced mutation displayed an increase in macro-deletions (in particular total gene deletions) and rearrangement events, some of which were further defined by either chromosome painting or direct DNA sequencing. The spectrum of (222)Rn irradiation-induced mutation was characterized by an increase in small alterations, especially multiple single base deletions/substitutions and micro-deletions. These studies define the specific response of human peripheral blood T cells to ionizing irradiation in vitro and form a basis for evaluating the genotoxic effects of human in vivo exposure.

LET and ion-species dependence for mutation induction and mutation spectrum on hprt locus in normal human fibroblasts.

We have been studying LET and ion species dependence of RBE in mutation frequency and mutation spectrum of deletion pattern of exons in hprt locus. Normal human skin fibroblasts were irradiated with heavy-ion beams, such as carbon- (290 MeV/u and 135 MeV/u), neon- (230 MeV/u and 400 MeV/u), silicon- (490 MeV/u) and iron- (500 MeV/u) ion beams, generated by Heavy Ion Medical Accelerator in Chiba (HIMAC) at national Institute of Radiological Sciences (NIRS). Mutation induction in hprt locus was detected to measure 6-thioguanine resistant colonies and deletion spectrum of exons was analyzed by multiplex PCR. The LET-RBE curves of mutation induction for carbon- and neon-ion beams showed a peak around 75 keV/micrometers and 155 keV/micrometers, respectively. On the other hand, there observed no clear peak for silicon-ion beams. The deletion spectrum of exons was different in induced mutants among different ion species. These results suggested that quantitative and qualitative difference in mutation occurred when using different ion species even if similar LET values.

Mutations induced by heavy charged particles.

The relative biological-effectiveness of radiation is increased when cells or tissue are exposed to densely ionizing (high-LET) radiation. A large number of studies focus on the following aspects of the biological effects of high-LET radiation: (i) basic understanding of radiation damage and repair; (ii) developing radiotherapy protocols for accelerated charged particles; and (iii) estimation of human risks from exposure to high-LET heavy charged particles. The increased lethal effectiveness (cell inactivation) of high-LET radiation contributes to new methods for using radiation therapy, but it is also necessary to study the enhanced mutagenic effect of high LET radiation, because higher frequencies of mutation can be expected to provide higher rates of carcinogenicity with human exposure. It is important to note that both measures of biological effectiveness (lethality and mutagenicity) depend on the quality of radiation, the dose, dose-rate effects, and the biological endpoints studied. This paper is intended to provide a review of current research on the mutagenic effects of high-LET radiation, and is organized into three sections. First, are descriptions of the induced mutations studied with various detection systems (section 1) because the detectable mutations induced by ionizing radiation, including heavy-ions, depend largely on the detection system used. Second is a discussion of the biological significance of the dependence of induced mutations on LET (section 2). This is related to the molecular nature of radiation lesions and to the repair mechanisms used to help cells recover from such damage. Finally, applications of mutation detection systems for studies in space (section 3) are described, in which the carcinogenic effects of space environmental radiation are considered.

Analysis of spontaneous, gamma ray- and ethylnitrosourea-induced hprt mutants in HL-60 cells with multiplex PCR.

AIM: To explore the molecular spectra and mechanism of human hypoxanthine guanine phosphoribosyl transferase (hprt) gene mutation induced by ethyluitrosourea (ENU) and (60)Co gamma-rays. METHODS: Independent human promyelocytic leukemia cells (HL-60) mutants at the hprt locus were isolated from untreated, ethyluitrosourea (ENU) and (60)Co gamma-ray-exposed cells, respectively, and verified by two-way screening. The genetic changes underlying the mutation were determined by multiplex polymerase chain reaction (PCR) amplification and electrophoresis technique. RESULTS: With dosage increased, survival rate of plated cell reduced (in the group with dosage of ENU with 100-200 micro g/ml, P<0.01; in the group with dosage of (60)Co gamma-ray with 2-4 Gy, P<0.05) and mutational frequency increased (in the group of ENU 12.5-200.0 micro g/ml, P<0.05; in the group of (60)Co gamma-ray with 1-4 Gy, P<0.05) significantly. In the 13 spontaneous mutants analyzed, 92.3 % of mutant clones did not show any change in number or size of exon, a single exon was lost in 7.7 %, and no evidence indicated total gene deletion occurred in nine hprt exons. However, deletions were found in 79.7 % of ENU-induced mutations (62.5-89.4 %, P<0.01) and in 61.7 % of gamma-ray-induced mutations (28.6-76.5 %, P<0.01). There were deletion mutations in all 9 exons of hprt gene and the most of induced mutations were chain deletion with multiplex exons (97.9 % in gamma-ray-induced mutants, 88.1 % in ENU-induced mutants). CONCLUSION: The spectra of spontaneous mutations differs completely from that induced by EUN or (60)Co gamma-ray. Although both ENU and gamma-ray can cause destruction of genetic structure, mechanism of mutagenesis between them may be different.

