Radiation-induced chromatid aberrations in the bone marrow and lymph nodes of the rat during the first 24 h after irradiation

Wistar rats of both sexes were exposed to 100 R of X-rays. Chromatid-type aberrations in metaphase figures of bone marrow and lymph node cells were scored after 2, 4, 6, 8 and 24 h and 3, 5, 7, 9 and 24 h, respectively.The shape of the curve for chromatid plus isochromatid breaks in bone marrow cells versus time is exponential. It is suggested that this shape is mainly a consequence of the continuous entrance into mitosis of cells irradiated while in S phase, in addition to those that were irradiated in G₂. For lymph nodes the frequency of chromatid plus isochromatid deletions increased up to the 5th h, then began to fall off in a manner similar to that for the bone marrow. The difference in the shape of the two curves is the consequence of the different dependence on time for chromatid and isochromatid breaks in each tissue. While the frequency of chromatid breaks fell steeply with time both for the bone marrow and for lymph nodes, the frequency of isochromatid breaks remained nearly constant for bone marrow, whereas it rose to a peak at the 5th h for the lymph nodes.These differences are tentatively explained by a shift in the phases of the cell cycle sampled owing to the greater mitotic delay of G₂ cells in lymph nodes, with the suggestion that in the late S phase the frequency of isochromatid breaks is lower than in all other phases of the cell cycle.     

Differential spermatogonial stem-cell survival and mutation frequency

One group of adult C3H×101 hybrid male mice was given 3 injections of 12.5 μCi of [³H]thymidine at 9-h intervals and irradiated 24 h after the last injection with X-ray doses of 100, 300, 500, 600, 1000 R or the first fraction of a split 1000-R dose given as two 500-R exposures 24 h apart. Mice were killed 207 and 414 h after irradiation. A second group of mice was given a single injection of 12.5 μCi of [³H]thymidine 1 h before irradiation with single exposures of 300, 500, 600, 1000 R, or the first fraction of a 1000-R exposure given as two 500-R fractions 24 h apart. Mice were killed 120 and 207 h after irradiation. In both experiments, parallel groups of mice were given X-ray only as a control for the effect of [³H]thymidine. Two sets of slides were prepared for each mouse receiving [³H]thymidine: one set was not autoradiographed and was used for scoring cell survival; the second set was coated with emulsion and used for scoring percentage of labeled cells. The dose-response curves for survival at 120 and 207 h were curvilinear, with no evidence of discontinuity over the 100–1000-R range. After multiple injections of [³H]thymidine and irradiation 24 h later, percentage of labeled cells at 207 h was comparable for controls, 100, 300, and 600 R; significantly lower than controls for 1000 R; and significantly above controls after 500 + 500 R. Thus the surviving stem-cell population was qualitatively the same for that portion of the dose-response curve giving a linear increase in mutation rate but was different for both 1000-R and 500 + 500-R exposures, and the single and fractionated 1000-R exposures differed from each other. This parallelism between survival of labeled cells and mutation frequency in spermatogonial stem cells suggests that a stage in the cell cycle 24–42 h after DNA synthesis is resistant to cell killing but sensitive to mutation induction. The mutation rate after a single 1000-R exposure is low because labeled, mutation-sensitive cells have been selectively killed. Mutation frequency after the 500 + 500-R dose is increased because of synchronization induced by the first dose combined with selective killing of unlabeled cells by the second fraction. Irradiation 1 h after labeling with [³H]-thymidine demonstrated that the S phase of the spermatogonial stem-cell cycle is sensitive to radiation-induced cell killing.     

