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In the studies reported here, the micronucleus assay, a common cytogenetic technique, was used to examine the dose-responses in fibroblasts from three ungulate species (white-tailed deer, woodland caribou, and Indian muntjac) exposed to high doses of ionizing radiation (1-4 Gy of (60)Co gamma radiation). This assay was also used to examine the effects of exposure to low doses (1-100 mGy) typical of what these species experience in a year from natural and anthropogenic environmental sources. An adaptive response, defined as the induction of resistance to a stressor by a prior exposure to a small "adapting" stress, was observed after exposure to low doses. This work indicates that very small doses are protective for the endpoint examined. The same level of protection was seen at all adapting doses, including 1 radiation track per cell, the lowest possible cellular dose. These results are consistent with other studies in a wide variety of organisms that demonstrate a protective effect of low doses at both cellular and whole-organism levels. This implies that environmental regulations predicated on the idea that even the smallest dose of radiation carries a quantifiable risk of direct adverse consequences to the exposed organism require further examination. Cytogenetic assays provide affordable and feasible biological effects-based alternatives that are more biologically relevant than traditional contaminant concentration-based radioecological risk assessment.

A tridentate single amino acid chelate (SAAC) derived from N-alpha-Fmoc-l-lysine was incorporated within a short peptide sequence using an automated peptide synthesizer. Novel derivatives of the chemotactic peptide fMLF were prepared such that the SAAC and its Re complex were selectively placed between a terminal glycine amino acid and the targeting fMLF sequence. The products, which were synthesized in parallel, were characterized by mass spectrometry and multi-NMR spectroscopy. The latter technique demonstrated that the structures of the targeting portions of the peptides are the same in the SAAC and Re-SAAC derivatives. The affinities of the reported compounds for the formyl peptide receptor were subsequently determined using flow cytometry and were found to be comparable to that of the parent peptide. The results of this work demonstrate the feasibility and numerous benefits of using the SAAC system to prepare peptide-targeted Tc(I) and Re(I) radiopharmaceuticals.

Electron paramagnetic resonance (EPR) dosimetry of human tooth enamel has been widely used in measuring radiation doses in various scenarios. However, there are situations that do not involve a human victim (e.g. tests for suspected environmental overexposures, measurements of doses to experimental animals in radiation biology research, or chronology of archaeological deposits). For such cases we have developed an EPR dosimetry technique making use of enamel of teeth extracted from mice. Tooth enamel from both previously irradiated and unirradiated mice was extracted and cleaned by processing in supersaturated KOH aqueous solution. Teeth from mice with no previous irradiation history exhibited a linear EPR response to the dose in the range from 0.8 to 5.5 Gy. The EPR dose reconstruction for a preliminarily irradiated batch resulted in the radiation dose of (1.4+/-0.2) Gy, which was in a good agreement with the estimated exposure of the teeth. The sensitivity of the EPR response of mouse enamel to gamma radiation was found to be half of that of human tooth enamel. The dosimetric EPR signal of mouse enamel is stable up at least to 42 days after exposure to radiation. Dose reconstruction was only possible with the enamel extracted from molars and premolars and could not be performed with incisors. Electron micrographs showed structural variations in the incisor enamel, possibly explaining the large interfering signal in the non-molar teeth.

We previously found that transgenic mice overexpressing growth hormone (TGM) have elevated and progressively increasing free radical processes in brain that strongly correlates with reduced survivorship. Young mature TGM, however, displayed vastly enhanced learning of an eight-choice cued maze and qualitatively different learning curves than normal controls. Here we document the age-related patterns in learning ability of TGM and normal mice. Learning appeared inferior in both genotypes of very young mice but TGM were confirmed to be superior to normal mice upon maturity. Older TGM, however, showed rapid age-related loss of their exceptional learning, whereas normal mice at 1 year of age showed little change. The cognitive decline of TGM was abolished by a complex "anti-aging" dietary supplement formulated to promote membrane and mitochondrial integrity, increase insulin sensitivity, reduce reactive oxygen and nitrogen species, and ameliorate inflammation. Results are discussed in the context of reactive oxygen and nitrogen species, long-term potentiation, learning, aging and neuropathology, based on known impacts of the growth hormone axis on the brain, and characteristics of TGM.

For radiation exposures below 100 mGy, the dosimetric signal in tooth enamel is too small to be measured by using the traditional dose reconstruction procedure. This is because low amplitude zero-added-dose signal can not be identified in an EPR spectrometer. A technique is presented wherein, zero-added-dose signal. when amplified by a proper known dose, can be measured in the EPR spectrometer. Mathematically, the accidental dose x is modified by a known amount of exposure, y (large enough so that the signal is now visible), and total exposure becomes x' = x + y, which is the modified-zero-added dose. The exposure x' is then quantified using the conventional backward extrapolation method and the accidental dose can be measured. In a laboratory controlled experiment, the feasibility of dose reconstruction in the 100 mGy range has been demonstrated. This may enable measurements of dose even due to suspected low exposure in tooth enamel.

