Classification of chronic radiation sickness cases using neural networks and classification trees.

Chronic radiation sickness is a deterministic radiation health effect observed among the Mayak Production Association workers in Russia. In this study, unsupervised neural networks were used to cluster hematological measurements in a subset (n = 88) of the Mayak Production Association population while excluding from the analysis the radiation dose and the historical clinical diagnosis. Clusters of observations that had lower average leukocyte and thrombocyte counts were labeled "affected" and those having higher average blood cell counts were labeled "unaffected." The class (cluster) membership for each individual was used subsequently as a dependent variable in a classification tree model in order to identify significant features of the underlying classification model. After re-classification of cases using this method, the results showed a better data separation between the blood cell counts for affected vs. unaffected groups compared to those based on historical classification, and a greater difference between group means for differential blood counts was observed than for the historical diagnosis. The reclassification of diagnostic groups changed the group mean radiation doses. The geometric means (and 95% CL) of cumulative radiation dose equivalent from external exposures, based on the historical diagnosis, are 0.31 (0.0035, 3.4) vs. 1.7 (0.0007, 18) Sv. After clustering and classification tree analyses, the group geometric means were 0.78 (0.0014, 8.6) vs. 1.5 (0.0007, 17) and 0.82 (0.0013, 9.0) vs. 1.4 (0.0008, 16) Sv, using (respectively) whole blood cell counts or differential counts as the independent variables. The approach presented here is useful as a diagnostic aid for both retrospective analyses and in the event of future radiation accidents.

Physicochemical and graph theoretical descriptors in developmental toxicity SAR: a comparative study.

Chemical insults to the developing fetus can lead to growth retardation, malformation, death, and functional deficits. The present study seeks to determine if physicochemical and/or graph theoretical parameters can be used to determine a structure-activity relationship (SAR) for developmental toxicity, and if consistency is observed among the selected features. The biological data utilized consists of a diverse series of compounds evaluated within the Chernoff-Kavlock in vivo mouse assay. Physicochemical parameters calculated correspond to electronic, steric, and transport properties. Graph theoretical parameters calculated include the simple, valence, and kappa indices. Both sets of parameters were independently applied to derive SARs in order to compare the quality of the respective models. Multiple random sampling, without replacement, was utilized to obtain ten training/test partitions. Models were built by linear discriminant analysis, decision trees, and neural networks respectively. Comparisons on identical sets of data were carried out to determine if any of the model building procedures had a significant advantage in terms of predictive performance. Furthermore, comparison of the features selected within and across the model building processes led to the determination of model consistency. Our results indicate that consistent features related to developmental toxicity are observed and that both physicochemical and graph theoretical parameters have equal utility.

Deterministic effects from occupational radiation exposures in a cohort of Mayak PA workers: data base description.

Project 2.3 of the Joint Coordinating Committee on Radiation Effects Research (JCCRER) is a study of deterministic health effects among a cohort of Russia nuclear workers. The preliminary study population includes a stratified random sample of 221 radiation workers who were employed in a cohort of 8,055 workers at the Mayak PA facilities for at least one year during the period from 1948 to 1958. High annual doses, approaching 1 Gy per year from external and internal radiation sources, were reported for a significant proportion of the workers in this cohort. The present data set includes 96 cases of chronic radiation sickness (CRS), 14 cases of acute radiation syndrome (ARS) and 13 cases of plutonium pneumosclerosis (PPn). The remainder of the sample consists of "uninjured workers" who had no known history of radiation illness or injury; however, the uninjured workers are not "controls" for radiation exposure. The data base is currently being expanded to 600 individuals sampled from the cohort of workers from 1948 to 1958 to allow a more complete analysis of the deterministic health effects and comparisons with existing health effect models. The final data base will be used with state-of-the-art modeling techniques to determine threshold doses and dose-response relationships for key clinical diagnostic variables.

Development, characterization and application of predictive-toxicology models.

