Temporal Dynamic Regulation of Autophagy and Senescence Induction in Response to Radiation Exposure.

Autophagy and senescence are closely related cellular responses to genotoxic stress, and play significant roles in the execution of cellular responses to radiation exposure. However, little is known about their interplay in the fate-decision of cells receiving lethal doses of radiation. Here, we report that autophagy precedes the establishment of premature senescence in normal human fibroblasts exposed to lethal doses of radiation. Activation of the p53-dependent DNA damage response caused sustained dephosphorylation of RB proteins and consequent cell cycle arrest, concurrently with Ulk1 dephosphorylation at Ser638 by PPM1D, which promoted autophagy induction 1-2 days after irradiation. In addition, mitochondrial fragmentation became obvious 1-2 days after irradiation, and autophagy was further enhanced. However, Ulk1 levels decreased significantly after 2 days, resulting in lower LC3-II levels. An autophagic flux assay using chloroquine (CQ) also revealed that the flux in irradiated cells gradually decreased over 30 days. In contrast, lysosomal augmentation started at 1 day, became significantly upregulated after 5 days, and continued for over 30 days. After a rapid decrease in autophagy, p16 expression increased and senescence was established, but autophagic activity remained reduced. These results demonstrated that X-ray irradiation triggered two processes, autophagy and senescence, with the former being temporary and regulated by DNA damage response and mitophagy, and the latter being sustained and regulated by persistent cell cycle arrest. The interplay between autophagy and senescence seems to be essential for the proper implementation of the cellular response to radiation exposure.

Application of unsupervised and supervised learning to a material attribute database of tablets produced at two different granulation scales.

The purpose of this study is to demonstrate the usefulness of machine learning (ML) for analyzing a material attribute database from tablets produced at different granulation scales. High shear wet granulators (scale 30 g and 1000 g) were used and data were collected according to the design of experiments at different scales. In total, 38 different tablets were prepared, and the tensile strength (TS) and dissolution rate after 10 min (DS10) were measured. In addition, 15 material attributes (MAs) related to particle size distribution, bulk density, elasticity, plasticity, surface properties, and moisture content of granules were evaluated. By using unsupervised learning including principal component analysis and hierarchical cluster analysis, the regions of tablets produced at each scale were visualized. Subsequently, supervised learning with feature selection including partial least squares regression with variable importance in projection and elastic net were applied. The constructed models could predict the TS and DS10 from the MAs and the compression force with high accuracy (R2= 0.777 and 0.748, respectively), independent of scale. In addition, important factors were successfully identified. ML can be used for better understanding of similarity/dissimilarity between scales, for constructing predictive models of critical quality attributes, and for determining critical factors.

Mitochondrial Metabolism in X-Irradiated Cells Undergoing Irreversible Cell-Cycle Arrest.

Irreversible cell-cycle-arrested cells not undergoing cell divisions have been thought to be metabolically less active because of the unnecessary consumption of energy for cell division. On the other hand, they might be actively involved in the tissue microenvironment through an inflammatory response. In this study, we examined the mitochondria-dependent metabolism in human cells irreversibly arrested in response to ionizing radiation to confirm this possibility. Human primary WI-38 fibroblast cells and the BJ-5ta fibroblast-like cell line were exposed to 20 Gy X-rays and cultured for up to 9 days after irradiation. The mitochondrial morphology and membrane potential were evaluated in the cells using the mitochondrial-specific fluorescent reagents MitoTracker Green (MTG) and 5,5',6,6'-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1), respectively. The ratio of the mean MTG-stained total mitochondrial area per unit cell area decreased for up to 9 days after X-irradiation. The fraction of the high mitochondrial membrane potential area visualized by JC-1 staining reached its minimum 2 days after irradiation and then increased (particularly, WI-38 cells increased 1.8-fold the value of the control). Their chronological changes indicate that the mitochondrial volume in the irreversible cell-cycle-arrested cells showed significant increase concurrently with cellular volume expansion, indicating that the mitochondria-dependent energy metabolism was still active. These results indicate that the energy metabolism in X-ray-induced senescent-like cells is active compared to nonirradiated normal cells, even though they do not undergo cell divisions.

Salvage of Ear Framework Exposure Following Autologous Microtia Reconstruction: Repair Strategy for Each Location of Exposure.

