Studies on Latent Derivatives of Aminoethanethiols As Potentially Selective Cytoprotectants I

[CANCER RESEARCH 27, 130-136, January 1967]

Studies on Latent Derivatives of Aminoethanethiols as Potentially Selective Cytoprotectants I. A Spectrophotometric Assay for Aminoethanethiols in Biologic Media1

KATHERINE A. HERRINGTON, KATHERINE POINTER,2 ALTON MEISTER,' AND ORRIE M. FRIEDMAN Collaborative Research, Inc., Wallham, Massachusetts

Summary been found (25) between the intracellular thiol concentration in rat liver tissue and the degree of protection. It has also been re A convenient Spectrophotometric method for determination of ported (23, 24) that the radioprotective action of cysteamine in aminoethanethiols in biologic materials is described. The method tissue culture systems is related to its concentration in the me is based on the reaction of aminoethanethiols with 3-fluoropyru- dium. Correlations have also been found between the degree of vate at pH 9 to produce products that absorb at 300 mjt. Disul- protection against the alkylating agent, Merophan, and the in fides formed by oxidation of aminoethanethiols can be determined crease in intracellular â€”SH concentrations in several rat tissues after their reduction by dithiothreitol (or dithioerythritol) or (Ref. 5, p. 94). cleavage with bisulfite. The method is suitable for studies of We have approached the problem of selective protection of "latent" protective compounds that can be converted by various normal tissues by attempting to prepare inactive derivatives of tissues to aminoethanethiols. protective agents that can be converted to active protectants in the tissues. If such conversion occurs selectively in the relatively more radiation-sensitive tissues (e.g., intestinal epithelium, bone Introduction marrow), increased doses of radiation or alkylating agents might It has been established that pretreatment of animals with then be used against tumors without increasing damage to the aminoethanethiols such as cysteamine and cysteine significantly host. In connection with oui' work on the development of latent reduces the toxicity of ionizing radiation (5, 6, 16, 20, 22). These protective agents, we have undertaken to determine the tissue compounds also protect against the damaging effects of radio- distribution of aminoethanethiols released in vivo and in certain mimetic alkylating agents such as the nitrogen mustards used in instances to determine by in vitro studies the mechanism by which cancer chemotherapy (9, 21). Various theories have been sug such release occurs. The results of these studies are reported in gested (5, 6, 20, 22) to explain the mechanism of action of pro the succeeding papers (15,17,18). This work has been facilitated tective compounds. These hypotheses are based on the belief by development of the analytic method reported here for the that the protective agent enters the cell before irradiation. It specific determination of aminoethanethiols in biologic media. has been repeatedly shown that for maximal protection there is Several Spectrophotometric methods for the determination of an optimum time interval between administration of protective thiols are based on their reactions with such reagents as nitroprus- agent and irradiation, and that the protective agent has no side (13), p-mercuribenzoate (8), and 5,5'-dithiobis(2-nitro- therapeutic effect when given after irradiation. It appears reason benzoic acid) (12); these reagents react both with aminoethane able to conclude that the protectant is localized at critical sites thiols as well as with other types of thiols. Thiols also react with within cells, and that the optimal time interval reflects a favor fluoropyruvate (4, 7) to yield products characterized by ultra able balance between such localization and metabolic destruction violet absorption maxima in the range 265-275 m/i; the maxima and excretion. Evidence for cellular localization of the protectant shift to 300 m/i in the presence of borate buffer at pH 8 or bi has come from a number of studies. A positive correlation has valent cations such as zinc (1-3). On the other hand, amino ethanethiols react with fluoropyruvate to yield products, believed to be thiazines, which absorb at 300 m/i (7). This characteristic 1Supported by Research Contract PH43-62-170, Cancer Chemo reaction forms the basis for the Spectrophotometric assay for therapy National Service Center, National Cancer Institute, NIH, aminoethanethiols described here. The ultraviolet absorption Bethesda, Md. 'Present address: Department of Biological Chemistry, Harvard spectrum of the cysteamine-fluoropyruvato adduct is shown in Medical School, Boston, Mass. Chart 1. 3Department of Biochemistry, Tufts University School of Aminoethanethiols such as cysteamine readily undergo oxida Medicine, Boston, Mass. tion in tissue preparations to yield cystamine as well as mixed Received May 3, 1966; accepted August 19, 1966. disulfides. Free cysteamine may be regenerated from disillude

