A bioanalytical approach for assessing the effects of soil extracts from solid waste dumpsite in Calabar (Nigeria) on lipid and estrogenic signaling of fish Poeciliopsis lucida hepatocellular carcinoma-1 cells in vitro and in vivo African catfish (Clarias gariepinus).

In applying bioanalytical approaches, the aim of this study was to determine the toxicity of contaminants derived from a solid waste dumpsite in Calabar (Nigeria), by investigating the alterations of lipid and estrogen signaling pathways in Poeciliopsis lucida hepatocellular carcinoma-1 (PLHC-1) cells and compared to in vivo African catfish (Clarias gariepinus), using polar, nonpolar and elutriate extraction methods. Cells were exposed for 48 hr period to different concentrations of the contaminant extracts. The PLHC-1 cells were evaluated for lipid responses as follows adipoRed assay, retinoid x receptor (rxr), peroxisome proliferator-activated receptor isoforms (ppar-α and γ), estrogen receptor (er-α) and vitellogenin (vtg) transcripts. The lipid signaling activation was also assessed in vivo using C. gariepinus, where hepatic levels of ppar-α were determined at both transcript and functional proteins levels. Data showed variable-, extract type and concentration-specific elevations in mRNA and protein levels for lipidomic and estrogenic effects. These effects were either biphasic at low and high concentrations, depending upon extract type, or concentration-dependent elevations. In general, these toxicological responses may be attributed to soil organic and inorganic contaminants burden previously derived from the dumpsite. Thus, our data demonstrate a unique lipid and endocrine-disruptive chemical (EDC) effects of each soil extract, suggesting multiple and complex contaminant interactions in the environment and biota. Analysis of numerous soil- or sediment-bound contaminants have numerous limitations and cost implications for developing countries. Our approach provides a bioanalytical protocol and endpoints for measuring the metabolic and EDC effects of complex environmental matrices for ecotoxicological assessment and monitoring.

Effects of Exposure Timing on cyp1a Expression, PAH Elimination, and Lipid Utilization in Lumpfish Embryos Exposed to Produced Water.

Intentional discharges of produced water from oil production platforms to the marine environment contain a complex mixture of toxicants, including polycyclic aromatic hydrocarbons (PAHs). Early life stages of fish are highly sensitive to petrogenic exposure, and short-term exposure during critical periods of embryonic development may have detrimental effects on larvae health and survival. However, why different periods are more sensitive to exposure than others are not fully understood. Three identical exposure experiments (48 h, approx. 30 µg/L tPAH, sum 42 PAHs) on lumpfish (Cyclopterus lumpus) embryos were conducted where only exposure timing was varied: 0-48 h post fertilization (hpf, starting before chorion hardening), 36-84 hpf (starting after chorion hardening), and 240-288 hpf (during organogenesis). Total PAH (tPAH) uptake at the end of exposure was 5× higher when exposed during fertilization than when exposed late (during organogenesis). The first evidence of cyp1a induction in lumpfish during embryogenesis was observed after 84 hpf. Early exposure affected lipid droplet coagulation, indicating altered lipid utilization during embryogenesis. Although no significant impacts of exposure were observed on hatching success, hatching was delayed when exposed at the latest time point. This study shows that chorion properties, lipid content, biotransformation potential, and timing of produced water exposure during lumpfish embryogenesis affected PAH uptake and elimination.

Microfluidic dual picoinjection based encapsulation of hemoglobin in alginate microcapsules reinforced by a poly(L-lysine)-g-poly(ethylene glycol).