DNA signals for G2 checkpoint response in diploid human fibroblasts

DNA (deoxyribonucleic acid) signals that induce the G2 checkpoint response were examined using proliferative secondary cultures of diploid human fibroblasts. Treatments that generated DNA double-strand breaks (DSBs) directly were effective inducers of checkpoint response, generally producing >80% inhibition of mitosis (G2 delay) and the kinase activity of M-phase-promoting factor within 2 h of treatment. Effective inducers of G2 checkpoint response included γ-irradiation and the cancer chemotherapeutic drugs, bleomycin and etoposide. Treatments that produced DNA single-strand breaks, directly or indirectly through nucleotide excision repair, were not effective inducers of G2 delay. Ineffective treatments included incubation with camptothecin, an inhibitor of topoisomerase I (topo I), and irradiation with sublethal fluences of UVC, followed by incubation with aphidicolin. Transient severe inhibition of DNA synthesis with aphidicolin did not affect mitosis substantially, suggesting that the replication arrest input to the G2 checkpoint required more than brief inhibition of DNA synthesis. In contrast, moderate camptothecin-induced inhibition of DNA synthesis was associated with a strong inhibition of mitosis that developed 4–12 h after drug treatment. This result suggested that G2 delay was not expressed until the cells that were in S-phase at the time of treatment with camptothecin proceeded into G2. DNA damage was not necessary for induction of mitotic delay. An inhibitor of topoisomerase II (topo II), ICRF-193, which inhibits chromatid decatenation in G2 cells without damaging DNA, induced a severe inhibition of mitosis and M-phase-promoting factor kinase activity. The results suggest that DNA double-strand breaks and insufficiency of chromatid decatenation effectively induce the G2 checkpoint response, but DNA single-strand breaks do not.     

Determination of free energies in reaction centers of Rb. sphaeroides

Magnetic field-dependent recombination measurements together with magnetic field-dependent triplet lifetimes (Chidsey, E.D., Takiff, L., Goldstein, R.A. and Boxer, S.G. (1985) Proc. Natl. Acad. Sci USA 82, 6850–6854) yield a free energy change ΔG(P⁺H⁻ − ³P*) = 0.165 eV ±0.008 at 290 K. This does not depend on whether nuclear spin relaxation in the state ³P* is assumed to be fast or slow compared to the lifetime of this state. This value, being (almost) temperature independent, indicates ΔG(P⁺H⁻ − ³P*) ΔH(P⁺H⁻ − ³P*) and is consistent with ΔG(¹P* − P⁺H⁻) and ΔH(¹P* − ³P*) from previous delayed fluorescence and phosphorescence data, implying ΔG ΔH for all combinations of these states.     

Differential effects of sulphur mustard on S-phase cells of primary fibroblast cultures from Syrian hamsters

Sulphur mustard (SM) (5 × 10⁻⁸ M) given to primary Syrian hamster fibroblasts growing short-term in vitro products a very sharp peak of chromatid aberrations 12–16 h after treatment.

The composition of this peak has been investigated in relation to the cell cycle using the facility to divide S-phase into 5 cytologically defined sub-phases on the basis of replication band patterns following bromodeoxyuridine incorporation. It is shown that: (1) Considerable delay and perturbation of the cycle is present. (2) A major contribution to the aberration peak comes from pre-S cells, and the highest aberration frequencies are observed in such cells. (3) The bulk of contribution from S-cells comes from the last subphase, SkV. (4) The majority of early S cells (sub-phases SkI–IV) fail to reach division within 36 h. Of the total S-cells scored, 54% are non-SkV controls but only 14% after SM. (5) Aberrations are localized to late-replicating regions in SkV but are random in pre-S. (6) No measurable perturbation of replication programme was found in chromosome arms synthesizing in late SkV after SM. (7) The rapid fall in aberration frequency at later sampling times is consistent with a quick and efficient repair of DNA lesions which is known to occur after SM alkylation.

The preferential loss of early S cells seems most likely to result from selective lethality, though differential delay and perturbation may make a contribution. It is interesting to note that the subphases missing are just those where the euchromatin replicates (early replicating R and G bands). The late-replicating chromatin, some of which is known to be dispensable in Syrian hamster, is confined to SkV, the sub-phase which appears to survive this dose of SM.     

Critical review of the methods used to measure the apparent dissociation constant and ligand purity in Ca2+ and Mg2+ buffer solutions

Using simulated Ca²⁺ and Mg²⁺ buffers, methods proposed to measure both ligand purity and the apparent dissociation constant (K_app) were investigated regarding (1) predicted accuracy of both parameters and (2) generality of the solution.

