Occam's broom and the dirty DSB: cytogenetic perspectives on cellular response to changes in track structure and ionization density.

Given equal doses, it is well-known that densely ionizing radiations are more potent in causing a number of biological effects compared to sparsely ionizing radiations, such as x- or gamma rays. According to classical models of radiation action, this results from differences in the spatial distribution of lesions along charged particle tracks. In recent years investigators have been barraged with the alternative narrative that this is instead due to 'qualitative' differences in the types of molecular lesions that each type of radiation produces. The present review discusses, mainly from a cytogenetic perspective, the merits and shortcomings of these seemingly contradictory viewpoints. There may be a kernel of truth to the idea that qualitative differences in the types of molecular lesions produced at the nanometer level affect RBE/LET relationships, but to ignore the fact that such differences result from longer-range spatial distributions of lesions produced along charged particle tracks is an unjustifiably narrow stance tantamount to employing Occam's Broom. Not only are such spatial considerations indispensable in explaining the impact of ionization density upon higher-order biological endpoints, particularly chromosome aberrations, the explanations they provide render arguments based principally on the quality of IR damage largely superfluous.

In Silico Models of DNA Damage and Repair in Proton Treatment Planning: A Proof of Concept.

There is strong in vitro cell survival evidence that the relative biological effectiveness (RBE) of protons is variable, with dependence on factors such as linear energy transfer (LET) and dose. This is coupled with the growing in vivo evidence, from post-treatment image change analysis, of a variable RBE. Despite this, a constant RBE of 1.1 is still applied as a standard in proton therapy. However, there is a building clinical interest in incorporating a variable RBE. Recently, correlations summarising Monte Carlo-based mechanistic models of DNA damage and repair with absorbed dose and LET have been published as the Manchester mechanistic (MM) model. These correlations offer an alternative path to variable RBE compared to the more standard phenomenological models. In this proof of concept work, these correlations have been extended to acquire RBE-weighted dose distributions and calculated, along with other RBE models, on a treatment plan. The phenomenological and mechanistic models for RBE have been shown to produce comparable results with some differences in magnitude and relative distribution. The mechanistic model found a large RBE for misrepair, which phenomenological models are unable to do. The potential of the MM model to predict multiple endpoints presents a clear advantage over phenomenological models.

A Bi-Exponential Repair Algorithm for Radiation-Induced Double-Strand Breaks: Application to Simulation of Chromosome Aberrations.

BACKGROUND: Radiation induces DNA double-strand breaks (DSBs), and chromosome aberrations (CA) form during the DSBs repair process. Several methods have been used to model the repair kinetics of DSBs including the bi-exponential model, i.e., N(t) = N1exp(-t/τ1) + N2exp(-t/τ2), where N(t) is the number of breaks at time t, and N1, N2, τ1 and τ2 are parameters. This bi-exponential fit for DSB decay suggests that some breaks are repaired rapidly and other, more complex breaks, take longer to repair. METHODS: The bi-exponential repair kinetics model is implemented into a recent simulation code called RITCARD (Radiation Induced Tracks, Chromosome Aberrations, Repair, and Damage). RITCARD simulates the geometric configuration of human chromosomes, radiation-induced breaks, their repair, and the creation of various categories of CAs. The bi-exponential repair relies on a computational algorithm that is shown to be mathematically exact. To categorize breaks as complex or simple, a threshold for the local (voxel) dose was used. RESULTS: The main findings are: i) the curves for the kinetics of restitution of DSBs are mostly independent of dose; ii) the fraction of unrepaired breaks increases with the linear energy transfer (LET) of the incident radiation; iii) the simulated dose-response curves for simple reciprocal chromosome exchanges that are linear-quadratic; iv) the alpha coefficient of the dose-response curve peaks at about 100 keV/µm.

From Energy Deposition of Ionizing Radiation to Cell Damage Signaling: Benchmarking Simulations by Measured Yields of Initial DNA Damage after Ion Microbeam Irradiation.