130 CANCER RESEARCH VOL. 27

Cyclo Chemical Corporation, Los Angeles, California. The cyclic disulfide was prepared from this material by Cleland's procedure (10). Sodium fluoropyruvate was purchased from Columbia Chem ical Corporation, Columbia, South Carolina. The compound is highly hygroscopic and, if allowed to absorb water, slowly de composes to yield a strongly oxidizing colored substance. The salt was stored in a desiccator at â€”18Â°C;under these conditions it was stable for at least 6 months. Ar-n-Butyl-cysteamine and N-n-octyl-cysteamine were kindly supplied by Dr. Thomas Sweeney, Walter Reed Army Institute of Research, Washington, D. C. All other compounds were of analytic reagent grade.

Methods Absorbancy was determined in a Beckman model DU spectro- o -Q photometer using silica cuvets of 1-cm light path. Solutions of O in fluoropyruvate absorb significantly at 300 mju; therefore this -0 reagent was added to the reference solutions used in the spectrophotometric assays. Fluoropyruvate solutions (0.08 M or 0.16 M) in 0.05 M disodium ethylenediaminetetraacetate (EDTA) ad justed to pH 5.5 with hydrochloric acid were stable at 26Â°Cforat least 8 hr. It has been reported4 that fluoropyruvate solutions at pH 5-6 are stable for a few days, after which significant amounts of inorganic fluoride and mesoxaldehydic acid are noted. Thiol compounds were routinely assayed by the 5,5'-dithiobis- (2-nitrobenzoic acid) method (12). Solutions of cysteamine (0.1 M) were prepared daily in 0.1 N hydrochloric acid; these were stable to oxidation for several hr. For use as standards in the determination of cysteamine, such solutions were diluted to 260 280 300 320 340 0.001 M immediately before use. Wavelength (mp) Tris-HCl buffer (tris(hydroxymethyl)aminomethane) (pH 9; CHART 1. Ultraviolet absorption spectrum of cysteamine- 1 M) was employed. fluoropyruvate product (10~4Mcysteamine; experimental details Solutions of 0.02 Msodium bisulfite were prepared in 0.001 M are given in the text under Direct Determination of Cysteamine). EDTA just before use. Stock solutions of 0.1 M dithiothreitol were stored at 4Â°Cand linkage by cleavage with sodium bisulfite or by reduction with kept for periods not exceeding 1 week. The stock solutions were dithiothreitol or dithioerythritol (10). Methods are described diluted to 0.01 M before use. here for the separate determination of (a) cysteamine, (6) cystamine and low molecular weight mixed disulfides of cysteamine, Results and Discussion and (c) mixed disulfides of cysteamine with protein. Reaction of Cysteamine with Fluoropyruvate Materials and Methods The reaction of cysteamine with fluoropyruvate occurs in the pH range 7.0-9.7 (Table 1). Identical results were obtained in Materials 0.4 M and 0.04 M Tris-HCl buffer (pH 9). This finding is of im Cysteamine, cystamine, and other aminothiols were obtained portance in applying the method to biologic material, since 0.4 M Tris-HCl buffer has sufficient capacity to buffer the trichloro- commercially as the corresponding hydrochlorides and were dried to constant weight in vacuo before use. In some experiments acetic acid needed for deproteinization. A substantial excess of cystamine was prepared by air oxidation of an aqueous cystea fluoropyruvate is required for complete reaction of cysteamine. The concentration of fluoropyruvate recommended below is mine solution adjusted to pH 9 by addition of glycine-NaOH about twice that required for complete reaction of 10~4Mcystea buffer. Dithiothreitol (10) was obtained from Calbiochem, Los mine under the conditions of the assay. Angeles, California. We wish to acknowledge a generous gift from Direct DÃ©termination of Cysteamine Dr. W. W. Cleland, University of Wisconsin, of the tetraacetate Samples containing cysteamine (0.1-0.4 /Â¿mole)ina volume of of dithiothreitol from which we prepared the compound before 1.1 ml were prepared in silica cuvets. The fluoropyruvate reagent it became available commercially. We are also indebted to Dr. Cleland for a sample of the recrystallized and sublimed cyclic 4Arjeh B. Galun, "Zion" Chemical Products, Ltd., Yavne, disulfide of dithiothreitol. Dithioerythritol was purchased from Israel (private communication).

