Biomedical subjects

C J Koch

Publications and source records attributed to C J Koch.

At least 91 records · Page 5Linked to original sources

PubMed1994

Enhanced radiation-sensitivity by preincubation with nitroimidazoles: effect of glutathione depletion.

PURPOSE: The mechanism of enhanced radiosensitization by nitroheterocyclics after a preincubation period under hypoxic conditions was investigated. The hypothesis that this phenomenon was caused by glutathione depletion was tested. METHODS AND MATERIALS: The phenomenon of enhanced radiosensitization by nitroheterocyclics after a preincubation period under hypoxic conditions is potentially of importance therapeutically because essentially nonlethal preradiation exposures to the electron affinic drugs cause a much larger radiation sensitization than would otherwise be expected. We have investigated this interesting property of several 2-nitroimidazoles to determine its possible cause and to test various hypotheses about maximizing its possible therapeutic benefit. In view of many observations that thiols are depleted by incubation of cells with nitroimidazoles under hypoxic conditions, we have specifically investigated this aspect of the preincubation effect. Depletion of glutathione was either enhanced by an overnight incubation with buthionine sulfoximine or minimized by preincubation with a 2-nitroimidazole which is sterically inhibited from causing thiol depletion. RESULTS: When conditions were chosen which minimized variations in cellular glutathione content during the preincubation period, no preincubation effect was observed. At low, therapeutically relevant radiation doses, where 2-nitroimidazoles are less efficient sensitizers, the preincubation effect may be even more important, but thiol depletion still minimizes its impact in this region of the dose-response curve. CONCLUSION: These results suggest that the preincubation effect is caused by a "self-sensitization" involving the known enhancement of radiation sensitization by thiol depletion.

Animals↗

PubMed1994

Bioreductive metabolism of SR-4233 (WIN 59075) by whole cell suspensions under aerobic and hypoxic conditions: role of the pentose cycle and implications for the mechanism of cytotoxicity observed in air.

PURPOSE: Measurement of pentose cycle (PC) activity is shown to be a noninvasive means for monitoring the reduction of SR-4233 in whole cells. Comparing these measurements to the actual measurements of drug loss under aerobic and hypoxic conditions helps to define the mechanism for the associated aerobic toxicity. METHODS AND MATERIALS: SR-4233 is activated to a toxic species by bioreductive metabolism. NADPH is required for the activation of the drug by purified enzymes, cell homogenates and whole cells. In vivo the NADPH:NADP+ ratio is maintained by the oxidation of glucose via the oxidative limb of the pentose cycle. By measuring radiolabeled 14CO2 released as a product of this oxidation one can get an accurate measurement of the rate of drug metabolism in whole cells. These results are compared to measurements of drug consumption under aerobic and hypoxic conditions using an HPLC assay. RESULTS: SR-4233 stimulates pentose cycle activity to a greater extent in air then under hypoxia, however, in the presence of added catalase, pentose cycle activity is stimulated to a similar extent under both conditions. The higher levels of PC activity observed in air are due to the production of hydrogen peroxide by the nitroxide free radical undergoing futile redox cycling. The contribution of H2O2 to the observed aerobic cytotoxicity of SR-4233 is minimal however, since toxicity is only slightly reduced in the presence of exogenous catalase and antioxidants such as vitamin E. The level of PC stimulation by SR-4233 suggests that the rate of electron addition to the drug is independent of O2 concentration. The loss of drug from the incubation medium, i.e., conversion to a stable intermediate species, occurs approximately five times faster under nitrogen than in air for A549 cells. It is the rate of drug loss from the cell and not the rate of reduction which best correlates with the observed aerobic and hypoxic toxicity. CONCLUSION: Toxicity in air and in nitrogen is directly related to the rate of drug reduction, i.e., at equivalent levels of drug loss we observe equal levels of cytotoxicity.

Aerobiosis↗

PubMed1994

The alkaline elution technique for measuring DNA single strand breaks: increased reliability and sensitivity.

