Atorvastatin causes developmental and behavioral toxicity in yellowstripe goby (Mugilogobius chulae) embryos/larvae via disrupting lipid metabolism and autophagy processes.

Atorvastatin (ATV) is one of the most commonly prescribed lipid-lowering drugs detected frequently in the environment due to its high use and low degradation rate. However, the toxic effects of residual ATV in the aquatic environment on non-target organisms and its toxic mechanisms are still largely unknown. In the present study, embryos of a native estuarine benthic fish, Mugilogobius chulae, were employed to investigate the developmental and behavioral toxic effects of ATV including environmentally relevant concentrations. The aim of this study was to provide a scientific basis for ecological risk assessment of ATV in the aquatic environment by investigating the changes of biological endpoints at multiple levels in M. chulae embryos/larvae. The results showed that ATV had significantly lethal and teratogenic effects on M. chulae embryos/larvae and caused abnormal changes in developmental parameters including hatch rate, body length, heart rate, and spontaneous movement. ATV exposure caused oxidative stress in M. chulae embryos/larvae subsequently inhibited autophagy and activated apoptosis, leading to abnormal developmental processes and behavioral changes in M. chulae embryos/larvae. The disruptions of lipid metabolism, autophagy, and apoptosis in M. chulae embryos/larvae caused by ATV exposure may pose a potential ecological risk at the population level.

Toxic effects of environmentally relevant concentrations of naproxen exposure on Daphnia magna including antioxidant system, development, and reproduction.

Naproxen (NPX) is one of common non-prescription non-steroidal anti-inflammatory drugs (NSAIDs) which is widely detected in aquatic environments worldwide due to its high usage and low degradation. NPX exerts anti-inflammatory and analgesic pharmacological effects through the inhibition of prostaglandin-endoperoxide synthase (PTGS), also known as cyclooxygenase (COX). Given its evolutionarily relatively conserved biological functions, the potential toxic effects of NPX on non-target aquatic organisms deserve more attention. However, the ecotoxicological studies of NPX mainly focused on its acute toxic effects under higher concentrations while the chronic toxic effects under realistic concentrations exposure, especially for the underlying molecular mechanisms still remain unclear. In the present study, Daphnia magna, being widely distributed in freshwater aquatic environments, was selected to investigate the toxic effects of environmentally relevant concentrations of NPX via determining the response of the Nrf2/Keap1 signaling pathway-mediated antioxidant system in acute exposure, as well as the changes in life-history traits, such as growth, reproduction, and behavior in chronic exposure. The results showed that the short-term exposure to NPX (24 h and 48 h) suppressed ptgs2 expression while activating Nrf2/Keap1 signaling pathway and its downstream antioxidant genes (ho-1, sod, cat and trxr). However, with prolonged exposure to 96 h, the opposite performance was observed, the accumulation of malondialdehyde (MDA) indicated that D. magna suffered from severe oxidative stress. To maintain homeostasis, the exposed organism may trigger ferroptosis and apoptosis processes with the help of Silent mating type information regulation 2 homologs (SIRTs). The long-term chronic exposure to NPX (21 days) caused toxic effects on D. magna at the individual and population levels, including growth, reproduction and behavior, which may be closely related to the oxidative stress induced by the drug. The present study suggested that more attention should be paid to the ecological risk assessment of NSAIDs including NPX on aquatic non-target organisms.

Response of lipid metabolism, energy supply, and cell fate in yellowstripe goby (Mugilogobius chulae) exposed to environmentally relevant concentrations atorvastatin.

The usage of typical pharmaceuticals and personal care products (PPCPs) such as cardiovascular and lipid-modulating drugs in clinical care accounts for the largest share of pharmaceutical consumption in most countries. Atorvastatin (ATV), one of the most commonly used lipid-lowering drugs, is frequently detected with lower concentrations in aquatic environments owing to its wide application, low removal, and degradation rates. However, the adverse effects of ATV on non-target aquatic organisms, especially the molecular mechanisms behind the toxic effects, still remain unclear. Therefore, this study investigated the potentially toxic effects of ATV exposure (including environmental concentrations) on yellowstripe goby (Mugilogobius chulae) and addressed the multi-dimensional responses. The results showed that ATV caused typical hepatotoxicity to M. chulae. ATV interfered with lipid metabolism by blocking fatty acid ß-oxidation and led to the over-consumption of lipids. Thus, the exposed organism was obliged to alter the energy supply patterns and substrates utilization pathways to keep the normal energy supply. In addition, the higher concentration of ATV exposure caused oxidative stress to the organism. Subsequently, M. chulae triggered the autophagy and apoptosis processes with the help of key stress-related transcriptional regulators FOXOs and Sestrins to degrade the damaged organelles and proteins to maintain intracellular homeostasis.

