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Attenuation of Cytogenetic Damage by 2-Mercaptoethanesulfonate in Cultured Human Lymphocytes Exposed to Cyclophosphamide and Its Reactive Metabolites¹

Department of Genetic Toxicology, Chemical Industry Institute of Toxicology, Research Triangle Park, North Carolina 27709

Cyclophosphamide (CP) is metabolized to the reactive intermediates, phosphoramide mustard (PAM) and acrolein (AC), which have generally different molecular binding targets. Sodium-2-mercaptoethanesulfonic acid (MESNA) has been used clinically to alleviate hemorrhagic cystitis caused by CP chemotherapy, has exhibited anticarcinogenic effects in rats exposed to CP during a long-term bioassay, and acts in the urogenital tract by reacting with 4'-OH-CP and AC. The purpose of this study was to: (a) compare the relative abilities of PAM and AC to induce cytogenetic damage and cytotoxicity in cultured human lymphocytes; (b) assess the efficacy of MESNA to attenuate the cytogenetic damage and cytotoxicity induced by CP, AC, PAM, and diethyl-4'-hydroperoxycyclophosphamide (DEHP-CP), an activated AC-generating compound; and (c) determine if concanavalin A-stimulated T-lymphocytes, which differentiate into suppressor cells upon lectin activation, exhibit any heightened cytogenetic sensitivity compared to a variety of cultured mammalian cells during exposure to PAM or AC as reported by other investigators.

Purified mononuclear leukocytes were stimulated with concanavalin A and exposed to CP (0.5–2.0 mM) without an exogenous activation system, AC (0.001–40.0 µM), PAM (0.0014–27.1 µM), or DEHP-CP (0.1–100.0 µM) in the presence or absence of MESNA (1, 5, or 10 mM). All four compounds induced significant concentration-related increases in the SCE frequency, but only PAM was clastogenic. On an induced SCE/µM basis, PAM was about 130 and 193 times more potent than were DEHP-CP and AC, respectively. MESNA protected against the cytogenetic damage and cytotoxicity induced by the four compounds, but it was particularly effective against AC and DEHP-CP by abolishing SCE induction completely. SCEs and chromosome aberrations differed considerably in their induction kinetics in lymphocytes exposed to PAM, and these disparities suggested an uncoupling of the two phenomena. Although SCE induction was not consistently associated with cytotoxicity with the four agents, chromosome aberration induction coincided with an inhibition of cell cycle kinetics in PAM-treated cells. The exceptionally high SCE frequency of up to 21 times baseline in cells exposed to PAM indicates that T-suppressor lymphocytes stimulated with concanavalin A may be particularly sensitive to the DNA-damaging effects of PAM. Finally, these data suggest that the anticarcinogenicity of MESNA correlates with its ability to attenuate cytogenetic damage and cytotoxicity induced by reactive CP metabolites.

¹ This research was funded by the Chemical Industry Institute of Toxicology, a privately funded institute, and was presented at the Environmental Mutagen Society meetings on February 19–23, 1984 (Montreal, Canada) and February 25–March 1, 1985 (Las Vegas, NV).

² To whom requests for reprints should be addressed, at Bristol Laboratories, Pathology and Toxicology Department, P. O. Box 4755, Syracuse, NY 13221.

³ Current address: Environmental Health Research and Testing, Inc., P. O. Box 12199, Research Triangle Park, NC 27709.

Received 6/28/85. Revised 9/17/85. Accepted 10/ 4/85.