The adoption of SAR techniques for risk assessment purposes requires that the predictive performance of models be characterized and optimized. The development of such methods with respect to CASE/MULTICASE are described. Moreover, the effects of size, informational content, ratio of actives/inactives in the model on predictivity must be determined. Characterized models can provide mechanistic insights: nature of toxicophore, reactivity, receptor binding. Comparison of toxicophores among SAR models allows a determination of mechanistic overlaps (e.g., mutagenicity, toxicity, inhibition of gap junctional intercellular communication vs. carcinogenicity). Methods have been developed to combine SAR submodels and thereby improve predictive performance. Now that predictive toxicology methods are gaining acceptance, the development of Good Laboratory Practices is a further priority, as is the development of graduate programs in Computational Toxicology to adequately train the needed professional.

Nitric oxide prevents myoglobin/tert-butyl hydroperoxide-induced inhibition of Ca2+ transport in skeletal and cardiac sarcoplasmic reticulum.

Interaction of hydrogen peroxide or organic hydroperoxides with hemoproteins is known to produce oxoferryl hemoprotein species that act as very potent oxidants. Since skeletal and cardiac muscle cells contain high concentrations of myoglobin this reaction may be an important mechanism of initiation or enhancement of oxidative stress, which may impair their Ca2+ transport systems. Using skeletal and cardiac sarcoplasmic reticulum (SR) vesicles, we demonstrated by EPR the formation of alkoxyl radicals and protein-centered peroxyl radicals in the presence of myoglobin (Mb) and tert-butyl hydroperoxide (t-BuOOH). The low temperature EPR signal of the radicals was characterized by major feature at g = 2.016 and a shoulder at g = 2.036. In the presence of SR vesicles, the magnitude of the protein-centered peroxyl radical signal decreased, suggesting that the radicals were involved in oxidative modification of SR membranes. This was accompanied by SR membrane oxidative damage, as evidenced by accumulation of 2-thiobarbituric acid-reactive substances (TBARS) and the inhibition of Ca2+ transport. We have shown that nitric oxide (NO), reacting with redox-active heme iron, can prevent peroxyl radical formation activated by Mb/t-BuOOH. Incubation of SR membranes with an NO donor, PAPA/NO (a non-thiol compound that releases NO) at 200-500 microM completely prevented the t-BuOOH-dependent production of peroxyl radicals and formation of TBARS, and thus protected against oxidative inhibition of Ca2+ transport.

Nitric oxide protects cardiomyocytes against tert-butyl hydroperoxide-induced formation of alkoxyl and peroxyl radicals and peroxidation of phosphatidylserine.

We studied protective effects of nitric oxide against tert-butyl hydroperoxide-induced oxidative damage to cardiac myocytes. Two distinct free radicals species--alkoxyl radicals associated with non-heme iron catalytic sites and myoglobin protein-centered peroxyl radicals--were found in low-temperature EPR spectra of cardiac myocytes exposed to t-BuOOH. The t-BuOOH-induced radical formation was accompanied by site-specific oxidative stress in membrane phospholipids (peroxidation of phosphatidylserine) assayed by fluorescence HPLC after metabolic labeling of cell phospholipids with oxidation-sensitive cis-parinaric acid. An NO-donor, (Z)-1-[N-(3-ammonio-propyl)-N-(n-propyl) amino]-diazen-1-ium-1,2-diolate], protected cardiac myocytes against tert-butyl hydroperoxide-induced: (i) formation of non-protein- and protein-centered free radical species and (ii) concomitant peroxidation of phosphatidylserine. Thus nitric oxide can act as an effective antioxidant in live cardiomyocytes.

Phantom-derived estimation of effective dose equivalent from X rays with and without a lead apron.

Organ dose equivalents were measured in a humanoid phantom in order to estimate effective dose equivalent (H(E)) and effective dose (E) from low-energy x rays and in the presence or absence of a protective lead apron. Plane-parallel irradiation conditions were approximated using direct x-ray beams of 76 and 104 kVp and resulting dosimetry data was adjusted to model exposures conditions in fluoroscopy settings. Values of H(E) and E estimated under-shielded conditions were compared to the results of several recent studies that used combinations of measured and calculated dosimetry to model exposures to radiologists. While the estimates of H(E) and E without the lead apron were within 0.2 to 20% of expected values, estimates based on personal monitors worn at the (phantom) waist (underneath the apron) underestimated either H(E) or E while monitors placed at the neck (above the apron) significantly overestimated both quantities. Also, the experimentally determined H(E) and E were 1.4 to 3.3 times greater than might be estimated using recently reported "two-monitor" algorithms for the estimation of effective dose quantities. The results suggest that accurate estimation of either H(E) or E from personal monitors under conditions of partial body exposures remains problematic and is likely to require the use of multiple monitors.