Acetaminophen exposure alters the DNA methylation pattern of Mugilogobius chulae, along with the changes in the Nrf2-Keap1 signaling pathway.

DNA methylation can dynamically regulate multiple physiological processes in organisms in response to changes of the external environment. The effects of acetaminophen (APAP) on DNA methylation in aquatic organisms and its toxic mechanisms is an interesting issue. In the present study, Mugilogobius chulae (Approximately 225 individual), a small benthic native fish, were employed to assess the toxic effects of APAP-exposure on non-target organisms. First, under APAP exposure (0.5 µg/L and 500 µg/L) for 168 h, 17,488 and 14,458 differentially methylated regions (DMRs) were identified in liver of M. chulae, respectively, which were involved in energy metabolism, signaling transduction, and cellular processes etc. The modification of lipid metabolism by DNA methylation was particularly prominent and the increased fat vacuoles in the sections were observed. Some key nodes associated with oxidative stress and detoxification such as Kelch-1ike ECH-associated protein l (Keap1) and fumarate hydratase (FH) were modified by DNA methylation. Meanwhile, changes in DNA methyltransferase and Nrf2-Keap1 signaling pathways at different concentrations of APAP (0.5 µg/L, 5 µg/L, 50 µg/L and 500 µg/L) for different time (24 h and 168 h) were addressed at the transcriptional level. Results showed that ten eleven translocation enzymes 2 (TET2) transcript expression was upregulated 5.7-folds after being exposed to 500 µg/L APAP for 168 h, indicating the urgent need for active demethylation in the exposed organism. The elevated DNA methylation levels of Keap1 led to repression of its transcriptional expression so as to promote recovery or reactivation of Nrf2, which displayed negatively relationship with Keap1 gene. Meanwhile, P62 was significantly positively correlated with Nrf2. Downstream genes in the Nrf2 signaling pathway changed synergistically except for Trx2, in which GST and UGT were highly significantly upregulated. This work illustrated that APAP exposure altered the DNA methylation processes, together with the Nrf2-Keap1 signaling pathway, and affected the stress responses of M. chulae to pharmaceuticals exposure.

Effects of atorvastatin on the Sirtuin/PXR signaling pathway in Mugilogobius chulae.

Atorvastatin (ATV) is a hypolipidemic drug widely detected in the aquatic environment. Nevertheless, limited information is provided about the toxic effects of ATV on estuary or coastal species and the underlying mechanisms. In the present study, the responses of genes expression in pregnane X receptor (PXR) signaling pathway and enzymatic activities in the liver of the estuarine benthic fish (Mugilogobius chulae) were investigated under acute and sub-chronic ATV exposure. Results showed that PXR was significantly inhibited in the highest exposure concentration of ATV for a shorter time (24 h, 500 µg L-1) but induced in a lower concentration (72 h, 5 µg L-1). The downstream genes in PXR signaling pathway such as CYP3A, SULT, UGT, and GST showed similar trends to PXR. P-gp and MRP1 were repressed in most treatments. GCLC associated with GSH synthesis was mostly induced under ATV exposure for a long time (168 h), suggesting that reactive oxygen species (ROS) were generated under ATV exposure. Similarly, GST and SOD enzymatic activities significantly increased in most exposure treatments. Under ATV exposure, SIRT1 and SIRT2 displayed induction to some extent in most treatments, suggesting that SIRTs may affect PXR expression by regulating the acetylation levels of PXR. The investigation demonstrated that ATV exposure affected the expression of the Sirtuin/PXR signaling pathway, thus further interfered adaption of M. chulae to the environment.

Hypoxia aggravates the burden of yellowstripe goby (Mugilogobius chulae) under atorvastatin exposure.