JANUARY 1967 131

TABLE 1 EFFECTOFpH ONTHECYSTEAMINE-FLUOROPYRUVATEREACTION"

BufferTris-HClÂ»Glycine-NaOHPotassium at300 0.8 mit0.6680.6820.7040.7080.6900.6450.6550.6700.6750.6750.2120.5630.5880.688 O O ro 0.6

c 0.4 o

-Q 0.2 <

0.05 0.10 phosphatepH7.58.18.59.09.58.48.79.09.49.76.07.07.38.0Absorbancy Cysteamine (pmole/ml) CHART2. Standard curve for Cysteamine (the conditions are described in the text under Direct Determination of Cysteamine) . 0 Cysteamine (final concentration, 10~4M) was assayed as de scribed in the text under Direct Determination of Cysteamine; cystamine formation. In studies on tissue homogenates it is pos the final concentration of the buffers was 0.4 M. sible to distinguish between soluble disulfides and trichloroacetic *Tris, tris(hydroxymethyl)aminomethane. acid-insoluble (i.e., protein-bound) disulfides. Thus, soluble disulfide can be determined provided that the protein is precipi (0.7 ml, 0.08 M)was added, followed by Tris-HCl buffer (1.2 ml) tated and removed before disulfide reduction or cleavage is The reference cuvet was identical except that Cysteamine was carried out. If reduction or cleavage is carried out before the omitted. The solutions were mixed, and the absorbancy at 300 mju protein is precipitated, the total Cysteamine bound as disulfide was read 5 min later. (Maximum absorbancy is reached after 2 can be determined, and hence the protein-bound Cysteamine can min; after standing for 6-7 min, there is a detectable decrease in be obtained by difference. absorbancy.) The absorbancy was linear with Cysteamine con centration over the range studied (Chart 2). Ten solutions of Disulfide Cleavagewith Sodium Bisulfite Cysteamine, separately prepared and assayed by the method of In the reaction Ellman (12), gave an average value for the molar extinction co efficient (at 300 nift) of 6800 Â±200. Under the same conditions, Râ€”Sâ€”Sâ€”R+ Râ€”S-+ Râ€”Sâ€”S03- the molar extinction of the cysteine-fluoropyruvate product was Removal of Râ€”S~ by reaction with fluoropyruvate ultimately 5800 Â±150. results in complete disulfide cleavage by forcing the reaction to The use of pure reagents in these procedures is critical. For ac the right. Thus, one can obtain the expected amount of cys- curate determinations of free aminoethanethiol in tissue homog- teamine from cystamine as described below. The Cysteamine enates or even in buffer alone, oxidative losses must be kept to a concentrations determined by this method must be doubled to minimum. Commercial preparations of trichloroacetic acid, for obtain the value for the total cysteamine oxidized to cystamine. example, may contain traces of metal ions which can catalyze Although this reaction is well known, certain experimental con the oxidation of thiols, especially at alkaline values of pH. We ditions, e.g., the order of addition of reagents, may critically affect found that when EDTA was omitted from the fluoropyruvate the results. reagent, the molar extinction coefficients determined under the Samples containing cysteamine, cystamine, or their mixtures conditions described above were about 10% lower. Furthermore, (0.5 ml) were added to cuvets, followed by bisulfite reagent (0.6 in the absence of EDTA the absorbancy at 300 mju declines more ml) and fluoropyruvate reagent (0.7 ml, 0.08 M). The reaction rapidly than in its presence. was initiated by addition of Tris buffer (1.2 ml); the solutions were mixed and the absorbancy at 300 m/n was read after 5 min Recovery of Cysteamine from Cystamine against a reference solution containing water (0.5 ml) in place of Aminoethanethiols disappear rapidly from alkaline solutions sample. Standard curves carried out with freshly prepared cys and in tissue homogenates (see below). In buffer such disappear teamine were identical whether or not bisulfite reagent was added ance is due to oxidation to the corresponding symmetric disulfide, (Chart 3). When such curves were repeated after overnight air while in tissue homogenates mixed disulfides with endogenous oxidation of the same cysteamine solution (buffered at pH 9 with thiols are also formed. Applications of the procedures described glycine-NaOH) no free thiol was detected unless bisulfite was below in which treatment with bisulfite and dithiothreitol was added. In the presence of added bisulfite exactly J of the initial carried out have shown conclusively that the loss of free cys- cysteamine was recovered (Chart 3). teamine in alkaline solution is associated almost exclusively with When mixtures containing cysteamine and cystamine are ana-

