Sources, spatial variation, and speciation of heavy metals in sediments of the Tamagawa River in Central Japan.

Sediments of the Tamagawa River in central Japan were studied to explain the spatial variation, to identify the sources of heavy metals, and to evaluate the anthropogenic influence on these pollutants in the river. Sediment samples were collected from 20 sites along the river (five upstream, four midstream, and 11 downstream). Heavy metal concentrations, viz. chromium, nickel, copper, zinc, lead, cadmium, and molybdenum, in the samples were measured using inductively coupled plasma-mass spectroscopy. The chemical speciations of heavy metals in the sediments were identified by the widely used five-step Hall method. Lead isotopes were analyzed to identify what portion is contributed by anthropogenic sources. The total heavy metal concentrations were compared with global averages for continental crust (shale) and average values for Japanese river sediments. The mean heavy metal concentrations were higher in downstream sediments than in upstream and midstream samples, and the concentrations in the silt samples were higher than those in the sand samples. Speciation results demonstrate that, for chromium and nickel, the residual fractions were dominant. These findings imply that the influence of anthropogenic chromium and nickel contamination is negligible, while copper, zinc, and lead were mostly extracted in the non-residual fraction (metals in adsorbed/exchangeable/carbonate forms or bound to amorphous Fe oxyhydroxides, crystalline Fe oxides, or organic matter), indicating that these elements have high chemical mobility. The proportion of lead (Pb) isotopes in the downstream silt samples indicates that Pb accumulation is primarily derived from anthropogenic sources.

A model for analysis of the yield and the level of clustering of radiation-induced DNA-strand breaks in hydrated plasmids.

Although it is widely accepted that the spatial distribution of strand breaks is highly relevant to the biological consequences of radiation, the extent to which strand breaks are clustered is not usually demonstrated directly from the experimental data. To evaluate the spatial distribution of radiation-induced strand breaks, the authors have developed a model for the generation of strand breaks after irradiation. The model assumes that (1) a radiation track has a certain probability of 'hitting' a plasmid, (2) the radiation generates strand break(s) by chance within a relatively small region that could produce double-strand breaks and (3) the number of strand breaks generated within the region follows a Poisson distribution. To find out whether the model is valid, the authors compared the calculated values with the experimental data obtained by a plasmid DNA assay. Taking into account the inherent bias of the plasmid assay, the model described well the experimental results of hydrated plasmids exposed to radiation.

Studies of soft X-ray-induced Auger effect on the induction of DNA damage.

PURPOSE: To understand the characteristics of DNA damage induced by Auger effect in DNA by ultrasoft X-irradiation. In situ electron paramagnetic resonance (EPR) spectroscopy as well as biochemical analysis has been applied to examine the DNA damage induction in both viewpoints of intermediate species and final products. MATERIALS AND METHODS: Unpaired electron species induced in a calf thymus DNA film irradiated with monochromatic ultrasoft X-rays (270-580 eV) was observed using an X-band EPR spectrometer installed in a synchrotron beamline. To determine the yield of single strand break (SSB), pUC18 plasmid DNA was irradiated and then analyzed by agarose gel electrophoresis. To analyze molecular change in a single strand DNA, a new technique using DNA-denaturation-treatment has been applied to quantify multiple SSB arising in both DNA strands. RESULTS: Short-lived EPR spectra were observed during irradiation. The intensity of transient EPR spectrum shows the similar energy dependence with that of the SSB yield around oxygen K-edge in particular. The fraction of the single-strand plasmid DNA (SS-DNA) after irradiation could be determined using a low-temperature-denaturation condition. The obtained slope of the dose-response for SS-DNA shows half of that of closed circular DNA as expected under the diluted solution condition. CONCLUSION: The availability of an EPR apparatus installed in a synchrotron beamline is demonstrated by detecting very short-lived unpaired electron species. Transient EPR spectra of DNA show the similar energy dependence to that of the SSB yield. The proposed DNA-denaturation assay works as expected using the low-temperature-denaturation condition.

Yields of strand breaks and base lesions induced by soft X-rays in plasmid DNA.

The yields of soft-X-ray-induced DNA damages have been measured by using closed-circular plasmid DNA. Several DNA solutions with three kinds of radical scavenger capacity and also fully hydrated DNA samples were irradiated to compare the contribution by indirect reaction of diffusible water radicals, such as OH*, with those by direct action of secondary electrons. The yields of prompt single- (SSBs) and double-strand breaks (DSBs) decrease with increasing scavenging capacity. The SSB yields for soft X-rays are approximately midway those between gamma-ray and ultrasoft X-ray data previously reported. Heat labile sites are observed only in the low scavenger condition. The yields of the base lesions revealed by post irradiation treatment with base excision repair enzymes showed a similar value for Nth and Fpg protein except in the hydrated sample. These results indicate that the direct effect of soft X-rays induces the damages with different efficiency from those by indirect effect.

LET dependence of lethality of carbon ion irradiation to single tobacco cells.