In the present study, an estuarine benthic fish, Mugilogobius chulae (M. chulae), was exposed to hypoxia, atorvastatin (ATV), a highly used and widely detected lipid-lowering drug in aquatic environment, and the combination of hypoxia and ATV for 7 days, respectively, so as to address and compare the effects of the combination of hypoxia and ATV exposure on M. chulae. The results showed that lipid metabolism in M. chulae was greatly affected: lipid synthesis was blocked and catabolism was enhanced, exhibiting that lipids content were heavily depleted. The combined exposure of hypoxia and ATV caused oxidative stress and induced massive inflammatory response in the liver of M. chulae. Signaling pathways involving in energy metabolism and redox responses regulated by key factors such as HIF, PPAR, p53 and sirt1 play important regulatory roles in hypoxia-ATV stress. Critically, we found that the response of M. chulae to ATV was more sensitive under hypoxia than normoxia. ATV exposure to aquatic non-target organisms under hypoxic conditions may make a great impact on the detoxification and energy metabolism, especially lipid metabolism, and aggravate the oxidative pressure of the exposed organisms.

The responses of Scrippsiella acuminata to the stresses of darkness: antioxidant activities and formation of pellicle cysts.

In order to understand the strategy of Scrippsiella acuminata to cold dark environment, the antioxidant responses and the formation of pellicle cysts of S. acuminata to darkness at 8°C and 20°C were investigated. Cell densities decreased significantly after 96 h dark treatment, and no live cells were observed after 9-days dark treatments. The darkness stress generally resulted in an increase of antioxidant defenses, including soluble protein, superoxide dismutase (SOD), glutathione (GSH), and malondialdehyde (MDA). Cellular soluble protein and SOD activity increased sharply under 20°C darkness, which protected algal cells against the oxidative stress from darkness, and resulted in relatively lower MDA levels. Soluble protein and SOD activity were enhanced under 8°C darkness as well however not in a sharp rise, and higher levels of MDA and GSH were recorded. The results suggested high SOD and protein levels protected cells against harsh darkness stress, while high GSH not only helped algae cells resist dark stress, but also played an important role in low temperature stress. Darkness promoted the formation of pellicle cysts of S. acuminata, and the maximum formation rates were 16.06% to 21.74% at 8°C and 20°C, respectively. Germination of pellicle cysts occurred within 24 h after light exposure, however pellicle cysts could not withstand long-time darkness stress, and all pellicle cysts died after 9-days darkness exposure. The results of this study suggest that S. acuminata is able to overcome temporary cold darkness through forming pellicle cysts.

Simvastatin as an emerging pollutant on non-target aquatic invertebrates: effects on antioxidant-related genes in Daphnia magna.

Simvastatin (SIM) is one of the most widely used lipid-lowering drugs and consequently has been frequently detected in various waters. However, its potential adverse effects and toxic mechanisms on non-target organisms such as Daphnia magna (D. magna) remain still unclear. In the present study, the expressions of Nrf2 and antioxidant genes including Keap1, HO-1, GCLC, GST, SOD, CAT, GPx5, GPx7, GRx, TRX, TrxR, and Prx1 in D. magna exposed to SIM for 24 h, 48 h, and 96 h were investigated. The changes of SOD, CAT, GST, and GPx enzymatic activities, and the GSH and MDA content under SIM for 48-h exposure were also addressed. Results showed that the expression of Nrf2 was inhibited at 24 h but induced at 96 h, displaying a time- and/or dose-dependent relationship under SIM exposure. In contrast, Keap1 exhibited induction at 24 h. HO-1 showed significant induction under SIM exposure for different time. SOD generally displayed an induction trend under SIM exposure for different periods. GPX5 expression showed significant induction under SIM exposure, particularly at 24 h in 5 µg L-1 increasing 15 folds of the control. But GPX7 expression generally displayed inhibition except in 5 µg L-1. Trx and TrxR showed different induction or inhibition, which was depended on the exposure time and concentration. Prx1 displayed significant induction in most SIM groups. In addition, the decreasing GSH and increasing MDA content also indicated oxidative stress of SIM exposure. Overall, SIM exposure affected the expression of Nrf2 and antioxidant-related genes and altered the redox homeostasis of D. magna, even may cause the morphological changes such as shorten spine and abnormal development eye.

Comparison of toxic effects of atorvastatin and gemfibrozil on Daphnia magna.