Teratogenesis induced by radiation in fetal mice has been closely linked to Trp53-dependent apoptosis. This study examined teratogenesis in tails and limb digits of fetal mice with varying Trp53 status after a 4-Gy radiation exposure, with and without a prior 40.5 degrees C, 60-min heat stress. Irradiation earlier in gestation (day 11) produced greater effects than later (day 12) exposure, but in both cases the maximum teratogenic effect of radiation occurred in Trp53 normal fetuses, the minimum in Trp53 null fetuses, and intermediate effects in Trp53 heterozygotes, indicating dominance of Trp53-dependent apoptosis. Heat stress 24 h prior to irradiation on day 11 did not alter the teratogenic effects in Trp53 normal or heterozygous fetuses, but it reduced effects in the Trp53 null fetuses. Conversely, heat stress immediately before irradiation on day 11 amplified teratogenesis in Trp53 null fetuses, still with only a small or no effect on fetuses with full or partial Trp53 function, respectively. These results indicate little effect of mild heat on Trp53-dependent apoptosis after irradiation, but they also suggest heat-induced amplification of Trp53-independent processes that led to apoptosis when heat was delivered near the time of radiation exposure, and heat-induced protection of that process when sufficient expression time was allowed. However, Trp53-dependent apoptosis, when functional, acted as the ultimate determinant of radiation-induced teratogenic effects during early organogenesis. On gestation day 12, radiation effects were diminished, but heat stress 24 h prior to radiation exposure had a large amplifying effect in Trp53 normal or heterozygous fetuses. In the absence of functional Trp53, the sensitizing effect of the heat was diminished. The results may suggest that at later times in organ development, DNA repair is more active, allowing some cells to escape radiation-induced Trp53-dependent apoptosis. However, heat may be able to significantly inhibit this active repair and increase the teratogenic effect of radiation. A diminished effect in the absence of functional Trp53 is consistent with an influence of heat on inhibiting DNA repair, but with a diminished probability of apoptosis.

Teratogenesis in tails and limb digits of fetal mice with varying Trp53 status was examined after exposure of pregnant females to 4 Gy gamma radiation with and without a prior 30-cGy exposure. Prior low-dose exposure modified the teratogenic effects of radiation in a manner dependent upon Trp53 status and gestation time. A 4-Gy exposure on gestation day 11 resulted in tail shortening and digit abnormalities. A 30-cGy exposure 24 h prior to a 4-Gy radiation exposure on day 11 reduced the extent of both digit abnormalities and the tail-shortening effects in Trp53(+/+) fetuses and also reduced tail shortening in Trp53(+/-) fetuses, but to a lesser extent. However, the pre-exposure enhanced the tail-shortening effects of 4 Gy in Trp53(-/-) fetuses. In contrast, a 30-cGy exposure given 24 h prior to a 4-Gy exposure on gestation day 12 had no effect on the reduced tail length resulting from the 4-Gy exposure of Trp53(+/+) or Trp53(+/-) fetuses, but it partly protected Trp53(-/-) fetuses against reduced tail length. A 4-Gy exposure alone on day 12 did not result in any increase in the frequency of digit abnormalities in Trp53(-/-) fetuses so any protective effect of the preirradiation could not be detected. However, the preirradiation did result in protection against in digit abnormalities in Trp53(+/-) fetuses. We conclude that radiation-induced teratogenesis reflects both Trp53-dependent and independent processes that lead to apoptosis, and these respond differently to prior adapting doses.

Hyperthermia can be teratogenic in fetal mice exposed during organogenesis, an effect considered to be due to heat-induced apoptosis of cells in the developing organs. We exposed pregnant mice carrying Trp53(+/+), Trp53(+/-) and Trp53(-/-) fetuses to mild whole-body hyperthermia that raised their core temperature to 40.5 degrees C for 60 min on either day 10 or 11 of gestation. On day 18 of gestation, the fetuses were removed from control and hyperthermia-treated mice and genotyped, and tail length was measured. Limb digits were examined for abnormalities. Tail length in unheated control fetuses was influenced by Trp53 status. A complete lack of functional Trp53 (Trp53(-/-)) but not partial lack of function (Trp53(+/-)) resulted in shorter tails compared to Trp53(+/+) fetuses, indicating a role for Trp53 in the regulation of tail lengthening in mouse fetuses. In all three genotypes, hyperthermia on gestation day 10 resulted in tails shorter than unheated controls, and hyperthermia on day 11 resulted in tails longer than controls. There was no effect on limb digit abnormalities. The data suggest that Trp53-dependent or independent apoptosis may not be directly involved in heat-induced teratogenesis, but that the primary teratogenic effect of heat results from the disruption of another tail length-regulating process that is independent of Trp53. However, the nature of the teratogenic outcome of that disruption depends on the gestation time. The ability of Trp53 to additionally regulate the tail lengthening process was also sensitive to the effects of heat, but that sensitivity again depended on the time of the heat stress during gestation.