132 CANCER RESEARCH VOL. 27

0.05 0.05 0.10 0.10 Cysteamine (jjmole/ml) Cysteamine (pmole/ml) CHART-L Standard curvesforfreshlyprepared (â€¢)and oxidized CHART 3. Standard curves for freshly prepared and oxidized (O) solution of cysteamine analyzed by the dithiothreitol-fluoro- solution of cysteamine assayed in the presence (O) and absence pyruvate method. (â€¢)of 0.004 M sodium bisulfite. (The conditions are described in the text under Disulfide Cleavage with Sodium Bisulfite.) cystamine (0.05-0.20 gniole; 0.9 ml) were treated with 0.2 ml of dithiothreitol solution (0.01 M). Reduction was initiated by addi lyzed, e.g., in homogenates of tissues from animals treated with tion of 1.2 ml of Tris-HCl buffer, and after 5 min, 0.7 ml of the cysteamine, the total cysteamine concentration is determined as fluoropyruvate reagent (0.16 M) was added with mixing. The follows: Assay is carried out (a) in the presence of bisulfite (to absorbancy at 300 m/Â¿wasread 10 min later against a reference yield the free thiol plus J of the thiol present as the disulfide) and solution lacking cystamine. For mixtures of cysteamine and (6) without bisulfite (to give free thiol). The difference between cystamine, the disulfide concentration can be determined by these values is a measure of the disulfide present; the difference difference by assaying samples with and without dithiothreitol. value multiplied by 2, plus the value for free thiol, gives the total When dithiothreitol is omitted, fluoropyruvate must be added concentration of cysteamine present in the free form and as before the Tris-HCl buffer and the absorbancy recorded after 5 cystamine. However, when this procedure is used for samples min as described above. When L-cystine was determined by this containing considerable amounts of mixed disulfides, this ap procedure, the preincubation period with dithiothreitol was in proach isnot valid since sulfite does not cleave all mixed disulfides creased to 10 min. in a random manner. This difficulty is obviated by reduction with The solution of cysteamine used in the studies described in dithiothreitol carried out as described below. Chart 3 was analyzed before and after overnight air oxidation using the dithiothreitol-fluoropyruvate procedure; identical Disidfide Reduction with Dithiothreitol values were obtained (Chart 4). Dithiothreitol and its diastereoisomer, dithioerythritol, were The reaction product of dithiothreitol and fluoropyruvate in shown by Cleland (10) to be effective reagents for reducing Tris-HCl buffer at pH 9 exhibits an absorption maximum at 272 disulfides and for maintaining thiols in the reduced form. For mju ( e = 610) with relatively less absorbancy at 300 mju (e = example, the quantitative reduction of cystine at pH 8 was 180). The cyclic disulfide of dithiothreitol does not react with demonstrated. The effectiveness of these compounds may be fluoropyruvate but exhibits a weak absorbancy at 300 m/Â¿(e = ascribed to the ease with which they are converted to stable cyclic 190) under the assay conditions. Consequently, at the concentra disulfides; thus, other thiols are maintained in the reduced form tion of dithiothreitol used (0.67 jumole/ml), the absorbancy at by the favorable thiol-disulfide equilibrium. 300 mju due to partial oxidation of dithiothreitol to the cyclic disulfide is negligible and can be ignored. When samples of cys Râ€”Sâ€”Sâ€”R+ HSâ€”CH2â€”CH(OH)-CH(OH)â€”CH2â€”SH ^Â±Râ€”Sâ€”Sâ€”CH2â€”CH(OH)â€”CH(OH)-CH2â€”SH + Râ€”SH tamine were preincubated with dithiothreitol for 5 min in 0.1 M Tris-HCl buffers at pH 8, 8.5, or 9, quantitative recovery of Râ€”Sâ€”Sâ€”CH2â€”CH(OH)â€”CH(OH)â€”CH2-SH-+ cysteamine was obtained. Standard curves for fresh solutions of Sâ€”CH2â€”CH(OH)â€”CH(OH)â€”CH2â€”S+ Râ€”SH cysteamine in the presence and absence of dithiothreitol or its cyclic disulfide at 0.67 /Â¿mole/mlwere identical. When dithio Since disulfide reduction by dithiothreitol occurs at the pH used threitol concentrations greater than 0.67 jumole/ml were used, for the fluoropyruvate assay, it was possible to carry out both the fluoropyruvate reaction did not reach completion in 10 min. the reduction of disulfide and assay of liberated aminoethanethiol The effect of dithiothreitol concentration on the absorbancy simultaneously. Dithioerythritol may be substituted for dithio measured 10 min after the addition of fluoropyruvate is shown in threitol in the assay. Chart 5. It can be seen that 0.3-0.7 Â¿imole/mlof dithiothreitol The following procedure was employed. Samples containing was sufficient for complete reduction of 0.2 /Â¿moleofcystamine.

