Aquatic Toxicology 78 (2006) 66–73 Toxin release in response to oxidative stress and programmed cell death in the cyanobacterium Microcystis aeruginosa Cliff Ross a,∗ , Lory Santiago-V´ azquez b , Valerie Paul a a Smithsonian Marine Station at Fort Pierce, 701 Seaway Drive, Ft. Pierce, FL 34949, United States b Department of Chemistry and Biochemistry, Florida Atlantic University, 777 Glades Road, Boca Raton, FL 33431, United States Received 7 December 2005; received in revised form 14 February 2006; accepted 15 February 2006 Abstract An unprecedented bloom of the cyanobacterium Microcystis aeruginosa K¨ utz. occurred in the St. Lucie Estuary, FL in the summer of 2005. Samples were analyzed for toxicity by ELISA and by use of the polymerase chain reaction (PCR) with specific oligonucleotide primers for the mcyB gene that has previously been correlated with the biosynthesis of toxic microcystins. Despite the fact that secreted toxin levels were relatively low in dense natural assemblages (3.5 gl -1 ), detectable toxin levels increased by 90% when M. aeruginosa was stressed by an increase in salinity, physical injury, application of the chemical herbicide paraquat, or UV irradiation. The application of the same stressors caused a three-fold increase in the production of H 2 O 2 when compared to non-stressed cells. The application of micromolar concentrations of H 2 O 2 induced programmed cell death (PCD) as measured by a caspase protease assay. Catalase was capable of inhibiting PCD, implicating H 2 O 2 as the inducing oxidative species. Our results indicate that physical stressors induce oxidative stress, which results in PCD and a concomitant release of toxin into the surrounding media. Remediation strategies that induce cellular stress should be approached with caution since these protocols are capable of releasing elevated levels of microcystins into the environment. © 2006 Elsevier B.V. All rights reserved. Keywords: Caspase; Cellular stress; Hydrogen peroxide; Microcystis aeruginosa; Microcystins; Programmed cell death 1. Introduction The prevalence of toxic cyanobacterial blooms in the state of Florida has received considerable attention in the past 20 years since first being recorded in Lake Okeechobee and Lake Istokpoga (Carmichael, 1992; Burns et al., 2002). Long-term studies in three major marine ecosystems (Florida Bay, Indian River Lagoon, and the Suwannee Estuary) and five freshwa- ter ecosystems (Lake Okeechobee, the St. Johns River, Lake Griffin, the Rainbow River, and the Suwannee River) have pro- vided informative data on trophic states, water exchange rates, light availability, and measurements of growth-limiting nutri- ents of planktonic assemblages (Phlips et al., 1993, 2002; Phlips, 2002). Aside from increasing anthropogenic input, cyanobacte- rial blooms can form in eutrophic water masses simply from abiotic natural sources such as surface or ground water input ∗ Corresponding author. Tel.: +1 772 465 6630; fax: +1 772 461 8154. E-mail address: Ross@sms.si.edu (C. Ross). from naturally nutrient-rich sediments (Phlips et al., 2002). As urbanization and agricultural expansion have led to increases in nutrient effluxes into Florida water systems, cyanobacteria have been quite opportunistic in exploiting these available nutrients (Canfield et al., 1989). Lake Okeechobee is one of best known sources of cyanobac- terial blooms in the United States (Phlips et al., 2002). The Florida hurricane season of 2004 resulted in a major sediment disturbance to the lake resulting in the release of high levels of inorganic phosphorus. Periods of high rainfall followed by the release of water from district canals most likely resulted in a washout of freshwater cyanobacteria into the St. Lucie River (SLR) Estuary. The SLR Estuary is one of the largest brackish water systems on the east coast of Florida. Encompassing 780 square miles, the estuary represents an indispensable asset both biologically and economically. Sporadic colonies of Microcystis aeruginosa were first iden- tified in the SLR Estuary in June 2005. By July 2005, M. aeruginosa abruptly emerged as a dense bloom covering the St. Lucie waterway within both St. Lucie and Martin counties, Florida (Fig. 1A and B). 0166-445X/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2006.02.007