One of the most common complications of total auricular reconstruction is exposure of the ear framework. Various reconstruction methods have been reported depending on the location and size of exposed cartilage. This report describes a safe reconstruction method for each exposed part of the grafted ear framework. From January 2019 to August 2021, 2 cases (4 areas) of framework exposure were observed following autologous microtia reconstruction. The first case developed 2 small areas of skin necrosis on the anterior helix and lower antihelix to concha. The former was reconstructed with a temporal fascia flap and the latter with a local transposition flap. The second case also developed 2 small areas of skin necrosis on the posterior helix and lower antihelix to concha. The former was sutured directly and the latter with a local transposition flap. However, both wounds recurred due to flap necrosis and the cartilage was exposed again. The 3rd operation was performed by covering both wounds with a posterior auricular turnover flap and skin graft. In both cases, the exposed framework was completely covered with the flaps, and the reconstructed ears showed well-defined convolutions. Covering exposed cartilage with a local flap with a random pattern of blood circulation is convenient because no additional skin grafts are required. However, the blood circulation of the flaps is inadequate when an elongated flap is required; consequently, flap necrosis may occur. On the other hand, a temporal fascia flap and posterior auricular flap, which have axillary pattern blood circulation, are considered to be safer. We believe that it is safe to use a temporal fascia flap for cartilage exposure in the upper half of the auricle, and a posterior auricular turnover flap for the lower half.

The Efficacy of Salvage Intervention with Emergency Transient External Arterial Bypass for Traumatic Artery Occlusion of Main Extremities.

Even if the vascular repair is successful, the frequency of limb loss is still high when popliteal artery injury is associated with postischemic syndrome due to blunt trauma or a prolonged ischemic time. Because prolonged ischemia interferes with an injured foot rescue, shortening of the ischemic time is a major aim of surgeons. We present two types of transient external arterial bypass and two cases of ischemic extremities due to main arterial injury. Even though the injured extremities had no circulation for more than 6 h, a transient external arterial bypass supplied circulation immediately, and they were reconstructed successfully. Although transient external arterial bypass is a dated technique, it is a recommended option, especially in the management of acute traumatic ischemia of the extremities to shorten the ischemic time and provide immediate reperfusion, which will bring the opportunity to save the ischemic limbs.

Fournier's Gangrene With Subcutaneous Emphysema of the Thigh Caused by Air Inflow Associated With a Rectovaginal Fistula: A Case Report of Pseudo-gas Gangrene.

INTRODUCTION: Rectovaginal fistulas (RVFs) are abnormal connections between the rectum and vagina. CASE REPORT: A 61-year-old female patient was admitted to the authors' hospital with swelling, extending from the left thigh to the left lower abdomen and crepitus. An axial computed tomography scan showed air in the soft tissue of the left thigh, left buttock, perineal region, and left lower abdomen. Gas gangrene was suspected. Accordingly, the patient was administered meropenem, clindamycin, and vancomycin and underwent emergency debridement. An intraoperative examination revealed necrotizing fasciitis in the left buttock but no inflammatory signs in the thigh. On postoperative day 8, fecal matter was discharged from the patient's vagina, and an RVF was detected by colon fiberscopy. The patient underwent resurfacing surgery with a free skin graft, and a colon stoma was fashioned 15 days after the primary surgery. The patient was discharged on day 14 following surgery with wound healing. CONCLUSION: The existence of free air in subcutaneous tissue combined with an infection, particularly in the extremities, is generally suggestive of gas gangrene. In the present case, subcutaneous gas was not caused by gas gangrene but rather by air inflow from an RVF. Appropriate treatment of the RVF was necessary to avoid the exacerbation of Fournier's gangrene and prevent necrosis spreading to the thigh.

Lethal DNA Lesions Caused by Direct and Indirect Actions of X rays are Repaired via Different DSB Repair Pathways under Aerobic and Anoxic Conditions.

We examined lethal damages of X rays induced by direct and indirect actions, in terms of double-strand break (DSB) repair susceptibility using two kinds of repair-deficient Chinese hamster ovary (CHO) cell lines. These CHO mutants (51D1 and xrs6) are genetically deficient in one of the two important DNA repair pathways after genotoxic injury [homologous recombination (HR) and non-homologous end binding (NHEJ) pathways, respectively]. The contribution of indirect action on cell killing can be estimated by applying the maximum level of dimethylsulfoxide (DMSO) to get rid of OH radicals. To control the proportion of direct and indirect actions in lethal damage, we irradiated CHO mutant cells under aerobic and anoxic conditions. The contributions of indirect action on HR-defective 51D1 cells were 76% and 57% under aerobic and anoxic conditions, respectively. Interestingly, these percentages were similar to those of the wild-type cells even if the radiosensitivity was different. However, the contributions of indirect action to cell killing on NHEJ-defective xrs6 cells were 52% and 33% under aerobic and anoxic conditions, respectively. Cell killing by indirect action was significantly affected by the oxygen concentration and the DSB repair pathways but was not correlated with radiosensitivity. These results suggest that the lethal damage induced by direct action is mostly repaired by NHEJ repair pathway since killing of NHEJ-defective cells has significantly higher contribution by the direct action. In other words, the HR repair pathway may not effectively repair the DSB by direct action in place of the NHEJ repair pathway. We conclude that the type of DSB produced by direct action is different from that of DSB induced by indirect action.