Bystander effects: intercellular transmission of radiation damage signals.

Biological effects were examined in confluent cultures of fibroblasts and epithelial cells exposed to very low mean doses of alpha radiation, doses by which only 1-2% of the cells were actually traversed by an alpha particle. Enhanced frequencies of sister chromatid exchanges and HPRT mutations occurred in the non-irradiated, 'bystander' cells associated with a similar increase in the frequency of micronuclei, indicating the induction of DNA damage in these cells. In order to gain information concerning molecular pathways, changes in gene expression were examined in bystander cells by western analysis and in situ immunofluorescence staining. The expression levels of p53, p21 and MDM2 were significantly modulated in bystander cells; the damage signals leading to these changes were transmitted from irradiated to bystander cells by gap junction mediated intercellular communication. The bystander response was suppressed by incubation with superoxide dismutase as well as an inhibitor of NADPH oxidase, suggesting the effect may be mediated by oxidative stress. To examine other signalling pathways responsive to oxidative stress, the activation of stress-related kinases and their downstream transcription factors were analysed in bystander cells by western blotting and electrophoretic mobility shift assays; a 2-4-fold increase in the phosphorylation levels of JNK, ERK1/2, p90RSK, Elk-1 and ATF2 was observed. These changes were detected by 15 min after irradiation and persisted for at least 1 h. These findings indicate the activation of multiple signal transduction pathways in bystander cells, involving signals arising from the plasma membrane as well as from DNA damage.

Mutagenicity of gamma-radiation, mitomycin C, and etoposide in the Hprt and Tk genes of Tk(+/-) mice.

The recently developed Tk(+/-) mouse detects in vivo somatic cell mutation in the endogenous, autosomal Tk gene. To evaluate the sensitivity of this model, we have treated Tk(+/-) mice with three agents that induce DNA damage by different mechanisms, and determined spleen lymphocyte mutant frequencies (MFs) in the autosomal Tk gene and in the X-linked Hprt gene. gamma-Radiation, which produces single- and double-strand breaks by nonspecific oxidative stress, efficiently increased Hprt MF, but not Tk MF. Mitomycin C, which produces bulky DNA monoadducts and crosslinks, was mutagenic in both the Hprt and Tk genes, but the response was greater in the Tk gene. An inhibitor of the ligase function of DNA topoisomerase II, etoposide, did not increase Hprt MF, and induced a small, but nonsignificant increase in Tk MF. Combined with previous data, the results indicate that the two genes are differentially sensitive to many agents, and that the Tk gene is more sensitive than the Hprt gene to some, but not all types of DNA damage.

Uneven distributions of naïve and memory T cells in the CD4 and CD8 T-cell populations derived from a single stem cell in an atomic bomb survivor: implications for the origins of the memory T-cell pools in adulthood.