Advances in accelerator technology, which have enabled conforming radiotherapy with charged hadronic species, have brought benefits as well as potential new risks to patients. To better understand the effects of ionizing radiation on tumor and surrounding tissue, it is important to investigate and quantify the relationship between energy deposition at the nanometric scale and the initial biological events. Monte Carlo track structure simulation codes provide a powerful tool for investigating this relationship; however, their success and reliability are dependent on their improvement and development accordingly to the dedicated biological data to which they are challenged. For this aim, a microbeam facility that allows for fluence control, down to one ion per cell nucleus, was used to evaluate relative frequencies of DNA damage after interaction between the incoming ion and DNA according to radiation quality. Primary human cells were exposed to alpha particles of three different energies with respective linear energy transfers (LETs) of approximately 36, 85 or 170 keV·µm-1 at the cells' center position, or to protons (19 keV·µm-1). Statistical evaluation of nuclear foci formation (53BP1/γ-H2AX), observed using immunofluorescence and related to a particle traversal, was undertaken in a large population of cell nuclei. The biological results were adjusted to consider the factors that drive the experimental uncertainties, then challenged with results using Geant4-DNA code modeling of the ionizing particle interactions on a virtual phantom of the cell nucleus with the same mean geometry and DNA density as the cells used in our experiments. Both results showed an increase of relative frequencies of foci (or simulated DNA damage) in cell nuclei as a function of increasing LET of the traversing particles, reaching a quasi-plateau when the LET exceeded 80-90 keV·µm-1. For the LET of an alpha particle ranging from 80-90 to 170 keV·µm-1, 10-30% of the particle hits did not lead to DNA damage inducing 53BP1 or γ-H2AX foci formation.

Relating Linear Energy Transfer to the Formation and Resolution of DNA Repair Foci After Irradiation with Equal Doses of X-ray Photons, Plateau, or Bragg-Peak Protons.

Proton beam therapy is increasingly applied for the treatment of human cancer, as it promises to reduce normal tissue damage. However, little is known about the relationship between linear energy transfer (LET), the type of DNA damage, and cellular repair mechanisms, particularly for cells irradiated with protons. We irradiated cultured cells delivering equal doses of X-ray photons, Bragg-peak protons, or plateau protons and used this set-up to quantitate initial DNA damage (mainly DNA double strand breaks (DSBs)), and to analyze kinetics of repair by detecting γH2A.X or 53BP1 using immunofluorescence. The results obtained validate the reliability of our set-up in delivering equal radiation doses under all conditions employed. Although the initial numbers of γH2A.X and 53BP1 foci scored were similar under the different irradiation conditions, it was notable that the maximum foci level was reached at 60 min after irradiation with Bragg-peak protons, as compared to 30 min for plateau protons and photons. Interestingly, Bragg-peak protons induced larger and irregularly shaped γH2A.X and 53BP1 foci. Additionally, the resolution of these foci was delayed. These results suggest that Bragg-peak protons induce DNA damage of increased complexity which is difficult to process by the cellular repair apparatus.

Low- and High-LET Ionizing Radiation Induces Delayed Homologous Recombination that Persists for Two Weeks before Resolving.

Genome instability is a hallmark of cancer cells and dysregulation or defects in DNA repair pathways cause genome instability and are linked to inherited cancer predisposition syndromes. Ionizing radiation can cause immediate effects such as mutation or cell death, observed within hours or a few days after irradiation. Ionizing radiation also induces delayed effects many cell generations after irradiation. Delayed effects include hypermutation, hyper-homologous recombination, chromosome instability and reduced clonogenic survival (delayed death). Delayed hyperrecombination (DHR) is mechanistically distinct from delayed chromosomal instability and delayed death. Using a green fluorescent protein (GFP) direct repeat homologous recombination system, time-lapse microscopy and colony-based assays, we demonstrate that DHR increases several-fold in response to low-LET X rays and high-LET carbon-ion radiation. Time-lapse analyses of DHR revealed two classes of recombinants not detected in colony-based assays, including cells that recombined and then senesced or died. With both low- and high-LET radiation, DHR was evident during the first two weeks postirradiation, but resolved to background levels during the third week. The results indicate that the risk of radiation-induced genome destabilization via DHR is time limited, and suggest that there is little or no additional risk of radiation-induced genome instability mediated by DHR with high-LET radiation compared to low-LET radiation.