The Bers’ Ca²⁺ macroelectrode method [Bers, D. M., 1982 A simple method for the determination of free [Ca] in Ca-EGTA solutions Am. J. Physiol. 242, C404–C408] cannot be used with Mg²⁺-macroelectrodes and is partly arbitrary since the linear part of the Scatchard plot is judged subjectively. Iterative methods have therefore been introduced. Iteration based on the Bers’ method or the lumped interference in the Nicolsky–Eisenman equation also failed with Mg²⁺ macroelectrodes. The Oiki et al., method [Oiki, S., Yomamoto, T., Okada, Y., 1994. Apparent stability constants and purity of Ca-chelating agents evaluated using Ca-sensitive electrodes by the double-log optimization method Cell Calcium 15, 209–46.] cannot be applied to Mg²⁺ macroelectrodes. The pH titration method of Moisescu and Pusch (Pflügers, Arch., 355, R122, 1975) predicted EGTA purity and Ca²⁺ contamination, but K_app values for EGTA were approximate. It cannot be applied to Mg²⁺ binding. The partition method [Godt, R.E., 1974. Calcium-activated tension of skinned muscle fibres of the frog. Dependence on magnesium adenosine triphosphate concentration J. Gen. Physiol. 63, 722–739.] only approximately estimated the K_app. Calibration, maintaining contaminating [Ca²⁺]/[Mg²⁺] at <1 μmol l⁻¹, and setting standards by dilution, is the ultimate check of calculated ionised concentrations, although technically difficult. The macroelectrode method of Lüthi et al. [1997. Calibration of Mg²⁺-selective macromolecules down to 1 μmol l⁻¹ in intracellular and Ca⁺- containing extracellular solutions. Exp. Physiol. 82, 453–467] accurately predicted purity and K_app at pK_app values >4 and was independent of electrode characteristics. It is considered the method of choice.

Macroelectrode primary calibration should be carried out in solutions varying from 0.5 to 10 mmol l⁻¹ combined with either Ca–EGTA or Mg–EDTA buffers; the [Ca²⁺] and [Mg²⁺] in other buffer ligands can be measured in a secondary calibration.     

Relationship of DNA repair to chromosome aberrations, sister-chromatid exchanges and survival during liquid-holding recovery in X-irradiated mammalian cells

The repair of X-ray induced DNA single strand breaks and DNA—protein cross-links was investigated in stationary phase, contact-inhibited mouse cells by the alkaline-elution technique. Approx. 90% of X-ray induced single strand breaks were rejoined during the first hour of repair, whereas most of the remaining breaks were rejoined more slowly during the next 5 h. At early repair times, the number of residual non-rejoined sungle strand breaks was approx. proportional to the X-ray dose. DNA—protein cross-links were removed at a slower rate (T1/2 approx. 10–12 h). Cells were held in stationary growth for various periods of time after irradiation before subculture at low density to score for colony survival (potentially lethal damage repair), chromosome aberrations in the first mitosis, and sister-chromatid exchanges in the second mitosis. Both cell killing and the frequency of chromosome aberrations decreased during the first several hours of recovery, reaching a minimum level by 6 h; this decrease correlated temporally with the repair of the slowly rejoining DNA-strand breaks. Relatively few sister-chromatid exchanges were observed when the cells were subcultured immediately after X-ray. The exchange frequency rose to maximum levels after a 4-h recovery interval, and returned to control levels after 12 h of recovery. The possible relationship of DNA repair to these changes in survival, chromosome aberrations, and sister-chromatid exchanges during liquid-holding recovery is discussed.     

Ternary complexes in solution with hydrogen phosphate and methyl phosphate as ligands

The stability constants of the 1:1 complexes formed between Cu(Arm)²⁺, where Arm = 2,2′-bipyridyl or 1,10-phenanthroline, and methyl phosphate, CH₃OPO₃²⁻, or hydrogen phosphate, HOPO₃²⁻, were determined by potentiometric pH titration in aqueous solution (25°C; l = 0.1 M, NaNO₃). On the basis of previously established log K versus pK_a straight-line plots (D. Chen et al., J. Chem. Soc., Dalton Trans. (1993) 1537–1546) for the complexes of simple phosphate monoesters and phosphonate derivatives, R-PO₃²⁻, where R is a non-coordinating residue, it is shown that the stabilities of the Cu(Arm) (CH₃OPO₃) complexes are solely determined by the basicity of the -PO₃²⁻ residue. In contrast, the Cu(Arm) (HOPO₃) complexes are slightly more stable (on average by 0.15 log unit) than expected on the basicity of HPO₄²⁻; this is possibly due to a more effective solvation including hydrogen bonding, an interaction not possible with coordinated CH₃OPO₃²⁻ species. Regarding biological systems the observation that HOPO₃²⁻ is somewhat favored over R-PO₃²⁻ species in metal ion interactions is meaningful.     