The processes that lead to the establishment and maintenance of memory T-cell pools in humans are not well understood. In this study, we examined the emergence of naïve and memory T cells in an adult male who was exposed to an atomic bomb radiation dose of approximately 2 Gy in 1945 at the age of 17. The analysis presented here was made possible by our earlier observation that this particular individual carries a hematopoietic stem cell mutation at the hypoxanthine phosphoribosyltransferase (HPRT) locus that is almost certainly a result of his exposure to A-bomb radiation. Our key finding is that we detected a very much higher HPRT mutant frequency in the naive (CD45RA(+)) cell component of this individual's CD4 and CD8 T-cell populations than in the memory (CD45RA(-)) cell component of his CD4 and CD8 T-cell populations. This stands in marked contrast to our finding that HPRT mutant frequencies are fairly similar in the naïve CD45RA(+) and memory CD45RA(-) components of the CD4 and CD8 T-cell populations of three unexposed individuals examined concurrently. In addition we found that the HPRT mutant frequencies were about 30-fold higher in the naïve (CD45RA(+)) CD4 T cells of the exposed individual than in his memory (CD45RA(-)) cell populations, but that the effect was a little less striking in his CD8 cell populations, where the HPRT mutant frequencies were only about 15-fold higher in his naïve T-cell pools than in his memory T-cell pools. We further found that 100% of the HPRT mutant cells in both his CD4 and CD8 naïve cell subsets appeared to have originated from repeated divisions of the initial HPRT mutant stem cell, whereas only 4 of 24 and 5 of 6 mutant cells in his CD4 and CD8 memory cell subsets appeared to have originated from that same stem cell. The most straightforward conclusion may be that the great majority of the T cells produced by this individual since he was 17 years old have remained as naïve-type T cells, rather than having become memory-type T cells. Thus the T cells that have been produced from the hematopoietic stem cells of this particular A-bomb-exposed individual seldom seem to enter and/or to remain in the memory T-cell pool for long periods. We speculate that this constraint on entry into memory T-cell pools may also apply to unirradiated individuals, but in the absence of genetic markers to assist us in obtaining evidential support, we must await clarifying information from radically different experimental approaches.

Induction and decline of HPRT mutants and deletions following a low dose radiation exposure at Chernobyl.

This study was conducted to evaluate the ability of mutation in the hypoxanthine-phosphoribosyltransferase gene (HPRT) to detect radiation-induced mutation in lymphocytes of Russian Chernobyl Clean-up workers, particularly as a function of time after exposure. It is part of a multi-endpoint study comparing HPRT mutation with chromosome translocation and glycophorin A mutation [Radiat. Res. 148 (1997) 463], and extends an earlier report on HPRT [Mutat. Res. 431 (1999) 233] by including data from all 9 years of our study (versus the first 6 years) and analysis of deletion size. Blood samples were collected from 1991 to 1999. HPRT mutant frequency (MF) as determined by the cloning assay was elevated 16% in Clean-up workers (N=300, the entire group minus one outlier) compared to Russian Controls (N=124) when adjusted for age and smoking status (P=0.028). Since exposures occurred over a short relative to the long sampling period, the year of sampling corresponded roughly to the length of time since exposure (correlation coefficient=0.94). When date of blood sample was considered, Control MF was not time dependent. Clean-up worker MF was estimated to be 47% higher than Control MF in 1991 (P=0.004) and to decline 4.4% per year thereafter (P=0.03). A total of 1123 Control mutants and 2799 Clean-up worker mutants were analyzed for deletion type and size by PCR assay for retention of HPRT exons and flanking markers on the X chromosome. There was little difference between the overall deletion spectra of Clean-up workers and Controls. However, there was a decline in the average size of deletions of Clean-up workers as time after exposure at Chernobyl increased from 6 to 13 years (P< or =0.05). The results illustrate the sensitivity of HPRT somatic mutation as a biomarker for populations with low dose radiation exposure, and the dependence of this sensitivity on time elapsed since radiation exposure.

Multiple manifestations of X-ray-induced genomic instability in Chinese hamster ovary (CHO) cells.

Carcinogenesis is postulated to follow a multistep cascade in which the first genetic event may destabilize cells and thereby facilitate the induction of subsequent mutations within the same cell. It has recently been shown that exposure to ionizing radiation can in itself induce a persistent, heritable genetic instability in cells. To further investigate this phenomenon, we utilized a mutationally unstable population derived from a single Chinese hamster ovary (CHO) cell that survived X irradiation. We exposed these cells to a second dose of radiation, selected hypoxanthine phosphoribosyl transferase (HPRT) mutant subclones, and identified the type of mutations involved. We found complete deletions, continuous tract partial deletions, single-exon deletions, discontinuous-exon deletions ("skip mutations"), and point mutations (changes of less than 100 bp) among the isolated HPRT mutants. We hypothesized that the skip mutation clones might be more likely to demonstrate genomic instability. To test this hypothesis, mutant subclones were screened for three markers of genetic instability: alteration of minisatellite sequences, change in telomere length, and induction of chromosomal aberrations. Clones with skip mutations and single-exon deletions possessed elevated frequencies of minisatellite alterations and chromosomal aberrations, particularly rings and dicentrics. All mutant clones showed longer telomere terminal restriction fragment lengths than did wild-type cells. These results are consistent with the hypothesis that irradiation may induce a global instability phenotype, since the multiple alterations observed are mechanistically distinct, heritable cellular modifications that arose in the clonogenic progeny of the irradiated cells. Skip mutations may be one manifestation of this instability, but their presence was not specifically associated with the other genetic alterations.

