Reactions of N,N-bis(2-chloroethyl)-p-aminophenylbutyric acid (chlorambucil) with 2'-deoxyguanosine.

N,N-Bis(2-chloroethyl)-p-aminophenylbutyric acid (chlorambucil, 1) was allowed to react in the presence of 2'-deoxyguanosine (16 mM) at physiological pH (cacodylic acid, 50% base), and the reactions were followed by HPLC/MS/MS techniques. Although the predominant reaction observed was chlorambucil hydrolysis, ca. 24% of 1 reacted with different heteroatoms of the nucleoside. As expected, the principal site of 2'-deoxyguanosine alkylation was N7. Alkylation of N7 caused spontaneous depurination, and N-(7-guaninylethyl)-N-hydroxyethyl-p-aminophenylbutyric acid (5) and the corresponding N7,N7-bis-adduct (6) were the major stable dGuo derivatives. Also several other adducts were detected and tentatively identified by means of MS/MS and UV. From them, the O(6-), N1-, N(2-), and O5'-derivatives can be biologically significant. Our results shed new light on DNA modifications caused by chlorambucil, which is an important chemotherapeutic drug and a known carcinogen.

Polymyxin permeabilization as a tool to investigate cytotoxicity of therapeutic aromatic alkylators in DNA repair-deficient Escherichia coli strains.

Chlorambucil (CLB; N,N-bis(2-chloroethyl)-p-aminophenylbutyric acid) and its biologically active beta-oxidation product phenylacetic acid mustard (PAM; N,N-bis(2-chloroethyl)-p-aminophenylacetic acid) are bifunctional aromatic alkylators. CLB is in wide clinical use as an anticancer drug and also as an immunosuppressant. The chemical structures indicate that CLB and PAM are mutagenic, teratogenic and carcinogenic, but the mode of action has remained obscure. We have investigated the biological effects of CLB and PAM with DNA repair-deficient Escherichia coli strains. In contrast to MNNG (N-methyl-N'-nitro-N-nitrosoguanine), CLB and PAM were not toxic to E. coli, but permeabilization of the outer membrane of the cells through use of polymyxin B nonapeptide (PMBN) rendered them susceptible to these compounds. The importance of DNA repair, shown by reversal of damage and attenuation of the toxicity of CLB and PAM, was indicated by the susceptibility of cells lacking O(6)-methylguanine-DNA methyltransferase I and II (ada ogt). Similarly, the protective role of base excision repair (BER) was substantiated by demonstration of an even more increased susceptibility to CLB and PAM of cells lacking 3-methyladenine-DNA glycosylase I and II (alkA1 tag-1). Cells deficient in mismatch repair (mutS) appeared to be slightly more sensitive than normal cells to CLB and PAM, although no such sensitivity to MNNG was observed. This implicates the role of mismatches in CLB- and PAM-related cytotoxicity. It is generally believed that bifunctional alkylating agents, like CLB and PAM, exert their cytotoxic action via DNA cross-linking. Our results with O(6)-methyltransferase- and 3-methyladenine-DNA glycosylase-deficient cells indicate that removal of the adducts prior to the formation of cross-links is an important mechanism maintaining cell viability. We conclude that PMBN permeabilization provides a valuable tool to investigate genetically engineered E. coli cells, whose outer membrane is not naturally permeable to mutagens or other interesting compounds.

Role of thiocyanate ion in detoxification of the anticancer agent chlorambucil.

N,N-Bis(2-chloroethyl)-p-aminophenylbutyric acid (chlorambucil, 1) is an orally administrated drug widely used in the chemotherapy of chronic lymphocytic leukemia. We have recently described a new metabolic path for the decomposition of 1 in human gastric juice based on its reactions with saliva-derived thiocyanate ion. We report here our quantitative data on the reactions of thiocyanate ion with CLB in various fluid matrixes at 37 degreesC. The rate of decomposition of 1 is zero-order with respect to SCN- concentration up to 100 mM. However, thiocyanate ion reacts ca. 18 300 times faster than water with the aziridinium ion derived from 1 at neutral and acidic pH. When the SCN- concentration was greater than 10 mM, practically no N,N-bis(2-hydroxyethyl)-p-aminophenylbutyric acid, 4, the product of chlorambucil hydrolysis, could be detected. Thiocyanate ion also effectively overcompensates for the rate retardation caused by Cl-; 10 mM SCN- is enough to decrease the effect of 0.5 M chloride ion to one-half. This is an important factor in human gastric juice where the chloride ion concentration is normally high.

Detoxification of an alkylating drug, N,N-bis(2-chloroethyl)-p-aminophenylbutyric acid (chlorambucil), in human gastric juice and saliva.

N,N-Bis(2-chloroethyl)-p-aminophenylbutyric acid (chlorambucil, 1) is an orally administered drug widely used in the chemotherapy of chronic lymphocytic leukemia. It is converted in gastric juice into three stable metabolites, which were characterized as N,N-bis(2-hydroxyethyl)-p-aminophenylbutyric acid (4), N-(2-hydroxyethyl)-N-[2-(thiocyano)ethyl]-p-aminophenylbutyric+ ++ acid (5), and N,N-bis[2-(thiocyano)ethyl]-p-aminophenylbutyric acid (6). 4 is the product of chloroambucil hydrolysis, while 5 and 6 are results of the reaction of 1 with saliva-derived thiocyanate ion. The destabilizing effect of low gastric oxonium ion concentration on 1 is also demonstrated.