Measurement of complex DNA damage induction and repair in human cellular systems after exposure to ionizing radiations of varying linear energy transfer (LET).

Detrimental effects of ionizing radiation (IR) are correlated to the varying efficiency of IR to induce complex DNA damage. A double strand break (DSB) can be considered the simpler form of complex DNA damage. These types of damage can consist of DSBs, single strand breaks (SSBs) and/or non-DSB lesions such as base damages and apurinic/apyrimidinic (AP; abasic) sites in different combinations. Enthralling theoretical (Monte Carlo simulations) and experimental evidence suggests an increase in the complexity of DNA damage and therefore repair resistance with linear energy transfer (LET). In this study, we have measured the induction and processing of DSB and non-DSB oxidative clusters using adaptations of immunofluorescence. Specifically, we applied foci colocalization approaches as the most current methodologies for the in situ detection of clustered DNA lesions in a variety of human normal (FEP18-11-T1) and cancerous cell lines of varying repair efficiency (MCF7, HepG2, A549, MO59K/J) and radiation qualities of increasing LET, that is γ-, X-rays 0.3-1 keV/µm, α-particles 116 keV/µm and 36Ar ions 270 keV/µm. Using γ-H2AX or 53BP1 foci staining as DSB probes, we calculated a DSB apparent rate of 5-16 DSBs/cell/Gy decreasing with LET. A similar trend was measured for non-DSB oxidized base lesions detected using antibodies against the human repair enzymes 8-oxoguanine-DNA glycosylase (OGG1) or AP endonuclease (APE1), that is damage foci as probes for oxidized purines or abasic sites, respectively. In addition, using colocalization parameters previously introduced by our groups, we detected an increasing clustering of damage for DSBs and non-DSBs. We also make correlations of damage complexity with the repair efficiency of each cell line and we discuss the biological importance of these new findings with regard to the severity of IR due to the complex nature of its DNA damage.

Time-Lapse Monitoring of DNA Damage Colocalized With Particle Tracks in Single Living Cells.

PURPOSE: Understanding the DNA damage and repair induced by hadron therapy (HT) beams is crucial for developing novel strategies to maximize the use of HT beams to treat cancer patients. However, spatiotemporal studies of DNA damage and repair for beam energies relevant to HT have been challenging. We report a technique that enables spatiotemporal measurement of radiation-induced damage in live cells and colocalization of this damage with charged particle tracks over a broad range of clinically relevant beam energies. The technique uses novel fluorescence nuclear track detectors with fluorescence confocal laser scanning microscopy in the beam line to visualize particle track traversals within the subcellular compartments of live cells within seconds after injury. METHODS AND MATERIALS: We designed and built a portable fluorescence confocal laser scanning microscope for use in the beam path, coated fluorescence nuclear track detectors with fluorescent-tagged live cells (HT1080 expressing enhanced green fluorescent protein tagged to XRCC1, a single-strand break repair protein), placed the entire assembly into a proton therapy beam line, and irradiated the cells with a fluence of ∼1 × 10(6) protons/cm(2). RESULTS: We successfully obtained confocal images of proton tracks and foci of DNA single-strand breaks immediately after irradiation. CONCLUSIONS: This technique represents an innovative method for analyzing biological responses in any HT beam line at energies and dose rates relevant to therapy. It allows precise determination of the number of tracks traversing a subcellular compartment and monitoring the cellular damage therein, and has the potential to measure the linear energy transfer of each track from therapeutic beams.

Misrepair of DNA double-strand breaks after exposure to heavy-ion beams causes a peak in the LET-RBE relationship with respect to cell killing in DT40 cells.