PURPOSE: To determine the radiation sensitivity and relationship between linear energy transfer (LET) and relative biological effectiveness (RBE) in single plant cells irradiated with heavy ions. MATERIALS AND METHODS: Single cells were isolated from the tobacco BY-2 cell line and irradiated with carbon ions (78.6-309 keV microm(-1)) and gamma-rays (0.2 keV microm(-1)). Two weeks after irradiation, colonies with 16 cells or more derived from the irradiated cells were counted as survivors. The surviving fraction was fitted using the single-hit, multitarget theory. RESULTS: The doses needed to reduce the surviving fraction of the cells to 0.1 (D10) of gamma-rays and carbon ions were 47.2 and 10.5-12.6 Gy, respectively. The RBE based on the D10 peaked at an LET of 247 keV microm(-1). The inactivation cross-section of carbon ions reached a plateau of 11.3 microm2 at an LET of 247 keV microm(-1). CONCLUSIONS: The radiation sensitivity of single tobacco cells was much lower than that of mammalian cells, although the mean number of base pairs per chromosome in the two cell types was similar. The RBE peak based on the D10 of carbon ions in single tobacco cells occurred at a higher LET than it does in other organisms.

Sexual modification of female spinach seeds (Spinacia oleracea L.) by irradiation with ion particles.

The female seeds of a spinach plant (Spinacia orelacea L.) were exposed to He (12.5 MeV/n) and C (18.3 MeV/n) ions in order to investigate the effects of ion particles on sex expression. He ions did not affect germination rates or flowering at doses up to 50 Gy. C ions did not affect germination rates or flowering at doses up to 15 Gy, but a dose of 25 Gy resulted in many plants with morphological aberrations. When unexposed female plants were grown without cross-fertilization for 10 weeks after sowing, 5.6-14.3% of the plants produced anthers from female flowers. These sex-modified plants could self-pollinate and form seeds, which expressed only female organs. Conversely, gynomonoecious plants were induced from these female seeds by exposure to He ions (5-50 Gy) and C ions (5-25 Gy) without any difference in the rates of flowered progeny. Moreover, andromonoecious plants were induced from female seeds by exposure to He ions at 50 Gy. These results suggest that the sex of a spinach plant is expressed as a flexible phenotype, converging from female to gyno- and andromonoecy after exposure to ion particles.

Rearrangements of the DNA in carbon ion-induced mutants of Arabidopsis thaliana.

To elucidate the nature of structural alterations in plants, three carbon ion-induced mutations in Arabidopsis thaliana, gl1-3, tt4(C1), and ttg1-21, were analyzed. The gl1-3 mutation was found to be generated by an inversion of a fragment that contained GL1 and Atpk7 loci on chromosome 3. The size of the inverted fragment was a few hundred kilobase pairs. The inversion was found to accompany an insertion of a 107-bp fragment derived from chromosome 2. The tt4(C1) mutation was also found to be due to an inversion. The size of the intervening region between the breakpoints was also estimated to be a few hundred kilobase pairs. In the case of ttg1-21, it was found that a break occurred at the TTG1 locus on chromosome 5, and reciprocal translocation took place between it and chromosome 3. From the sequences flanking the breakpoints, the DNA strand breaks induced by carbon ions were found to be rejoined using, if present, only short homologous sequences. Small deletions were also observed around the breakpoints. These results suggest that the nonhomologous end-joining (NHEJ) pathway operates after plant cells are exposed to ion particles.

Effect of fractionated exposure to carbon ions on the frequency of chromosome aberrations in tobacco root cells.

The induction of chromosome aberrations in tobacco root tip cells was measured after exposure to either 18 MeV/n carbon ions or 2 MeV electrons. The RBE value for acute exposure was found to be about 10. Splitting the dose into two fractions did not produce any significant effect on the yield of aberrations following carbon-ion exposure, whereas a clear decrease was observed after exposure to electrons thereby indicating an induction/activation of error-free repair after the first fraction. Moreover, this decrease appeared to be independent of the types of chromosome aberrations. On the other hand, it was suggested that the lack of any significant effect on the yield of aberrations is either due to a lack of error-free repair or to a less efficient damage repair after exposure to carbon ions.

Molecular analysis of carbon ion-induced mutations in Arabidopsis thaliana.

In order to elucidate the characteristics of the mutations induced by ion particles at the molecular level in plants, mutated loci in carbon ion-induced mutants of Arabidopsis were investigated by PCR and Southern blot analyses. In the present study, two lines of gl1 mutant and two lines of tt4 mutant were isolated after carbon ion-irradiation. Out of four mutants, one had a deletion, other two contained rearrangements, and one had a point-like mutation. From the present result, it was suggested that ion particles induced different kinds of alterations of the DNA and therefore they could produce various types of mutant alleles in plants.

Nucleotide sequence of the GLABROUS1 gene of Arabidopsis thaliana ecotype Columbia.