Extrapolation of the dna fragment-size distribution after high-dose irradiation to predict effects at low doses.

The patterns of DSBs induced in the genome are different for sparsely and densely ionizing radiations: In the former case, the patterns are well described by a random-breakage model; in the latter, a more sophisticated tool is needed. We used a Monte Carlo algorithm with a random-walk geometry of chromatin, and a track structure defined by the radial distribution of energy deposition from an incident ion, to fit the PFGE data for fragment-size distribution after high-dose irradiation. These fits determined the unknown parameters of the model, enabling the extrapolation of data for high-dose irradiation to the low doses that are relevant for NASA space radiation research. The randomly-located-clusters formalism was used to speed the simulations. It was shown that only one adjustable parameter, Q, the track efficiency parameter, was necessary to predict DNA fragment sizes for wide ranges of doses. This parameter was determined for a variety of radiations and LETs and was used to predict the DSB patterns at the HPRT locus of the human X chromosome after low-dose irradiation. It was found that high-LET radiation would be more likely than low-LET radiation to induce additional DSBs within the HPRT gene if this gene already contained one DSB.

Dimethyl sulfoxide protects against thermal and epithermal neutron-induced cell death and mutagenesis of Chinese hamster ovary (CHO) cells.

PURPOSE: To investigate the protective effects of dimethyl sulfoxide (DMSO) on cell killing and mutagenicity at the HPRT locus in Chinese hamster ovary (CHO) cells against thermal and epithermal neutrons produced at the Kyoto University Research (KUR) reactor. METHODS AND MATERIALS: DMSO was added to cells 15 min before irradiation and removed 15 min after irradiation. Cells were irradiated by thermal and epithermal neutrons with or without boron at 10 ppm. The biological endpoint of cell survival was measured by colony formation assay. The mutagenicity was measured by the mutant frequency in the HPRT locus. A total of 378 independent neutron-induced mutant clones were isolated in separate experiments. The molecular structure of HPRT mutations was determined by analysis by multiplex polymerase chain reaction of all nine exons. RESULTS: The D(0) values of epithermal and thermal neutrons in three different modes, i.e., thermal, epithermal, and mixtures of thermal and epithermal, were 0.8-1.2 Gy. When cells were treated with DMSO, the D(0) values increased to 1.0-2.3, especially in the absence of boron. DMSO showed a protective effect against mutagenesis of the HPRT locus induced by epithermal and thermal neutron irradiation. After DMSO treatment, the mutagenicity was decreased, especially when the cells were irradiated in epithermal neutron mode. Molecular structure analysis indicated that total and partial deletions were dominant and the incidence of total deletions was increased in the presence of boron in the thermal neutron and mixed modes. In the epithermal neutron mode, more than half of the mutations were total deletions. When cells were treated with DMSO, the incidence of total deletions by thermal neutron irradiation with boron and epithermal irradiation decreased. CONCLUSIONS: Our results suggest that DMSO has various protective effects against cytotoxic and mutagenic effects of thermal and epithermal neutrons, and that the extent of protection is reflected by the percentage of absorbed dose distribution for each neutron irradiation mode.

Laser pyrolysis products-genotoxic, clastogenic and mutagenic effects of the particulate aerosol fractions.

Laser therapy has gained wide acceptance and application in many medical disciplines. Nevertheless, during surgical procedures, the thermal destruction of tissue creates a smoke plume. Recent research data indicate that pyrolysates liberated during vaporisation of tissue induce DNA damage. However, assessing potential health hazards during medical laser treatment requires comprehensive insight into the cytotoxic, genotoxic, clastogenic and mutagenic capacity of laser pyrolysis products (LPP). Therefore, the aim of this study was to evaluate the cytotoxic, genotoxic, clastogenic and mutagenic potential of substances resulting from laser irradiation. Four different types of porcine tissues were irradiated with a surgical CO2 laser, the aerosols were sampled under defined conditions and subjected to the SCE test, micronucleus test and the HPRT test. The results showed that the pyrolysis products are strong inducers of cytotoxic effects. The pyrolysis products induced positive effects in the SCE test, micronucleus test and the HPRT test. The ability and extent to induce genotoxic and mutagenic effects turned out to be dependent on the type of tissue that had been irradiated. In general, the effects were most pronounced with liver pyrolysate. In all test systems, a clear dose relationship could be established. In conclusion, we were able to prove that the particulate fraction of laser pyrolysis aerosols originating from biological tissues undoubtedly have to be classified as cytotoxic, genotoxic, clastogenic and mutagenic. Therefore, they could be potential health hazards for humans.