JANUARY 1967 133

Cysteamine + Homogenate O 0.6 O ro

0.4 u C o -O W O V) 0.2 f Cysteamine

0.5 1.0 1.5 Dithiothreitol (jjmoles/ml) 20 40 60 CHART5. Effect of dithiothreitol concentration on the cystea- Minutes mine-fluoropyruvate reaction. The experimental details are given CHART7. Disappearance of cysteamine in the presence of rat in the text. kidney homogenate. The experimental details are given in the text. The values are corrected for absorbancy due to the homogenate alone (about 0.030 absorbancy unit).

at intervals of 0, 10, 20, 30, 45, and 60 min and added to 1-ml E 08 portions of ice-cold 10% trichloroacetic acid. The precipitated O protein was removed by centrifugation, and the supernatant O solutions were assayed in duplicate for cysteamine as described rO 0.6 above under Direct Determination of Cysteamine. The results are shown in Chart 7. In the absence of homogenate 92% of the cysteamine disappeared after 60 min; in its presence the disap e 0.4 o pearance of cysteamine was slower. Thus 60% of the thiol disap .o peared after 60 min. An antioxidant effect was also observed with -o 0.2- homogenates of rat duodenum. The dithiothreitol-fluoropyruvate and bisulfite-fluoropyruvate methods were then applied to the determination of cystamine in mixtures containing rat kidney homogenate. The concentration of cystamine was varied from 20 40 2.5 X 10~4 M to 1.0 X 10~3 M in mixtures containing glycine- Fluoropyruvate (jjmoles/ml) NaOH buffer (0.1 M, pH 8.7) and rat kidney homogenate (2% w/v). Such mixtures, together with the appropriate controls, CHART6. Effect of fluoropyruvate concentration on the cystea- were incubated at 37Â°Cfor 5 min. After deproteinization, the mine-fluoropyruvate reaction in the presence of dithiothreitol. The experimental details are given in the text. acid supernatant solutions (0.3 ml) were assayed; the results are given in Chart 8. The recovery of cysteamine by both the dithio threitol-fluoropyruvate and the bisulfite-fluoropyruvate proce A final concentration of 0.67 jumole/ml was therefore used for dures was about 93 %. When reduction of cystamine was carried assay. It should be noted that the concentration of fluoropyruvate reagent used in the dithiothreitol-fluoropyruvate assay (0.16 M) out with dithiothreitol prior to precipitation of the protein, a is twice that recommended for the direct determination of cys- quantitative recovery of cysteamine was obtained. In these