Acceleration of CHO cells into mitosis and reduction of X-ray-induced G2 delay by cordycepin

The mitotic cell selection technique was used to monitor the effect of cordycepin and/or 100 rad of X-rays on the entry of asynchronous or synchronous Chinese hamster ovary cells into mitosis. Continuous exposure of asynchronous cells to 5–50 μg/ml of cordycepin caused a rapid increase in the relative numbers of cells entering mitosis. In irradiated cells, cordycepin also reduced a 120-min mitotic delay by about 80 min and shifted the X-ray transition point about 10 min farther away from mitosis. Further studies showed that synchronous cells, treated continuously with 15 μg/ml of cordycepin starting at mid-to-late S phase, proceeded into mitosis approx. 40 min ahead of controls. This acceleration was associated with a 30-min lengthening of S phase and a reduction in the length of G2 from 80 to about 10 min. Furthermore, cordycepin reduced the 70-min mitotic delay observed for cells irradiated in S phase by 20 min. In contrast to the results for treatment at mid-S phase, continuous treatment during G2 of unirradiated synchronous cells with 15 μg/ml of cordycepin had little effect on accelerating cells into mitosis, yet did reduce by about 60 min the 170-min mitotic delay observed for cells irradiated in G2. Unirradiated synchronous cells treated with cordycepin starting before mid-S did not reach mitosis. Thus, there are the following transition points or intervals for cordycepin: for treatment prior to mid-S phase, cell cycle progression through S is blocked; for treatment between mid-S and late S, progression through S continues but progression through G2 is accelerated; and for treatment during G2, the rate of progression in accelerated only if the cells have been irradiated. These results are discussed in relation to the synthesis during late S and G2 of critical protein molecules essential for mitosis.     

Toxicity and mutagenicity of X-rays and [125I]dUrd or [3H]TdR incorporated in the DNA of human lymphoblast cells

We measured the toxicity and mutagenicity induced in human diploid lymphoblasts by various radiation doses of X-rays and two internal emitters. [¹²⁵I]iododeoxyuridine ([¹²⁵I]dUrd) and [³H]thymidine ([³H]TdR), incorporated into cellular DNA. [¹²⁵I]dUrd was more effective than [³H]TdR at killing cells and producing mutations to 6-thioguanine resistance (6TG^R). No ouabain-resistant mutants were induced by any of these agents. Expressing dose as total disintegrations per cell (dpc), the D₀ for cell killing for [¹²⁵I]dUrd was 28 dpc and for [³H]TdR was 385 dpc. The D₀ for X-rays was 48 rad at 37°C. The slopes of the mutation curves were approximately 75 × 10⁻⁸ 6TG^R mutants per cell per disintegration for [¹²⁵I]dUrd and 2 × 10⁻⁸ for [³H]TdR. X-Rays induced 8 × 10⁻⁸ 6TG^R mutants per cell per rad. Normalizing for survival, [¹²⁵I]dUrd remained much more mutagenic at low doses (high survival levels) than the other two agents. Treatment of the cells at either 37°C or while frozen at −70°C yielded no difference in cytotoxicity or mutation for [¹²⁵I]dUrd or [³H]TdR, whereas X-rays were 6 times less effective in killing cells at −70°C.

Assuming that incorporation was random throughout the genome, the mutagenic efficiencies of the radionuclides could be calculated by dividing the mutation rate by the level of incorporation. If the effective target size of the 6TG^R locus is 1000–3000 base pairs, then the mutagenic efficiency of [¹²⁵I]dUrd is 1.0–3.0 and of [³H]TdR is 0.02–0.06 total genomic mutations per cell per disintegration. ¹²⁵I disintegrations are known to produce localized DNA double-strand breaks. If these breaks are potentially lethal lesions, they must be repaired, since the mean lethal dose (D₀) was 28 dpc. The observations that a single dpc has a high probability of producing a mutation (mutagenic efficiency 1.0–3.0) would suggest, however, that this repair is extremely error-prone. If the breaks need not be repaired to permit survival, then lethal lesions are a subset of or are completely different from mutagenic lesions.     

