Eggshell and egg yolk proteins in fish: hepatic proteins for the next generation: oogenetic, population, and evolutionary implications of endocrine disruption

The oocyte is the starting point for a new generation. Most of the machinery for DNA and protein synthesis needed for the developing embryo is made autonomously by the fertilized oocyte. However, in fish and in many other oviparous vertebrates, the major constituents of the egg, i.e. yolk and eggshell proteins, are synthesized in the liver and transported to the oocyte for uptake. Vitellogenesis, the process of yolk protein (vitellogenin) synthesis, transport, and uptake into the oocyte, and zonagenesis, the synthesis of eggshell zona radiata proteins, their transport and deposition by the maturing oocyte, are important aspects of oogenesis. The many molecular events involved in these processes require tight, coordinated regulation that is under strict endocrine control, with the female sex steroid hormone estradiol-17β in a central role. The ability of many synthetic chemical compounds to mimic this estrogen can lead to unscheduled hepatic synthesis of vitellogenin and zona radiata proteins, with potentially detrimental effects to the adult, the egg, the developing embryo and, hence, to the recruitment to the fish population. This has led to the development of specific and sensitive assays for these proteins in fish, and the application of vitellogenin and zona radiata proteins as informative biomarkers for endocrine disrupting effects of chemicals and effluents using fish as test organisms. The genes encoding these important reproductive proteins are conserved in the animal kingdom and are products of several hundred million years of evolution

Ecotoxicological properties of ketoprofen and the S(+)‐enantiomer (dexketoprofen): Bioassays in freshwater model species and biomarkers in fish PLHC‐1 cell line

The increased use of non-steroidal anti-inflammatory drugs (NSAIDs) has resulted in their ubiquitous presence in the environment. The toxicological properties of these two widely prescribed NSAIDs, namely - racemic ketoprofen (rac-KP) and its enantiomer S(+)-ketoprofen (dexketoprofen, DKP) were evaluated. Firstly, by acute and chronic toxicity tests using three representative model organisms (Vibrio fischeri, Pseudokirchneriella subcapitata and Ceriodaphnia dubia). Secondly, by evaluating the responses of biotransformation systems and multidrug resistance associated proteins (MRP1/MRP2) using the PLHC-1 fish hepatic cell-line. Toxicity data from both acute and chronic DKP exposure indicated higher sensitivity through inhibition of bioluminescence and algal growth and through increased mortality/immobilization compared to rac-KP exposure. The growth inhibition test showed that rac-KP and DKP exhibited different values for EC50 (240.2 µg/L and 65.6 µg/L, respectively). Furthermore, rac-KP and DKP did not exert cytotoxic effects in PLHC-1 cells, and produced compound-, time- and concentration-specific differential effects on CYP1A and GST levels. For CYP1A, the effects of rac-KP and DKP differed at transcriptional and catalytic level. Exposure to rac-KP and DKP modulated MRP1 and MRP2 mRNA levels and these effects were also dependent on compound, exposure time and concentration of the individual drug. The present study revealed for the first time, the interactions between these NSAIDs and key detoxification systems, and different sensitivity to the racemic mixture compared to its enantiomer. This article is protected by copyright. All rights reserved

Novel organ-specific effects of Ketoprofen and its enantiomer, dexketoprofen on toxicological response transcripts and their functional products in salmon.

Racemic ketoprofen (RS-KP) and its enantiomer, dexketoprofen (S(+)-KP) are widely used non-steroidal anti-inflammatory drugs (NSAIDs), and commonly detected in the aquatic environment. The present study has evaluated the toxicological effects of RS-KP and S(+)-KP on biotransformation and oxidative stress responses in gills and liver of Atlantic salmon. Fish were exposed for 10 days using different concentrations of RS-KP (1, 10 and 100 μg/L) and S(+)-KP (0.5, 5 and 50 μg/L). Biotransformation and oxidative stress responses were analysed at both transcript and functional levels. In the gills, significant inhibitory effect at transcriptional and enzymatic levels were observed for biotransformation and oxidative stress responses. On the contrary, biotransformation responses were significantly increased at transcriptional and translational levels in the liver, while the associated enzymatic activities did not parallel this trend and were inhibited and further demonstrated by principal component analysis (PCA). Our findings showed that both compounds produced comparable toxicological effects, by producing organ-specific effect differences. RS-KP and S(+)-KP did not bioaccumulate in fish muscle, either due to rapid metabolism or excretion as a result of their hydrophobic properties. Interestingly, the inhibitory effects observed in the gills suggest that these drugs may not undergo first pass metabolism, that might result to downstream differences in toxicological outcomes