To determine the radiobiological mechanisms underlying relative biological effectiveness (RBE) and the repair efficiencies of DNA double-strand breaks (DSBs) as a function of linear energy transfer (LET), we exposed cells of the chicken B-lymphocyte cell line DT40 and its DSB repair pathway-deficient derivatives to heavy-ion beams produced at the Heavy-Ion Medical Accelerator in Chiba (HIMAC) at the National Institute of Radiological Sciences (NIRS), Chiba, Japan. The relationship between LET and cell lethality was investigated in the DNA DSB repair gene knockouts Ku70(-/-), Rad54(-/-), and Ku70(-/-)Rad54(-/-), and in the wild-type cells. We found that cell-cycle stage and activity of the DNA DSB repair pathways influence LET-mediated biological effects. An expected LET-RBE relationship was observed in the cells capable of DNA repair, but no peak was found in the RBE with respect to cell survival in the Ku70(-/-)Rad54(-/-) cells or in Ku70(-/-) cells in the G1 and early S cell-cycle phases (when no sister chromatids were present and homologous recombination could not occur). These findings suggest that the peak in RBE is caused by deficient repair of the DNA DSBs.

Comparison of the repair of potentially lethal damage after low- and high-LET radiation exposure, assessed from the kinetics and fidelity of chromosome rejoining in normal human fibroblasts.

Potentially lethal damage (PLD) and its repair (PLDR) were studied in confluent human fibroblasts by analyzing the kinetics of chromosome break rejoining after X-ray or heavy-ion exposures. Cells were either held in the non-cycling G0 phase of the cell cycle for 12 h, or forced to proliferate immediately after irradiation. Fusion premature chromosome condensation (PCC) was combined with fluorescence in situ hybridization (FISH) to study chromosomal aberrations in interphase. The culture condition had no impact on the rejoining kinetics of PCC breaks during the 12 h after X-ray or heavy-ion irradiation. However, 12 h after X-ray and silicon irradiation, cycling cells had more chromosome exchanges than non-cycling cells. After 6 Gy X-rays, the yield of exchanges in cycling cells was 2.8 times higher than that in non-cycling cells, and after 2 Gy of 55 keV/µm silicon ions the yield of exchanges in cycling cells was twice that of non-cycling cells. In contrast, after exposure to 2 Gy 200-keV/µm or 440-keV/µm iron ions the yield of exchanges was similar in non-cycling and cycling cells. Since the majority of repair in G0/G1 occurs via the non-homologous end joining process (NHEJ), increased PLDR in X-ray and silicon-ion irradiated cells may result from improved cell cycle-specific rejoining fidelity through the NHEJ pathway, which is not the case in high-LET iron-ion irradiated cells.

Studies on biological effects of ion beams on lethality, molecular nature of mutation, mutation rate, and spectrum of mutation phenotype for mutation breeding in higher plants.

Recently, heavy ions or ion beams have been used to generate new mutants or varieties, especially in higher plants. It has been found that ion beams show high relative biological effectiveness (RBE) of growth inhibition, lethality, and so on, but the characteristics of ion beams on mutation have not been clearly elucidated. To understand the effect of ion beams on mutation induction, mutation rates were investigated using visible known Arabidopsis mutant phenotypes, indicating that mutation frequencies induced by carbon ions were 20-fold higher than by electrons. In chrysanthemum and carnation, flower-color and flower-form mutants, which are hardly produced by gamma rays or X rays, were induced by ion beams. Novel mutants and their responsible genes, such as UV-B resistant, serrated petals and sepals, anthocyaninless, etc. were induced by ion beams. These results indicated that the characteristics of ion beams for mutation induction are high mutation frequency and broad mutation spectrum and therefore, efficient induction of novel mutants. On the other hand, PCR and sequencing analyses showed that half of all mutants induced by ion beams possessed large DNA alterations, while the rest had point-like mutations. Both mutations induced by ion beams had a common feature that deletion of several bases were predominantly induced. It is plausible that ion beams induce a limited amount of large and irreparable DNA damage, resulting in production of a null mutation that shows a new mutant phenotype.