Purpose: To determine whether yeast cells (Saccharomyces cerevisiae) defective in damage-inducible cell-cycle arrest can invoke an adaptive response and become resistant to normally lethal doses of ionizing radiation., Materials and Methods: Wild-type yeast cells, cells defective for DNA-damage-responsive G1 and G2 cell-cycle arrest (rad9delta), and cells defective for recombinational repair of DNA damage (rad50, 51, 52) were subjected to adapting treatments of heat or radiation and subsequently exposed to normally lethal doses of radiation. Survival, as measured by colony-forming ability, was compared with non-adapted, control cells., Results: Wild-type and rad9delta cells became more resistant to potentially lethal doses of radiation after exposure to conditions that are known to elicit the adaptive response. Further, the relative magnitude of resistance developed by the normal, wild-type and rad9delta yeast cells was similar, with a dose modifying factor (at D1) for radiation-induced radiation resistance of 1.3 for both strains. Dose modifying factors (at D1) for heat-induced radiation resistance were 1.7 and 1.6 for wild-type and rad9delta cells, respectively. In contrast, none of the recombinational repair-defective cells exhibited radiation resistance after an adapting treatment., Conclusions: The ability of yeast cells to arrest in cell-cycle gap phases did not appear to contribute significantly to radiation resistance induced by radiation or heat. Instead, it is suggested that the adaptive response was due mainly to the existence and enhancement of cellular recombinational repair capacity, which was sufficient to repair any DNA damage without the requirement of a detectable cell-cycle delay.

We have compared dose-rate effects for gamma-radiation-induced apoptosis and micronucleus formation in human lymphocytes. Long-term assessment of individual radiation-induced apoptosis showed little intraindividual variation but significant interindividual variation. The effectiveness of radiation exposure to cause apoptosis or micronucleus formation was reduced by low-dose-rate exposures, but the reduction was apparent at different dose rates for these two end points. Micronucleus formation showed a dose-rate effect when the dose rate was lowered to 0.29 cGy/min, but there was no accompanying cell cycle delay. A further increase in the dose-rate effect was seen at 0.15 cGy/min, but was now accompanied by cell cycle delay. There was no dose-rate effect for the induction of apoptosis until the dose rate was reduced to 0.15 cGy/min, indicating that the mechanisms or signals for processing radiation-induced lesions for these two end points must be different at least in part. There appear to be two mechanisms that contribute to the dose-rate effect for micronucleus formation. One of these does not affect binucleate cell frequency and occurs at dose rates higher than that required to produce a dose-rate effect for apoptosis, and one affects binucleate cell frequency, induced only at the very low dose rate which coincidentally produces a dose-rate effect for apoptosis. Since the dose rate at which cells showed reduced apoptosis as well as a further reduction in micronucleus formation was very low, we conclude that the processing of the radiation-induced lesions that induce apoptosis, and some micronuclei, is very slow in quiescent and PHA-stimulated lymphocytes, respectively.

We have investigated the effect of the adaptive response on acute myeloid leukemia (AML) induced in CBA/Harwell mice by a chronic radiation exposure. Groups of mice irradiated with a total dose of 1. 0 Gy at two different chronic dose rates (0.5, 0.004 Gy/h) had similar frequencies of AML. Compared to control animals that did not develop AML, irradiation at either of these dose rates did not change the longevity of the mice that did not die of leukemia. The survival rates of irradiated mice that did develop leukemia in the two groups were not different from each other, indicating that the dose rates produced similar responses and therefore were both chronic exposures. We then tested the ability of a chronic 10-cGy (0. 5 Gy/h) exposure to ionizing radiation, mild hyperthermia (40.5 degrees C whole-body, 60 min) or treatment with interleukin-1 (1500 U i.p.) to induce an adaptive response and modify the frequency or latency of AML which resulted from a subsequent (24 h later) 1.0-Gy (0.5 Gy/h) chronic radiation exposure. The frequency of radiation-induced leukemia was not changed in mice given any of the three adapting treatments 24 h prior to the chronic 1.0-Gy dose that induced leukemia. However, the latent period for development of AML was significantly increased by both the prior low radiation dose and mild hyperthermia treatment. Injection of interleukin-1, in contrast, may have reduced the latent period. Similar to the single 1.0-Gy chronic exposure alone, none of the adapting treatments prior to that exposure influenced the survival of animals that did not develop AML. These results indicate that an earlier exposure to a small adapting dose of radiation or to a mild heat stress can influence secondary steps in radiation-induced carcinogenesis.