Atorvastatin (ATV) and gemfibrozil (GEM) are two typical lipid-lowering pharmaceuticals with different action modes, which are frequently detected in various water bodies owning to their wide usage. However, there is limited information about their effects on Daphnia magna. The present study addressed and compared the toxic effects of ATV and GEM on D. magna through determining the responses of the stress related genes (including Nrf2, Keap1, HO-1, GCLC, p53 and PIG3) in D. magna for 24 h and 48 h acute exposure and the changes of life history traits and swimming behaviors in a 21 days chronic exposure under different concentrations of ATV and GEM exposure (5 µg L-1, 50 µg L-1, 500 µg L-1 and 5000 µg L-1). Results showed that the expression of Nrf2, Keap1, HO-1, GCLC, p53 and PIG3 were induced to various degrees under the ATV exposure. There were similar performances for GEM. ATV and GEM caused the delay of first brooding and hatching time and decrease of eggs production number, especially in GEM exposure, reproduction of Daphnia was significantly inhibited, decreasing 38.51% compared to the control. ATV and GEM increased the heart rate of D. magna, and changed swimming behaviors of D. magna. In summary, two lipid-lowering pharmaceuticals caused oxidative stress on D. magna, subsequently brought about alterations in physiological traits. Comparatively, ATV pose more higher risks to D. magna than GEM, but the detailed action mechanisms of ATV and GEM on D. magna needs more investigations in future.

Simvastatin affects the PPARα signaling pathway and causes oxidative stress and embryonic development interference in Mugilogobius abei.

Simvastatin (SV) is a common hypolipidemic drug in clinical medicine that can reduce endogenous cholesterol biosynthesis by inhibiting hydroxyl-methyl-glutaryl coenzyme A reductase. SV took a large market share in the lipid-lowering drugs and it is frequently detected in various water bodies due to its increasing consumption in past years. In the present investigation, we selected a native fish species in the Pearl River Basin in China, Mugilogobius abei (M. abei), to study the effects of SV on non-target aquatic organisms. Results showed that a significant decrease in the volume of adipocytes under SV exposure were observed on oil red O section, and the expression of HMG-CoAR decreased significantly. The mRNA and protein expression of PPARα were significantly up-regulated, the expressions of other genes related to lipid metabolism were up-regulated to varying degrees as well. There was a positive correlation between the concentrations of SV and the protein expressions of plasma phospholipid transfer protein (PLTP) and cholesterolester transfer protein (CETP). In addition, the frozen sections showed that SV led to ROS accumulation in liver in a time and concentration dependent manner. The mRNA and protein expressions of Nrf2 were significantly up-regulated after 24 hours of SV exposure. Some biomarkers associated with antioxidant such as Trx2, TrxR and MDA content were positively correlated with the exposure concentration and time, while the content of GSH decreased sharply. It is noteworthy that the environmentally relevant concentration (0.5 µg/L) of SV exposure caused delayed embryonic development and deformations, decreased hatching rates. We conclude that SV promotes fat metabolism, gives rise to oxidative stress and has significant toxicity on embryo development in M. abei.

Atorvastatin causes oxidative stress and alteration of lipid metabolism in estuarine goby Mugilogobius abei.

The potential effects of the environmental residues of Atorvastatin (ATV) as a widely used antilipemic agent on aquatic organisms deserve more investigations because of its high detection frequency in environment. The responses of Nrf2/Keap1 signaling pathway (including the transcriptional expression of Nrf2, Keap1, GCLC, GPx, GST, SOD, CAT, Trx2, TrxR, HMG-CoAR and PGC-1α) in Mugilogobius abei were investigated under acute and sub-chronic exposure of ATV in the simulated laboratory conditions. The changes of related enzymatic activity (GST, GPx, SOD, CAT and TrxR) and the content of GSH and MDA combining with the observation of histology sections of liver in M. abei were also addressed. The results show Nrf2 and its downstream antioxidant genes were induced to different degrees under ATV exposure. The activities of antioxidant enzymes were inhibited at 24 h and 72 h but induced/recovered at 168 h. Correspondingly, negatively correlated to GSH, MDA increased first but reduced then. Notably, with the increase of exposure concentration/time, the volume of lipid cells in liver decreased, suggesting more lipid decomposition. Therefore, lipid metabolism was suppressed (down-regulation of PGC-1α) and cholesterol biosynthesis was induced (up-regulation of HMG-COAR) at 168 h. In short, ATV brings oxidative stress to M. abei in the initial phase. However, with the increase of exposure time, ATV activates Nrf2/Keap1 signaling pathway and improves the antioxidant capacity of M. abei to reverse this adverse effect. ATV also affects lipid metabolism of M. abei by reducing cholesterol content and accelerating lipid decomposition.

Simvastatin affects Nrf2/MAPK signaling pathway and hepatic histological structure change in Gambusia affinis.