Non-random peroxidation of different classes of membrane phospholipids in live cells detected by metabolically integrated cis-parinaric acid.

Quantitative assays of lipid peroxidation in intact, living cells are essential for evaluating oxidative damage from various sources and for testing the efficacy of antioxidant interventions. We report a novel method based on the use of cis-parinaric acid (PnA) as a reporter molecule for membrane lipid peroxidation in intact mammalian cells. Using four different cell lines (human leukemia HL-60, K562 and K/VP.5 cells, and Chinese hamster ovary (CHO) fibroblasts), we developed a technique to metabolically integrate PnA into all major classes of membrane phospholipids, i.e., phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and cardiolipin, that can be quantified by HPLC with fluorescence detection. Integrated PnA constituted less than 1% of lipid fatty acid residues, suggesting that membrane structure and characteristics were not significantly altered. Low concentrations (20-40 microM) of tert-butyl hydroperoxide (t-BuOOH) caused selective oxidation of PnA residues in phosphatidylserine and phosphatidylethanolamine of K562 cells and K/VP.5 cells while cell viability was unaffected. At higher t-BuOOH concentrations (exceeding 100 microM), however, a progressive, random oxidation of all major phospholipid classes occurred and was accompanied by significant cell death. In HL-60 cells, phosphatidylethanolamine, phosphatidylserine and cardiolipin were sensitive to low concentrations of t-BuOOH, while phosphatidylcholine and phosphatidylinositol were not affected. Phosphatidylinositol was the only phospholipid that responded to the low concentrations of t-BuOOH in CHO cells. At high t-BuOOH concentrations, again, all phospholipid classes underwent extensive oxidation. All phospholipids were nearly equally affected by peroxidation induced by a initiator of peroxyl radicals, 2,2'-azobis-(2,4-dimethylvaleronitrile) AMVN), in K562 cells. In gamma-irradiated (4-128 Gy) CHO cells, phosphatidylserine was the most affected phospholipid class (34% peroxidation) followed by phosphatidylinositol (24% peroxidation) while the other three phospholipid classes were apparently unaffected. Since loss of PnA fluorescence is a direct result of irreparable oxidative loss of its conjugated double bond system, the method described allows for selective and sensitive monitoring of oxidative stress in live cells without interference from cell repair mechanisms.

Detection and characterization of the electron paramagnetic resonance-silent glutathionyl-5,5-dimethyl-1-pyrroline N-oxide adduct derived from redox cycling of phenoxyl radicals in model systems and HL-60 cells.

The antioxidant function of glutathione includes enzymatic reduction of hydrogen peroxide by glutathione peroxidase and nonenzymatic reduction of organic radicals and reactive oxygen species. The glutathionyl S-centered radical, formed by the nonenzymatic reduction process, is a marker of oxidative reactions proceeding by radical mechanisms. Spin-adducts of glutathionyl radicals with the spin trap DMPO, 5,5-dimethyl-1-pyrroline N-oxide, are not sufficiently stable and can be detected only under steady-state conditions. We developed a novel HPLC method for the detection of an EPR-silent DMPO adduct of glutathionyl radicals in model systems and in cells. We synthesized a sufficient quantity of the adduct for characterization by UV spectrophotometry, ionspray mass spectrometry, and 1H NMR spectroscopy. The UV absorption lambda max of the adduct, 258 nm, was indicative of a 2-(S-alkylthiyl)pyrroline N-oxide chromophore. The molecular mass of the adduct was 418 amu. No signal for the C2 proton of the DMPO-derived portion of the adduct was evident in its 1H NMR spectrum. The results were consistent with the structure 2-(S-glutathionyl)-5,5-dimethyl-1-pyrroline N-oxide (GS-DMPO nitrone). We showed that this adduct accumulated in the course of peroxidase-dependent redox cycling of phenol in the presence of glutathione and DMPO as well as in HL-60 cells exposed to a phenol/H2O2/DMPO reaction mixture. The EPR-silent GS-DMPO nitrone was readily assayed by HPLC under conditions incompatible with the detection of the GS-DMPO nitroxide by EPR. This is to our knowledge the first direct experimental evidence for the redox cycling of phenol in this bone marrow-derived cell line. The method may prove useful in the study of radical-driven oxidations of glutathione in various pathophysiological processes associated with radical mechanisms.