The GLABROUS1 (GL1) gene from Arabidopsis thaliana ecotype Columbia was isolated and sequenced. Comparison of the nucleotide sequence with that of the gl1-2allele revealed that two sites were changed in gl1-2. One was a 14-bp deletion in exon 3, which seemed most likely to be the cause of the mutated phenotype. The other was a change from TC to CT in the 5' untranslated region. Since the 5' upstream sequence required for the GL1 gene expression has not been clearly specified, one cannot rule out the possibility that this change may contribute to the phenotype of gl1-2. A comparison was also made with the GL1 gene of ecotype Wassilewskija and revealed some lack of sequence conservation. The changes observed may have little influence on the function or the expression of the GL1 gene product.

Effects of heavy ions on the germination and survival of Arabidopsis thaliana.

Inhibition of germination and reduction in survival of Arabidopsis thaliana were investigated to study the effects of heavy ions on a multicellular system. Dry seeds of Col and Ler ecotypes were exposed to He, C, Ar and Ne ions with linear energy transfer (LET) in the range of 17-549 keV/micron and to electrons (LET = 0.2 keV/micron). The relative biological effectiveness (RBE) for the survival of both ecotypes showed the same pattern of variation with a maximum RBE of 11-12 at 252 keV/micron. For germination, RBE increased with increasing LET in Ler but not in Col, showing different sensitivities between the plant ecotypes. Inactivation cross sections of survival increased linearly in the range of 0.2-17 keV/micron and proceeded more steeply in the range of 113-252 keV/micron. At higher LET, cross sections appeared to reach a plateau at a little less than the size of the cell nucleus. When the value for survival was plotted against LET, it decreased steeply in the range about 113-252 keV/micron, indicating that heavy ions may have similar effects on both the shoulder and slope of the survival curve.

A new Arabidopsis mutant induced by ion beams affects flavonoid synthesis with spotted pigmentation in testa.

A new stable mutant of Arabidopsis thaliana with a spotted pigment in the seed coat, named anthocyanin spotted testa (ast), was induced by carbon ion irradiation. The spotted pigmentation of ast mutant was observed in immature seeds from 1-2 days after flowering (DAF), at the integument of the ovule, and spread as the seed coat formed. Anthocyanin accumulation was about 6 times higher in ast mutant than in the wild-type at 6 DAF of the immature seeds, but was almost the same in mature dry seeds. A higher anthocyanin accumulation was not observed in the seedlings, leaves or floral buds of ast mutant compared with the wild-type, which suggests that a high accumulation of anthocyanins is specific to the seed coat of the immature ast seeds. Reciprocal crosses between ast mutant and the wild-type indicated that ast is a single recessive gene mutation and segregates as a delayed inheritance. The results of crossing with tt7 and ttg mutants also confirmed that the AST gene is probably a regulatory locus that controls flavonoid biosynthesis. A mapping analysis revealed that the gene is located on chromosome I and is closely linked to the SSLP DNA marker nga280 with a distance of 3.2 cM. AST has been registered as a new mutant of Arabidopsis.

Reduced rejoining ability of DNA strand breaks with differentiation in barley root cells.

To investigate the relationship between differentiation and the rejoining ability of DNA strand breaks, DNA strand breaks induced by gamma-rays were analyzed in barley roots using the alkaline unwinding assay. The extent of unwinding in an alkaline solution containing 0.5 M NaCl was found to be significantly inhibited at the root tip consisting of meristematic cells but not in the remainder of the root consisting of differentiated cells immediately after 100 Gy of gamma-irradiation. The difference in the extent of unwinding was diminished when the alkaline solution contained 2 M NaCl, suggesting a difference of chromosome structure and/or cell skeleton between the two regions. The rejoining kinetics of DNA strand breaks consisted of a fast and a slow component in both regions. DNA strand breaks were rejoined to the level of unirradiated control at the root tip but remained partly unrejoined in the remainder of the root during 6 h post-irradiation incubation. The difference of rejoining ability observed between the two regions originated from the different efficiency of rejoining at the slow component. The rejoining at the slow component in the root tip was found to be inhibited in the presence of the protein synthesis inhibitor cycloheximide, suggesting that de novo synthesized proteins are involved in the rejoining of DNA strand breaks. In contrast, the slow component in the remainder of the root was not inhibited by cycloheximide. These results suggest that the reduced rejoining ability of DNA strand breaks of differentiated cells may result from a deficiency of rejoining DNA strand breaks by inducible repair at the slow component. In addition, the lack of an apparent correlation between rejoining ability and growth inhibition of the root is discussed.

Detection of DNA strand breaks in barley roots after gamma-ray irradiation using the alkaline unwinding assay.

The alkaline unwinding assay was applied to measure strand breaks in DNA of intact barley roots after gamma-ray irradiation without extracting DNA from the tissue. The most suitable condition for unwinding was determined to be 0.03 M NaOH containing 0.5 M NaCl for alkaline solution composition, and 15 min at 20 degrees C for unwinding time. Under this condition, DNA strand breaks were shown to be linearly increased with increasing dose, and further be rejoined during the post-irradiation incubation for 6 hr.