studies 1-ml aliquots of the incubation mixtures were added to teamine. An excess of fluoropyruvate is required in the presence 0.5 ml of 0.026 M dithiothreitol, and after 5 min, 0.5 ml of 20% of dithiothreitol; the effect of fluoropyruvate concentration in trichloroacetic acid was added. After centrifugation, the protein- the assay of cystamine is shown in Chart 6. free supernatant solutions (0.3 ml containing 1.95 /umoles of dithiothreitol) were analyzed. In this procedure the samples Determination of Cystamine as Cysteamine in Tissue Homogenates were added directly to cuvets containing 0.8 ml of water. Tris- In preliminary work the oxidation of cysteamine by tissue HC1 buffer (1.2 ml) was then added, followed by fluoropyruvate homogenates was studied. A mixture of cysteamine (0.002 M), (0.7 ml of 0.16 M). The results suggest that some of the added glycine-NaOH buffer (0.1 M, pH 9), and rat kidney homogenate cystamine undergoes disulfide interchange with protein sulfhy- (2% w/v) was prepared together with the appropriate controls dryl groups during incubation, and the findings indicate that such (i.e., those lacking homogenate, cysteamine, or both). The mix protein-bound cysteamine is quantitatively released in the pres tures were then incubated at 37Â°C.Aliquots (1 ml) were removed ence of dithiothreitol.

134 CANCER RESEARCH VOL. 27

of much greater intensity than expected for ^-substituted amino ethanethiols. Because of their low water solubilities, these com pounds were dissolved in absolute ethanol and assayed at a final ethanol concentration of 20%. (At this concentration of ethanol, Pretreated with O 0.6 formation of the cysteamine-fluoropyruvate product is normal.) O Dithiothreitol ro The results indicate that the present method can be satis factorily applied to the determination of other aminoethanethiols such as cysteine and to ./V-alkyl aminoethanethiols such as the 0.4 JV-n-butyl- and JV-n-octyl-derivatives. A procedure has also been devised for determination of either cysteamine or cysteine in o .0 dividually in mixtures of the 2. The method, to be reported later, ^ o is based on differences in the ultraviolet absorption spectra of the M -Q 0.2 fluoropyruvate adducts of the 2 compounds at different pH values. The results also indicate that the presence of N-acyl derivatives such as JV-acetylcysteine and glutathionine does not seriously interfere with the determination of aminoethanethiols.

