Identification of mutagenic electrophiles in chlorinated water and characterization of nucleoside adducts formed in DNA by phenanthrene-9,10-oxide

This research has been directed towards identifying mutagens that are present in chlorinated drinking water and investigating the addition reactions of two mutagens that may be present, 3-chloro-4-(dichloromethyl)-5-hydroxy-2-(5H)-furanone (MX) and phenanthrene-9,10-oxide (PhO), with nucleophiles such as thiols or DNA. An immobilized nucleophile, 5-mercapto-2-nitrobenzoic acid (MNB) attached by an ester linkage to a polysaccharide coating of controlled pore glass, was used to trap the unknown mutagenic electrophiles present in chlorinated drinking water, permitting the removal of the bulk of noncovalently bound matrix by washing. This was followed by the release, isolation and identification of the trapped unknowns. Overall recovery of model electrophiles from this process was up to 67%. MX was reacted with the nucleophile 4-nitrothiophenol (NTP) in order to investigate the types of addition products that might be formed. Attempts were made to determine the stability of MX in solution, optimize MX-NTP reaction conditions and isolate the addition products using HPLC. Characterization of the DNA adducts formed by a second mutagenic electrophile, phenanthrene-9,10-oxide (PhO), was performed. The reaction of PhO with DNA formed covalent nucleoside adducts at the exocyclic amino groups of 2$\sp\prime$-deoxyadenosine (dA, 42.9%) and 2$\sp\prime$-deoxyguanosine (dG, 52.2%), in addition to a minor 2$\sp\prime$-deoxycytidine (dC, 4.9%) adduct. In order to determine unequivocally the structures of the chiral pairs of cis and trans dA adducts formed from PhO, the adducts were chemically synthesized from chiral cis and trans 9-amino-10-hydroxy-9,10-dihydrophenanthrene of known configuration. Assignment of absolute configurations of the four PhO-dA adducts formed in DNA was accomplished by comparison of their HPLC retention times and circular dichroism spectra with those of synthesized adducts.