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.

Cell-killing efficiency and number of platinum atoms binding to DNA, RNA, and protein molecules of HeLa cells treated with combinations of hyperthermia and cis-diamine(glycolato)platinum(II).

HeLa S-3 cells were treated with 195mPt-radiolabeled cis-diamine(glylato)platinum(II) (254-S) for 60 min at various temperatures, and the relationship between the lethal effect and the number of Pt atoms binding to DNA, RNA, and proteins was examined. The mean lethal concentration (D0) of 254-S for a 60-min treatment at 0 degree C, 25 degrees C, 37 degrees C, 40 degrees C, 42 degrees C, and 44 degrees C was 233, 132, 61.1, 42.7, 25.6, and 9.9 microM, respectively. By using identically treated cells, the numbers of Pt atoms combined with DNA, RNA, and protein molecules were determined in the subcellular fractions. Thus, the D0 values given as drug concentrations were replaced with the number of Pt atoms combined in each fraction. The, the cell-killing efficiency of the Pt atom was expressed as the reciprocal of the number of Pt atoms combined and was calculated for each molecule. The efficiency for the DNA molecule was 0.61 x 10(4), 1.09 x 10(4), 1.88 x 10(4), 1.90 x 10(4), 2.66 x 10(4), and 5.88 x 10(4) nucleotides, respectively, for the conditions described. From 0 degree C to 44 degrees C, the cell-killing efficiency of Pt atoms increased by a factor of 9.6.

Enhancement of cisplatin sensitivity of quiescent cells in solid tumors by combined treatment with tirapazamine and low-temperature hyperthermia.

We examined the enhanced chemosensitivity of quiescent (Q) cells in solid tumors to cis-diamminedichloroplatinum (II) (cisplatin) by combined treatment with tirapazamine (TPZ) and mild heating. C3H/He and Balb/c mice bearing SCC VII and EMT6/KU tumors, respectively, received continuous administration of 5-bromo-2'-deoxyuridine (BrdU) for 5 days using implanted mini-osmotic pumps to label all proliferating (P) cells. TPZ was administered intraperitoneally 2 h before cisplatin injection and/or tumors were locally heated at 40 degrees C for 60 min immediately after cisplatin injection. Sixty minutes after cisplatin injection, the tumors were excised, minced and trypsinized. The tumor cell suspensions were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus (MN) frequency in cells without BrdU labeling (= Q cells) was determined using immunofluorescence staining for BrdU. The MN frequency in total (P+Q) tumor cells was determined from the tumors that were not pretreated with BrdU. The sensitivity to cisplatin was evaluated in terms of the frequency of induced micronuclei in binuclear tumor cells (MN frequency). Other groups of tumor-bearing C3H/He and Balb/c mice not given BrdU were injected with 195mPt-radiolabeled cisplatin. In both tumor systems, the MN frequency in Q cells was lower than that in the total cells. TPZ and mild heat treatment elevated the MN frequency in total and Q cells in both tumor systems, and to a higher extent in Q cells. The combination of TPZ and mild heat treatment increased the MN frequency more markedly than treatment with either TPZ or mild heating alone. In total tumor cells, TPZ and mild heat treatment increased the MN frequency in EMT6/KU tumor cells more markedly than in SCC VII tumor cells. 195mPt-labeled cisplatin uptake into total tumor cells was increased by mild heat treatment but not by TPZ. The cisplatin-sensitivity of Q cells was lower than that of total cells in both tumor systems. TPZ was thought to sensitize Q cells by killing the hypoxic cells without influencing tumor blood flow, and mild hyperthermia appeared to sensitize Q cells by distributing more cisplatin with an increase in blood flow in solid tumors.

Augmentation in chemosensitivity of intratumor quiescent cells by combined treatment with nicotinamide and mild hyperthermia.

C3H/He and Balb/c mice bearing SCC VII and EMT6/KU tumors, respectively, received continuous administration of 5-bromo-2'-deoxyuridine (BrdU) for 5 days using implanted mini-osmotic pumps to label all proliferating (P) cells. Nicotinamide was administered intraperitoneally before cisplatin injection and/or tumors were locally heated at 40 degrees C for 60 min immediately after cisplatin injection. The tumors were then excised, minced and trypsinized. The tumor cell suspensions 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. The MN frequency in total (P+Q) tumor cells was determined from tumors that had not been pretreated with BrdU labeling. The sensitivity to cisplatin was evaluated in terms of the frequency of induced micronuclei in binuclear tumor cells (MN frequency). In both tumor systems, the MN frequency in Q cells was lower than that in the total cell population. Nicotinamide treatment elevated the MN frequency in total SCC VII cells. Mild heating raised the MN frequency more markedly in Q cells than in total cells. The combination of nicotinamide and mild heat treatment increased the MN frequency more markedly than either treatment alone. In total SCC VII cells, nicotinamide increased 195mPt-cisplatin uptake. Mild heating elevated 195mPt-cisplatin uptake in total EMT6/KU cells. Cisplatin-sensitivity of Q cells was lower than that of total cells in both tumor systems. Nicotinamide sensitized tumor cells including a large acutely hypoxic fraction, such as those of SCC VII tumors, through inhibition of the fluctuations in tumor blood flow. Tumor cells including a large chronically hypoxic fraction such as Q cells were thought to be sensitized by mild heating through an increase in tumor blood flow.

