ON THE ROLE OF COPPER AND IRON IN DNA CLEAVAGE BY OCHRATOXIN-A - STRUCTURE-ACTIVITY-RELATIONSHIPS IN METAL-BINDING AND COPPER-MEDIATED DNA CLEAVAGE

Ochratoxin A (OTA, 1: X = Cl) is a fungal carcinogen that facilitates single-strand DNA cleavage and DNA adduction when metabolically activa ted. To determine if redox-active transition metals induce OTA-mediate d DNA damage, we have examined the toxin's ability to bind Cu(II) and Fe(III) in aqueous media and facilitate DNA cleavage in their presence using agarose gel electrophoresis and supercoiled plasmid DNA. Using fluorescence spectroscopy, 1 was found to bind Cu(II) readily at physi ological pH, while acidic conditions (pH 2.6) were employed to study F e(III) binding due to the formation of Fe-oxide precipitates at higher pH values. Structure-activity relationships employing synthetic deriv atives of 1 implied that 1 binds both Cu(II) and Fe(III) by its phenol ic oxygen, while the carboxylic acid of its phenylalanine moiety binds Cu(II), but does not appear to play a role in Fe(III) coordination at pH 2.6. In terms of metal-mediated DNA cleavage, no role for 1 could be detected in Fe-induced DNA strand scission. With Cu(II), DNA cleava ge by the 1:1 copper-bound complex of 1 could only be initiated by add ition of a suitable reducing agent (sodium ascorbate). However, 1 was found to facilitate DNA cleavage by the Cu(II) complex of 1,10-phenant hroline (Cu(OP)(2)); a prototypical Cu-mediated nuclease system that c leaves DNA upon activation by an external reducing agent. Structure-ac tivity relationships employing analogs lacking the chlorine atom, ochr atoxin B (2: X = H), and the lactone (12), indicated that the chlorine atom is essential for activity of the OTA in potentiating DNA cleavag e by Cu(OP)(2). The implications of our findings to the genotoxic prop erties of 1 are discussed.