Effects of employing a ¹°B-carrier and manipulating intratumour hypoxia on local tumour response and lung metastatic potential in boron neutron capture therapy.

OBJECTIVES: To evaluate the effects of employing a (10)B-carrier and manipulating intratumour hypoxia on local tumour response and lung metastatic potential in boron neutron capture therapy (BNCT) by measuring the response of intratumour quiescent (Q) cells. METHODS: B16-BL6 melanoma tumour-bearing C57BL/6 mice were continuously given 5-bromo-2'-deoxyuridine (BrdU) to label all proliferating (P) cells. The tumours received reactor thermal neutron beam irradiation following the administration of a (10)B-carrier [L-para-boronophenylalanine-(10)B (BPA) or sodium mercaptoundecahydrododecaborate-(10)B (BSH)] in combination with an acute hypoxia-releasing agent (nicotinamide) or mild temperature hyperthermia (MTH). Immediately after the irradiation, cells from some tumours were isolated and incubated with a cytokinesis blocker. The responses of the Q and total (P+Q) cell populations were assessed based on the frequency of micronuclei using immunofluorescence staining for BrdU. In other tumour-bearing mice, macroscopic lung metastases were enumerated 17 days after irradiation. RESULTS: BPA-BNCT increased the sensitivity of the total tumour cell population more than BSH-BNCT. However, the sensitivity of Q cells treated with BPA was lower than that of BSH-treated Q cells. With or without a (10)B-carrier, MTH enhanced the sensitivity of the Q cell population. Without irradiation, nicotinamide treatment decreased the number of lung metastases. With irradiation, BPA-BNCT, especially in combination with nicotinamide treatment, showed the potential to reduce the number of metastases more than BSH-BNCT. CONCLUSION: BSH-BNCT in combination with MTH improves local tumour control, while BPA-BNCT in combination with nicotinamide may reduce the number of lung metastases.

Reducing intratumour acute hypoxia through bevacizumab treatment, referring to the response of quiescent tumour cells and metastatic potential.

OBJECTIVES: The aim was to evaluate the influence of bevacizumab on intratumour oxygenation status and lung metastasis following radiotherapy, with specific reference to the response of quiescent (Q) cell populations within irradiated tumours. METHODS: B16-BL6 melanoma tumour-bearing C57BL/6 mice were continuously given 5-bromo-2-deoxyuridine (BrdU) to label all proliferating (P) cells. They received γ-ray irradiation following treatment with the acute hypoxia-releasing agent nicotinamide or local mild temperature hyperthermia (MTH) with or without the administration of bevacizumab under aerobic conditions or totally hypoxic conditions, achieved by clamping the proximal end of the tumours. Immediately after the irradiation, cells from some tumours were isolated and incubated with a cytokinesis blocker. The responses of the Q and total (P + Q) cell populations were assessed based on the frequency of micronuclei using immunofluorescence staining for BrdU. In the other tumour-bearing mice, macroscopic lung metastases were enumerated 17 days after irradiation. RESULTS: 3 days after bevacizumab administration, acute hypoxia-rich total cell population in the tumour showed a remarkably enhanced radiosensitivity to γ-rays, and the hypoxic fraction (HF) was reduced, even after MTH treatment. However, the hypoxic fraction was not reduced after nicotinamide treatment. With or without γ-ray irradiation, bevacizumab administration showed some potential to reduce the number of lung metastases as well as nicotinamide treatment. CONCLUSION: Bevacizumab has the potential to reduce perfusion-limited acute hypoxia and some potential to cause a decrease in the number of lung metastases as well as nicotinamide.

A phantom experiment for the evaluation of whole body exposure during BNCT using cyclotron-based epithermal neutron source (C-BENS).

At Kyoto University Research Reactor Institute (KURRI), cyclotron-based epithermal neutron source was installed in December 2008, and the supplementary construction works have been performed. As of December 2010, the various irradiation characteristics important for BNCT were mostly evaluated. The whole body exposure during BNCT medical irradiation is one of the important characteristics. In this article, measurements of absorbed dose for thermal and fast neutrons and gamma-ray at ten positions corresponding to important organs are reported.

Significance of manipulating tumour hypoxia and radiation dose rate in terms of local tumour response and lung metastatic potential, referring to the response of quiescent cell populations.