Alteration in the hypoxic fraction of quiescent cell populations by hyperthermia at mild temperatures.

We investigated oxygenation of quiescent (Q) tumour cells in vivo by mild heat treatment. C3H/He mice bearing SCC VII tumours received BrdU continuously for 5 days via implanted mini-osmotic pumps, to label all proliferating (P) cells. The tumours were then irradiated after treatment, and were excised, minced and trypsinized. The tumour cell suspension thus obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus (MN) frequency in cells without BrdU labelling was determined using immunofluorescence staining for BrdU. This MN frequency was then used to calculate the surviving fraction of unlabelled cells from the regression line for the relationship between the MN frequency and the surviving fraction of total (P + Q) tumour cells. Thus, a cell survival curve could be determined for the cells not labelled with BrdU, which can be regarded as the Q cells in a tumour for all practical purposes. The MN frequency in total tumour cell population was determined from the irradiated tumours that were not pretreated with BrdU. Assays performed immediately after irradiation of both normally aerated and hypoxic tumours showed that Q cells contained higher hypoxic fractions than the total tumour cell population. Mild heat treatment (40.0 degrees C, 60 min) before irradiation decreased the hypoxic fraction, even when is was combined with nicotinamide administration. In contrast, mild heating did not decrease the hypoxic fraction when the mice were placed in a circulating carbogen (95% O2/5% CO2) chamber. Therefore, mild heat treatment was thought to preferentially oxygenate the chronically hypoxic fraction.

Modification of the response of a quiescent cell population within a murine solid tumour to boron neutron capture irradiation: studies with nicotinamide and hyperthermia.

C3H/He mice bearing SCC VII tumours received 5-bromo-2'-deoxyuridine (BrdU) continuously for 5 days via implanted mini-osmotic pumps, to label all proliferating (P) cells. 20 min after intraperitoneal injection of sodium borocaptate-10B (BSH), or 3 h after oral administration of dl-p-boronophenylalanine-10B (BPA), the tumours were irradiated with thermal neutrons. To modify the uptake dose of 10B, nicotinamide (NA) was intraperitoneally injected 60 min before the administration of 10B-compounds and/or the tumours were heated to 41.5 degrees C for 20 min immediately before irradiation. After irradiation, the tumours were excised, minced and trypsinized. The tumour cell suspensions were then incubated with cytochalasin-B (a cytokinesis-blocker). The micronucleus (MN) frequency in cells not BrdU-labelled (quiescent (Q) cells) was determined using immunofluorescence staining for BrdU. With or without the administration of 10B-compounds, the sensitivity of Q cells was lower than that of total (P + Q) tumour cells. With thermal neutron irradiation in the presence of either BPA or BSH, the MN frequency in each cell population was increased. A greater increase in the MN frequency of total tumour cells was observed after thermal neutron irradiation in the presence of BPA than in the presence of BSH. The distribution of 10B from BPA into tumour cells was thought to be more dependent on the uptake ability of the tumour cells than that from BSH. Sufficient quantity of 10B from these two 10B-compounds to cause a highly lethal event inside the cancer cell with thermal neutron irradiation could not be delivered to Q cells. When NA and/or heat treatment were combined with 10B-compound administration, NA increased MN frequency in the BSH treated total cells, and heat treatment elevated MN frequency in Q cells. From the viewpoint of cell kill effect, the combined treatment with nicotinamide and heat treatment was more useful than treatment with either nicotinamide or heat treatment alone, not only in the total tumour cells but also in the Q cells.

Reduction of hypoxic cells in solid tumours induced by mild hyperthermia: special reference to differences in changes in the hypoxic fraction between total and quiescent cell populations.

C3H/He mice bearing SCC VII tumours received 5-bromo-2'-deoxyuridine (BrdU) continuously for 5 days via implanted mini-osmotic pumps in order to label all proliferating (P) cells. The tumours were then heated at 40 degrees C for 60 min. At various time points after heating, tumour-bearing mice were irradiated while alive or after being killed. Immediately after irradiation, the tumours were excised, minced and trypsinized. The tumour cell suspensions obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus (MN) frequency in cells without BrdU labelling, which could be regarded as quiescent (Q) cells, was determined using immunofluorescence staining for BrdU. The MN frequency in the total (P+Q) tumour cell population was determined from the irradiated tumours that were not pretreated with BrdU. The MN frequency of BrdU unlabelled cells was then used to calculate the surviving fraction of the unlabelled cells from the regression line for the relationship between the MN frequency and the surviving fraction of total (P+Q) tumour cells. In general, Q cells contained a greater hypoxic fraction (HF) than the total tumour cell population. Mild heating decreased the HF of Q cells more markedly than in the total cell population, and the minimum values of HFs of both total and Q cell populations were obtained 6 h after heating. Two days after heating, the HF of total tumour cells returned to almost that of unheated tumours. In contrast, the HF of Q cells did not return to the HF level of unheated tumours until 1 week after heating. It was thought that irradiation within 12 h after mild heating might be a potentially promising therapeutic modality for controlling radioresistant Q tumour cells.