Creation of novel large dataset comprising several granulation methods and the prediction of tablet properties from critical material attributes and critical process parameters using regularized linear regression models including interaction terms.

Our aim was to understand better the causal relationships between material attributes (MAs), process parameters (PPs), and critical quality attributes (CQAs) using an originally created large dataset and regularized linear regression models. In this study, we focused on the following three points: (1) creation of a dataset comprising several tablet production methods, (2) the influence of interaction terms of MAs and/or PPs, and (3) comparison of regularized linear regression models with partial least squares (PLS) regression. First, we prepared 44 kinds of tablets using direct compression and five kinds of granulation methods. We then measured 12 MAs and two model CQAs (tensile strength and disintegration time of tablet). Principal component analysis showed that the constructed dataset comprised a wide variety of particles. We applied regularized linear regression models, such as ridge regression, LASSO and Elastic Net (ENET), and PLS to our dataset to predict CQAs from the MAs and PPs. As a result of external validation, the prediction performance of the models was sufficiently high, although ENET was slightly better than the other methods. Moreover, in almost all cases, the models with interaction terms showed higher predictive ability than those without interaction terms, indicating that the interaction terms of MAs and/or PPs have a strong influence on CQAs. ENET also allowed the selection of critical factors that strongly affect CQAs. The results of this study will help to understand systematically knowledge obtained in pharmaceutical development.

Eccrine Porocarcinoma on the Lateral Nose Wall: A Rare Case Report.

Eccrine porocarcinoma (EPC) is an uncommon malignant tumor derived from the eccrine sweat glands. We present a case of EPC on the lateral nose wall, in which the tumor was excised, and the resultant defect was reconstructed using a nasolabial flap. A 66-year-old female was referred to the Department of Plastic and Reconstructive Surgery to receive treatment for a cutaneous tumor on her right lateral nose wall, which had been growing rapidly for 3 months. Histological analysis of a biopsy specimen of the tumor suggested that it was a squamous cell carcinoma. Surgical excision was performed with a 3-mm margin. The tumor was histologically diagnosed as an EPC. EPC exhibits various pathological features; therefore, it is often confused with other malignant cutaneous tumors. We consider that histologically examining surgical specimens obtained via total resection, rather than incisional biopsy specimens, is important for correctly diagnosing EPC.

Risk Factors for Postoperative Complications among the Elderly after Plastic Surgery Procedures Performed under General Anesthesia.

BACKGROUND: The frequency of surgery involving elderly patients has been increasing. The use of free tissue transfers in the elderly has been examined previously (Howard et al., 2005, Hwang et al., 2016, Grammatica et al., 2015, Serletti et al., 2000, and Sierakowski et al., 2017), whereas there have not been any such studies of plastic surgery procedures. We evaluated the risk factors for complications after plastic surgery procedures performed under general anesthesia in patients aged ≥75 years. METHODS: The cases of patients aged ≥75 years who underwent plastic surgery procedures under general anesthesia at the Department of Plastic and Reconstructive Surgery, National Hospital Organization Nagasaki Medical Center, between 2009 and 2016 were reviewed retrospectively. Multiple logistic regression analysis was used to identify the risk factors for postoperative complications. RESULTS: Two hundred and sixty-three cases were reviewed. Complications were seen in 137 patients. Age was not predictive of complications. The risk factors included a serum albumin level of <2.8 g/dl (odds ratio (OR): 2.96), an operative time of ≥120 min (OR: 6.22), and an American Society of Anesthesiologists performance status of ≥3 (OR: 2.39). CONCLUSIONS: Age is not contraindication for surgery in the elderly. It is important to assess comorbidities and perform surgical procedures as soon as possible to shorten the surgical period.

Cell cycle tracking for irradiated and unirradiated bystander cells in a single colony with exposure to a soft X-ray microbeam.