0.025 0.050 Conclusions Cystamine (^mole/ml) The usefulness of the present method for the determination of CHART 8. Determination of cystamine after incubation, with aminoethanethiols derives from the specificity of the fluoro rat kidney homogenate. Protein-free supernatant solutions were pyruvate reaction, the applicability of a simple spectrophotometric analyzed with fluoropyruvate alone (â€¢), with bisulfite/fluoro- method, and the lack of interference by various biologic com pyruvate (O), and with dithiothreitol/fluoropyruvate(B). Addi pounds. Prior or simultaneous reduction of disulfide bonds ex tional samples were pretreated with dithiothreitol before acid tends the usefulness of the method to the determination of amino precipitation (D). (The values are corrected for absorbancy due to the homogenate alone.) ethanethiols as the corresponding disulfides or as mixed disulfides with endogenous low molecular weight thiols or tissue proteins. Since oxidation does not normally proceed beyond the disulfide TABLE 2 stage, recovery of thiol after reduction by dithiothreitol is usually ABSORBANCYOPVARIOUS AMINOETHANETHIOL-FLUOROPYRUVATEquantitative. The bisulfite modification is of value in studies in ADDUCTS" which dithiothreitol cannot be used, e.g., in investigations with cysteamine-S-sulfate which reacts directly with dithiothreitol CompoundCysteamineL-Cys (16). For the determination of aminoethanethiols in tissue homogenates, the dithiothreitol procedure is preferable in that reduction teineJV-Acetyl-L-cys can be carried out at values of pH of 7 or higher before deproteini- teineDL-HomocysteineGlutathioneDL-PenicillamineJV-n-Butyl-cysteamineJV-n-Octylzation, thus liberating aminoethanethiols that may be bound to protein by disulfide bonds. The availability of the present method has facilitated other studies in this program (16, 18, 19) on latent cytoprotectants that can be used in conjunction with alkylating agents and radia -cysteamineAnne298300269300268303285286^680058008005200670530020006400tion for the treatment of cancer. The method may also be useful " The conditions were as described for Direct Determination in detecting mixed disulfide formation between proteins and low molecular weight mercaptans; such phenomena may be of signifi of Cysteamine in the text, except that the final fluoropyruvate concentration was 0.037 M. cance in the mechanism of action ot radiation protectants.

Acknowledgments Reaction of Other Aminoethanethiols with Fluoropyruvate The authors gratefully acknowledge the very capable technical Absorbancy data for several other aminoethanethiols that have assistance of Mrs. Judy R. Macnab and Mrs. Carolyn Small. been tested in the fluoropyruvate assay are shown in Table 2. The solutions were assayed for free thiol by Ellman's method References (12) and then tested as described above. Our values for the molar extinction coefficients for cysteamine, cysteine, and glutathione 1. Avi-Dor, Y. Effect of Bivalent Metal Ions on the Spectrum of are all somewhat higher than those reported at pH 8 by Avi-Dor the Interaction Product of Glutathione and Fluoropyruvate. Biochim. Biophys. Acta, 34: 266-67, 1959. and Mager (4). The product obtained with penicillamine formed 2. . A Study of the Effect of Particle-bound T-Gluta- relatively slowly; the maximum absorbancy was reached after 20 myltranspeptidase on the Product of Interaction of Fluoro min. This result may probably be ascribed to steric hindrance pyruvate with Glutathione. Biochem. J., 76: 370-74, 1960. introduced by the methyl groups. It is of interest that the prod 3. Avi-Dor, Y., and Lipkin, R. A Spectrophotometric Method for ucts formed from W-n-butyl- and jY-n-octyl-cysteamine are the Determination of Reduced Glutathione. J. Biol. Chetn., characterized by absorption maxima at longer wavelengths and 233: 69-72, 1958.