Cis-diamminedichloroplatinum II (cisplatin) is a DNA inter- and intrastrand crosslinking agent which can sensitize prokaryotic and eukaryotic cells to killing by ionizing radiation. The mechanism of radiosensitization is unknown but may involve cisplatin inhibition of repair of DNA damage caused by radiation. Repair proficient wild type and repair deficient (rad52, recombinational repair or rad3, excision repair) strains of the yeast Saccharomyces cerevisiae were used to determine whether defects in DNA repair mechanisms would modify the radiosensitizing effect of cisplatin. We report that cisplatin exposure could sensitize yeast cells with a competent recombinational repair mechanism (wild type or rad3), but could not sensitize cells defective in recombinational repair (rad52), indicating that the radiosensitizing effect of cisplatin was due to inhibition of DNA repair processes involving error free RAD52-dependent recombinational repair. The presence or absence of oxygen during irradiation did not alter this radiosensitization. Consistent with this result, cisplatin did not sensitize cells to mutation that results from lesion processing by an error prone DNA repair system. However, under certain circumstances, cisplatin exposure did not cause radiosensitization to killing by radiation in repair competent wild type cells. Within 2 h after a sublethal cisplatin treatment, wild type yeast cells became both thermally tolerant and radiation resistant. Cisplatin pretreatment also suppressed mutations caused by exposure to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a response previously shown in wild type yeast cells following radiation pretreatment. Like radiation, the cisplatin-induced stress response did not confer radiation resistance or suppress MNNG mutations in a recombinational repair deficient mutant (rad52), although thermal tolerance was still induced. These results support the idea that cisplatin adducts in DNA interfere with RAD52-dependent recombinational repair and thereby sensitize cells to killing by radiation. However, the lesions can subsequently induce a general stress response, part of which is induction of RAD52-dependent error free recombinational repair. This stress response confers radiation resistance, thermal tolerance, and mutation resistance in yeast.

Purpose: To investigate the repair of ionizing radiation-induced DNA lesions in human skin fibroblasts in the presence of cisplatin-DNA adducts and to determine the persistence of DNA repair inhibition by cisplatin., Materials and Methods: Normal human fibroblasts (AG 1522) treated with cisplatin were exposed to 4 Gy 60Co gamma-radiation and assayed for repair of radiation-induced damage under growth-permissive conditions. DNA damage was measured by the fluorescence analysis of DNA unwinding (FADU) and cytokinesis-blocked micronucleus assays., Results: Rejoining of strand breaks caused by 4 Gy radiation in cells without cisplatin pre-treatment appeared to be biphasic with an initial fast component (up to 15 min of repair time) followed by a slower component, and was completed by 90 min. Cisplatin treatment (10 microg/ml, 30 min) immediately before irradiation had no effect on the fast rejoining component, but inhibited the slow component (p<0.01). The same cisplatin treatment 24 h prior to irradiation inhibited both slow and fast components (p<0.01). In contrast, decreasing the cisplatin exposure to 1.0 microg/ml for 30 min, 24h prior to irradiation, resulted in an increased amount of strand break repair at each time point measured compared with irradiated control cells. This mild cisplatin treatment (95% survival) also resulted in a reduction of radiation-generated micronuclei indicating an adaptive response., Conclusions: Cisplatin used in combination with ionizing radiation can produce differential cellular responses depending upon the severity of the cisplatin treatment and the time interval between cisplatin and radiation exposures.

Chromosomes are located within the interphase nucleus in regions called domains. Using fluorescence in situ hybridization with whole chromosome paints, a pain of homologous chromosomes can be visualized as two discrete domains and their relative spatial location determined. This study examines the effects of an ionizing radiation exposure on the relative spatial location of chromosome 7 and 21 domains in human skin fibroblasts and lung endothelial cells. The distance between homologous chromosome domains was assessed for each nucleus, before and after exposure to ionizing radiation, using conventional epifluorescence and confocal laser scanning microscopy. Results from conventional microscopy indicated that homologous chromosome domains were re-positioned closer to each other within interphase nuclei after exposure to radiation. Analysis of three-dimensional data obtained from confocal microscopy confirmed these results. In control cells, and in cells examined immediately after irradiation, 66.2% +/- 2.1% of the homologous chromosome 21 domains within endothelial cell nuclei were located greater than 4.0 microns apart (33.8% +/- 1.9% were less than 4.0 microns apart). However, when cells were examined 2 h after a 4.0 Gy gamma-ray exposure, only 30.5% +/- 2.1% of the homologous chromosome domains were greater than 4.0 microns apart (69.5% +/- 2.1% were less than 4.0 microns apart). Similar results were obtained for chromosomes 7 and 21 in skin fibroblast nuclei. The results indicate that homologous chromosome domains rearranged and became closer together within the interphase nuclei in response to ionizing radiation. The exact mechanism of this response is unknown, but it may be related to DNA repair processes. It is speculated that chromosome domains are re-positioned to permit repair of radiation-induced DNA damage.