Hemoglobin (Hb) encapsulation inside polysaccharide hydrogels has been considered a possible red blood cell (RBC) surrogate in transfusiology. Here we report on the microfluidic dual picoinjection assisted synthesis of Hb encapsulated alginate-poly(L-lysine)-g-poly(ethylene glycol) beads. This process is realized by the on-chip injections of blended Hb alginate solutions in emulsified aqueous calcium chloride (CaCl2) droplets followed by a subsequent injection of an aqueous PLL-g-PEG into each emulsified aqueous droplet. The proposed fabrication approach was realized using a flow-focusing and two picoinjection sites in a single PDMS device. Aqueous CaCl2 solution was emulsified and infused with Hb-alginate solution as the squeezed droplet passed through the first picoinjection site. The injection of PLL-g-PEG to reinforce the microgel and minimize the protein leaching was realized in the second picoinjection site located downstream from the first in the same microfluidic channel. In this process, monodisperse Hb-alginate-PLL-g-PEG particles with a diameter around the size of RBCs (9 µm) were obtained with around 80% of the 7.5 mg ml-1 Hb included in the injected aqueous alginate retaining in the obtained microparticles. Microparticles with Hb loading (32.8 pg per bead) and retention (28.8 pg per bead) over a week of storage at 4 °C are in accordance with the average amount of Hb per RBC. The Hb-alginate-PLL-g-PEG microbeads fabricated in the size range of RBCs are significant for further exploration.

Estrogenicity of chemical mixtures revealed by a panel of bioassays.

Estrogenic compounds are widely released to surface waters and may cause adverse effects to sensitive aquatic species. Three hormones, estrone, 17ß-estradiol and 17α-ethinylestradiol, are of particular concern as they are bioactive at very low concentrations. Current analytical methods are not all sensitive enough for monitoring these substances in water and do not cover mixture effects. Bioassays could complement chemical analysis since they detect the overall effect of complex mixtures. Here, four chemical mixtures and two hormone mixtures were prepared and tested as reference materials together with two environmental water samples by eight laboratories employing nine in vitro and in vivo bioassays covering different steps involved in the estrogenic response. The reference materials included priority substances under the European Water Framework Directive, hormones and other emerging pollutants. Each substance in the mixture was present at its proposed safety limit concentration (EQS) in the European legislation. The in vitro bioassays detected the estrogenic effect of chemical mixtures even when 17ß-estradiol was not present but differences in responsiveness were observed. LiBERA was the most responsive, followed by LYES. The additive effect of the hormones was captured by ERα-CALUX, MELN, LYES and LiBERA. Particularly, all in vitro bioassays detected the estrogenic effects in environmental water samples (EEQ values in the range of 0.75-304 × EQS), although the concentrations of hormones were below the limit of quantification in analytical measurements. The present study confirms the applicability of reference materials for estrogenic effects' detection through bioassays and indicates possible methodological drawbacks of some of them that may lead to false negative/positive outcomes. The observed difference in responsiveness among bioassays - based on mixture composition - is probably due to biological differences between them, suggesting that panels of bioassays with different characteristics should be applied according to specific environmental pollution conditions.

Application of quantitative transcriptomics in evaluating the ex vivo effects of per- and polyfluoroalkyl substances on Atlantic cod (Gadus morhua) ovarian physiology.

Because of their global consumption and persistence, per- and polyfluoroalkyl substances (PFASs), are ubiquitously distributed in the environment, as well as in wildlife and humans. In the present study, we have employed an ex vivo organ culture technique, based on the floating agarose method, of Atlantic cod ovarian tissue to investigate the effects of three different concentrations of PFOS, PFOA (1, 5 and 25 µM) and PFNA (0.5, 5 and 50 µM), used singly and in also in combination (1×, 20× and 100×). In the 1× exposure mixture, concentrations were decided based on their proportional levels (in molar equivalents) relative to PFOS, which is the most abundant PFAS in cod liver from a 2013 screening project. To investigate the detailed underlying mechanisms and biological processes, transcriptome sequencing was performed on exposed ovarian tissue. The number of differentially expressed genes (DEGs) having at least 0.75 log2-fold change was elevated in high, compared to low and medium concentration exposures. The highest PFNA, PFOA and PFOS concentrations, and the highest (100×) mixture exposure, showed 40, 68, 1295, and 802 DEGs, respectively. The latter two exposure groups shared a maximum of 438 DEGs. In addition, they both shared the majority of functionally enriched pathways belonging to biological processes such as cellular signaling, cell adhesion, lipid metabolism, immunological responses, cancer, reproduction and metabolism. Shortlisted DEGs that were specifically annotated to reproduction associated gene ontology (GO) terms were observed only in the highest PFOS and mixture exposure groups. These transcripts contributed to ovarian key events such as steroidogenesis (star, cyp19a1a), oocyte growth (amh), maturation (igfbp5b, tgfß2, tgfß3), and ovulation (pgr, mmp2). Contrary to other PFAS congeners, the highest PFOS concentration showed almost similar transcript expression patterns compared to the highest mixture exposure group. This indicates that PFOS is the active component of the mixture that significantly altered the normal functioning of female gonads, and possibly leading to serious reproductive consequences in teleosts.