PURPOSE: To establish a new experimental technique to explore the photoelectric and subsequent Auger effects on the cell cycles of soft X-ray microbeam-irradiated cells and unirradiated bystander cells in a single colony. MATERIALS AND METHODS: Several cells located in the center of a microcolony of HeLa-Fucci cells consisting of 20-80 cells were irradiated with soft X-ray (5.35 keV) microbeam using synchrotron radiation as a light source. All cells in the colony were tracked for 72 h by time-lapse microscopy imaging. Cell cycle progression, division, and death of each cell in the movies obtained were analyzed by pedigree assay. The number of cell divisions in the microcolony was also determined. RESULTS: The fates of these cells were clarified by tracking both irradiated and unirradiated bystander cells. Irradiated cells showed significant cell cycle retardation, explosive cell death, or cell fusion after a few divisions. These serious effects were also observed in 15 and 26% of the bystander cells for 10 and 20 Gy irradiation, respectively, and frequently appeared in at least two daughter or granddaughter cells from a single-parent cell. CONCLUSIONS: We successfully tracked the fates of microbeam-irradiated cells and unirradiated bystander cells with live cell recordings, which have revealed the dynamics of soft X-ray irradiated and unirradiated bystander cells for the first time. Notably, cell deaths or cell cycle arrests frequently arose in closely related cells. These details would not have been revealed by a conventional immunostaining imaging method. Our approach promises to reveal the dynamic cellular effects of soft X-ray microbeam irradiation and subsequent Auger processes from various endpoints in future studies.

Determination of the relative biological effectiveness and oxygen enhancement ratio for micronuclei formation using high-LET radiation in solid tumor cells: An in vitro and in vivo study.

We determined the relative biological effectiveness (RBE) and oxygen enhancement ratio (OER) of micronuclei (MN) formation in clamped (hypoxic) and non-clamped (normoxic) solid tumors in mice legs following exposure to X-rays and heavy ions. Single-cell suspensions (aerobic) of non-irradiated tumors were prepared in parallel and used directly to determine the radiation response for aerobic cells. Squamous cell carcinoma (SCCVII) cells were transplanted into the right hind legs of syngeneic C3H/He male mice. Irradiation doses with either X-rays or heavy ions at a dose-averaged LET (linear energy transfer) of 14-192keV/µm were delivered to 5-mm diameter tumors and aerobic single-cells in sample-tubes. After irradiation, the tumors were excised and trypsinized to observe MN in single-cells. The single-cell suspensions were used for MN formation assays. The RBE values increased with increasing LET. The maximum RBE values for the three different oxygen conditions; hypoxic tumor, normoxic tumor, and aerobic cells, were 8.18, 5.30, and 3.76 at an LET of 192keV/µm, respectively. After X-irradiation, the OERh/n values (hypoxic tumor/normoxic tumor) were lower than the OERh/a (hypoxic tumor/aerobic cells), and were 1.73 and 2.58, respectively. We found that the OER for the in vivo studies were smaller in comparison to that for the in vitro studies. Both of the OER values at 192keV/µm were small in comparison to those of the X-ray irradiated samples. The OERh/n and OERh/a values at 192keV/µm were 1.12 and 1.19, respectively. Our results suggest that high LET radiation has a large biological effect even if a solid tumor includes substantial numbers of hypoxic cells. To conclude, we found that the RBE values under each oxygen state for non-MN fraction increased with increasing LET and that the OER values for both tumors in vivo and cells in vitro decreased with increasing LET.

The combination of Hsp90 inhibitor 17AAG and heavy-ion irradiation provides effective tumor control in human lung cancer cells.

Hsp90 inhibitors have become well-studied antitumor agents for their selective property against tumors versus normal cells. The combined treatment of Hsp90 inhibitor and conventional photon radiation also showed more effective tumor growth delay than radiation alone. However, little is known regarding the combined treatment of Hsp90 inhibitor and heavy-ion irradiation. In this study, SQ5 human lung tumor cells were used in vitro for clonogenic cell survival and in vivo for tumor growth delay measurement using a mouse xenograft model after 17-allylamino-17-demethoxygeldanamycin (17AAG) pretreatment and carbon ion irradiation. Repair of DNA double strand breaks (DSBs) was also assessed along with expressions of DSB repair-related proteins. Cell cycle analysis after the combined treatment was also performed. The combined treatment of 17AAG and carbon ions revealed a promising treatment option in both in vitro and in vivo studies. One likely cause of this effectiveness was shown to be the inhibition of homologous recombination repair by 17AAG. The more intensified G2 cell cycle delay was also associated with the combined treatment when compared with carbon ion treatment alone. Our findings indicate that the combination of Hsp90 inhibition and heavy-ion irradiation provides a new effective therapeutic alternative for treatment of solid tumors.