Novel organ-specific effects of Ketoprofen and its enantiomer, dexketoprofen on toxicological response transcripts and their functional products in salmon

Racemic ketoprofen (RS-KP) and its enantiomer, dexketoprofen (S(+)-KP) are widely used non-steroidal anti-inflammatory drugs (NSAIDs), and commonly detected in the aquatic environment. The present study has evaluated the toxicological effects of RS-KP and S(+)-KP on biotransformation and oxidative stress responses in gills and liver of Atlantic salmon. Fish were exposed for 10 days using different concentrations of RS-KP (1, 10 and 100 μg/L) and S(+)-KP (0.5, 5 and 50 μg/L). Biotransformation and oxidative stress responses were analysed at both transcript and functional levels. In the gills, significant inhibitory effect at transcriptional and enzymatic levels were observed for biotransformation and oxidative stress responses. On the contrary, biotransformation responses were significantly increased at transcriptional and translational levels in the liver, while the associated enzymatic activities did not parallel this trend and were inhibited and further demonstrated by principal component analysis (PCA). Our findings showed that both compounds produced comparable toxicological effects, by producing organ-specific effect differences. RS-KP and S(+)-KP did not bioaccumulate in fish muscle, either due to rapid metabolism or excretion as a result of their hydrophobic properties. Interestingly, the inhibitory effects observed in the gills suggest that these drugs may not undergo first pass metabolism, that might result to downstream differences in toxicological outcomes

Quantitative transcriptomics, and lipidomics in evaluating ovarian developmental effects in Atlantic cod (Gadus morhua) caged at a capped marine waste disposal site

In the present study, a previously capped waste disposal site at Kollevåg (Norway) was selected to study the effects of contaminant leakage on biomarkers associated with Atlantic cod (Gadus morhua) reproductive endocrinology and development. Immature cod were caged for 6 weeks at 3 locations, selected to achieve a spatial gradient of contamination, and compared to a reference station. Quantitative transcriptomic, and lipidomic analysis was used to evaluate the effects of the potential complex contaminant mixture on ovarian developmental and endocrine physiology. The number of expressed transcripts, with 0.75 log2-fold differential expression or more, varied among stations and paralleled the severity of contamination. Particularly, significant bioaccumulation of ∑PCB-7, ∑DDTs and ∑PBDEs were observed at station 1, compared to the other station, including the reference station. Respectively 1416, 698 and 719 differentially expressed genes (DEGs), were observed at stations 1, 2 and 3, compared to the reference station, with transcripts belonging to steroid hormone synthesis pathway being significantly upregulation. Transcription factors such as esr2 and ahr2 were increased at all three stations, with highest fold-change at Station 1. MetaCore pathway maps identified affected pathways that are involved in ovarian physiology, where some unique pathways were significantly affected at each station. For the lipidomics, sphingolipid metabolism was particularly affected at station 1, and these effects paralleled the high contaminant burden at this station. Overall, our findings showed a novel and direct association between contaminant burden and ovarian toxicological and endocrine physiological responses in cod caged at the capped Kollevåg waste disposal site.publishedVersio

Effect of diet on molecular relationships between Atlantic cod larval muscle growth dynamics, metabolism, and antioxidant defense system