Changes in MicroRNA expression patterns in human fibroblasts after low-LET radiation.

Exposure to radiation provokes cellular responses controlled in part by gene expression networks. MicroRNAs (miRNAs) are small non-coding RNAs which mostly regulate gene expression by degrading the messages or inhibiting translation. Here, we investigated changes in miRNA expression patterns after low (0.1 Gy) and high (2.0 Gy) doses of X-ray in human fibroblasts. At early (0.5 h) and late (6 and 24 h) time points, irradiation caused qualitative and quantitative differences in the down-regulation of miRNA levels, including miR-92b, 137, 660, and 656. A transient up-regulation of miRNAs was observed after 2 h post-irradiation following high doses of radiation, including miR-558 and 662. MicroRNA levels were inversely correlated with targets from mRNA and proteomic profiling after 2.0 Gy of radiation. MicroRNAs miR-579, 608, 548-3p, and 585 are noted for targeting genes involved in radioresponsive mechanisms, such as cell cycle checkpoint and apoptosis. We suggest here a model in which miRNAs may act as "hub" regulators of specific cellular responses, immediately down-regulated so as to stimulate DNA repair mechanisms, followed by up-regulation involved in suppressing apoptosis for cell survival. Taken together, miRNAs may mediate signaling pathways in sequential fashion in response to radiation, and may serve as biodosimetric markers of radiation exposure.

Induced telomerase activity in primary aortic endothelial cells by low-LET gamma-radiation is mediated through NF-kappaB activation.

Our objective was to understand the mechanism through which cells that initially survive irradiation could acquire survival advantage. In this study, we show evidence that low-linear energy transfer gamma-radiation can induce telomerase enzyme activity in primary aortic endothelial cells, and that an upstream regulator, nuclear factor kappa B (NF-kappaB), controls this activation. Telomeric repeat amplification protocol (TRAP) assay showed that cells exposed to a dose of 2 Gy induce telomerase activity. Subsequent analysis revealed that radiation-induced telomeric activity is regulated at the transcriptional level by triggering activation of the promoter of the telomerase catalytic subunit, telomerase reverse transcriptase (TERT). A mechanistic study revealed that NF-kappaB becomes functionally activated upon radiation exposure and mediates the upregulation of telomerase activity by binding to the kappaB-binding region in the promoter region of the TERT gene. More significantly, elimination of the NF-small ka, CyrillicB recognition site on the telomerase promoter or inhibition of NF-small ka, CyrillicB by ectopically expressing the inhibitor protein IkappaBalpha mutant (Ismall ka, CyrillicBalpha(S32A/S36A))) compromises radiation-induced telomerase promoter activation. Consistent with the notion that NF-kappaB mediates gamma-ray-induced telomerase responses, TRAP assay revealed that ectopically expressed IkappaBalpha(S32A/S36A)) also attenuated telomerase enzyme activity. These findings indicate that NF-kappaB activation following ionizing radiation exposure may elicit a survival advantage by upregulating and maintaining telomerase activity.

Susceptible genes and molecular pathways related to heavy ion irradiation in oral squamous cell carcinoma cells.

BACKGROUND AND PURPOSE: Heavy ion beams are high linear energy transfer (LET) radiation characterized by a higher relative biologic effectiveness than low LET radiation. The aim of the current study was to determine the difference of gene expression between heavy ion beams and X-rays in oral squamous cell carcinoma (OSCC)-derived cells. MATERIALS AND METHODS: The OSCC cells were irradiated with accelerated carbon or neon ion irradiation or X-rays using three different doses. We sought to identify genes the expression of which is affected by carbon and neon ion irradiation using Affymetrix GeneChip analysis. The identified genes were analyzed using the Ingenuity Pathway Analysis Tool to investigate the functional network and gene ontology. Changes in mRNA expression in the genes were assessed by real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). RESULTS: The microarray analysis identified 84 genes that were modulated by carbon and neon ion irradiation at all doses in OSCC cells. Among the genes, three genes (TGFBR2, SMURF2, and BMP7) and two genes (CCND1 and E2F3), respectively, were found to be involved in the transforming growth factor beta-signaling pathway and cell cycle:G1/S checkpoint regulation pathway. The qRT-PCR data from the five genes after heavy ion irradiation were consistent with the microarray data (P < 0.01). CONCLUSION: Our findings should serve as a basis for global characterization of radiation-regulated genes and pathways in heavy ion-irradiated OSCC.