Nutrient removal by thermophilic Fischerella (Mastigocladus laminosus) in a simulated algaculture process

A novel nutrient removal/waste heat utilization process was simulated using semicontinuous cultures of the thermophilic cyanobacterium Fischerella. Dissolved inorganic carbon (DIC)-enriched cultures, maintained with 10 mg l⁻¹ daily productivity, diurnally varying temperature (from 55°C to 26–28°C), a 12:12 light cycle (200 μE sec⁻¹ m⁻²) and 50% biomass recycling into heated effluent at the beginning of each light period, removed > 95% of NO₃⁻ + NO₂⁻−N, 71% of NH₃-N, 82% of PO₄³⁻ −P, and 70% of total P from effluent water samples containing approximately 400 μg l⁻¹ combined N and 60 μg l⁻¹ P. Nutrient removal was not severely impaired by an altered temperature gradient, doubled light intensity, or DIC limitation. Recycling 75% of the biomass at the end of each light period resulted in unimpaired NO₃⁻ + NO₂⁻ removal, 38–45% P removal and no net NH₃ removal. Diurnally varying P removal, averaging 50–60%, and nearly constant > 80% N removal, are therefore projected for a full-scale process with continuous biomass recycling.     

Mitochondrial superoxide production during oxalate-mediated oxidative stress in renal epithelial cells

Crystals of calcium oxalate monohydrate (COM) in the renal tubule form the basis of most kidney stones. Tubular dysfunction resulting from COM-cell interactions occurs by mechanism(s) that are incompletely understood. We examined the production of reactive oxygen intermediates (ROI) by proximal (LLC-PK1) and distal (MDCK) tubular epithelial cells after treatment with COM (25–250 μg/ml) to determine whether ROI, specifically superoxide (O₂^•−), production was activated, and whether it was sufficient to induce oxidative stress. Employing inhibitors of cytosolic and mitochondrial systems, the source of ROI production was investigated. In addition, intracellular glutathione (total and oxidized), energy status (ATP), and NADH were measured. COM treatment for 1–24 h increased O₂^•− production 3–6-fold as measured by both lucigenin chemiluminescence in permeabilized cells and dihydrorhodamine fluorescence in intact cells. Using selective inhibitors we found no evidence of cytosolic production. The use of mitochondrial probes, substrates, and inhibitors indicated that increased O₂^•− production originated from mitochondria. Treatment with COM decreased glutathione (total and redox state), indicating a sustained oxidative insult. An increase in NADH in COM-treated cells suggested this cofactor could be responsible for elevating O₂^•− generation. In conclusion, COM increased mitochondrial O₂^•− production by epithelial cells, with a subsequent depletion of antioxidant status. These changes may contribute to the reported cellular transformations during the development of renal calculi.     

Effect of nomegestrol acetate on estrone-sulfatase and 17β-hydroxysteroid dehydrogenase activities in human breast cancer cells

It is well recognized that estradiol (E₂) is one of the most important hormones supporting the growth and evolution of breast cancer. Consequently, to block this hormone before it enters the cancer cell or in the cell itself, has been one of the main targets in recent years. In the present study we explored the effect of the progestin, nomegestrol acetate, on the estrone sulfatase and 17β-hydroxy-steroid dehydrogenase (17β-HSD) activities of MCF-7 and T-47D human breast cancer cells. Using physiological doses of estrone sulfate (E₁S: 5 × 10⁻⁹ M), nomegestrol acetate blocked very significantly the conversion of E₁S to E₂. In the MCF-7 cells, using concentrations of 5 × 10⁻⁶ M and 5 × 10⁻⁵ M of nomegestrol acetate, the decrease of E₁S to E₂ was, respectively, −43% and −77%. The values were, respectively, −60% and −71% for the T-47D cells. Using E₁S at 2 × 10⁻⁶ M and nomegestrol acetate at 10⁻⁵ M, a direct inhibitory effect on the enzyme of −36% and −18% was obtained with the cell homogenate of the MCF-7 and T-47D cells, respectively. In another series of studies, it was observed that after 24 h incubation of a physiological concentration of estrone (E₁: 5 × 10⁻⁹ M) this estrogen is converted in a great proportion to E₂. Nomegestrol acetate inhibits this transformation by −35% and −85% at 5 × 10⁻⁷ M and 5 × 10⁻⁵ M, respectively in T-47D cells; whereas in the MCF-7 cells the inhibitory effect is only significant, −48%, at 5 × 10⁻⁵ M concentration of nomegestrol acetate. It is concluded that nomegestrol acetate in the hormone-dependent MCF-7 and T-47D breast cancer cells significantly inhibits the estrone sulfatase and 17β-HSD activities which converts E₁S to the biologically active estrogen estradiol. This inhibition provoked by this progestin on the enzymes involved in the biosynthesis of E₂ can open new clinical possibilities in breast cancer therapy.     