We studied molecular effects (RNAseq and qPCR) of first feeding prey types (copepods or rotifers/Artemia) on skeletal muscle myogenesis and growth dynamics (proliferation, differentiation), metabolism (glycolysis, gluconeogenesis, oxidative phosphorylation), and antioxidant defense system (production/regulation of reactive oxygen species (ROS) in cod (Gadus morhua) larval skeletal muscle. Larval somatic growth rates were significantly higher in copepod fed larvae, although shifts in gene expressions related to muscle growth dynamics between hypertrophy and hyperplasia and generation and regulation of ROS mostly occurred around 5-, 10-, and 15-mm standard length (SL) for both groups. Gene expression for cell membrane proteins (such as nox1 and igf1r) peaked at 7 mm SL in all larvae, corresponding with increased ROS expressions in cod muscle during the exponential stratified hyperplasia phase from 7 mm SL. Expression for muscle differentiation (mef2a) occurred continuously (strongest from 10 mm SL). Expressions for muscle proliferation (pcna) and hydrogen peroxide (H2O2) generation (sod1 and sod2) occurred in the 5 - 15 mm SL range, peaking at 10 mm SL in all larvae. A downregulation of sod1 and sod2 in skeletal muscle from 15 mm SL indicated the first response of the defense antioxidant system. Gene expressions related to glucose metabolism (slc2A11, pfk, fpb2, ldha) was 3 - 10 times higher in copepod-fed larvae than in rotifer/Artemia-fed larvae between 7 – 10 mm (live prey period). Copepods move faster than rotifers, and cod larvae will also gradually increase their active swimming periods, due to less viscous forces. Active swimming during the strongest muscle stratified hyperplasia phase (7 – 10 mm SL) could promote a better delivery and transport across the muscle membrane and intracellular flux through glycolysis and oxidative phosphorylation and would contribute to the observed earlier and more effective glucose metabolism in the larvae fed copepods. We suggest that active swimming is an important factor promoting cod larval muscle growth, especially during the strongest muscle hyperplasia phase between 7 and 10 mm SL. The rapid movements of copepods and better nutritional composition could play important roles in stabilizing ROS levels, promoting high swimming activities and enhancing long-term muscle growth in cod.publishedVersio

Genomic approach in evaluating the role of androgens on the growth of Atlantic cod (Gadus morhua) previtellogenic oocytes

Previous studies have suggested that androgens may play an integral role in early oocyte development in fish. This study evaluated the effects of androgens (11-ketotestosterone: 11-KT and testosterone: T) on gene expression patterns and growth of cod previtellogenic oocytes. cDNA libraries of androgen-responsive genes were generated using suppressive subtractive hybridization (SSH) of clones containing differentially expressed genes in oocytes separately exposed to different concentrations of 11-KT and T, in addition to a solvent control. Secondly, a targeted microarray was developed based on differentially expressed genes. In the experimental setup, tissue was cultured in vitro with different concentrations of 11-KT and T (0, 10 and 100 mu M). The array analyses showed 0.5-3.5-fold significant alterations in transcript levels for a number of genes. Real-time PCR and in -situ hybridization were also used to analyze the changes in expression for selected genes. Quantitative histological analyses showed a consistent stereological validation of oocyte growth and development after exposure to androgens. The present study reveals novel roles of androgens on the development of previtellogenic oocytes, suggesting androgen control of early oocyte growth in cod. The strong effects of 11-KT on oocyte growth support our hypothesis that non-aromatizable androgens may exert direct hormonal effects in previtellogenic oocytes, with possible consequences for overt fecundity. (C) 2008 Elsevier Inc. All tights reserved

The effects on steroidogenesis and histopathology of adult male Japanese quails (Coturnix coturnix japonica) testis following pre-pubertal exposure to di(n-butyl) phthalate (DBP)

In the present study, we have investigated the effects of 30-day dietary (pre-pubertal) exposure to different doses (0 (control), 1, 10, 50, 200 and 400 mg/kg bodyweight/day) of di(n-butyl) phthalate (DBP) on Leydig cells of adult male Japanese quails by quantifying the transcript levels for P450 side-chain cleavage (p450scc), P450c17 (CYP17), and 3β- and 17β-hydroxysteroid dehydrogenase (hsd) using quantitative (real-time) polymerase chain reaction (qRT-PCR). In addition, the plasma testosterone levels were analysed using radioimmunoassay (RIA) and testis was examined for evidence of gross pathology and histopathology. Our data showed that pre-pubertal exposure to DBP produced alterations in testicular architecture as evident by poorly developed or mis-shaped testis, and altered spermatogenesis due to tubular degeneration and atrophy of seminiferous tubules especially in the high DBP dose (200 and 400 mg/kg) treated groups. In addition, DBP altered several key enzymes involved in testicular steroidogenesis pathways in an apparent dose-dependent manner. For example, biphasic effects of DBP were observed for P450scc and 3β-hsd mRNA, that were generally increasing at low dose 10 mg/kg, and thereafter, an apparent dose-dependent decrease between 50 and 400 mg/kg. The steroidogenic acute regulatory (StAR) protein was at the lowest detectable limits and therefore not quantifiable. These effects did not parallel the non-significant changes observed for plasma testosterone levels. The present data is consistent with previous reports showing that DBP modulates Leydig cell steroidogenesis in several species, with a potential negative effect on reproduction in those avian species that are vulnerable to endocrine disrupting chemicals.The Norwegian Research Council (NRC) (Grant number 1964442/S40) to AA.http://www.elsevier.com/locate/cbpchj201

