Brevetoxin forms covalent DNA adducts in rat lung following intratracheal exposure.

Karenia brevis, formerly Gymnodinium breve, a brevetoxin-producing dinoflagellate, is recognized as the major harmful algae forming the red tide blooms in the Gulf of Mexico (Davis 1948). Two classes of brevetoxins were originally isolated from K. brevis by high pressure liquid chromatography (Risk et al. 1979). Structural analysis of these fractions identified two backbone structures, containing either 11 (brevetoxin-B) or 10 (brevetoxin-A) fused cyclic ether rings (Lin et al. 1981; Shimizu et al. 1986). Each backbone structure has similar functional groups and is subject to similar metabolic modifications (Wang et al. 2004). Brevetoxins bind to voltage-gated sodium channels to alter gating transitions interfering with the sensitive movements that transition the channel’s closed, open, and inactivated states (Catterall and Risk 1981). The initial actions of brevetoxins lead to nerve activation; within a few seconds of nerve activation, transient repetitive neuronal discharges are followed by action potential depression and eventually by a complete blockade of neuronal excitability (Huang et al. 1984). The direct consumption of brevetoxin-laden shellfish causes the food poisoning referred to as neurotoxic shellfish poisoning (NSP) in humans (McFarren et al. 1965). NSP has become a rare phenomenon, even with the frequent occurrence of red tide events, in large part due to effective monitoring and management of shellfish harvest. However, inhalation of airborne brevetoxins at beaches remains common, and the occupational and recreational hazards have been the subject of recent investigations (Backer et al. 2003, 2005). Experimental exposure of brevetoxin aerosols was first demonstrated by Woodcock (1948), and in the last decade several studies have characterized the toxicokinetics and adverse effects of brevetoxins after intratracheal exposure of rats or sheep (Abraham et al. 2005; Benson et al. 1999). During this time, various animal and tissue explant models have been used to discriminate the molecular actions of brevetoxins on voltage-gated sodium channels in lung smooth muscle, autonomic nerve, and central respiratory centers (Ramsdell 2008). Exposure to brevetoxin-containing aerosols has also been shown to cause severe allergic reactions in asthmatic patients (Fleming et al. 2007; Kirkpatrick et al. 2006), but to date the adverse effects of direct exposure to the respiratory tract have not been found to be severe in healthy subjects. A second potentially adverse mode of brevetoxin action may be oxidative damage as result of brevetoxin metabolism. Previous research has shown that the brevetoxin B (PbTx-2) is widely metabolized after systemic administration to rats, after exposure to freshly isolated rat hepatocytes, and after treatment with cDNA-expressed rat cytochrome P-450 enzymes (Radwan and Ramsdell 2006; Radwan et al. 2005). Metabolic reactions were observed on the A-ring, H-ring, and R-group of the terminal K-ring. They included hydrolysis (A-ring), epoxidation (H-ring), oxidation (R-group aldehyde), reduction (R-group aldehyde and α, β-unsaturated carbon), and peptide conjugation (R-group α,β-unsaturated aldehyde) (Figure 1). The epoxide metabolites are of interest because epoxides are highly reactive intermediates that react with nucleophilic sites to form covalent adducts with cellular macromolecules such as DNA and proteins (Koskinen and Plna 2000). Figure 1 Schematic diagram showing the intermediary metabolites generated after in vitro metabolic bioactivation pathways of brevetoxin-B in rat liver cells. The large variability of phase I or II enzymes among animal species and their target organs defines a key question concerning organ-specific toxicity. Whether the actual target has the capacity to activate (or efficiently deactivate) chemicals is a key factor in susceptibility to toxin adduction. Lung is a target organ for the toxicity of most inhaled toxins. Occupational, accidental, or prolonged pulmonary exposure to elevated concentrations of brevetoxins is now the primary route for toxicity in humans during red tide blooms. Yet the mechanism by which lung cells metabolize brevetoxin to reduce its potential toxicity is still unknown. Results of several studies indicate that a variety of xenobiotic-metabolizing cytochromes [such as cytochrome P450 (CYP) 1A and CYP3A families] in the human lung and in lung-derived cell lines contributes to the in situ activation of pulmonary toxins (Castell et al. 2005). Lung cells also express phase II enzymes such as epoxide hydrolase, UGT1A (glucuronyl transferase), and GST-P1 (glutathione S-transferase), which largely act as detoxifying enzymes. Adduct formation is a key step in inducing genotoxicity as a result of exposure to chemicals and metabolites that damage nucleic acid. The present study aimed to investigate brevetoxin adduction with nucleic materials in the lungs of exposed rats and thus to provide insight into DNA adduction by brevetoxins following metabolic activation. Detection of brevetoxin–nucleic acid adduction has pharmacological and risk assessment importance and should provide a foundation for biological monitoring of human exposure to such genotoxic environmental agents.