X-ray induction of 6-thioguanine-resistant mutants in division arrested, G0/G1 phase Chinese hamster ovary cells

The cytotoxic and mutagenic effect of X-irradiation was determined with Chinese hamster ovary cells arrested in the G₀/G₁ phase of the cell cycle through 9 days incubation in serum-free medium. In comparison with exponential phase cultures, the arrested cells showed increased cytotoxicity and mutation induction over the dose range of 50–800 rad. Exponential cultures showed a linear mutant frequency-survival relationship while the arrested cells showed a biphasic linear relationship. A post irradiation holding period of 24 h does not result in any change in the mutant frequency. The increased sensitivity of the arrested cells to the mutagenic effects of X-rays appears to be a cell-cycle phase phenomenon. Upon readdition of serum, the arrested cells re-enter the cell cycle in a synchronous manner, reaching S phase at 10–12 h. Cells irradiated at 5 h after serum addition, i.e. in G₁, show a similar does response for mutant frequency, while those irradiated at 10 h or later, i.e. in late G₁, S or G₂, show lower mutation induction. These observations are consistent with a chromosome interchange mechanism of mutation induction by X-rays, possibly through interactions between repairing regions of the DNA. Irradiation of cells in the G₀/G₁ phase allow more time for such interactions in the absence of semiconservative DNA replication.     

A negative resistance region underlies the triggering property of membrane potential in human T-lymphocytes

Steady-state current-voltage relationships (SSCVRs) of the plasma membrane of human T-lymphocytes were studied at the physiological temperature of 37°C by using the whole-cell patch-clamp technique. SSCVRs displayed a characteristic N-like shape with a negative resistance region (NRR) in a voltage range of −45 to −35 mV. The majority of cells assayed revealed SSCVR patterns crossing the V-axis at three points (in mV): V₁ = −55 to −45, V₂ = −40 to −35, V₃ = −30 to −10. SSCVRs of T-cells activated by phytohaemagglutinin (48–96 h) also displayed NRR, but crossed the V-axis at one point only (V₁ = −55 to −60 mV). It implies the possibility of two stable levels of membrane potential (V₁ and V₃) for the resting T-cells, but only one (V₁) for activated T-cells. These data thus account for the triggering property of T-cell membrane potential previously reported. The NRR can be explained on the basis of the Hodgkin-Huxley type n⁴j model of K⁺ channel kinetics. According to the model the possibility for a membrane to have on or two stable levels of membrane potential depends on the ratio of selective K⁺ conductance to non-selective leaky conductance (G_k/G_leak). The steady-state level of K⁺ conductance in resting T-lymphocytes proved to be sensitive to Ca²⁺. Buffering Ca²⁺ ions from either external or internal solution resulted in an appreciable increase in K⁺ conductance. The possibility for membrane potential have two stable levels of membrane potential in connection with the Ca²⁺ dependence of K⁺ conductance was supposed to be important for Ca²⁺-signalling during T-cell activation.     

Quantification of illegitimate V(D)J recombinase-mediated mutations in lymphocytes of newborns and adults

We used a direct polymerase chain reaction (PCR) method for quantification of HPRT exons 2+3 deletions and t(14;18) translocations as a measure of illegitimate V(D)J recombination. We determined the baseline frequencies of these two mutations in mononuclear leukocyte DNA from the umbilical cord blood of newborns and from the peripheral blood of adults. In an initial group of 21 newborns, no t(14;18) translocations were detected (<0.049×10⁻⁷). The frequency of HPRT exons 2+3 deletions was 0.10×10⁻⁷ per mononuclear leukocyte, lower than expected based on the T-cell proportion of this cell fraction (55%–70%) and previous results using the T-cell cloning assay (2–3×10⁻⁷ per clonable T-cell). Phytohemagglutinin (PHA), as used in the T-cell cloning assay, was examined for its effect on the frequencies of these mutation events in mononuclear leukocytes from an additional 11 newborns and from 12 adults. There was no significant effect of PHA on t(14;18) translocations which were rare among the newborns (1 detected among 2.7×10⁸ leukocytes analyzed), and which occurred at frequencies from <1×10⁻⁷ (undetected) to 1.6×10⁻⁴ among the adults. The extremely high frequencies of t(14;18)-bearing cells in three adults were due mainly to in vivo expansion of two to six clones. However, PHA appeared to stimulate a modest (although not significant) increase in the frequency of HPRT exons 2+3 deletions in the leukocytes of the newborns, from 0.07×10⁻⁷ to 0.23×10⁻⁷. We show that both the direct PCR assay and the T-cell cloning assay detect similar frequencies of HPRT exons 2+3 deletions when calculations are normalized to blood volume, indicating that the apparent discrepancy is probably due to the different population of cells used in the assays. This direct PCR assay may have utility in characterizing the effects of environmental genotoxic agents on this clinically important recombination mechanism.     