We have investigated heat- and radiation-induced apoptosis in human lymphocytes in vitro. We have previously shown that apoptosis was induced by radiation at doses as low as 0.05 Gy. Here we report that heat induced apoptosis in human lymphocytes in a temperature- and time-dependent manner. Temperatures at or below 42 degrees C, for up to 90 min, did not cause lymphocytes to undergo apoptosis, whereas temperatures at or above 43 degrees C, for 30 min and longer, did induce apoptosis. Lymphocytes were protected against apoptosis induced by 44 degrees C heat by a prior heat shock of 42 degrees C for 30 min. Heat-induced thermal tolerance developed immediately following the inducing heat shock, was greater after 4 h, and persisted for at least 24 h. While heat also induced radiation resistance, this change was minor and not apparent until about 24 h after the heat shock. Prior to the development of radiation resistance, heat shock sensitized lymphocytes to radiation-induced apoptosis. We have previously shown that radiation-induced apoptosis in lymphocytes varies between donors and therefore may be useful in assessing individual radiosensitivity. We report here that heat also induced variable levels of apoptosis in lymphocytes from different donors, although the range of responses was not as large as those observed with radiation-induced apoptosis. In summary, heat shock induces tolerance to heat-induced apoptosis and results first in sensitization and then protection of lymphocytes against radiation-induced apoptosis.

We have tested the possibility of using apoptosis (programmed cell death) in human peripheral blood lymphocytes as a short-term biological dosimeter. Lymphocytes isolated from whole blood were irradiated in culture with 250 kVp x-rays or 60Co gamma rays. Two assays were used to measure apoptosis in lymphocytes after irradiation: in situ terminal deoxynucleotidyl transferase assay and fluorescence analysis of DNA unwinding assay. Similar qualitative and quantitative results were produced by the assays, supporting the notion that the fluorescence analysis of DNA unwinding assay measured DNA fragmentation associated with apoptosis. Induction of apoptosis in lymphocytes irradiated in vitro was proportional to dose and could be detected following exposures as low as 0.05 Gy. Lymphocytes from individual donors had reproducible dose responses. There was, however, variation between donors. X-ray and gamma-ray exposures induced similar levels of apoptosis at similar doses. The induction kinetics of apoptosis in vitro indicate a maximum is reached about 72 h after irradiation. In conclusion, the in vitro experimental evidence indicates that radiation-induced apoptosis in human lymphocytes has the kinetics, sensitivity, and reproducibility to be a potential biological dosimeter.

Unrepaired or misrepaired radiation damage in mammalian chromosomes can result in micronucleus formation at the first cell division. This represents loss of genomic information which may cause cell death. To improve our understanding of the mechanism of radiation-induced micronucleus formation, we characterized micronucleus ultrastructure and identified the origin of micronucleus DNA. Immunofluorescence microscopy showed that micronuclei were structurally similar to main nuclei since they contained nuclear lamins A and C and were encapsulated by a network of vimentin intermediate filaments. The contents of radiation-induced micronuclei were characterized using fluorescence in situ hybridization to probe for DNA originating from chromosomes 2, 7, 11 and 16. We postulated that if incorporation of DNA into micronuclei were random, then the probability of chromosomal DNA in micronuclei would be related to the target, i.e. chromosome size. Our results demonstrated that incorporation of DNA from smaller chromosomes (11 and 16) was not different from expected values but incorporation of DNA from the larger chromosomes (2 and 7) was significantly greater than expected. Not all chromosomes in the human genome, therefore, were equally susceptible to genomic loss by micronucleus encapsulation. In conclusion, radiation-induced micronuclei have similar structural characteristics to main nuclei, chromosome damage and/or repair after ionizing radiation may be non-random, and micronucleus formation may reflect this variability.

The matrix metalloproteinase, matrilysin, is thought to play an important role in the early steps of tumor progression. We determined the chromosome location of the matrilysin gene (MMP7) by Southern and PCR analysis of two different panels of somatic cell hybrids and in situ hybridization of metaphase chromosomes. Matrilysin maps to the region, 11q21-->q22, adding MMP7 to the cluster of matrix metalloproteinase genes that have already mapped to this region.