OH radicals from the indirect actions of X-rays induce cell lethality and mediate the majority of the oxygen enhancement effect.

We examined OH radical-mediated indirect actions from X irradiation on cell killing in wild-type Chinese hamster ovary cell lines (CHO and AA8) under oxic and hypoxic conditions, and compared the contribution of direct and indirect actions under both conditions. The contribution of indirect action on cell killing can be estimated from the maximum degree of protection by dimethylsulfoxide, which suppresses indirect action by quenching OH radicals without affecting the direct action of X rays on cell killing. The contributions of indirect action on cell killing of CHO cells were 76% and 50% under oxic and hypoxic conditions, respectively, and those for AA8 cells were 85% and 47%, respectively. Therefore, the indirect action on cell killing was enhanced by oxygen during X irradiation in both cell lines tested. Oxygen enhancement ratios (OERs) at the 10% survival level (D10 or LD90) for CHO and AA8 cells were 2.68 ± 0.15 and 2.76 ± 0.08, respectively. OERs were evaluated separately for indirect and direct actions, which gave the values of 3.75 and 2.01 for CHO, and 4.11 and 1.32 for AA8 cells, respectively. Thus the generally accepted OER value of ∼3 is best understood as the average of the OER values for both indirect and direct actions. These results imply that both indirect and direct actions on cell killing require oxygen for the majority of lethal DNA damage, however, oxygen plays a larger role in indirect than for direct effects. Conversely, the lethal damage induced by the direct action of X rays are less affected by oxygen concentration.

Significance of DNA polymerase I in in vivo processing of clustered DNA damage.

We examined the biological consequences of bi-stranded clustered damage sites, consisting of a combination of DNA lesions, such as a 1-nucleotide gap (GAP), an apurinic/apyrimidinic (AP) site, and an 8-oxo-7,8-dihydroguanine (8-oxoG), using a bacterial plasmid-based assay. Following transformation with the plasmid containing bi-stranded clustered damage sites into the wild type strain of Escherichia coli, transformation frequencies were significantly lower for the bi-stranded clustered GAP/AP lesions (separated by 1bp) than for either a single GAP or a single AP site. When the two lesions were separated by 10-20bp, the transformation efficiencies were comparable with those of the single lesions. This recovery of transformation efficiency for separated lesions requires DNA polymerase I (Pol I) activity. Analogously, the mutation frequency was found to depend on the distance separating lesions in a bi-stranded cluster containing a GAP and an 8-oxoG, and Pol I was found to play an important role in minimising mutations induced as a result of clustered lesions. The mutagenic potential of 8-oxoG within the bi-stranded lesions does not depend on whether it is situated on the leading or lagging strand. These results indicate that the biological consequences of clustered DNA damage strongly depend on the extent of repair of the strand breaks as well as the DNA polymerase in lesion-avoidance pathways during replication.

Evaluation of SCCVII tumor cell survival in clamped and non-clamped solid tumors exposed to carbon-ion beams in comparison to X-rays.

The aim of this study was to measure the RBE (relative biological effectiveness) and OER (oxygen enhancement ratio) for survival of cells within implanted solid tumors following exposure to 290MeV/nucleon carbon-ion beams or X-rays. Squamous cell carcinoma cells (SCCVII) were transplanted into the right hind legs of syngeneic C3H male mice. Irradiation with either carbon-ion beams with a 6-cm spread-out Bragg peak (SOBP, at 46 and 80keV/µm) or X-rays was delivered to 5-mm or less diameter tumors. We defined three different oxygen statuses of the irradiated cells. Hypoxic and normoxic conditions in tumors were produced by clamping or not clamping the leg to avoid blood flow. Furthermore, single-cell suspensions were prepared from non-irradiated tumors and directly used to determine the radiation response of aerobic cells. Single-cell suspensions (aerobic condition) were fully air-saturated. Single-cell suspensions were prepared from excised and trypsinized tumors, and were used for in vivo-in vitro colony formation assays to obtain cell survival curves. The RBE values increased with increasing LET in SOBP beams. The maximum RBE values in three different oxygen conditions; hypoxic tumor, normoxic tumor and aerobic cells, were 2.16, 1.76 and 1.66 at an LET of 80keV/µm, respectively. After X-ray irradiation the OERh/n values (hypoxic tumor/normoxic tumor) were lower than the OERh/a (hypoxic tumor/aerobic cells), and were 1.87±0.13 and 2.52±0.11, respectively. The OER values of carbon-ion irradiated samples were small in comparison to those of X-ray irradiated samples. However, no significant changes of the OER at proximal and distal positions within the SOBP carbon-ion beams were observed. To conclude, we found that the RBE values for cell survival increased with increasing LET and that the OER values changed little with increasing LET within the SOBP carbon-ion beams.

