The peroxidation of polyunsaturated lipids generates a range of substances that possess DNA damaging potential. This includes lipid hydroperoxides and various species that contain unpaired electrons, such as the alkoxyl and peroxyl radicals. In addition, a range of genotoxic carbonyl-containing compounds are formed, such as malondialdehyde, various 4-hydroxy-2-alkenals such as 4-hydroxynonenal and a number of 2-alkenals. It has previously been assumed that the antioxidants and electrophile scavenging enzymes existing in mammalian cells effectively protect the genetic material against these substances. However, thanks to recent analytical advances in the detection of low levels of DNA adducts, it is now evident that DNA adducts formed from a range of lipid peroxidation products are abundant in both rodent and human genomes. This suggests that the cellular defence system is not 100% efficient and that a proportion of endogenously produced lipid peroxidation products escape detoxification and cause DNA damage. This review surveys the genotoxic properties of the major classes of lipid peroxidation products, focusing on their chemistry of DNA adduction, the mutagenic properties of such damage and the evidence that it occurs in intact biological systems. Furthermore, avenues of future research that will clarify the significance of such damage to spontaneous mutagenesis and carcinogenesis are proposed and discussed.

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Cylindrospermopsin (CYN) is a cyanobacterial toxin found in drinking-water sources world wide. It was the likely cause of human poisonings in Australia and possibly Brazil. Although CYN itself is a potent protein synthesis inhibitor, its acute toxicity appears to be mediated by cytochrome p-450 (CYP450)-generated metabolites. CYN also induces genotoxic effects both in vitro and in vivo, and preliminary evidence suggests that tumors are generated by oral exposure to CYN. To understand the role of CYP450-activated CYN metabolites on in vitro genotoxicity, this study quantified the process in primary mouse hepatocytes using the COMET assay in both the presence and absence of CYP450 inhibitors known to block acute CYN cytotoxicity. CYN was cytotoxic at concentrations above 0.1 microM (EC50 = 0.5 microM) but produced significant increases in Comet tail length, area, and tail moment at 0.05 microM and above; hence genotoxicity is unlikely to be secondary to metabolic disruption due to toxicity. The CYP450 inhibitors omeprazole (100 microM) and SKF525A (50 microM) completely inhibited the genotoxicity induced by CYN. The toxin also inhibits production of glutathione (GSH), a finding confirmed in this study. This could potentiate cytotoxicity, and by implication genotoxicity, via reduced reactive oxygen species (ROS) quenching. The lipid peroxidation marker, malondialdehyde (MDA) was quantified in CYN-treated cells, and the effect of the reduced glutathione (GSSG) reductase (GSSG-rd.) inhibitor 1,3-bis(chloroethyl)-l-nitrosourea (BCNU) on both MDA production and lactate dehydrogenase (LDH) leakage was examined. MDA levels were not elevated by CYN treatment, and block of GSH regeneration by BCNU did not affect lipid peroxidation or cytotoxicity. It therefore seems likely that CYP450-derived metabolites are responsible for both the acute cytotoxicity and genotoxicity induced by CYN.

The toxicology of the cyanobacterial alkaloid cylindrospermopsin (CYN), a potent inhibitor of protein synthesis, appears complex and is not well understood. In exposed mice the liver is the main target for the toxic effects of CYN. In this study primary mouse hepatocyte cultures were used to investigate the mechanisms involved in CYN toxicity. The results show that 1-5 microM CYN caused significant concentration-dependent cytotoxicity (52%-82% cell death) at 18 h. Protein synthesis inhibition was a sensitive, early indicator of cellular responses to CYN. Following removal of the toxin, the inhibition of protein synthesis could not be reversed, showing behavior similar to that of the irreversible inhibitor emetine. In contrast to the LDH leakage, protein synthesis was maximally inhibited by 0.5 microM CYN. No protein synthesis occurred over 4-18 h at or above this concentration. Inhibition of cytochrome P450 (CYP450) activity with 50 microM proadifen or 50 microM ketoconazole diminished the toxicity of CYN but not the effects on protein synthesis. These findings imply a dissociation of the two events and implicate the involvement of CYP450-derived metabolites in the toxicity process, but not in the impairment of protein synthesis. Thus, the total abolition of protein synthesis may exaggerate the metabolite effects but cannot be considered a primary cause of cell death in hepatocytes over an acute time frame. In cell types deficient in CYP450 enzymes, protein synthesis inhibition may play a more crucial role in the development of cytotoxicity.