The alkaline elution procedure is noted, on the one hand, for its sensitivity in the detection of DNA damage, but on the other hand, for its extreme variability and inconsistency. These deficiencies in the technique have been traced to incomplete exchanges of the various solutions used in the cell rinse and lysis and DNA rinse and elution portions of the procedure. Solutions to the above indicated problems involve several changes to the standard procedure, including, at the cellular stage, the complete removal of rinsing solutions followed by the addition of lysis solution at 0 degrees C. After standard cell lysis and alkaline rinse of the DNA, the alkaline rinse solution is replaced by elution solution at high flow rate to allow a uniform starting time for the elution, which then proceeds as a nearly first-order function of time (not elution volume). Using gamma-rays as the damaging agent, reproducibility within and between experiments is easily comparable to radiation survival itself, and typically, duplicate elution channels within an experiment provide nearly identical information without the need for internal standards and complex ratiometric analysis techniques. The procedures described allow the reproducible assessment of radiation damage to cellular DNA at doses much below 1 Gy and allow the study of repair processes down to similar levels of residual damage. This unexpected increase in technique sensitivity may be caused by maintenance of near-freezing temperatures during cell manipulation and addition of lysis solutions or to more complete and uniform lysis.

Alkalies↗

PubMed1994

Recognition of platinum-induced DNA damage by nuclear proteins: screening for mechanisms.

The association of high mobility group (HMG) proteins with cisplatin-damaged DNA has attracted considerable interest because of a possible relationship with drug-acquired resistance and the repair of DNA damage caused by this important chemotherapeutic agent. We have further characterized the binding of HMG proteins to cisplatin-damaged DNA using a modification of the damaged DNA affinity precipitation assay (DDAP) and proteins isolated from the nuclei of V79 cells. HMG proteins recognized cisplatin adducts only in double-stranded DNA sequences. Pre-treatment of cells with cisplatin (1 microM) prior to protein extraction decreased the apparent yield of HMG proteins. However, the pre-treatment of isolated protein did not prevent recognition of the DNA adducts. To investigate the possible role of HMG proteins in toxicity and resistance, we have extended the DDAP method to study other platinum agents, some of which are active in cisplatin-resistant cells. A comparison of trans- and cis-[PtCl2(NH3)quinoline] is presented as an example. HMG proteins recognized DNA damage caused by the cis, but not the trans isomer. However, the trans isomer is known to be significantly more toxic and is highly active in cisplatin-resistant cells, suggesting a mechanism of action different from cisplatin. Therefore, the toxicity of trans-[PtCl2(NH3)quinoline] appears to be unrelated to the recognition of damage by HMG proteins. The DDAP assay may provide an additional screen for new mechanisms of cytotoxic platinum agents.

Animals↗

PubMed1994

Do low doses of radiation induce a response modulating induction or repair of DNA single-strand breaks?

Many recent reports have indicated interesting structure in the low-dose region of survival curves for mammalian cells (e.g., variations in the effects of oxygen, high-order modulation of the dose-response function, induction of repair, activation of protein-modifying enzymes, influences of cytokines, etc.). Since we have recently identified and corrected several problems causing variability in the results of the alkaline elution methodology, we felt that it was important to investigate the formation and repair of single-strand breaks (SSBs) in this low-dose region. To date we have asked three questions relevant to the structure features noted above: (1) Is the dose response linear at very low radiation doses? (2) Is repair of SSBs complete? (3) Do low "priming" doses of radiation induce subsequent variations in the sensitivity, or rate or extent of repair? To date, we have found no basis in SSB induction and repair for the interesting substructure noted in the survival responses above. At doses from 0.25 Gy to 4 Gy in air, no significant deviations from a linear dose response were observed, and a "priming" dose of 2 Gy has a minimal effect on subsequent sensitivity of SSB formation. Repair of SSBs appears complete at clinical doses of 2 Gy, but substantial unrepaired SSB damage remains 1 h after doses (11 Gy) corresponding to a surviving fraction of 0.01. No significant change in repair of SSBs at 4 h after a 2-Gy priming dose has yet been determined.

Animals↗

PubMed1993

Detection of hypoxic cells by monoclonal antibody recognizing 2-nitroimidazole adducts.

