[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 —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 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1967 American Association for Cancer Research. Latent Derivatives of Aminoethanethiols. I 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 —18°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 spectro- photometric 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 26°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 4°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) cysta- mine 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 Détermination of Cysteamine Dr. W. W. Cleland, University of Wisconsin, of the tetraacetate Samples containing cysteamine (0.1-0.4 /¿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 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1967 American Association for Cancer Research. K. A. Herrington, K. Pointer, A. Meister, and O. M. Friedman TABLE 1 EFFECTOFpH ONTHECYSTEAMINE-FLUOROPYRUVATEREACTION" BufferTris-HCl»Glycine-NaOHPotassium