Phenoxyl radicals of etoposide (VP-16) can directly oxidize intracellular thiols: protective versus damaging effects of phenolic antioxidants.

Phenolic compounds can act as radical scavengers due to their ability to donate a mobile hydrogen to peroxyl radicals producing a phenoxyl radical if the phenoxyl radical formed in the radical scavenging reaction efficiently interacts with vitally important biomolecules, then this interaction may result in cytotoxic effects rather than in antioxidant protection. In the present work we have chosen two model compounds--a phenolic antitumor drug, VP-16, known to be highly cytotoxic, and a homolog of vitamin E, 2,2,5,7,8-pentamethyl-6-hydroxychromane (PMC)--as typical representatives of phenoxyl radicals to study interactions of their phenoxyl radicals with intracellular thiols. Using a water-soluble source of peroxyl radicals, the azo-initiator 2,2'-azobis(2-aminodinopropane) (AAPH), we found that both PMC and VP-16 are very efficient scavengers of peroxyl radicals as evidenced by their ability to inhibit AAPH-induced chemiluminescence of luminol and oxidation of PnA incorporated into DOPC liposomes. Both PMC and VP-16 were also able to protect against AAPH-induced oxidative degradation of DNA in nuclei from human leukemic K562 cells. In contrast, there was a dramatic difference in the ability of VP-16 and PMC to protect GSH against AAPH-induced oxidation: while PMC inhibited AAPH-induced oxidation of GSH in a concentration-dependent manner, VP-16 did not protect GSH against oxidation. We hypothesized that this was due to different reactivities of the phenoxyl radicals formed by AAPH-derived peroxyl radicals from VP-16 and PMC toward GSH. To substantiate this hypothesis, we compared interactions of the phenoxyl radicals generated from VP-16 and PMC with intracellular thiols in K562 cell homogenates. While the PMC phenoxyl radicals were only slightly affected by thiols, the VP-16 phenoxyl radicals were reduced by thiols. This is evidenced by (i) a significant inhibition of the tyrosinase-induced VP-16 consumption upon addition of K562 cell homogenates, (ii) a depletion of endogenous thiols in K562 cell homogenates induced by VP-16+tyrosinase, (iii) a transient disappearance of the VP-16 phenoxyl radical signal from the ESR spectra and its reappearance after depletion of endogenous thiols, and (iv) elimination of the lag period for the appearance of the VP-16 phenoxyl radical ESR signal subsequent to depletion of thiols by mersalyl acid. To evaluate the contribution of GSH and protein thiols to reduction of the VP-GSH-peroxidase + cumeme hydroperoxide to specifically deplete endogenous GSH.(ABSTRACT TRUNCATED AT 400 WORDS)

Phenoxyl radical-induced thiol-dependent generation of reactive oxygen species: implications for benzene toxicity.