Hypoxic cells in tissue pose many medical problems, and there is a need for more accurate measurements of tissue hypoxia. However, measurement of the pO2 and the extent of hypoxia within normal and tumor tissue have proven difficult. One of the most sensitive of the currently available methodologies involves the oxygen-dependent metabolic activation of nitroheterocyclic drugs, leading to adducts between the drugs and cellular macromolecules. Limitations of the present drugs and adduct-detection methods prompted the present studies. A pentafluorinated derivative [EF5; 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetam ide] of etanidazole was synthesized with the expectation of lessening some of the non-oxygen-dependent variability in adduct formation observed previously with other nitroaromatic compounds. EF5-protein conjugates, prepared by radiochemical reduction, were found to be immunogenic and allowed the development of monoclonal antibodies. One of these antibodies, ELK2-4, has been characterized and found to be highly specific for the EF5 adducts whether produced radiochemically or by cellular bioreductive metabolism. 9L rat glioma cells pretreated with EF5 under hypoxic, compared with aerobic, conditions were readily discriminated immunochemically using fluorochrome-conjugated secondary antibodies which recognize the ELK2-4 antibody subtype (IgG1). Similarly, the central region of multicenter spheroids, composed of EMT6 mouse mammary sarcoma cells, was selectively visualized by immunohistochemistry after the spheroids were incubated for 4 h in 0.5 mM EF5. Tumor biopsy, preparation, and immunohistochemical staining 24 h after treatment of tumor-bearing animals with drug also demonstrated high contrast regions within EMT6 mouse or Morris 7777 hepatoma rat tumors. The use of this new compound and its highly specific monoclonal antibody may allow elucidation of bioreductive metabolism of the nitroheterocyclics and significantly improve technologies for the quantitation of tissue pO2.

Animals↗

PubMed1993

Bioreductive metabolism of AF-2[2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide] combined with 2-nitroimidazoles. Implications for use as hypoxic cell markers.

Metabolism of misonidazole under hypoxic conditions depletes the parent drug and causes about 4% of the reduced-drug-products to form adducts with cellular macromolecules (binding), and this process has been used to detect hypoxia in cells and tissues. The nitrofuran, AF-2 [2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide] has been shown to increase both the metabolic depletion of misonidazole and its binding. In the present study, factors which might affect this process have been examined, in an in vitro system, to test the hypothesis that metabolic depletion of misonidazole could limit its ability to diffuse freely to the hypoxic cell population. Drastic reductions in glucose concentrations from their normal value of 5-10 mM to less than 0.5 mM had no significant effect on the metabolism of either misonidazole or AF-2. Similarly, glucose concentration did not influence the binding of misonidazole, even when concentrations of both oxygen (extreme hypoxia) and glucose were near zero--a very toxic biochemical environment. Similarly, the metabolism of the nitroheterocyclics had no effect on glucose consumption. The bioreductive depletion of misonidazole in extreme hypoxia appeared to be independent of drug concentration between 25 and 100 microM: this nearly zero-order rate of drug metabolism prevented the possibility of working at constant drug concentration. AF-2 exacerbated this effect by greatly enhancing the metabolic depletion of misonidazole. AF-2 was found to increase both the metabolic depletion and binding of misonidazole by the same factor. An unexpected finding was that metabolism of etanidazole, a 2-nitroimidazole closely related to misonidazole, was not enhanced by AF-2. Micromolar amounts of oxygen inhibited the reductive activation of AF-2, and also the interaction between AF-2 and misonidazole. Our results suggest that metabolic depletion of nitroheterocyclics could influence their ability to diffuse adequately to hypoxic tissues, particularly at the low drug concentrations that have been used to measure tissue hypoxia in vivo.

Animals↗

PubMed1993

Unusual oxygen concentration dependence of toxicity of SR-4233, a hypoxic cell toxin.

Toxicity from drugs activated by bioreductive metabolism has been suggested as a means to eliminate the treatment resistance caused by hypoxic tumor cells. In general, drugs have been selected to maximize the hypoxic cytotoxicity ratio [exposure (drug concentration x time) in air:exposure in nitrogen] to cause equal toxicity. On this basis, two recently developed drugs have very similar characteristics; an aziridine derivative of misonidazole (RSU1069) and a benzotriazine di-N-oxide (SR4233). The oxygen dependence of the toxic response has not previously been characterized. This report shows that the toxicity from SR4233 extends over a much greater range of oxygen concentrations than does that of RSU1069. Furthermore, unlike all previous drugs studied, the toxicity of SR4233 does not level off at high oxygen concentrations, but continues to decrease as the oxygen concentration increases. For 1 mM oxygen (the solubility of oxygen in medium at 37 degrees C equilibrated with 100% oxygen and water vapor) the toxicity from SR4233 is at least 2000-fold less than that for hypoxia. Modeling the effect of oxygen on combined radiation and toxicity shows that radiation plus SR4233 should be much more effective in eliminating hypoxic cells than radiation plus RSU1069. The unusual oxygen dependence of toxicity by SR4233 may indicate a unique biochemical activation process.

Animals↗

PubMed1992

Distribution of misonidazole adducts in 9L gliosarcoma tumors and spheroids: implications for oxygen distribution.