The purpose of this study was to evaluate the influence of manipulating intratumour oxygenation status and radiation dose rate on local tumour response and lung metastases following radiotherapy, referring to the response of quiescent cell populations within irradiated tumours. B16-BL6 melanoma tumour-bearing C57BL/6 mice were continuously given 5-bromo-2'-deoxyuridine (BrdU) to label all proliferating (P) cells. They received gamma-ray irradiation at high dose rate (HDR) or reduced dose rate (RDR) following treatment with the acute hypoxia-releasing agent nicotinamide or local hyperthermia at mild temperatures (MTH). Immediately after the irradiation, cells from some tumours were isolated and incubated with a cytokinesis blocker. The responses of the quiescent (Q) and total (proliferating + Q) cell populations were assessed based on the frequency of micronuclei using immunofluorescence staining for BrdU. In other tumour-bearing mice, 17 days after irradiation, macroscopic lung metastases were enumerated. Following HDR irradiation, nicotinamide and MTH enhanced the sensitivity of the total and Q-cell populations, respectively. The decrease in sensitivity at RDR irradiation compared with HDR irradiation was slightly inhibited by MTH, especially in Q cells. Without gamma-ray irradiation, nicotinamide treatment tended to reduce the number of lung metastases. With gamma-rays, in combination with nicotinamide or MTH, especially the former, HDR irradiation decreased the number of metastases more remarkably than RDR irradiation. Manipulating both tumour hypoxia and irradiation dose rate have the potential to influence lung metastasis. The combination with the acute hypoxia-releasing agent nicotinamide may be more promising in HDR than RDR irradiation in terms of reducing the number of lung metastases.

The usefulness of a continuous administration of tirapazamine combined with reduced dose-rate irradiation using {gamma}-rays or reactor thermal neutrons.

We clarified the usefulness of the continuous administration of tirapazamine (TPZ) in combination with reduced dose-rate irradiation (RDRI) using gamma-rays or reactor thermal neutrons. Squamous cell carcinoma (SCC) VII tumour-bearing mice received a continuous administration of 5-bromo-2'-deoxyuridine (BrdU) to label all proliferating (P) cells. Then, they received a single intraperitoneal injection or 24 h continuous subcutaneous infusion of TPZ in combination with conventional dose-rate irradiation (CDRI) or RDRI using gamma-rays or thermal neutrons. After irradiation, the tumour cells were isolated and incubated with a cytokinesis blocker, and the micronucleus (MN) frequency in cells without BrdU labelling ( = quiescent (Q) cells) was determined using immunofluorescence staining for BrdU. The MN frequency in the total tumour cells was determined using tumours that were not pre-treated with BrdU. The sensitivity of both total and Q cells, especially of Q cells, was significantly reduced with RDRI compared with CDRI. Combination of TPZ increased the sensitivity of both populations, with a slightly more remarkable increase in Q cells. Furthermore, the continuous administration of TPZ raised the sensitivity of both total and Q cell populations, especially the former, more markedly than the single administration, whether combined with CDRI or RDRI using gamma-rays or thermal neutrons. From the viewpoint of solid tumour control as a whole, including intratumour Q-cell control, the use of TPZ, especially when administered continuously, combined with RDRI, is useful for suppressing the reduction in the sensitivity of tumour cells caused by the decrease in irradiation dose rate in vivo.

Mutations within the tyrosine kinase domain of EGFR gene specifically occur in lung adenocarcinoma patients with a low exposure of tobacco smoking.

Somatically acquired mutations in the epidermal growth factor receptor (EGFR) gene in lung cancer are associated with significant clinical responses to gefitinib, a tyrosine kinase inhibitor that targets EGFR. We screened the EGFR in 469 resected tumours of patients with lung cancer, which included 322 adenocarcinomas, 102 squamous cell carcinomas, 27 large cell carcinomas, 13 small cell carcinomas, and five other cell types. PCR with a specific condition was performed to identify any deletion in exon 19, while mutant-allele-specific amplification was performed to identify a mutation in codon 858 of exon 21. EGFR mutations were found in 136 cases (42.2%) with adenocarcinoma, in one case with large cell carcinoma, and in one case with pleomorphic carcinoma. An in-frame deletion in exon 19 was found in 62 cases while an L858R mutation was found in 77 cases. In the 322 cases with adenocarcinoma, these mutations were more frequently found in women than in men (P=0.0004), in well differentiated tumours than in poorly differentiated tumours (P=0.0014), and in patients who were never smokers than in patients who were current/former smokers (P<0.0001). The mutation was more frequently observed in patients who smoked

Hyperbaric oxygenation therapy enhances the protective effect of moderate hypothermia against forebrain ischemia in the gerbil hippocampus.