Mechanisms of phenoxyl radical-induced generation of oxygen radicals potentially involved in toxicity of benzene were studied. We hypothesized that phenoxyl radical intermediates formed from phenolic metabolites of benzene by oxidative enzymes (e.g., peroxidases, tyrosinase) are able to damage biomolecules via (i) oxidation of low-molecular-weight thiols and protein thiols and (ii) thiol-dependent generation of oxygen radicals and subsequent oxidation of DNA. Phenoxyl radicals were generated by the oxidation of phenol by myeloperoxidase+H2O2, horseradish peroxidase+H2O2, or tyrosinase. The reaction of phenolphenoxyl radicals with GSH and dihydrolipoic acid was studied. Our HPLC measurements showed that both thiols reduced the phenoxyl radical back to phenol. This reaction was accompanied by the formation of thiyl radicals (detected by ESR as 5,5-dimethyl-1-pyrroline-N-oxide/glutathione thiyl radical spin adducts) and of superoxide radicals (measured by their chemiluminescence response in the presence of lucigenin). Hydroxylation of 2'-deoxyguanosine to 8-oxo-7,8-dihydro-2'-deoxyguanosine was demonstrated in the course of the tyrosinase-catalyzed oxidation of phenol in the presence of dihydrolipoic acid and Fe(III)-EDTA. Redox-cycling of phenoxyl radicals by thiols produces oxygen radicals which can be responsible for the oxidative damage of DNA by radical intermediates of benzene metabolism.

Plutonium-catalyzed oxidative DNA damage in the absence of significant alpha-particle decay.

Plutonium is considered to be a carcinogen because it emits alpha particles that may result in the irradiation of stem cell population. In the present study we show that plutonium can also catalyze reactions that induce hydroxyl radicals in the absence of significant alpha-particle irradiation. Using the low specific activity isotope, 242Pu, experiments were performed under conditions in which chemical generation of hydroxyl radicals was expected to exceed the radiolytic generation by one hundred thousand-fold. The results showed that markers of oxidative DNA base damage, thymine glycol and 8-oxoguanine could be induced from plutonium-catalyzed reactions of hydrogen peroxide and ascorbate similarly to those occurring in the presence of iron catalysts. Plutonium-242, as a neutralized nitrate in phosphate buffer, was 4.8-fold more efficient than iron at catalyzing the oxidation of ascorbate at pH 7. The results suggest that plutonium complexes could participate in reactions at pH 7 that induce oxidative stress--a significant tumor-promoting factor in generally accepted models of carcinogenesis.

DNA excision repair as a component of adaptation to low doses of ionizing radiation in Escherichia coli.

In this study we examined whether or not DNA excision repair is a component of adaptation induced by very low-dose ionizing radiation in Escherichia coli, a well-characterized prokaryote, and investigated the relationship between enhanced excision repair and the SOS response. Competent E. coli cells were irradiated using low doses (0.1-10 Gy) of 137Cs gamma-rays, allowed to recover for 2 h and were then transformed using pUC18 DNA containing approximately 22 oxidized thymine residues (thymine glycols) per molecule. Successful transformants were identified by recovery of plasmid-borne ampicillin resistance and the resulting data (colony counts) were used to calculate ratios of plasmid recovery in irradiated to control cells. Results showed that cells irradiated with very low doses (0.1-0.5 Gy) were up to 30-40% more efficient at utilizing thymine glycol-containing pUC18 DNA. The enhanced excision repair by very low doses (< 0.5 Gy) of gamma-rays was shown to be independent of the recA-controlled SOS response in experiments using recA cells or cells carrying recA-lacZ gene fusions. The stimulating effect in AB1157 prototype cells were subsequently confirmed using a DNA precipitation assay in which DNA incision events were accumulated by inhibiting DNA ligation with ethidium bromide. Our data suggest that there seems to be narrow 'windows' of dose-effect for the induction of SOS-independent DNA excision repair. Being similar to mammalian cell studies, the dose range for this effect was about 200-fold less than D37 for radiation survival.

Background and radiation-induced 8-hydroxy-2'-deoxyguanosine in gamma-irradiated Escherichia coli.