The 9L rat gliosarcoma is a widely used experimental brain tumor which has an unusual radiation response. The radiation sensitivity of cells in subcutaneous tumors has been shown to be intermediate between those of aerobic and hypoxic 9L cells in vitro, and cells in spheroids and intracranial tumors appear to be as sensitive as fully aerobic cells. The hypoxic marker misonidazole was used to investigate the distribution of oxygen in these 9L systems. In vitro calibration of binding of [3H]-misonidazole as a function of oxygen concentration demonstrated an inverse relationship similar to those of several other experimental tumors. In autoradiograms of sections from tumors labeled in vivo the grain density rose gradually from the periphery of the tumor to the center. Over millimeter distances the distribution of grains was remarkably uniform, in contrast to the substantial variation reported for several other tumors. The grain density as a function of distance from capillaries was essentially constant. Several interpretations of this result are possible, including the postulate that intermittent blood flow occurs in all 9L tumor capillaries, which results in the majority of the binding of misonidazole occurring during the periods of transient hypoxia. Cells adjacent to the necrotic center in 9L spheroids and the rare necrotic regions in tumors appeared to be severely hypoxic, based on the quantity of misonidazole they bound. Spheroid "cure" experiments demonstrated that these cells were not clonogenic.

Animals↗

PubMed1992

Metabolism and radiosensitization of 4,5-dimethylmisonidazole, a ring-substituted analog of misonidazole.

4,5-Dimethylmisonidazole (DMM) is a ring-substituted derivative of the 2-nitroimidazole, misonidazole. 2-Nitroimidazoles are able to sensitize radioresistant hypoxic cells, and to kill them outright through bioreductive metabolism. The toxic process is believed to reflect the consequences of reductively activated drugs forming adducts with cellular (macro)molecules. Both this process and the radiosensitizing activity are thought to correlate with the electron affinity of radiosensitizing agents. In the present study, methyl groups were added to the imidazole ring of misonidazole in order to hinder adduct formation with cellular molecules after reductive-activation of the compound. It was anticipated that this would substantially decrease the hypoxic-cell toxicity of the parent drug. The presence of the two methyl groups reduced the half-wave reduction potential of DMM by about 70 mV, so we expected that its radiosensitizing ability would also decrease. In direct comparison with misonidazole, DMM, at equimolar concentrations, showed dramatically reduced binding to cellular macromolecules under bioreductive conditions, both in vivo, using a liver perfusion system, and in vitro, using tissue culture cells incubated under extreme hypoxia. However, DMM was only moderately less toxic than the parent compound, and showed greatly diminished radiation sensitization capacity. Since the decrease in toxicity was much less than expected, and the decrease in radiosensitization was much more than expected, this compound may be an important drug for continuing studies on the mechanisms of radiation sensitization, binding and cytotoxicity caused by electron affinic drugs.

Animals↗

PubMed1992

Comparisons of cellular radiation response using absolute rather than relative parameters.

One important goal of radiobiology is to describe the response to radiation damage in quantitative terms. Because the dose response is nonlinear, this typically involves the comparison of several dose-dependent parameters. Past attempts at simplifying this process have often involved manipulations of these dose-dependent parameters to derive a single comparative number. Unfortunately, the advantages of a single comparative number, often a ratio or even a ratio of ratios, can be outweighed by the loss of significant biological information. Examples are given in four areas of research: (1) definition of radiation response at clinically relevant radiation doses, (2) modification of radiation sensitivity by oxygen, (3) effects of combined radiation modifiers (e.g., sensitizers and protectors), and (4) comparisons of radiation modifiers in different dose/response regions. In each area, to define and compare dose response, we propose the use of consistent, simple, and absolute radiation response parameters: the inverse dose (or dose) required to produce a given effect. The use of absolute sensitivity (or resistance) avoids the use of many other parameters, ratios, and definitions and permits a uniform and unambiguous description of "radiation response."

Animals↗

PubMed1992

Radioprotection of DNA in isolated nuclei by naturally occurring thiols at intermediate oxygen tension.