Acrolein is a highly toxic aldehyde involved in a number of diseases as well as drug-induced toxicities. Its pronounced toxicity reflects the readiness with which it forms adducts in proteins and DNA. As a bifunctional electrophile, initial reactions between acrolein and protein generate adducts containing an electrophilic center that can participate in secondary deleterious reactions (e.g., cross-linking). We hypothesize that inactivation of these reactive protein adducts with nucleophilic drugs may counteract acrolein toxicity. Because we previously observed that 1-hydrazinophthalazine (hydralazine) strongly diminishes the toxicity of the acrolein precursor allyl alcohol, we explored the possibility that hydralazine targets reactive acrolein adducts in proteins. We report that hydralazine abolished the immunoreactivity of an acrolein-modified model protein (bovine serum albumin), but only if the drug was added to the protein within 30 min of commencing modification by acrolein. The ability of a range of carbonyl-trapping drugs to interfere with "early" events in protein modification strongly correlated with their protective potencies against allyl alcohol toxicity in hepatocytes. In mass spectrometry studies using a model lysine-containing peptide, hydralazine rapidly formed hydrazones with Michael adducts generated by acrolein. Using an antibody raised against such ternary drug-acroleinprotein complexes in Western blotting experiments, clear adduct-trapping was evident in acrolein-preloaded hepatocytes exposed to cytoprotective concentrations of hydralazine ranging from 2 to 50 M. These novel findings begin to reveal the molecular mechanisms whereby hydralazine functions as an efficient "protein adduct-trapping" drug.

Several clinically relevant anticancer drugs induce genomic mutations and cell death by increasing topoisomerase II-mediated DNA breakage. To determine whether endogenous DNA damage also affects this cleavage event, the effects of abasic sites (the most commonly formed spontaneous DNA lesion) on topoisomerase II activity were investigated. The presence of 3 abasic sites/plasmid stimulated enzyme-mediated DNA breakage >6-fold, primarily by enhancing the forward rate of cleavage. This corresponds to a potency that is >2000-fold higher than that of the anticancer drug, etoposide. These findings suggest that abasic sites represent endogenous topoisomerase II poisons and imply that anticancer drugs mimic the cleavage-enhancing actions of naturally occurring DNA lesions.Topoisomerase II is the cellular target for some of the most active agents currently used for the treatment of human cancers (1-4). These drugs, which include etoposide, doxorubicin, and mitoxantrone, elicit their antineoplastic effects by a mechanism that is markedly different from that of other enzymetargeted agents. Rather than acting by inhibiting the catalytic activity of the enzyme, anticancer drugs dramatically increase levels of covalent topoisomerase II-cleaved DNA complexes that are normal, but fleeting intermediates in the catalytic cycle of the enzyme (1-4). When present at high concentrations, a portion of these transient enzyme-associated DNA breaks are converted to permanent untethered breaks during replication and transcription (5-7). Thus, these agents act as poisons and convert topoisomerase II, an essential enzyme, into a physiological toxin that produces protein-linked breaks in the genome of treated cells (1-4).Consistent with the ability to generate DNA breaks, topoisomerase II-targeted drugs produce mutagenic side effects. Insertions, deletions, and chromosomal aberrations have been observed in treated cells and animals (8, 9). Furthermore, levels of sister chromatid exchange and illegitimate recombination are increased significantly (8, 9). Finally, 11q23 chromosomal translocations that produce acute myelogenous leukemia have been reported in patients following treatment with etoposide-based regimens (8, 10 -13).Approximately 80% of infant leukemias share the same 11q23 chromosomal translocations as are found in the druginduced leukemias despite the fact that the affected children have never been exposed to anticancer agents (14). Coupled with the fact that topoisomerase II poisons act by exploiting the natural ability of the enzyme to cleave DNA, this finding raises two possibilities. First, there may be cellular factors that induce topoisomerase II-mediated DNA breakage in vivo and trigger DNA recombination, mutagenesis, or cell death pathways. Second, anticancer agents targeted to this enzyme may actually represent exogenous counterparts of these factors.Since anticancer agents presumably act at the topoisomerase II/nucleic acid interface (1-4, 15), we questioned whether specific lesions generated by spontaneous DNA damage fu...

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Hydralazine strongly suppresses the toxicity of acrolein, a reactive aldehyde that contributes to numerous health disorders. At least two mechanisms may underlie the cytoprotection, both of which involve the nucleophilic hydrazine possessed by hydralazine. Under the simplest scenario, hydralazine directly scavenges free acrolein, decreasing intracellular acrolein availability and thereby suppressing macromolecular adduction. In a second "adduct-trapping" mechanism, the drug forms hydrazones with acrolein-derived Michael adducts in cell proteins, preventing secondary reactions of adducted proteins that may trigger cell death. To identify the most important mechanism, we explored these two pathways in mouse hepatocytes poisoned with the acrolein precursor allyl alcohol. Intense concentration-dependent adduct-trapping in cell proteins accompanied the suppression of toxicity by hydralazine. However, protective concentrations of hydralazine did not alter extracellular free acrolein levels, cellular glutathione loss, or protein carbonylation, suggesting that the cytoprotection is not due to minimization of intracellular acrolein availability. To explore ways whereby adduct-trapping might confer cytoprotection, the effect of hydralazine on acrolein-induced protein crosslinking was examined. Using bovine pancreas ribonuclease A as a model protein, acrolein caused rapid time-and concentration-dependent cross-linking, with dimerized protein detectable within 45 min of commencing protein modification. Lysine adduction in monomeric protein preceded the appearance of oligomers, whereas reductive methylation of protein amine groups abolished both adduction and oligomerization. Hydralazine inhibited cross-linking if added 30 min after commencing acrolein exposure but was ineffective if added after a 90-min delay. Adduct-trapping closely accompanied the inhibition of cross-linking by hydralazine. These findings suggest that cross-link blocking may contribute to hydralazine cytoprotection.