Moderate hypothermia may have a beneficial effect on the neurological outcome. However, ischemic deterioration such as brain swelling during rewarming has been reported as a notable complication after successful therapeutic cerebral hypothermia. In this study, we investigated the effects of hyperbaric oxygenation during rewarming. Forebrain ischemia was produced in 24 gerbils and sham ischemia in 8 animals. Then ischemia-treated animals were divided into 3 groups, whole-body moderate hypothermia (31 degrees C for 60 min) and hyperbaric oxygenation (HBO2) (2- atmosphere absolute for 60 min using 100% oxygen) during rewarming group (n = 8), moderate hypothermia without HBO2 group (n = 8), and sham treatment without hypothermia and without HBO2 group (n = 8). Both the hypothermia group (77.9 +/- 48.1 neurons per mm, mean +/- SD) and hypothermia + HBO2 group (127.6 +/- 29.7 neurons per mm,) showed significant preservation of CA1 pyramidal neurons in the hippocampus compared to that in the sham treatment group (6.4 +/- 2.7) (p < 0.01). Furthermore, the hypothermia + HBO2 group showed significantly greater preservation of CA1 pyramidal neurons than the hypothermia group (p < 0.05). These results suggest that HBO2 during rewarming preserves the protective effect of hypothermia against ischemic neuronal damage.

The usefulness of continuous administration of hypoxic cytotoxin combined with mild temperature hyperthermia, with reference to effects on quiescent tumour cell populations.

To evaluate the usefulness of continuous administration of hypoxic cytotoxins in terms of targeting acute hypoxia in solid tumours and the significance of combination with mild temperature hyperthermia (MTH) (40 degrees C, 60 min), the cytotoxic effects of singly or continuously administered tirapazamine (TPZ) and TX-402 were examined in combination with or without MTH in vivo. Further, the effects were also analysed on total (=proliferating (P)+quiescent (Q)) and Q cell populations in solid tumours with the method for selectively detecting the Q cell response. C3H/He mice bearing SCC VII tumours received a continuous administration of 5-bromo-2'-deoxyuridine (BrdU) for 5 days to label all P cells. The tumour-bearing mice then received a single intra-peritoneal injection or 24 h continuous subcutaneous infusion of hypoxic cytotoxin, TPZ or TX-402, with or without MTH. On the other hand, to detect the changes in the hypoxic fraction (HF) in the tumours by MTH, another group of mice with or without MTH received a series of test doses of gamma-rays while alive or after tumour clamping. After each treatment, the tumour cells were isolated and incubated with a cytokinesis blocker (=cytochalasin-B) and the micronucleus (MN) frequency in cells without BrdU labelling (=Q cells) was determined using immunofluorescence staining for BrdU. The MN frequency in total tumour cells was determined from the tumours that were not pre-treated with BrdU. The sensitivity to TX-402 was slightly higher than that to TPZ in both total and Q tumour cells. Continuous administration elevated the sensitivity of both total and Q cells, especially total cells. MTH raised the sensitivity of Q cells more remarkably than that of total cells in both single and continuous administrations. It was thought to be probably because of the higher dose distribution of hypoxic cytotoxin in intermediately hypoxic areas derived mainly from chronic hypoxia through MTH. From the viewpoint of tumour control as a whole including both total and Q tumour cells, the continuous administration of hypoxic cytotoxin combined with MTH may be useful for sensitizing tumour cells in vivo.

Molecular cloning of a class IV chitinase allergen from Japanese cedar (Cryptomeria japonica) pollen and competitive inhibition of its immunoglobulin E-binding capacity by latex C-serum.