Incubation of isolated Chinese hamster ovary cell nuclei, equilibrated in an atmosphere containing 2% O2, with glutathione, cysteine, or cysteamine resulted in a decrease in the number of X-ray-induced DNA double-strand breaks (DSBs), determined by pH 9.0 filter elution. In the absence of exogenous thiol, no sensitization was observed with the addition of N-ethylmaleimide, indicating that endogenous thiols were not present at significant levels. Protection by 0.3 mM glutathione was not enhanced by the addition of exogenous glutathione S-transferases or by glutathione peroxidase. The data were analyzed according to a simple competition model with various hypotheses. Cysteamine was more than an order of magnitude more effective than the other thiols tested, on a molar basis, in preventing DSB formation. Depending on the hypothesis used to evaluate the data, glutathione was either much less effective, on a molar basis, in preventing the bulk of the DSBs or was capable of preventing only approximately 55% of the damage, regardless of concentration. These data suggest that natural thiols other than glutathione may contribute to cellular radioprotection even if their concentration is much lower than that of glutathione. The data also suggest that despite the relative inefficiency of glutathione as a radioprotector, some areas of oxygenated tissues--where the oxygen tension falls below 2%--may be protected by glutathione concentrations in the physiological range of 3-20 mM.

Animals↗

PubMed1991

Radiolytic reduction of protein and nonprotein disulfides in the presence of formate: a chain reaction.

We have reported recently that the disulfide groups in bovine serum albumin can be reduced by a radiolytic chain reaction which occurs in deoxygenated solutions containing formate ions. This reaction, which involves the reduction of disulfide groups by hydrated electrons and carbon dioxide radical anions, has now been studied in greater detail and compared with an analogous reaction in small, disulfide containing molecules over a range of pH values and substrate concentrations. A two-step reaction is proposed to account for the reduction of disulfides in reactions which can have chain lengths of 20 or more. Thiols produced by the disulfide reduction are stable to the conditions of the reaction. For example, a biological assay showed that the integrity of glutathione was maintained even at radiation doses much larger than those required to achieve complete reduction of glutathione disulfide. It was found that the extent of disulfide reduction could easily be controlled by varying the radiation dose delivered to the solutions. Radiolytic reduction is a very useful way of reducing protein and low molecular weight disulfides without the use of excess quantities of reagents such as dithiothreitol. In many cases, the reaction solutions could be used directly for subsequent reactions and this may be of considerable value in modifying the structure of hormones, enzymes, membrane receptors, and other disulfide containing proteins. If ammonium formate is used, freeze drying is an effective way to remove the formate salt, should this be required.

Cesium Radioisotopes↗

PubMed1991

Pharmacokinetics and cytotoxicity of RSU-1069 in subcutaneous 9L tumours under oxic and hypoxic conditions.

The acute toxicity, pharmacokinetics and hypoxic cytotoxicity of RSU-1069 were investigated using the subcutaneous (sc) rat 9L tumour model. The pharmacokinetics were studied after i.p. injection of RSU-1069 (20 mg kg-1 or 100 mg kg-1). For both doses, the elimination of RSU-1069 followed first-order kinetics in both plasma and unclamped tumours. After 100 mg kg-1, the peak plasma concentration of RSU-1069 was 40 micrograms ml-1; the elimination t1/2 was 39.3 +/- 11.1 min. After 20 mg kg-1, the peak plasma concentration was 3 micrograms ml-1; the elimination t1/2 was 47.8 +/- 6.3 min. In unclamped tumours, the peak concentration was 50 micrograms g-1 with an elimination t1/2 of 36.1 +/- 9.6 min for the 100 mg kg-1 dose, and 4 micrograms g-1 with an elimination t1/2 of 41.9 +/- 6.1 min for the 20 mg kg-1 dose. The tumour and plasma elimination half-times were not significantly different (P greater than 0.2) for the two doses. Clamping the tumour 30 min after administration of 100 mg kg-1 of RSU-1069 decreased the tumour elimination t1/2 to 10.9 +/- 1.4 min. After releasing the clamp, RSU-1069 returned rapidly to the unclamped tumour concentration. The unclamped tumour/plasma ratio reached a maximum of 4-6, then decreased to a constant value of about 2 for both doses, indicating that RSU-1069 accumulates in these 9L tumours. RSU-1069 kills hypoxic sc 9L cells more efficiently than oxic sc 9L cells; at a surviving fraction of 0.5, the SER was 4.8. For in vitro 9L cells, the SER was approximately 50 when the comparison was between those treated in 2.1% 0(2) and those treated in less than 7.5 x 10(-3)% 0(2); it was approximately 100 when the comparison was between those treated in 21% 0(2) and those treated in less than 7.5 x 10(-3)% 0(3). Tumours treated with RSU-1069 and clamped for various times exhibited biphasic cell-kill kinetics; at 50 mg kg-1, little additional cell kill was achieved after 40 min of clamping. Our data also indicate that RSU-1069 is 300-1000 fold more efficient than misonidazole or SR2508 for killing hypoxic sc 9L tumour cells in situ.