A model for the induction of chromosome aberrations through direct and bystander mechanisms.

A state vector model (SVM) for chromosome aberrations and neoplastic transformation has been adapted to describe detrimental bystander effects. The model describes initiation (formation of translocations) and promotion (clonal expansion and loss of contact inhibition of initiated cells). Additional terms either in the initiation model or in the rate of clonal expansion of initiated cells, describe detrimental bystander effects for chromosome aberrations as reported in the scientific literature. In the present study, the SVM with bystander effects is tested on a suitable dataset. In addition to the simulation of non-linear effects, a classical dataset for neoplastic transformation in C3H 10T1/2 cells after alpha particle irradiation is used to show that the model without bystander features can also describe LNT-like dose responses. A published model for bystander induced neoplastic transformation was adapted for chromosome aberration induction and used to compare the results obtained with the different models.

Genomic instability and the role of radiation quality.

Genomic instability (GI) is a hallmark of tumorigenic progression and is observed as delayed genetic damage in the progeny of irradiated and unirradiated bystander cells. The expression of GI can be influenced by genotype, cell type and radiation quality. While several studies have demonstrated the induction of GI by high and low-linear energy transfer (LET) radiation, our work on human and mouse primary cell systems has shown LET-dependent differences in the induction and expression of GI. These differences might be attributed to differences in radiation track structure, dose rate, contribution of bystander cells and radiation dose. This paper reviews the role of radiation quality in the induction of GI and describe the possible mechanisms underlining the observed differences between radiation types on its induction. The experimental results presented suggest that dose might be the most significant factor in determining induction of GI after low-LET radiation.

Distances between tropomyosin sites across the muscle thin filament using luminescence resonance energy transfer: evidence for tropomyosin flexibility.

To obtain information about the interaction of tropomyosin (Tm) with actin associated with the regulatory states of the muscle thin filament, we used luminescence resonance energy transfer (LRET) between Tb(3+) as a donor and rhodamine as an acceptor. A novel Tb(3+) chelator, S-(2-nitro-5-thiobenzoate)cysteaminyl-DTPA-Cs124, was synthesized, which specifically labels Cys groups in proteins. With the Tb chelate as the donor and tetramethylrhodamine-5-maleimide as the acceptor, both bound to specific Cys groups of Tm, we obtained 67 A as the distance between Tm's across the actin filament, a much shorter value than that obtained from structural studies (72-86 A). The difference appears to be due to submillisecond motion associated with Tm flexibility, which brings the probes closer during the millisecond lifetime of the donor. Ca(2+) did not change the energy transfer with the reconstituted thin filament, but myosin subfragment 1 decreased the transfer, consistent with either a 5-6 A increase in distance or, more likely, a decrease in flexibility.

Structure of the transition state of gating in the acetylcholine receptor channel pore: a phi-value analysis.

