Thiol and disulfide metabolites of the radiation protector and potential chemopreventive agent WR-2721 are linked to both its anti-cytotoxic and anti-mutagenic mechanisms of action.
The ability of the potential chemopreventive agent S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721) to protect against radiation-induced mutagenesis at the hprt locus and cell killing was studied using CHO-AA8 cells incubated for 30 min at 37 degrees C in growth medium containing its active thiol 2-[(aminopropyl)amino]ethane-thiol (WR-1065). In parallel experiments, the thiol and disulfide forms of the drug present in cells and incubation medium were determined in order to identify which, if either, of the components were associated with the observed protective effects. Treatment with 4 mM WR-1065 produced significant intracellular levels of the thiol (WRSH) and disulfide (WRSS) forms of the drug, but also caused dramatic elevation of cellular glutathione (GSH) and cysteine levels, accompanied by marked protection against 60Co gamma-photon- and neutron-induced cell killing and mutagenesis. When drug-treated cells were transferred to drug-free medium and incubated for 4 h at 37 degrees C, levels of WRSH and WRSS and protection against cell killing decreased markedly, whereas levels of GSH and cysteine and protection against mutagenesis showed little change. GSH and cysteine levels were not associated with protection against radiation-induced mutagenesis, as established by experiments performed with buthionine sulfoximine to block GSH synthesis. These data do not support the hypothesis that modulation of GSH or cysteine levels by WR-1065 is a major mechanism accounting for protection. Protection against mutagenesis was seen for cells incubated in medium with concentrations of added WR-1065 as low as 10 microM, where cellular levels of WRSH and WRSS became difficult to measure (< or = 5 microM) and no protection against cell killing was found. An unexpected observation was that cells incubated in 40 microM WR-1065 incorporated the drug much more rapidly than expected for uptake by passive diffusion and concentrated the drug to a marked degree; this indicates that a cell-mediated transport system is involved in the uptake of WR-1065 at low drug concentrations.
Grdina
D J
DJ
Center for Mechanistic Biology and Biotechnology, Argonne National Laboratory, IL 60439-4833, USA.
Shigematsu
N
N
Dale
P
P
Newton
G L
GL
Aguilera
J A
JA
Fahey
R C
RC
D004999:Amifostine / Q000378:metabolism / Q000493:pharmacokinetics* / Q000494:pharmacology*; D000818:Animals; D016587:Antimutagenic Agents / Q000378:metabolism / Q000493:pharmacokinetics* / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000378:metabolism / Q000493:pharmacokinetics* / Q000494:pharmacology*; D001711:Biotransformation; D016466:CHO Cells / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D006224:Cricetinae; D003545:Cysteine / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism* / Q000494:pharmacology*; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism; D019803:Glutathione Disulfide; D007041:Hypoxanthine Phosphoribosyltransferase / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D016296:Mutagenesis; D013438:Sulfhydryl Compounds / Q000378:metabolism* / Q000494:pharmacology*
D016587:Antimutagenic Agents; D000970:Antineoplastic Agents; D004220:Disulfides; D013438:Sulfhydryl Compounds; D007041:Hypoxanthine Phosphoribosyltransferase; D005978:Glutathione; D003545:Cysteine; D004999:Amifostine; D019803:Glutathione Disulfide