Zebrafish neurotoxicity from aphantoxins—cyanobacterial paralytic shellfish poisons (PSPs) from Aphanizomenon flos‐aquae DC‐1

Abstract

Aphanizomenon flos-aquae (A. flos-aquae), a cyanobacterium frequently encountered in water blooms worldwide, is source of neurotoxins known as PSPs or aphantoxins that present a major threat to the environment and to human health. Although the molecular mechanism of PSP action is well known, many unresolved questions remain concerning its mechanisms of toxicity. Aphantoxins purified from a natural isolate of A. flos-aquae DC-1 were analyzed by high-performance liquid chromatography (HPLC), the major component toxins were the gonyautoxins1 and 5 (GTX1 and GTX5, 34.04% and 21.28%, respectively) and the neosaxitoxin (neoSTX, 12.77%). The LD50 of the aphantoxin preparation was determined to be 11.33 μg/kg (7.75 μg saxitoxin equivalents (STXeq) per kg) following intraperitoneal injection of zebrafish (Danio rerio). To address the neurotoxicology of the aphantoxin preparation, zebrafish were injected with low and high sublethal doses of A. flos-aquae DC-1 toxins 7.73 and 9.28 μg /kg (5.3 and 6.4 μg STXeq/kg, respectively) and brain tissues were analyzed by electron microscopy and RT-PCR at different timepoints postinjection. Low-dose aphantoxin exposure was associated with chromatin condensation, cell-membrane blebbing, and the appearance of apoptotic bodies. High-dose exposure was associated with cytoplasmic vacuolization, mitochondrial swelling, and expansion of the endoplasmic reticulum. At early timepoints (3 h) many cells exhibited characteristic features of both apoptosis and necrosis. At later timepoints apoptosis appeared to predominate in the low-dose group, whereas necrosis predominated in the high-dose group. RT-PCR revealed that mRNA levels of the apoptosis-related genes encoding p53, Bax, caspase-3, and c-Jun were upregulated after aphantoxin exposure, but there was no evidence of DNA laddering; apoptosis could take place by pathways independent of DNA fragmentation. These results demonstrate that aphantoxin exposure can cause cell death in zebrafish brain tissue, with low doses inducing apoptosis and higher doses inducing necrosis.

Extracellular polymeric substance from Aphanizomenon flos-aquae induces apoptosis via the mitochondrial pathway in A431 human epidermoid carcinoma cells

“…Numerous bioactive extracellular polymeric substances with noteworthy functional properties have been discovered in marine organisms, and studies that focus on marine natural products, particularly marine algae EPS, are increasingly attracting worldwide attention (8,13). Natural products that have been isolated from cyanobacteria, as well as their derivatives, have been proven to be a valuable chemical resource for finding promising drugs that could assist the prevention and treatment of cancer (14).…”

“…According to methods of Zhang (13), with minor modifications. A. flos-aquae were cultured in 50 ml sterilized BG11 medium for 30 days at 25±1˚C, with a 12 h light/dark cycle under a photon irradiance of 40 µE/m 2 /s, which was provided by daylight fluorescent lamp.…”

Antioxidative responses in zebrafish liver exposed to sublethal doses Aphanizomenon flos-aquae DC-1 aphantoxins

“…In zebrafish larvae, PSPs are reported to affect development, increase dysmorphosis, and lead to mortality (Lefebvre et al, 2004). In adults, PSPs accumulate in muscle (Bakke et al, 2010), and apoptotic gene upregulation was reported in zebrafish liver (Zhang et al, 2011(Zhang et al, , 2013a. In addition, PSPs also adversely affect swimming ability in both larval and adult fish (Samson et al, 2008;Zhang et al, 2013b).…”

“…Waterblooms predominated by A. flos-aquae DC-1 have been recorded in Dianchi Lake, Yunnan Province, China, in most years in recent decades, reflecting environmental conditions favorable for bloom development (Wu et al, 2010). The prevalent organism in these waterblooms has been shown to secrete neurotoxic PSPs (Liu et al, 2006) which are known to damage the morphological ultrastructure and biochemical parameters of zebrafish (Danio rerio) liver and adversely affect swimming capacity (Zhang et al, 2013a(Zhang et al, , 2013b(Zhang et al, , 2013c. The Dianchi Lake is an important source of freshwater for drinking, agriculture, and tourism activities, serving a population of over 5 million in the vicinity of Kunming city, Yunnan Province (Liu et al, 2006;Zhang et al, 2013a).…”

“…The prevalent organism in these waterblooms has been shown to secrete neurotoxic PSPs (Liu et al, 2006) which are known to damage the morphological ultrastructure and biochemical parameters of zebrafish (Danio rerio) liver and adversely affect swimming capacity (Zhang et al, 2013a(Zhang et al, , 2013b(Zhang et al, , 2013c. The Dianchi Lake is an important source of freshwater for drinking, agriculture, and tourism activities, serving a population of over 5 million in the vicinity of Kunming city, Yunnan Province (Liu et al, 2006;Zhang et al, 2013a). The waterblooms are therefore considered to pose a significant threat to human health and environmental safety, and the danger of paralytic shellfish poisoning has had a negative impact on the local economy and other developments.…”