JANUARY 1967 135

4. Avi-Dor, Y., and Mager, J. A Spect rophotometric Method for 16. Kalkwarf, D. R. Chemical Protection from Radiation Effects. Determination of Cysteine and Related Compounds. Ibid., Nucleonics, 18: 76-81, 1960. m: 249-58, 1956. 17. Kelley, J. J., Hamilton, N. F., and Friedman, O. M. Studies 5. Bacq, Z. M. Chemical Protection Against Ionizing Radiation. on Latent Derivatives of Aminoethanethiols as Potentially Springfield, 111.: Charles C Thomas, 1965. Selective Cytoprotectants. III. Reactions of Cysteamine-S- 6. Balabukha, V. S. (ed.). Chemical Protection of the Body sulfate in Biologic Media. Cancer Res., 27: 143-47, 1967. Against Ionizing Radiation. New York: Macmillan Co., 1963. 18. Kelley, J. J., Herrington, K. A., Ward, S. P., Meister, A., and 7. Bergmann, E. D., and Mielewitz, A. Studies on Organic Friedman, O. M. Studies on Latent Derivatives of Amino Fluorine Compounds. Part XXX. Some Reactions of Fluoro- ethanethiols as Potentially Selective Cytoprotectants. II. pyruvic Acid. J. Chem. Soc., 3736-39, 1963. In Vivo Distribution of Cysteamine Liberated in Rat Tissues. 8. Boyer, P. D. Spectrophotometric Study of the Reactions of Cancer Res., 27: 137-42, 1967. Protein Sulfhydryl Groups with Organic Mercurials. J. Am. 19. Peters, R. A., and Hall, R. J. Note upon the Reaction of Fluo- Chem. Soc., 76: 4331-13, 1954. ropyruvate with someâ€”SH Substances. Biochim. Biophys. 9. Cima, L., and Pozza, F. The Influence of Radioprotective Acta, 26:433-34,1957. Compounds on the Toxicity of Nitrogen Mustard. Rie. Sci., 20. Pihl, A., and Eldjarti, L. Pharmacological Aspects of Ionizing 30: 680-85, 1960. Radiation and of Chemical Protection in Mammals. Phar 10. Cleland, W. W. Dithiothreitol, a New Protective Reagent for macol. Rev., 10: 437-74, 1958. SH Groups. Biochemistry, 3: 480-82, 1964. 21. Therkelsen, A. J. Studies of the Mechanism of the Protective 11. Contractor, S. F. Protection Against Nitrogen Mustard by Action of Sulfhydryl Compounds and Amines Against Nitro Cysteine and Related Substances Investigated Using [3H] gen Mustard (HN2) and Roentgen Irradiation in Mice. Bio Methyl-di-(2-chloroethyl) Amine. Biochem. Pharmacol., 13: chem. Pharmacol., /: 258-66, 1958. 821-32, 1963. 22. Thomson, J. F. Radiation Protection in Mammals. London: 12. Ellman, G. L. Tissue Sulfhydryl Groups. Arch Biochem. Reinhold Publishing Corporation, 1962. Biophys., 82: 70-77, 1959. 23. Vergroesen, A. J., Budke, L., and Cohen, J. A. Factors In 13. Grunert, II. R., and Phillips, P. II. A Modification of the fluencing the Radioprotection of Tissue Culture Cells by Nitroprusside Method of Analysis for Glutathione. Ibid., 30: Sulfhydryl Compounds. Nature, 204: 246-47, 1964. 217-25, 1951. 24. Vergroesen, A. J., Budke, L., and Vos, O. Protection of Tissue- 14. Hermann, P. Zur Reaktion von Halogenbrenztrabensaure mit Culture Cells Against Ionizing Radiation. III. Influence of Thiolaminen. Chem. Ber., 94: 442-45, 1961. Anoxia on the Radioprotection of Tissue-Culture Cells by 15. Herrington, K. A., Small, C. J., Meister, A., and Friedman, Cysteamine. Intern. J. Radiation Biol., 6: 117-26, 1963. O. M. Studies on Latent Derivatives of Aminoethanethiols as 25. Yakovlev, V. G., and Isupova, L. S. Mechanism of the Protec Potentially Selective Cytoprotectants. IV. Enzymatic Hy tive Action of Certain Thiol Compounds. In: V. S. Balabukha drolysis of Cysteamine-S-phosphate. Cancer Res., 37: 148-51, (ed.), Chemical Protection of the Body Against Ionizing Radi 1967. ation, p. 39. New York: Macmillan Co., 1963.

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1967 American Association for Cancer Research. Studies on Latent Derivatives of Aminoethanethiols as Potentially Selective Cytoprotectants: I. A Spectrophotometric Assay for Aminoethanethiols in Biologic Media

Katherine A. Herrington, Katherine Pointer, Alton Meister, et al.

Cancer Res 1967;27:130-136.

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