An eight week trial, involving superficial hyperthermia delivered biweekly via simple water bath immersion, was tested for its ability to clear mild to moderate psoriatic lesions. Seven patients were treated and three cases rapidly improved. In the remaining patients, the treatment frequency was increased to alternate days; two cases improved significantly, one patient showed a partial response, and the fourth had no visible change (this was the only patient taking concurrent drug therapy--etretinate). In addition to resolving psoriatic lesions, water bath hyperthermia also reduced edema (swelling) and relieved pruritus (itching) in all patients, both during the treatment period and for up to several months after lesions had returned. Lesion reappearance occurred within one to three months after the last heat treatment. We retreated one patient and produced a second complete remission. These results indicate that simple repetitive water bath hyperthermia alone is effective in the treatment of psoriatic lesions in heatable locations. An unexpected side effect was enhanced melanin content (tanning) in all areas where hyperthermia treated skin was exposed to sunlight.

We have investigated the influence of the cellular adaptive response to ionizing radiation on radiation-induced apoptosis in human cells. The adaptive response is believed to be a protective mechanism that confers resistance to the detrimental effects of ionizing radiation and that can be induced by different agents, including hyperthermia and radiation. We have used fluorescence analysis of DNA unwinding (FADU) to assay the induction of apoptosis in human peripheral blood lymphocytes by ionizing radiation. Using the FADU assay, we have observed the initial radiation-induced DNA damage, its subsequent disappearance due to enzymatic repair, and its time- and dose-dependent reappearance. We believe this reappearance of DNA damage to be indicative of the DNA fragmentation event associated with apoptosis. This interpretation has been supported at the individual cell level using an in situ terminal deoxynucleotidyl transferase (TDT) assay (Apoptag, Oncor Inc.), which detects the 3'-hydroxyl ends of fragmented DNA, and by fluorescence analysis of nuclear morphology in Hoechst 33258 stained cells. Pretreatment of cells with low-dose gamma-radiation (0.1 Gy) or mild hyperthermia (40 degrees C for 30 min) altered the extent of radiation-induced (3 Gy) apoptosis. Both pretreatments sensitized lymphocytes to become apoptotic after the 3-Gy radiation exposure. This sensitization may represent an adaptive response mechanism that reduces the risk that genetically damaged cells will proliferate. The ability to modify the probability of radiation-induced apoptosis may lower the cancer risk from a radiation exposure.

We have investigated the role of heat-shock protein 104 (hsp-104) in the induction of ionizing radiation resistance in yeast. Yeast defective in the production of hsp-104 are constitutively sensitive to killing by heat and are known to be compromised for the induction of thermotolerance. We confirmed that hsp104 yeast mutants are altered in their ability to become thermotolerant by a heat stress. However, we found that the mutant strain did respond to heat shock and increased its resistance to a second lethal heat stress, although the magnitude of the increase was much lower than for the parental wild-type strain. Here we report that the constitutive level of ionizing radiation resistance was higher in the hsp104 mutant compared to wild-type cells and that heat shock induced increased radioresistance in both strains. When radiation was used as the stressing agent, thermal resistance was fully induced in wild-type cells and only slightly induced in the hsp104 mutant. In comparison, radioresistance was induced in both cell strains by a radiation stress. The increased radiation resistance was always slightly higher in the hsp104 mutant but developed with similar kinetics in both cell strains. Entry into stationary growth phase is another event known to induce both thermal tolerance and radiation resistance in yeast. In these experiments, entry into stationary phase induced similar levels of thermal tolerance in the two strains but higher levels of radioresistance in hsp104 mutants. The activation of both resistance mechanisms occurred earlier in the hsp104 mutants compared to wild-type cells. These results show that hsp-104 is not essential for either the process by which the cell recognizes and responds to any of these stresses, or the mechanisms by which the cell actually elevates its resistance to heat and radiation. We conclude, however, that hsp-104 is important in regulating the development and magnitude of induced radiation resistance as well as induced thermal tolerance in yeast.