Modulation of neuro-dopamine homeostasis in juvenile female Atlantic cod (Gadus morhua) exposed to polycyclic aromatic hydrocarbons and perfluoroalkyl substances

The dopaminergic effect of PAH and PFAS mixtures, prepared according to environmentally relevant concentrations, has been studied in juvenile female Atlantic cod (Gadus morhua). Benzo[a]pyrene, dibenzothiophene, fluorene, naphthalene, phenanthrene, and pyrene were used to prepare a PAH mixture, while PFNA, PFOA, PFOS, and PFTrA were used to prepare a PFAS mixture. Cod were injected intraperitoneally twice, with either a low (1×) or high (20×) dose of each compound mixture or their combinations. After 2 weeks of exposure, levels of plasma 17β-estradiol (E2) were significantly elevated in high PAH/high PFAS treated group. Brain dopamine/metabolite ratios (DOPAC/dopamine and HVA+DOPAC/dopamine) changed with E2 plasma levels, except for high PAH/low PFAS and low PAH/high PFAS treated groups. On the transcript levels, th mRNA inversely correlated with dopamine/metabolite ratios and gnrh2 mRNA levels. Respective decreases and increases of drd1 and drd2a after exposure to the high PAH dose were observed. Specifically, high PFAS exposure decreased both drds, leading to high plasma E2 concentrations. Other studied end points suggest that these compounds, at different doses and combinations, have different toxicity threshold and modes of action. These effects indicate potential alterations in the feedback signaling processes within the dopaminergic pathway by these contaminant mixtures.acceptedVersio

Changes in testicular histomorphometry and ultrastructure of Leydig cells in adult male Japanese quail exposed to di (n-butyl) phthalate (DBP) during the prepubertal period

DATA AVAILABILITY : The data that support the findings of this study are available from the corresponding author (UMB), upon reasonable request.Phthalate esters, such as di(n-butyl) phthalate, (DBP), are synthetic chemical pollutants commonly used as plasticizers in the manufacture of plastics. In the present study, we investigated the effects of DBP in the testes of adult male quails (Coturnix cortunix japonica) exposed by oral gavage to variable doses of DBP (0 [control], 1, 10, 50, 200, and 400 mg/ kgbw−d), for 30 days during the prepubertal period, using histo-morphometric and ultrastructural techniques. Generally, significant decreases in seminiferous tubular diameter (STD) and epithelial height (SEH) were observed predominantly at the highest DBP doses (200 and 400 mg/kg), as compared to medium (50 mg/kg), and lowest doses (1 and 10 mg/kg) as well as the control group. Ultrastructurally, apparent dose-specific degenerative changes were observed in the Leydig cells. The lowest DBP doses (1 and 10 mg/kg) did not produce significant effects on Leydig cell ultrastructure, whereas, at the highest doses (200 and 400 mg/kg), the Leydig cells were remarkably conspicuous in the interstitium and appeared foamy. There was a preponderance of electron-lucent lipid droplets which crowded out the normal organelles of the cell, as well as increases in the number of dense bodies in the cytoplasm. The smooth endoplasmic reticulum (sER) was less obvious, compacted, and wedged between the abundant lipid droplets and mitochondria. Taken together, these findings indicate that pre-pubertal exposure of precocious quail birds to DBP, produced parameter-specific histometric tubular changes, as well as dose-dependent cyto-structural derangement of the Leydig cells; which consequently may lead to overt reproductive impairments in the adult bird in the environment.FUNDING : Open access funding provided by University of Pretoria. This study was supported by research grants to UMB, from the University of Pretoria and South African Veterinary Foundation (SAVF)/Norvatis Wildlife Foundation fund.https://www.springer.com/journal/11356am2024Anatomy and PhysiologySDG-03:Good heatlh and well-beingSDG-09: Industry, innovation and infrastructur