Animals↗

PubMed1990

Importance of thiols in the reductive binding of 2-nitroimidazoles to macromolecules.

Reductive activation of 2-nitroimidazoles in the presence of bovine serum albumin (BSA) led to binding of the nitroheterocycles to the protein. The binding was most efficient to BSA in which protein disulfides had been reduced to thiol groups. Protein thiols were at least twenty times more efficient than other protein, RNA or DNA nucleophiles in binding the reductively-activated nitroheterocycles. This result is of practical importance in the development of immunoassays for 2-nitroimidazoles as hypoxia markers in normal and tumor tissue.

Glyoxal↗

PubMed1990

Adsorption of creatinine to Fuller's earth.

In search for a secondary reference method for the determination of creatinine, the adsorption of creatinine to seven different commercial batches of Fuller's earth have been investigated. Mineralogical investigation of the batches broadly divided them into two subgroups with quite different cation exchange capacities (CEC): dominant smectitic with and without calcium carbonate and dominant palygorskitic with calcium carbonate. Adsorption experiments using 14C-creatinine show, for all Fuller's earth samples, an incomplete adsorption of creatinine. The amount of creatinine adsorbed depends on pH, temperature, concentration of cations as Ca2+ and Na+, and other conditions. Weakly acidic suspensions of Fuller's earth in the course of time change their ability to adsorb creatinine. Compared to a synthetic strong acidic cation exchanger, Fuller's earth is more selective to creatinine. Because of the sensitivity of the adsorption to the various experimental conditions a determination of creatinine in biological fluids using Fuller's earth is too variable. Therefore, we cannot recommend this procedure as a secondary reference method for the determination of creatinine.

Adsorption↗

PubMed1989

Quantitation of hypoxia in multicellular spheroids by video image analysis.

Polyclonal antibodies to a series of radiation chemically produced 2-nitroimidazole-protein adducts have been used in the indirect immunofluorescent detection of hypoxic cells in EMT6/Ed spheroids. The spheroids were incubated with the radioactive 2-nitrioimidazoles under atmospheres of air, several intermediate oxygen concentrations, and nitrogen. The fluorescence intensity of the marker across radii of spheroids labeled at various oxygen tensions, as measured by video image analysis, paralleled the autoradiographic grain density. Thus, we conclude that the immunohistochemical approach provides a quantitative measure of the distribution of 2-nitroimidazole adducts in spheroids. The advantages of speed, technical simplicity, economy, and independence from the requirement of radiolabeled precursor render the fluorescent marker of potential use in the clinical detection of cellular hypoxia and tissue ischemia.

Autoradiography↗

PubMed1989

Combined radiation-protective and radiation-sensitizing agents. IV: Measurement of intracellular protector concentrations.

Radiosensitization of hypoxic V79 Chinese hamster cells by 0.5 mM misonidazole at approximately 0-4 degrees C is substantially enhanced by pretreating the cells overnight with 0.1 mM buthionine sulfoximine, which lowers the cellular glutathione content to 5% of control values (from 4 mM to approximately 0.2 mM). The enhanced sensitization is reversed by concentrations of exogenous cysteine that are much lower (0.02 mM) than the original glutathione content. Reduced Co-enzyme A affords reversal of the enhancing effect at concentrations of about 1 mM. Sodium ascorbate gives no protection at all even at concentrations of 2 mM. The intracellular concentration of the reducing agents was measured using a spin-through oil technique. There was no diffusion of Co-A (MW greater than 750) or ascorbate (excluded by charge) into the cells. In contrast, cysteine was rapidly concentrated by factors of 4-10, even at the low temperatures used. Extracellular ascorbate's inability to radioprotect argues against electron transfer across the cell membrane as a mechanism for radioprotection. This mechanism could have explained the ability of exogenous thiols to radioprotect in former studies using glutathione, and in the present studies using Co-A. The potential of cysteine to be concentrated by cells poses a problem in the interpretation of "exogenous protection" by non-diffusing thiols, since trace contamination by cysteine could lead to the actual protection observed. Cysteine could also be formed by exchange reactions of exogenous thiols with the disulfide of cysteine, present in all media formulations.

Animals↗