BACKGROUND: Japanese cedar (Cryptomeria japonica) pollinosis is one of the most prevalent allergic diseases in Japan. Only three C. japonica allergens, Cry j 1, Cry j 2, and CJP-6, have been characterized. The full IgE-binding spectrum of C. japonica pollen allergens demonstrates that many allergens remain to be identified. OBJECTIVE: The aim of this study was to characterize a novel allergen with a high frequency of IgE binding. METHODS: The cDNA coding for a high-frequency IgE-binding protein, designated CJP-4, was cloned from the total mRNA of C. japonica pollen. The corresponding native allergen was purified by affinity precipitation with colloidal chitin and gel chromatography. The IgE-binding ability of purified native CJP-4 was characterized by ELISA and ELISA inhibition. RESULTS: The CJP-4 cDNA encoded 281 amino acids with significant sequence homology to class IV chitinases. Purified native CJP-4, migrated as a homogeneous 34-kDa protein on SDS-PAGE, revealed endochitinase activity on native PAGE. The purified protein displayed the ability to bind IgE from all patients tested (31/31) in ELISA, whereas Cry j 1 bound to IgE at a 71% frequency (22/31). Pre-incubation with latex C-serum completely inhibited the reaction of pooled sera IgE from patients with C. japonica pollinosis and/or latex allergy to purified CJP-4. CONCLUSION: We identified CJP-4 as a novel and fourth C. japonica chitinase allergen with high IgE-binding frequency. The competitive IgE-binding profile between C. japonica chitinase and latex C-serum indicated that C. japonica chitinase should be an important pan-allergen in C. japonica pollen.

Allergens in Japanese patients with allergic conjunctivitis in autumn.

PURPOSE: The purpose of the current study is to evaluate the relation between various specific class E immunoglobulins (IgE) in the serum and allergic conjunctivitis in autumn. METHODS: Total IgE and specific IgE to 12 inhalant allergens were measured using the CAP system in 32 patients with allergic conjunctivitis in spring (spring group), 27 patients with allergic conjunctivitis in autumn (autumn group), and 40 healthy volunteers (control group). RESULTS: Specific IgE levels caused by house dust, Dermatophagoides pteronyssinus, and orchard grass were higher in the autumn group than in the spring group. The highest positivity rate for a specific allergen was 51.9% for house dust, followed by D. pteronyssinus(48.1%) in the autumn group, while the highest rate was 68.8 % for cedar pollen, followed by cypress pollen (59.4%) in the spring group. Correlation analysis showed that house dust was significantly correlated with animal epithelia, D. pteronyssinus, acarus, and Alternaria tenuis in the autumn group (P<0.001). CONCLUSIONS: These results suggest that house dust is the main cause of allergic conjunctivitis during autumn. In spring, cypress pollen is the largest cause of allergic conjunctivitis, while indoor allergens such as house dust, animal epithelia, D. pteronyssinus, and acarus are not causative allergens in Japan.

DAMGO suppresses both excitatory and inhibitory synaptic transmission in supraoptic neurones of mouse hypothalamic slice preparations.

Opioid effects on synaptic transmission in the mouse supraoptic nucleus (SON) were investigated using whole-cell, patch-clamp techniques. The mu-opioid receptor agonist, [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO) decreased the amplitude of both evoked excitatory postsynaptic currents (eEPSCs) and inhibitory postsynaptic currents (eIPSCs), and also decreased the frequency of both miniature EPSCs and IPSCs without effect on the amplitude. The selective mu-opioid receptor antagonist, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH(2), and the nonselective antagonist naloxone, antagonized these inhibitory effects. The application of DAMGO suppressed the amplitude of both the first and second evoked postsynaptic currents with a paired-pulse stimulus protocol, but increased the paired-pulse ratios (second ePSC/first ePSC). DAMGO induced neither inward nor outward currents, and had no significant changes in either glutamate- or GABA-induced currents. When compared with the relatively selective kappa- and delta-opioid receptor agonists dynorphin and [D-Pen(2), D-Pen(5)]-enkephalin, DAMGO showed the most potent inhibitory effects on evoked and miniature postsynaptic currents. Taken together, these results imply that DAMGO strongly suppresses the release of glutamate and GABA via mu-opioid receptors in the mouse SON, and support the involvement of presynaptic regulation by opioids in the control of magnocellular neurosecretory neurones.

Molecular cloning and characterization of a new Japanese cedar pollen allergen homologous to plant isoflavone reductase family.

