Chronic environmentally relevant levels of Simvastatin induces non-monotonic responses in Zebrafish (Danio rerio)

Abstract

Simvastatin (SIM), a hypocholesterolaemic compound, is among the most prescribed pharmaceuticals for cardiovascular disease prevention worldwide. Several studies have shown that acute exposure to SIM is able to produce multiple adverse effects in aquatic organisms. However, uncertainties still remain regarding the chronic effects of SIM in aquatic ecosystems. Therefore, the present study aimed to investigate the effects of SIM in the model freshwater teleost zebrafish (Danio rerio) following a chronic exposure (90 days) to environmentally relevant concentrations ranging from 8 ng/L to 1000 ng/L. This study used a multi-parametric approach integrating distinct ecological-relevant endpoints, i.e. survival, growth, reproduction and embryonic development, with biochemical markers (cholesterol and triglycerides). Furthermore, Real Time PCR was used to analyse the transcription levels of key genes involved in the mevalonate pathway (hmgcra, cyp51, and dhcr7). Globally, SIM induced several non-monotonic dose-responses; embryonic development, biochemical and molecular markers, were significantly impacted in the low-intermediate concentrations, 40 ng/L and 200 ng/L, whereas no effects were recorded for the highest tested SIM levels (1000 ng/L). Taken together, these findings expand our understanding of statins effects in teleost’s, demonstrating significant impacts at environmentally relevant concentrations. The findings highlight the importance of addressing the effects of chemicals under chronic low-level concentrations.Graphical abstractHighlightsSeveral uncertainties exist regarding simvastatin mode of action in non-target organismsThis work integrates D. rerio multi-level responses after long-term exposure to simvastatinSimvastatin impacted cholesterol/triglycerides levels and transcript levels of genes related to mevalonate pathway.Parental exposure to simvastatin induced offspring embryonic malformations.Embryonic abnormalities, biochemical and molecular data did follow a non-monotonic curve.

Chronic exposure to environmentally relevant levels of simvastatin disrupts zebrafish brain gene signaling involved in energy metabolism

“…The concentration of SIM in the aquaria was analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) once during the bioassay, at T0h after the first SIM addition of the day and 8 hours later, before the second SIM addition of the day (T8h). At T8h, the minimum and maximum decay of SIM was 27.9% and 57.3% in the 8 and 1000 ng/L SIM treatments, respectively (see Barros et al 2018).…”

“…The assay was carried out at the Aquatic Animal Facilities of the Interdisciplinary Centre A broad description of the experimental conditions of the chronic toxicity test was presented in the first part of this study, which aimed at evaluating the effects of SIM in ecological relevant endpoints (morphometric endpoints, reproduction and embryonic development), biochemical (cholesterol and triglyceride levels in liver) and molecular markers (transcription of genes related to the cholesterol branch of the mevalonate pathway in the liver) (Barros et al, 2018). Briefly, the experiment consisted of five treatments with two replicates each: the control (0.0002% acetone), and four increasing nominal concentrations of SIM (8,40,200, and 1000 ng/L, prepared in 0.0002% acetone).…”

“…Brains from both males and females (n = 8/sex) from each treatment were individually sampled, preserved in RNALater and stored at -80 °C until gene expression analysis. Liver was also sampled and used in the first part of the study (Barros et al, 2018).…”

“…Consequently, fish can be continually exposed to SIM, and statins in general, in their natural habitats and several studies have reported multiple detrimental effects of SIM in fish (Crespo and Solé, 2016;Cunha, Santos and Ferreira, 2016;Cunha et al, 2017;Thorpe et al, 2004). The effects described in zebrafish (Danio rerio) include morphological and physiological alterations in embryos, movement and heartbeat reduction, disruption of detoxification mechanisms, alteration in the transcription of genes related to the cholesterol biosynthesis pathway, as well as, genes encoding nuclear receptors implicated in lipid homeostasis (RXR) and development (RAR) (Al-Habsi, Massarsky and Moon, 2016;Barros et al, 2018;Campos et al, 2016;Cunha, Santos and Ferreira, 2016;Ribeiro et al, 2015). However, despite the diversity of studies regarding the adverse effects of statins in aquatic vertebrates, very few mechanistic studies are available regarding the effects of statins besides the cholesterol branch of the mevalonate 4 (MVA) pathway.…”

“…This study is a component of a larger investigation that consisted on the assessment of the effects of environmentally relevant concentrations of SIM (8ng/L to 1000ng/L) in zebrafish following a chronic exposure of 90 days. Here we focus on the effects of SIM in the regulation of molecular pathways involved in the brain energy metabolism, while key ecological responses (survival, growth, reproduction and embryonic development), biochemical and molecular markers of lipid homeostasis in liver were reported in a previous publication - Barros et al (2018).…”