Suppressed expression of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) in an irradiation-attenuated Plasmodium berghei XAT strain.

Plasmodium berghei XAT (XAT) is a non-reversible, non-lethal type malaria parasite strain derived from the highly virulent lethal P. berghei NK65 (NK65) by X-irradiation. The difference in polypeptide expression between NK65 and XAT was examined in this study. Western blot patterns of the parasite polypeptides showed that a 30-kDa polypeptide was not detected in XAT. In the present paper, we focused the study on the difference in the expression of the 30-kDa polypeptide between XAT and NK65. Although several other significant differences were noted in the spots shown by two-dimensional gel electrophoresis, the 30-kDa polypeptide was isolated by means of preparative 2D-gel electrophoresis followed by HPLC, and N-terminal amino acid sequence of the polypeptide was eventually determined. Complementary DNA clones encoding the 30-kDa polypeptide were isolated and characterized. Full-length cDNA clones from XAT encoded a protein of 231 amino acid residues with a 693-bp open reading frame. The deduced amino acid sequence exhibited 67% identity with that for P. falciparum hypoxanthine-guanine phosphoribosyltransferase (HGPRT; EC 2.4.2.8), suggesting that this protein is P. berghei HGPRT. Northern blot analysis revealed that expression of HGPRT in XAT was only one-eighth of that in NK65. This finding indicates that HGPRT gene expression is markedly suppressed in XAT. The amino acid sequence of HGPRT from NK65 was identical to that from XAT. This finding showed that the amino acid sequence of XAT-HGPRT was not mutated and had not undergone deletion.

Total gene deletions and mutant frequency of the HPRT gene as indicators of radiation exposure in Chernobyl liquidators.

This study was conducted to determine the utility of deletion spectrum and mutant frequency (MF) of the hypoxanthine phosphoribosyl transferase gene (HPRT) as indicators of radiation exposure in Russian Liquidators who served in 1986 or 1987 in the clean up effort following the nuclear power plant accident at Chernobyl. HPRT MF was determined using the cloning assay for 117 Russian Controls and 122 Liquidators whose blood samples were obtained between 1991 and 1998. Only subjects from whom mutants were obtained for deletion analysis are included. Multiplex PCR analysis was performed on cell extracts of 1080 thioguanine resistant clones from Controls and 944 clones from Liquidators. Although the deletion spectra of Liquidators and Controls were similar overall, the Liquidator deletion spectrum was heterogeneous over time. Most notable, the proportion of total gene deletions was higher in 1991-1992 Liquidators than in Russian Controls (chi 2 = 10.5, p = 0.001) and in 1993-1994 Liquidators (chi 2 = 8.3, p = 0.004), and was marginally elevated relative to 1995-1996 Liquidators (chi 2 = 3.3, p = 0.07). This type of mutations has been highly associated with radiation exposure. Total gene deletions were not increased after 1992. Band shift mutations were also increased in the 1991-1992 Liquidators but were associated with increased MF of both Liquidators and Controls (p = 0.009), not with increased MF in 1991-1992 Liquidators (p = 0.7), and hence are not believed to be associated with radiation exposure. Regression analysis demonstrated that relative to Russian Controls HPRT MF was elevated in Liquidators overall when adjusted for age and smoking status (37%, p = 0.0001), and also was elevated in Liquidators sampled in 1991-1992 (72%, p = 0.0076), 1993-1994 (22%, p = 0.037), and 1995-1996 (62%, p = 0.0001). In summary, HPRT MF was found to be the more sensitive and persistent indicator of radiation exposure, but the specificity of total gene deletions led to detection of probable heterogeneity of radiation exposure within the exposed population.