Bystander response in human lymphoblastoid TK6 cells

The mechanisms of the medium-mediated bystander response induced by γ-rays in non-irradiated TK6 cells were investigated. Cell cultures were irradiated and the culture medium discarded immediately after irradiation and replaced with a fresh one. In cells incubated with conditioned medium from irradiated cells (CM), a significant decrease in cell viability and cloning efficiency was observed, together with a significant increase in apoptosis, also in directly irradiated cells. To examine whether bystander apoptosis involved the extrinsic pathway, an inhibitor of caspase-8 was added to CM cultures, which significantly decreased apoptosis to control levels. The addition to CM of ROS scavengers, Cu–Zn superoxide dismutase and N-acetylcysteine did not affect the induction of apoptosis. To assess whether CM treatment activates a DNA damage response, also the formation of γ-H2AX foci, as markers of double-strand breaks and their colocalisation with 53-binding protein 1 (53BP1) and the protein mutated in the Nijmegen breakage syndrome 1 (NBS1) was analysed. In cultures treated for 2 h with CM, 9–11% of cells showed γ-H2AX foci, which partially or totally lacked colocalisation with 53BP1 and NBS1 foci. About 85% of irradiated cells were positive for γ-H2AX foci, which colocalised with 53BP1 and NBS1 proteins. At 24 h from irradiation, very few irradiated cells retained foci, fitting DNA repair kinetics. The number of foci-positive bystander cells also decreased to background values 24 h after CM incubation. Our results suggest that irradiated TK6 cells release into the medium some soluble factors, not ROS, which are responsible for the cytotoxic effects induced in bystander cells. In our experimental system, the role of ROS appeared to be of minor importance in inducing cell mortality, but probably critical in activating the DNA damage response in the responsive fraction of bystander cells.     

Mutational spectrum of the lacI gene in Escherichia coli K12 induced by low-energy ion beam

The mutational spectrum of the genomic lacI gene induced by low-energy nitrogen ion irradiation in wild type Escherichia coli strain W3110 were compared with the spontaneous and the vacuum controls. The mutant frequency of irradiated group was dose-dependent and reached 26.3 × 10⁻⁶ at dose of 31.2 × 10¹⁴ ions/cm², which was about 18-fold over the background (1.5 × 10⁻⁶) and 10-fold over the vacuum controls (2.6 × 10⁻⁶). This result indicated that the low-energy ion irradiation was one of many effective mutagens, though the vacuum condition of low-energy ions contributed some low-level gene mutations. It was found that the difference between the spontaneous and the vacuum control was the increases of base-pair substitutions in the vacuum control group. The spectra of irradiated group were quite similar to that of oxygen free-radical induced in the same strain, suggesting free-radicals and other adducts generated by low-energy ions might play an important role in the mutagenesis in vivo. When the spontaneous and the vacuum control group were compared, base-pair substitutions, deletions and additions of the irradiated group were significantly increased, and the +TGGC or −TGGC at hot spot was decreased from 82 to 48%. But the remarkable increase in absolute MF of the +TGGC or −TGGC at hot spot in the irradiated group suggested that low-energy ions did induce the mutations of this type. The spectra of our irradiated group had relative low-level base-pair substitutions, high-level ±TGGC and high proportion additions than those of γ-radiation induced, implying there were some different effects or processes between them.     