BACKGROUND: Japanese cedar (Cryptomeria japonica) pollen is a major cause of seasonal pollinosis, and more than 10% of Japanese people suffer from this allergic disorder. However, only two major pollen allergens, Cry j 1 and Cry j 2, have been identified and exclusively characterized. OBJECTIVE: The aim of this study was to explore and identify important Japanese cedar pollen allergens other than Cry j 1 or Cry j 2. METHODS: C. japonica cDNA library was immunoscreened by rabbit antiserum raised against a partially purified cedar pollen allergen fraction. An isolated cDNA clone was inserted into a glutathione S-transferase (GST)-tagged Escherichia coli expression vector to obtain recombinant GST fusion protein. Non-fusion recombinant protein was purified by glutathione Sepharose affinity chromatography in conjunction with factor Xa cleavage of the GST moiety. IgE-binding ability of the recombinant protein was then evaluated by western blot analysis and enzyme-linked immunosorbent assay (ELISA). RESULTS: The cDNA encodes 306 amino acids with significant sequence similarity to those of plant isoflavone reductase-like proteins, which include a recently identified birch pollen allergen Bet v 5. Western blot analysis demonstrated that recombinant protein was recognized by cedar pollinosis patient IgE. In contrast to Bet v 5 being reported as a minor allergen, the recombinant protein exhibited 76% IgE binding frequency (19/25) against pollinosis patients. CONCLUSION: Here we identified the third member of Japanese cedar pollen allergen homologous to isoflavone reductase. Its high IgE-binding frequency implicates that the isoflavone reductase homologue might be an additional major pollen allergen in C. japonica.

Neutralization of interleukin-1beta in the acute phase of myocardial infarction promotes the progression of left ventricular remodeling.

OBJECTIVES: We sought to examine the role of the pro-inflammatory cytokine, interleukin-1-beta (IL-1beta), in the process of left ventricular (LV) remodeling in the early phase after myocardial infarction (MI). BACKGROUND: Studies have shown that pro-inflammatory cytokines are closely related to the progression of LV remodeling after MI. METHODS: Mice underwent coronary artery ligation, and the time course of LV remodeling was followed up to 20 weeks. The gene expression level of IL-1beta was examined. In a second set of experiments, the mice underwent coronary artery ligation followed by treatment with anti-IL-1beta antibody (100 microg, intravenously), versus control immunoglobulin G (100 microg, intravenously) immediately after the operation. RESULTS: Rapid hypertrophy of noninfarcted myocardium was observed by four weeks, and interstitial fibrosis progressed steadily up to 20 weeks. Anti-IL-1beta treatment increased the occurrence of ventricular rupture and suppressed collagen accumulation in the infarct-related area. At four and eight weeks after the operation, total heart weight and LV end-diastolic dimension were significantly greater in the anti-IL-1beta-treated mice than in the other groups. In the infarct-related area, collagen accumulation was suppressed, whereas in the noninfarcted area, pro-collagen gene expression levels, particularly type III, were decreased in the anti-IL-1beta-treated mice. CONCLUSIONS: Anti-IL-1beta treatment suppressed pro-collagen gene expression and delayed wound healing mechanisms-properties that are likely to lead to progression of LV remodeling. In the acute phase of MI, IL-1beta appears to play a protective role.

Evaluation of the potential of p-boronophenylalaninol as a boron carrier in boron neutron capture therapy, referring to the effect on intratumor quiescent cells.