Saccharomyces cerevisiae and Chlamydomonas reinhardtii respond to a sublethal exposure of ionizing radiation by increasing their resistance to killing by a second exposure. We demonstrate here that the two lower eukaryotes apparently achieve this by different mechanisms. We have shown that induced radioresistance in yeast results from increased capacity for recombinational repair, which we believe to occur in G2-phase haploid cells by recombination between homologous chromosomes. This is not possible in G1-phase haploid cells, which lack a second copy of DNA. Haploid C. reinhardtii cells, however, show induced resistance when irradiated asynchronously or in the G1 phase of the cell cycle. We have shown previously that the development of radiation resistance in yeast is proportional to the magnitude of the inducing dose and clearly demonstrates an oxygen effect. There was no oxygen effect for induced radiation resistance in C. reinhardtii cells, but induction remained proportional to dose. In yeast we have reported that both increased radioresistance and thermotolerance are inducible by a heat shock. Here, C. reinhardtii showed induced thermotolerance but no induced radioresistance in response to a heat stress. We have also determined previously that the induced recombinational DNA repair system in yeast recognizes alkylation lesions and therefore confers increased resistance to mutation by MNNG. In these experiments, C. reinhardtii induced for radioresistance were not more resistant to MNNG mutagenesis. These data indicate that haploid C. reinhardtii has a unique DSB repair mechanism. We propose that one possible mechanism may involve chloroplast DNA in a cooperative chloroplast/nuclear recombinational repair process.

DNA recombinational repair, and an increase in its capacity induced by DNA damage, is believed to be the major mechanism that confers resistance to killing by ionizing radiation in yeast. We have examined the nature of the DNA lesions generated by ionizing radiation that induce this mechanism, using two different end points: resistance to cell killing and ability of the error-free recombinational repair system to compete for other DNA lesions and thereby suppress chemical mutation. Under the various conditions examined in this study, the "maximum" inducible radiation resistance was increased approximately 1.5- to 3-fold and suppression of mutation about 10-fold. DNA lesions produced by low-LET gamma rays at doses greater than about 20 Gy given in oxygen were shown to be more efficient, per unit dose, at inducing radioresistance to killing than were lesions produced by neutrons (high-LET radiation). This suggests that DNA single-strand breaks are more important lesions in the induction of radioresistance than DNA double-strand breaks. Oxygen-modified lesions produced by gamma rays (low-LET radiation) were particularly efficient as induction signals. DNA damage due to hydroxyl radicals (OH.) derived from the radiolytic decomposition of H2O produced lesions that strongly induced this DNA repair mechanism. Similarly, OH. derived from aqueous electrons (e-aq) in the presence of N2O also efficiently induced the response. Cells induced to radioresistance to killing with high-LET radiation did not suppress N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-generated mutations as well as cells induced with low-LET radiation, supporting the conclusion that the type of DNA damage produced by low-LET radiation is a better inducer of recombinational repair. Surprisingly, however, cells induced with gamma radiation in the presence of N2O that became radioresistant to killing were unable to suppress MNNG mutations. This result indicates that OH. generated via e-aq (in N2O) may produce unusual DNA lesions which retard normal repair and render the system unavailable to compete for MNNG-generated lesions. We suggest that the repairability of these unique lesions is restricted by either their chemical nature or topological accessibility. Attempted repair of these lesions has lethal consequences and accounts for N2O radiosensitization of repair-competent but not incompetent cells. We conclude that induction of radioresistance in yeast by ionizing radiation responds variably to different DNA lesions, and these affect the availability of the induced recombinational repair system to deal with subsequent damage.

Either an ionizing radiation exposure or a heat shock is capable of inducing both thermal tolerance and radiation resistance in yeast. Yeast mutants, deficient in topoisomerase I, in topoisomerase II, or in DNA polymerase I, were used to investigate the mechanism of these inducible resistances. The absence of either or both topoisomerase activities did not prevent induction of either heat or radiation resistance. However, if both topoisomerase I and II activities were absent, the sensitivity of yeast to become thermally tolerant (in response to a heat stress) was markedly increased. The absence of only topoisomerase I activity (top1) resulted in the constitutive expression of increased radiation resistance equivalent to that induced by a heat shock in wild-type cells, and the topoisomerase I-deficient cells were not further inducible by heat. This heat-inducible component of radiation resistance (or its equivalent constitutive expression in top1 cells) was, in turn, only a portion of the full response inducible by radiation. The absence of polymerase I activity had no detectable effect on either response. Our results indicate that the actual systems that confer resistance to heat or radiation are independent of either topoisomerase activity or DNA polymerase function, but suggest that topoisomerases may have a regulatory role during the signaling of these mechanisms. The results of our experiments imply that maintenance of correct DNA topology prevents induction of the heat-shock response, and that heat-shock induction of a component of the full radiation resistance in yeast may be the consequence of topoisomerase I inactivation.

