Effect-directed analysis reveals inhibition of zebrafish uptake transporter Oatp1d1 by caulerpenyne, a major secondary metabolite from the invasive marine alga Caulerpa taxifolia.

Caulerpa taxifolia is a marine alga of tropical and subtropical distribution and a well-known invasive species in several temperate regions. Its invasiveness mainly stems from the production of secondary metabolites, some of which are toxic or repellent substances. In this study we investigated the possible inhibitory effects of C. taxifolia secondary metabolites on the activity of two zebrafish (Danio rerio) uptake transporters that transport organic anions (Oatp1d1) and cations (Oct1). Both transporters were transiently transfected and overexpressed in human embryonic kidney HEK293T cells. Transport activity assays using lucifer yellow (LY) and 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP+) as model substrates were applied for the determination of Oatp1d1 and Oct1 interactors. A two-step Effect-Directed Analysis (EDA) procedure was applied for the separation and identification of compounds. We identified caulerpenyne (CYN) as the major metabolite in C. taxifolia and reveal its potent inhibitory effect towards zebrafish Oatp1d1 as well as weak effect on zebrafish Oct1 transport. The observed effect was confirmed by testing CYN purified from C. taxifolia, resulting in an IC50 of 17.97 µM, and a weak CYN interaction was also determined for the zebrafish Oct1 transporter. Finally, using Michaelis-Menten kinetics experiments, we identified CYN as a non-competitive inhibitor of the zebrafish Oatp1d1. In conclusion, this study describes a novel mechanism of biological activity in C. taxifolia, shows that CYN was a potent non-competitive inhibitor of zebrafish Oatp1d1, and demonstrates that EDA can be reliably used for characterization of environmentally relevant complex biological samples.

Chemical indicators of anthropogenic impacts in sediments of the pristine karst lakes.

The anthropogenic impact on the pristine karst lakes was investigated using combination of specific parameters, including multielemental analysis of major inorganic constituents (Al, K, Fe) and trace metals (Li, Ag, Cd, Sn, Pb, Bi, Cr, Co, Ni, Cu, Zn and Sb), polycyclic aromatic hydrocarbons (PAHs) and anionic surfactants of linear alkylbenzene sulfonate (LAS) type. The study was performed in the Plitvice Lakes National Park, situated in a sparsely populated area of the northwestern Dinarides, central Croatia. Dated cores of recent sediments from the two biggest lakes, Lake Prosce and Lake Kozjak, were analysed for the selected contaminants using highly specific methods, involving inductively coupled plasma mass spectrometry (ICP/MS), gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). The concentration of inorganic constituents reflected primarily the geological background of the area as well as geomorphological and geochemical characteristics of the Plitvice Lakes. Due to the higher terrigenous input, the concentration of all elements was significantly higher in the Lake Prosce. The concentration of toxic metals was relatively low in both lakes, except for Cd (>1 mg kg(-1)) and Pb (up to 40 mg kg(-1)). The vertical profiles of these metals suggested that elevated concentrations of Cd were of natural origin, derived from the erosion of the Jurassic dolomite bedrock, while Pb was predominately of recent anthropogenic origin. A similar distribution pattern, suggesting the same prevailing mechanism of input, was observed for pyrolytic PAHs. The characteristic diagnostic PAH ratios revealed that higher PAHs prevailingly originated from the combustion of biomass and fossil fuels. LAS, which represent highly specific indicators of untreated wastewaters, were found in rather high concentrations in the recent sediment layers (up to 4.7 mg kg(-1)), suggesting that contaminated household and hotel wastewaters reach the Lakes, very probably by leaking through the porous karst rocks.