C57BL mice bearing EL4 tumors and C3H / He mice bearing SCC VII tumors received 5-bromo-2'-deoxyuridine (BrdU) continuously for 5 days via implanted mini-osmotic pumps to label all proliferating (P) cells. Three hours after oral administration of l-p-boronophenylalanine-(10)B (BPA), or 30 min after intraperitoneal injection of sodium borocaptate-(10)B (BSH) or l-p-boronophenylalaninol (BPA-ol), a newly developed (10)B-containing alpha-amino alcohol, the tumors were irradiated with thermal neutron beams. For the combination with mild temperature hyperthermia (MTH) and / or tirapazamine (TPZ), the tumors were heated at 40 degrees C for 30 min immediately before neutron exposure, and TPZ was intraperitoneally injected 30 min before irradiation. The tumors were then excised, minced and trypsinized. The tumor cell suspensions thus obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus (MN) frequency in cells without BrdU labeling ( = quiescent (Q) cells) was determined using immunofluorescence staining for BrdU. Meanwhile, 6 h after irradiation, tumor cell suspensions obtained in the same manner were used for determining the apoptosis frequency in Q cells. The MN and apoptosis frequency in total (P + Q) tumor cells were determined from tumors that were not pretreated with BrdU. Without TPZ or MTH, BPA-ol increased both frequencies most markedly, especially for total cells. However, as with BPA, the sensitivity difference between total and Q cells was much larger than with BSH. On combined treatment with both MTH and TPZ, this sensitivity difference was markedly reduced, similarly to when BPA was used. MTH increased the (10)B uptake of all (10)B-compounds into both tumor cells. BPA-ol has good potential as a (10)B-carrier in neutron capture therapy, especially when combined with both MTH and TPZ.

Prediction of sensitivity of esophageal tumors to adjuvant chemotherapy by cDNA microarray analysis of gene-expression profiles.

We applied cDNA microarray analyses of 9216 genes to establish a genetic method for predicting the outcome of adjuvant chemotherapy to esophageal cancers. We analyzed expression profiles of 20 esophageal cancer tissues from patients who were treated with the same adjuvant chemotherapy after removal of tumor by operation, and we attempted to find genes associated with the duration of survival after surgery. By comparing expression profiles of those cancer tissues, we identified by statistical analysis 52 genes that were likely to be correlated with prognosis and possibly with sensitivity/resistance to the anticancer drugs. We also developed a drug response score based on the differential expression of these genes, and we found a significant correlation between the drug response score and individual patients' prognoses. Our results indicated that this scoring system, based on microarray analysis of selected genes, is likely to have great potential for predicting the prognosis of individual cancer patients with the adjuvant chemotherapy.

Synthesis and antimalarial activity of novel medium-sized 1,2,4,5-tetraoxacycloalkanes.

CsOH- or Ag(2)O-mediated cycloalkylation of (alkylidene)bisperoxides 3 and 1,n-dihaloalkanes (n = 3-8) provided the corresponding medium-sized 1,2,4,5-tetraoxacycloalkanes 4-8 in moderate yields. Subsequent evaluation of the antimalarial activity of the cyclic peroxides 4-8 in vitro and in vivo revealed that 1,2,6,7-tetraoxaspiro[7.11]nonadecane 4a has considerable potential as a new, inexpensive, and potent antimalarial drug.

Organization of projections from the medial agranular cortex to the superior colliculus in the rat: a study using anterograde and retrograde tracing methods.

The organization of corticotectal projections from the medial agranular cortex (AGm), which has been considered to contain rat's frontal eye field, was examined using anterograde and retrograde tracing techniques. When biotinylated dextranamine (BDA) injections were made into the rostral part of the AGm, small numbers of BDA-labeled axons were found in the rostral two-thirds of the superior colliculus (SC) while some labeled axons were seen in the caudal one-third of the SC. These labeled axons were distributed mainly in the lateral part of the stratum griseum intermediale. On the other hand, after BDA injections into the caudal part of the AGm, moderate to dense plexuses of labeled axons were found in the rostral two-thirds of the SC while some labeled axons were seen in the caudal one-third of the SC. These labeled axons were distributed in the ventromedial and dorsolateral marginal zones of the stratum griseum intermediale as well as in the stratum griseum profundum. The corticotectal projections were largely uncrossed. After combined injections of BDA into the caudal part of the AGm on one side and cholera toxin B subunit (CTb) into the paramedian pontine reticular formation on the opposite side or into the interstitial nucleus of Cajal on the same side, the overlapping distributions of BDA-labeled axons and CTb-labeled neurons were found in the ventromedial marginal zone of the stratum griseum intermediale ipsilateral to the site of BDA injection. These results suggest that the caudal part of the AGm plays a more significant role in the oculomotor function than does the rostral part of the AGm.

Allixin induction and accumulation by light irradiation.

Allixin, a phytoalexin isolated from garlic, was induced by irradiating fresh garlic cloves with sunlight or UV light. Induced allixin was analyzed by HPLC, and the accumulated amounts of allixin were 3.1-6.3 microg/g under experimental conditions.

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

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