A single oral dose (200 mg) of an equimolar mixture of potassium canrenoate and its 20,20,21,21-tetradeutero analogue was administered to three healthy men. The steroids in urine collected for 24 hr after dosage were isolated on XAD-2 resin, and purified and fractionated into groups by lipophilic gel chromatography before and after hydrolysis of conjugates. GC/MS analysis of these fractions allowed the detection and identification of canrenone, canrenoic acid and its ester glucuronide, 3 beta-hydroxy-3-deoxocanrenone, 3beta-hydroxy-4,5alpha-dihydro-3-deoxocanrenone and a 3epsilon-hydroxy-4,5,6,7-tetrahydro-3-deoxocanrenone. In addition a number of di- and trihydroxy compounds formed by reduction and hydroxylation were partially identified from their E1 and C1 mass spectra. The results provide information on the metabolism of oral potassium canrenoate in man, and demonstrate the utility of combining stable isotope labeling, lipophilic gel chromatography, and GC/MS in studies of steroidal spirolactones.

Electron impact and chemical ionization mass spectra are reported for several steroidal spirolactones and their TMS ethers. The electron impact spectra were characterized generally by low abundance molecular ions and large numbers of fragment ions. Methane chemical ionization spectra exhibited high intensity [M+H]+ and/or [M+H-H2O]+ or [M+H-TMSOH]+ ions with few other fragment ions. Ammonia chemical ionization spectra had intense [M+H]+ and/or [M+NH4]+ ions with a few fragment ions generally formed by loss of H2O or TMSOH from these parent ions. Ammonia chemical ionization gave intense parent ions even for polyhydroxy compounds and their TMS ethers in contrast to methane chemical ionization. The results of this study suggest that a combination of electron impact with ammonia chemical ionization mass spectrometry would offer the best techniques for detection and identification of these compounds in biological fluids.

Chromatography of steroidal spirolactones on DEAE-Sephadex A-25 under selected pH conditions allowed efficient separation of these compounds from other steroids and many of the endogenous components of human urine. The spirolactones were recovered in high yield, mostly over 90%. Lipophilic-gel chromatography provided a useful method for group fractionation of mixtures of these spirolactones with high recoveries (generally over 90%), unaffected by the presence of endogenous material from normal human urine.

1. The metabolism of [1-3H]canrenone, a primary metabolite of spironolactone and potassium canrenoate, by rat liver preparations in vitro has been investigated. 2. Canrenone was metabolized by 3-oxo-delta 4-reduction to give 3 alpha-hydroxy-5 beta-spirolactones, and also by a number of O2 and NADPH-dependent microsomal hydroxylation reactions. 3. A major metabolic route requiring the presence of a microsomal fraction, but apparently independent of oxygen and NADPH, led to the formation of a number of compounds tentatively identified as trihydroxy-spirolactones.

Metabolism of spironolactone in man is extensive and complex. For many years the dethioacetylated metabolite, canrenone, was assumed to be the major metabolite. However, recent studies using specific high performance liquid chromatography (HPLC) have demonstrated the presence of spironolactone and the sulfur-containing metabolites 7 alpha-thiomethylspirolactone (IV) and 6 beta-hydroxy-7 alpha-thiomethylspirolactone (V), in addition to canrenone, in the serum after a single oral dose of spironolactone. The importance of spironolactone and metabolites IV and V relative to canrenone at steady state remains unknown and was the subject of the present investigation. Twelve healthy males received 100 mg spironolactone, once daily, for 15 days. Repeated blood samples were taken on days 1, 8 and 15 for estimation of spironolactone and its metabolites. Peak serum levels [mean (SD)] of spironolactone, canrenone, and sulfur-containing metabolites IV and V were 72 (45), 155 (43), 359 (106) and 101 (26) ng/ml, respectively on day 1 and 80 (20), 181 (39), 391 (118) and 125 (24) ng/ml, respectively on day 15. The AUC (0-24) values of these compounds on day 15 were 231 (50), 2173 (312), 2804 (777) and 1727 (367) ng.hr/ml, respectively and the post-steady state elimination half-life (t1/2) values were 1.4 (0.5), 16.5 (6.3), 13.8 (6.4), and 15.0 (4.0) hours, respectively. It was concluded that unmetabolized spironolactone is present in the serum and that the sulfur-containing metabolite IV rather than canrenone is the major metabolite in serum following single or repeated doses of spironolactone.

1. A method for the estimation of gentisic acid in urine has been devised which is based on thin-layer chromatography and fluorimetry.2. In man the urinary excretion of gentisic acid accounted for 0.6% of a 0.32 g dose of aspirin and 1.1% of a 1.28 g dose. The increase in the percentage of the dose excreted as gentisic acid provides further evidence that the elimination of salicylic acid cannot be entirely described by first order kinetics.3. Equations are presented which describe the amount of gentisic acid formed from various doses of salicylic acid in a model system, when elimination proceeds partly by simultaneous first order and zero order kinetics. The close agreement of the experimental and theoretical results indicates that the model provides an acceptable interpretation of salicylic acid elimination in man.