The DNA base damage product, 8-hydroxy-2'-deoxyguanosine (7-hydro-8-oxo-2'-deoxyguanosine, or 8-OHdG) was measured in 2'-deoxyribonucleoside digests of DNA from irradiated E. coli or model DNA using HPLC separation and electrochemical detection (LCED). The LCED technique enabled the specific quantitation of 25 fmol of 8-OHdG (signal: noise ratio = 4:1) among a variety of DNA base lesions within the sample. A radiation dose-yield relationship for 8-OHdG was observable in cells that were rapidly lysed after irradiation using a chloroform-saturated, sarkosyl and EDTA solution at 4 degrees C (Tilby and Loverock 1983). The observed yield of 8-OHdG was 7.05 +/- 0.27 pmol J-1 and was against a background of 120 8-OHdG per E. coli genome. This yield translates to an average of 12 additional 8-OHdG lesions per genome at the mean lethal dose. The low radiochemical yield and high background suggests that factors other than the toxicity of 8-OHdG per se--such as its participation in clusters of base lesions (e.g. Ward 1985)--must contribute significantly to cell killing. Although exposure of DNA to phenol mixtures has often been reported to increase 8-OHdG, we found that a more significant effect of phenol mixtures was the potentiation of the radiochemical yields of 8-OHdG in model DNA from 5.26 +/- 0.48 to 158 +/- 12.2 nmol J-1. Other factors, such as the exposure of dried 2'-deoxyribonucleoside digests to ambient humidity in the presence of buffer components, were shown to contribute more significantly to the background of 8-OHdG.

Phenol sensitization of DNA to subsequent oxidative damage in 8-hydroxyguanine assays.

The DNA base adduct, 8-hydroxyguanine (8-OHGua), has been reported to be a key biomarker relevant to carcinogenesis and cellular oxidative stress important in tumor promotion. Although investigators often report artificially high levels of 8-OHGua in DNA samples that have been exposed to phenol solutions and/or air during processing, few quantitative results are available. We show that routine phenol-based DNA purification procedures can increase 8-hydroxydeoxyguanosine (8-OHdG) levels 20-fold in samples that are exposed to air after the phenol is removed from the solutions. Surprisingly, air exposure alone accounts for a significant portion of this increase (4-fold) when compared to dG or DNA samples that have been solubilized in buffers purged with nitrogen. Most importantly, phenol treatments of DNA are shown to sensitize DNA to 8-OHdG formation by subsequent exposures to air. The sensitization of DNA occurs even though extensive dialysis is used between phenol treatment and enzymatic DNA digestion. Alternate procedures, including chloroform:isoamyl-alcohol extractions, also yield air-sensitive DNA samples. Other artifacts of organic extraction prior to air exposure include alterations in DNA base ratios after nuclease digestions. Overall, these results strongly suggest that studies of 8-OHdG in carcinogenesis should avoid dry conditions, such as lyophilization followed by exposure to air, and that all four of the bases should be monitored before 8-OHdG concentrations are normalized by undamaged deoxynucleoside concentrations. Failure to heed these precautions can lead to 2- to 20-fold overestimates of 8-OHdG in target tissues or in vitro models.

Oxidative stress effects on conjugational recombination and mutation in catalase-deficient Escherichia coli.

The objective of the present investigation was to determine the effects on genetic recombination and mutation in Escherichia coli of either endogenous increases in oxygen radicals resulting from catalase deficiencies, or exogenous increases resulting from H2O2 treatment. Using the classical paradigm of Escherichia coli bacterial conjugation, strains deficient in the production of hydroperoxidase I (HPI) and/or hydroperoxidase II (HPII) were used as recipients in Hfr x F- matings. 'Background' recombination rates, measured by the rate of appearance of threonine prototrophs, was similar to wild-type levels in the HPI-deficient (katG) strain, but were significantly decreased in HPII- (katE) mutants. The addition of relatively nontoxic H2O2 concentrations (0.25 mmoles dm-3) to the mating mixtures stimulated recombination rates in wild-type and katE strains, but decreased rates in katG and katEkatG strains. A 0.5 mmoles dm-3 concentration of H2O2 inhibited recombination rates in all strains. In order to gauge the level of recA-dependent 'SOS' processes occurring under the experimental conditions, 'background' mutation rates were determined in both fluctuation and forward mutation (thyA) assays. Mutation rates in aerobically-grown cultures were increased up to 2.2-fold in katG and katEkatG strains. Treatment with relatively nontoxic H2O2 concentrations elevated the thyA mutagenesis up to 8-fold in catalase-deficient cultures. Furthermore, these studies along with data presented elsewhere show that the SOS phenotype of katEkatG is more resistant than that of katG strains. These studies clearly show that cellular oxidative stress occurring from catalase deficiency interferes with normal DNA metabolism.