The mutagenic potential of 8-oxoG/single strand break-containing clusters depends on their relative positions.

The biological consequences of clusters containing a single strand break and base lesion(s) remain largely unknown. In the present study we determined the mutagenicities of two- and three-lesion clustered damage sites containing a 1-nucleotide gap (GAP) and 8-oxo-7,8-dihydroguanine(s) (8-oxoG(s)) in Escherichia coli. The mutation frequencies (MFs) of bi-stranded two-lesion clusters (GAP/8-oxoG), especially in mutY-deficient strains, were high and were similar to those for bi-stranded clusters with 8-oxoG and base lesions/AP sites, suggesting that the GAP is processed with an efficiency similar to the efficiency of processing a base lesion or an AP site within a cluster. The MFs of tandem two-lesion clusters comprised of a GAP and an 8-oxoG on the same strand were comparable to or less than the MF of a single 8-oxoG. The mutagenic potential of three-lesion clusters, which were comprised of a tandem lesion (a GAP and an 8-oxoG) and an opposing single 8-oxoG, was higher than that of a single 8-oxoG, but was no more than that of a bi-stranded 8-oxoGs. We suggest that incorporation of a nucleotide opposite 8-oxoG is less mutagenic when a GAP is present in a cluster than when a GAP is absent. Our observations indicate that the repair of a GAP is retarded by an opposing 8-oxoG, but not by a tandem 8-oxoG, and that the extent of GAP repair determines the biological consequences.

A novel technique using DNA denaturation to detect multiply induced single-strand breaks in a hydrated plasmid DNA molecule by X-ray and 4He2+ ion irradiation.

To detect multiple single-strand breaks (SSBs) produced in plasmid DNA molecules by direct energy deposition from radiation tracks, we have developed a novel technique using DNA denaturation by which irradiated DNA is analysed as single-strand DNA (SS-DNA). The multiple SSBs that arise in both strands of DNA, but do not induce a double-strand break, are quantified as loss of SS-DNA using agarose gel electrophoresis. We have applied this method to X-ray and (4)He(2+) ion-irradiated samples of fully hydrated pUC18 plasmid DNA. The fractions of both SS-DNA and closed circular DNA (CC-DNA) exponentially decrease with the increasing dose of X rays and (4)He(2+) ions. The efficiency of the loss of SS-DNA was half that of CC-DNA for both types of irradiation, indicating that one of two strands in DNA is not broken when one SSB is produced in CC-DNA by irradiation. Contrary to our initial expectation, these results indicate that SSBs are not multiply induced even by high linear energy transfer radiation distributed in both strands.

Contributions of direct and indirect actions in cell killing by high-LET radiations.

The biological effects of radiation originate principally in damages to DNA. DNA damages by X rays as well as heavy ions are induced by a combination of direct and indirect actions. The contribution of indirect action in cell killing can be estimated from the maximum degree of protection by dimethylsulfoxide (DMSO), which suppresses indirect action without affecting direct action. Exponentially growing Chinese hamster V79 cells were exposed to high-LET radiations of 20 to 2106 keV/mum in the presence or absence of DMSO and their survival was determined using a colony formation assay. The contribution of indirect action to cell killing decreased with increasing LET. However, the contribution did not reach zero even at very high LETs and was estimated to be 32% at an LET of 2106 keV/mum. Therefore, even though the radiochemically estimated G value of OH radicals was nearly zero at an LET of 1000 keV/mum, indirect action by OH radicals contributed to a substantial fraction of the biological effects of high-LET radiations. The RBE determined at a survival level of 10% increased with LET, reaching a maximum value of 2.88 at 200 keV/mum, and decreased thereafter. When the RBE was estimated separately for direct action (RBE(D)) and indirect action (RBE(I)); both exhibited an LET dependence similar to that of the RBE, peaking at 200 keV/mum. However, the peak value was much higher for RBE(D) (5.99) than RBE(I) (1.89). Thus direct action contributes more to the high RBE of high-LET radiations than indirect action does.