The gating mechanism of the acetylcholine receptor channel (AChR) was investigated by using rate equilibrium linear free energy relationships (LFERs) to probe the transition state between the closed and open conformations. The properties of the transition state of gating in the second transmembrane segment (M2) of the delta subunit, one of the five homologous pore-lining segments, was measured on a residue-by-residue basis. Series of point mutations were engineered at individual positions of this domain, and the corresponding constructs were characterized electrophysiologically, at the single-channel level. Fully liganded AChR opening and closing rate constants were estimated, and Phi-values (which are a measure of the extent of the conformational change realized at the transition state) were calculated for each reaction series as the slope of the Brønsted relationship (log rate constant versus log equilibrium constant). Our results indicate that, at the transition state of gating, the extracellular half of deltaM2 partly resembles the open state (Phi-values between 0.24 and 0.38) while the intracellular half completely resembles the closed state (Phi-values between -0.18 and 0.03), with a break point near the middle of the M2 segment. This suggests that during gating the two halves of deltaM2 move asynchronously, with the rearrangement of the extracellular portion preceding (following) that of the intracellular part of deltaM2 during opening (closing). This particular sequence of molecular events indicates that the gating conformational change, which starts at the extracellular acetylcholine-binding sites (when opening), does not propagate exclusively along the primary sequence of the protein. In addition, our data are consistent with the deltaM2 segment bending or swiveling around its central residues during gating. We also elaborate on unsettled aspects of the analysis such as the accuracy of two-point LFERs, the physical interpretation of fractional Phi-values, and the existence of single versus parallel transition states for the gating reaction.

Biomarkers specific to densely-ionising (high LET) radiations.

There have been several suggestions of biomarkers that are specific to high LET radiation. Such a biomarker could significantly increase the power of epidemiological studies of individuals exposed to densely-ionising radiations such as alpha particles (e.g. radon, plutonium workers, individuals exposed to depleted uranium) or neutrons (e.g. radiation workers, airline personnel. We discuss here a potentially powerful high LET biomarker (the H value) which is the ratio of induced inter-chromosomal aberrations to intra-arm aberrations. Both theoretical and experimental studies have suggested that this ratio should differ by a factor of about three between high LET radiation and any other likely clastogen, and will yield more discrimination than the previously suggested F value (ratio of inter-chromosomal aberrations to intra-chromosomal inter-arm aberrations). Evidence of the long-term stability of such chromosomal biomarkers has also been generated. Because these stable intra-arm anld inter-chromosomal aberrations are (1) frequent and (2) measurable at long times after exposure, this H value appears to be a practical biomarker of high LET exposure, and several in vitro studies have confirmed the approach for unstable aberrations. The approach is currently being tested in a population of Russian radiation workers exposed several decades ago to high- or low LET radiation.

Mutation of Arg-166 of alkaline phosphatase alters the thio effect but not the transition state for phosphoryl transfer. Implications for the interpretation of thio effects in reactions of phosphatases.

It has been suggested that the mechanism of alkaline phosphatase (AP) is associative, or triester-like, because phosphorothioate monoesters are hydrolyzed by AP approximately 10(2)-fold slower than phosphate monoesters. This "thio effect" is similar to that observed for the nonenzymatic hydrolysis of phosphate triesters, and is the inverse of that observed for the nonenzymatic hydrolysis of phosphate monoesters. The latter reactions proceed by loose, dissociative transition states, in contrast to reactions of triesters, which have tight, associative transition states. Wild-type alkaline phosphatase catalyzes the hydrolysis of p-nitrophenyl phosphate approximately 70 times faster than p-nitrophenyl phosphorothioate. In contrast, the R166A mutant alkaline phosphatase enzyme, in which the active site arginine at position 166 is replaced with an alanine, hydrolyzes p-nitrophenyl phosphate only about 3 times faster than p-nitrophenyl phosphorothioate. Despite this approximately 23-fold change in the magnitude of the thio effects, the magnitudes of Bronsted beta(lg) for the native AP (-0.77 +/- 0.09) and the R166A mutant (-0.78 +/- 0. 06) are the same. The identical values for the beta(lg) indicate that the transition states are similar for the reactions catalyzed by the wild-type and the R166A mutant enzymes. The fact that a significant change in the thio effect is not accompanied by a change in the beta(lg) indicates that the thio effect is not a reliable reporter for the transition state of the enzymatic phosphoryl transfer reaction. This result has important implications for the interpretation of thio effects in enzymatic reactions.