Contaminant accumulation and biological responses in Atlantic cod (Gadus morhua) caged at a capped waste disposal site in Kollevåg, Western Norway.

The aim of this study was to assess whether fish in Kollevåg, a sheltered bay on the western coast of Norway, previously utilized as a waste disposal site, could be affected by environmental contaminants leaking from the waste. Farmed, juvenile Atlantic cod (Gadus morhua) were caged for six weeks at three different locations in Kollevåg bay and at one reference location. Sediments and cod samples (bile and liver) were analyzed for polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs), brominated flame retardants (BFRs), per-and polyfluoroalkyl substances (PFASs) and polycyclic aromatic hydrocarbon (PAH) metabolites, revealing a contamination gradient at the four stations. Furthermore, hepatosomatic index (HSI) and Fulton's condition factor (CF) were significantly lower in cod caged closest to the disposal site. Levels and activities of biomarker proteins, such as vitellogenin (Vtg), metallothionein (Mt), and biotransformation and oxidative stress enzymes, including cytochrome P450 1a and 3a (Cyp1a, Cyp3a), glutathione s-transferase (Gst) and catalase (Cat), were quantified in blood plasma and liver tissue. Hepatic Cat and Gst activities were significantly reduced in cod caged at the innermost stations in Kollevåg, indicating modulation of oxidative stress responses. However, these results contrasted with reduced hepatic lipid peroxidation. Significant increases in transcript levels were observed for genes involved in lipid metabolism (fasn and acly) in cod liver, while transcript levels of ovarian steroidogenic enzyme genes such as p450scc, cyp19, 3ß-hsd and 20ß-hsd showed significant station-dependent increases. Cyp1a and Vtg protein levels were however not significantly altered in cod caged in Kollevåg. Plasma levels of estradiol (E2) and testosterone (T) were determined by enzyme immunoassay (EIA) and showed elevated E2 levels, but only at the innermost station. We conclude that the bay of Kollevåg did not fullfill adequate environmental condition based on environmental quality standards (EQSs) for chemicals in coastal waters. Following a six weeks caging period, environmental contaminants accumulated in cod tissues and effects were observed on biomarker responses, especially those involved in reproductive processes in cod ovary.

Quantitative proteomics analysis of zebrafish exposed to sub-lethal dosages of ß-methyl-amino-L-alanine (BMAA).

The non-protein amino acid ß-methylamino-L-alanine (BMAA) is a neurotoxin present in microalgae and shown to accumulate in the food web. BMAA has been linked to the complex neurodegenerative disorder of Guam and to increased incidents sporadic ALS. Two main neurotoxic routes are suggested; an excitotoxic by acting as an agonist towards glutamate receptors and a metabolic by misincorporating into cellular proteins. We have used zebrafish, an increasingly used model for neurodegenerative diseases, to further identify signaling components involved in BMAA-induced toxicity. Zebrafish embryos were exposed to sub-lethal dosages of BMAA and a label-free proteomics analysis was conducted on larvae 4 days post fertilization. The exposed larvae showed no developmental abnormalities, but a reduced heart rate and increased expression of GSK3 isoforms. Search towards a reviewed database containing 2968 entries identified 480 proteins. Only 17 of these were regulated 2-fold or more in the exposed larvae. Seven of these proteins could be associated to glutamate receptor signaling and recycling. The remaining nine have all been linked to disturbance in protein homeostasis, reactive oxygen species (ROS) development or neuronal cell death. We also found that BMAA influenced the endocannabinoid system by up-regulation of fatty acid amide hydrolase (FAAH) and that FAAH inhibitor URB597 reduced the BMAA effect on heart rate and GSK3 expression.