Effect of hyperthermia on the number of platinum atoms binding to DNA of HeLa cells treated with 195mPt-radiolabelled cis-diaminedichloroplatinum(II).

HeLa S-3 cells were treated with 195mPt-radiolabelled cis-diaminedichloroplatinum II) (CDDP) for 60 min at various temperatures to examine the relationship between the lethal effect and the number of Pt atoms binding to DNA, RNA and protein molecules. The mean lethal concentration (Do) of CDDP for 60-min treatment at 0, 25, 37, 40, 42 and 44 degrees C was 233, 69.9, 15.9, 11.7, 8.3 and 4.7 microM respectively. By using identically treated cells, the number of Pt atoms combined with DNA, RNA and protein molecules was determined in the subcellular fractions prepared by the method of Schneider (1961). Thus, the Do's given as the drug concentrations were substituted for the number of Pt atoms combined with each fraction. Then the efficiency of the Pt atom to kill the cells was expressed as the reciprocal of the number of Pt atoms combined and was calculated for each molecule. The efficiency for DNA was 2.47, 2.75, 9.49, 9.66, 10.53 and 15.00 x 10(4) nucleotides respectively for the conditions described above. A detailed comparison of the Do's and efficiencies suggested that the supra-additive effect of the combination treatment could be explained by two mechanisms, i.e. the increased drug level in DNA (from 37 to 42 degrees C) and the increased efficiency of the Pt atoms to kill the cells (> 42 degrees C).

Middle dose rate irradiation in combination with carbogen inhalation selectively and more markedly increases the responses of SCCVII tumors.

PURPOSE: Carbogen increases the radiation response of tumors and reduced dose rate irradiation spares the damage of normal tissues. The purpose in this paper is to investigate the possibility of selective radiosensitization of tumors by reduced dose rate irradiation in combination with carbogen inhalation. METHODS AND MATERIALS: SCCVII tumors in C3H/He mice were irradiated at middle dose rate (0.1 Gy/min) or high dose rate irradiation (3.0 Gy/min) in combination with carbogen inhalation. The mice were enclosed in a box with carbogen flushing at 1.01/min. The tumor response was measured by a cytokinesis block micronucleus assay. The effects on intestinal crypt cells and bone marrow cells were investigated by microcolony assay or Hendry's method, respectively. RESULTS: The anti-tumor effect of middle dose rate irradiation was equal to that of a high dose rate irradiation. Carbogen inhalation, more efficiently, increased the antitumor effect when combined with middle and high dose rate irradiation, and yielded enhancement ratios of 1.6 at around 2 Gy. Middle dose rate irradiation produced less damage on intestinal crypt cells and bone marrow cells in comparison with high dose rate irradiation, and carbogen inhalation never enhanced the responses of these normal tissues in combination with middle dose rate irradiation. Dose modifying factors were 1.3-2.0. CONCLUSION: Since middle dose rate irradiation in combination with carbogen inhalation gave the therapeutic gain factors of 2.0-3.2, which were much larger than those obtained with any other radiosensitizers, this combination has a potential as a new modality for improving the results of cancer radiotherapy.

Scintigraphic study on the distribution of radiolabeled cis-diamminedichloroplatinum (II) in the tumor-bearing rabbit: a comparison between intra-arterial injection with lipiodol and intravenous injection.

To compare the distribution of cis-diamminedichloroplatinum (II) (CDDP) on scintigraphic images between intra-arterial injection with lipiodol and intravenous injection, we obtained serial scintigraphic images using radiolabeled CDDP (Pt-195m CDDP), which had a high specific activity (7.4 x 10(6) Bq [200 microCi]/mg-CDDP), in the rabbit VX-2 tumor system. A dose of 9.25 x 10(6) Bq (250 microCi) of Pt-195m CDDP was injected at one shot via an ear vein, while 7.4 x 10(6) Bq (200 microCi of Pt-195m CDDP mixed with 1 ml of lipiodol was injected at one shot via a saphenous artery. Mean pixel count, which was corrected for the total dose injected, in the arterially injected tumor always exceeded than that in the intravenously injected tumor. Pt-195m CDDP uptake by the rabbit tumor at 2 hr was 7.8% of the total injected dose with intravenous injection and 16.0% with intra-arterial injection. These data suggest that intra-arterial injection with lipiodol is a more useful method to increase CDDP uptake by tumors than intra-venous injection.