The dependence of thiol-inducible radiation resistance in Escherichia coli K12 on the medium and catalytic metal.

Radiation protection by thiols in procaryotes and lower eucaryotes has been demonstrated repeatedly to require a competent DNA repair phenotype, suggesting that simple chemical radical scavenging and hydrogen donation are only a portion of the mechanism of radiation protection by thiols. In the present report, thiol-induced radiation resistance--a model in which cells are pretreated with dithiothreitol and then irradiated in the absence of thiol--is shown to be a medium-dependent process. Wild-type log-phase cells treated with dithiothreitol in minimal-glucose medium are induced to radioresistance that persists after the thiol has been removed. Although the thiol pretreatment affected the antioxidants (catalase, superoxide dismutase, and glutathione) in cells at the time of irradiation, various antioxidant levels did not predict radiation resistance. Thiol-induced radioresistance is not expressed in rich medium-treated cells or in DNA repair (recA)-deficient cells. Addition of the efficient chelator, DETAPAC, to the thiol treatment medium leads to additional radioresistance in the case of minimal medium and a moderate expression of resistance in rich medium. Experiments using the intracellular chelator, 1,10-phenanthroline, in the presence of thiol led to inhibition of thiol-induced resistance in minimal medium and radiosensitization in rich medium. These results can be explained by a "site-specific" mechanism of thiol oxidation in which the chelators control the site(s) and rate of thiol oxidation, subsequently determining the type of cellular response.

Superoxide dismutase and media dependence of far-UV radiation resistance in thiol-treated cells.

Pretreatment of wild-type Escherichia coli K12 cells with dithiothreitol (DTT) induces far-UV radiation resistance after the thiol is removed (Claycamp 1988). The present study shows that a 1 h treatment of cells with DTT in minimal medium followed by a 0.5 h incubation in buffer (37 degrees C) results in a dose reduction factor (DRF) calculated at F37 of 1.81. When the thiol pretreatment was in rich medium, sensitization occurs with DRF = 0.729. This sensitization could be reversed to protection by inhibiting extracellular thiol oxidation in rich medium with the chelator, DETAPAC, such that the thiol oxidation rate was equivalent to that of DTT in minimal medium. Both thiol-induced resistance and sensitization produced changes predominantly in the shoulders of the survival curves. Furthermore, for either protection or sensitization, at least one form of endogenous superoxide dismutase (SOD) was required: in SOD-deficient cells (sodAsodB) the DRFs were 1.08 and 0.882 for minimal and rich media, respectively. These results suggest that different targets are involved in thiol-induced UV protection and sensitization: DNA and extracellular targets (e.g. the membrane), respectively. The results augment observations of alternate and multiple repair pathways inducible by oxygen radicals and may help understanding non-physicochemical thiol protection mechanisms.

Thiol and hydrogen peroxide modification of recA induction in UV-irradiated wild-type and catalase-deficient Escherichia coli K12.

Induction of recA in Escherichia coli, monitored as beta-D-galactosidase (beta-Gal) activity in recA-lacZ fusion strains, was shown to be elevated and prolonged by dithiothreitol (DTT) treatment after far-UV radiation. Pretreatment of UV-irradiated cultures using DTT led to a shortened recA response and little increase of beta-Gal yield. Similar studies were performed using a catalase-deficient recA-lacZ strain in which the major feature was elevated levels of recA-lacZ induction. Catalase activity in UV-irradiated wild-type cells was reduced by DTT treatment to levels as low as in a katE mutant strain, leading to similar recA-lacZ induction patterns between the strains. Neither DTT nor H2O2 treatment of cells could induce significant recA transcription in the absence of UV-radiation, implying that both agents modify recA activity primarily by interfering with repair of recA-inducing DNA lesions. The results confirm previous studies suggesting that modification of DNA repair is probably a significant portion of thiol radiation protection.