Bradykinin counteracts the stimulatory effect of angiotensin-(1-7) on the proximal tubule Na+ -ATPase activity through B2 receptor

Recently, we demonstrated that angiotensin-(1–7) (Ang-(1–7)) stimulates the Na⁺-ATPase activity through a losartan-sensitive angiotensin receptor, whereas bradykinin inhibits the enzyme activity through the B₂ receptor [Regul. Pept. 91 (2000) 45; Pharmacol. Rev. 32 (1980) 1]. In the present paper, the effect of bradykinin (BK) on Ang-(1–7)-stimulated Na⁺-ATPase activity was evaluated. Preincubation of Na⁺-ATPase with 10⁻⁹ M Ang-(1–7) increases enzyme activity from 7.9±0.9 to 14.1±1.5 nmol Pi mg⁻¹ min⁻¹, corresponding to an increase of 79% (p<0.05). This effect is reverted by bradykinin in a dose-dependent manner (10⁻¹⁴–10⁻⁸ M), reaching maximal inhibitory effect at 10⁻⁹ M. Des-Arg⁹ bradykinin (DABK), an agonist of B₁ receptor, at the concentrations of 10⁻⁹–10⁻⁷ M, does not mimic the BK inhibitory effect, and des-Arg⁹-[Leu⁸]-BK (DALBK), a B₁ receptor antagonist, at the concentrations of 10⁻¹⁰–10⁻⁷ M, does not prevent the inhibitory effect of BK on Ang-(1–7)-stimulated enzyme. On the other hand, HOE 140, an antagonist of B₂ receptor, abolishes the inhibitory effect of BK on the Ang-(1–7)-stimulated enzyme in a dose-dependent manner, reaching maximal effect at 10⁻⁷ M. Taken together, these data indicate that stimulation of B₂ receptors by BK can counteract the stimulatory effect of Ang-(1–7) on the proximal tubule Na⁺-ATPase activity.     

Chromosomal instability, reproductive cell death and apoptosis induced by O-methylguanine in Mex, Mex and methylation-tolerant mismatch repair compromised cells: facts and models

O⁶-Methylguanine (O⁶-MeG) is induced in DNA by methylating environmental carcinogens and various cytostatic drugs. It is repaired by O⁶-methylguanine-DNA methyltransferase (MGMT). If not repaired prior to replication, the lesion generates gene mutations and leads to cell death, sister chromatid exchanges (SCEs), chromosomal aberrations and malignant transformation. To address the question of how O⁶-MeG is transformed into genotoxic effects, isogenic Chinese hamster cell lines either not expressing MGMT (phenotypically Mex⁻), expressing MGMT (Mex⁺) or exhibiting the tolerance phenotype (Mex⁻, methylation resistant) were compared as to their clastogenic response. Mex⁻ cells were more sensitive than Mex⁺ cells to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)-induced chromosomal breakage, with marked differences in sensitivity depending on recovery time. At early recovery time, when cells out of the first post-treatment mitosis were scored, aberration frequency was about 40% reduced in Mex⁺ as compared to Mex⁻ cells. At later stages of recovery when cells out of the second post-treatment mitosis were analyzed, the frequency of aberrations increased strongly in Mex⁻ cells whereas it dropped to nearly control level in Mex⁺ cells. From this we conclude that, in the first post-treatment replication cycle of Mex⁻ cells, only a minor part of aberrations (<40%) was due to O⁶-MeG whereas, in the second post-treatment replication cycle, the major part of aberrations (>90%) was caused by the lesion. Thus, O⁶-MeG is a potent clastogenic DNA damage that needs two DNA replication cycles in order to be transformed with high efficiency into aberrations. The same holds true for sister chromatid exchanges (SCEs). MNNG is highly potent in inducing SCEs in Mex⁻ cells in the second replication cycle after alkylation. Under these conditions, SCE induction is nearly completely prevented by the expression of MGMT. This is opposed to SCE induction in the first post-treatment replication cycle, where higher doses of MNNG were required to induce SCEs and no protective effect of MGMT was observed. This indicates that SCEs induced in the first replication cycle after alkylation are due to other lesions than O⁶-MeG. In methylation tolerant cells, which are characterized by impaired G–T mismatch binding and MSH2 expression, aberration frequency induced by MNNG was weakly reduced in the first and strongly reduced in the second post-treatment mitoses, as compared to CHO wild-type cells. The results indicate that mismatch repair of O⁶-MeG–T mispairs is decisively involved in O⁶-MeG born chromosomal instability and recombination. We also show that Mex⁺ and methylation tolerant cells are more resistant than Mex⁻ cells with regard to induction of apoptosis, indicating O⁶-MeG to be also an apoptosis-inducing lesion. The data are discussed as to the mechanism of cytotoxicity, aberration and SCE formation in cells treated with a methylating agent.