Simvastatin (SV) is a typical lipid-lowering agent detected widely in waters, so its latent toxic effects to fish are deserved of concern. The purposes of this study aim at revealing the responses of antioxidant system in mosquitofish (Gambusia affinis) under SV exposure. Transcriptional expressions of oxidative stress-related key transcriptional factor Nrf2 and its downstream genes in mosquitofish were determined under SV exposure for different time. Partly related enzymatic activities, Nrf2 and MAPK protein expressions were also addressed in the same conditions, and histological changes in liver tissues were investigated too. Results showed that Nrf2 mRNA increased with the rising SV concentrations at 3 d and 7 d, displaying typical dose-dependent relationship, and Nrf2 protein by WB showed consistency with transcriptional changes to some degree. Comparatively, responses of gene expressions were more sensitive than enzymatic changes. The histological changes in the mosquitofish liver exposed to SV for 7 d indicated the potential adverse effects of statins. This work demonstrated that SV in aquatic environment could affect the transcriptional expression of antioxidant system, partly related enzymatic activity, and hepatic structure in the mosquitofish, revealing its potential risk on non-target organisms and environmental safety.

Effects of triclosan on the RedoximiRs/Sirtuin/Nrf2/ARE signaling pathway in mosquitofish (Gambusia affinis).

Triclosan (TCS) has been widely used in daily life for its broad-spectrum antibacterial property and subsequently detected frequently in aquatic waterborne. Environmental relevant concentrations of TCS in water (ng-µg/L) may pose potential unexpected impact on non-target aquatic organisms. In the present work, we investigated the transcriptional responses of Nrf2 as well as its downstream genes, sirtuins and redox-sensitive microRNAs (RedoximiRs) in livers of the small freshwater fish mosquitofish (Gambusia affinis) which were exposed to environmental relevant concentrations of TCS (0.05 µg/L, 0.5 µg/L and 5 µg/L for 24 h and 168 h). Results showed there were similar up-regulations in Nrf2 and its target genes (e. g. NQO1, CAT and SOD) at transcriptional, enzymatic and protein levels, reflecting oxidative stress of TCS to mosquitofish. Meanwhile, up-regulations of Sirt1, Sirt2 and down-regulations of miR-34b, miR-200b-5p and miR-21 could modulate antioxidant system via the Nrf2/ARE signaling pathway by the post-transcriptional regulations. Some oxidative stress-related biomarkers displayed in concentration-dependent manners (e. g. NQO1 mRNA, CAT mRNA) and/or time-dependent manners (e. g. GSH contents). This study indicated that the RedoximiRs/Sirtuin/Nrf2/ARE signaling pathway played a crucial role in mosquitofish exposed to TCS, and there might be potentially profound effects for TCS on the aquatic ecological safety.

Adverse effects in Daphnia magna exposed to e-waste leachate: Assessment based on life trait changes and responses of detoxification-related genes.

The sharp increase in e-waste derived from great consumption of electronic products has become a potential global environmental challenge. Limited information is available about the potential detrimental impact of e-waste on aquatic organisms. The present study investigated the expression of detoxification-related genes and life-history parameter changes in Daphnia magna exposed to e-waste leachate, simultaneously integrating with the chemical analysis of typical pollutants from e-waste leachate. The study aims at assessing impacts of e-waste to aquatic invertebrates and providing insights into its toxic mechanisms. The results showed high concentration of heavy metals like Cu (1657.14 ± 259.3 µg g-1, DW) and persistent organic compounds like polybrominated diphenyl ethers (7831.32 ± 1273.86 ng g-1, DW) in stream sediments near e-waste dismantling areas. Chronic exposure to these pollutants can affect the growth and reproduction of D. magna, resulting in significant development retardation, decreased total egg production per female, and even smaller body size. Expression of some detoxification and reproduction-related genes including DappuHR96, CYP360a, P-gp, EcR, CYP314 and Vtg exhibited different response patterns depending on the e-waste leachate concentration. E-waste leachate may affect the expression of detoxification-related and growth and reproduction-related genes and disrupt the growth and reproduction processes of D. magna.

Paracetamol affects the expression of detoxification- and reproduction-related genes and alters the life traits of Daphnia magna.

Paracetamol (APAP) is a widely used non-steroidal anti-inflammatory drug and has been frequently detected in aquatic environment. However, limited information is provided about the toxic effects and detoxification mechanism of APAP in aquatic invertebrates. In the present study, the change of life traits of Daphnia magna (e.g., body length, growth rate and reproduction) was investigated under the chronic APAP exposure (0-5000 µg/L) for 21 day, and the effects of APAP on the expression of the detoxification- and reproduction-related genes including HR96, CYP360A8, CYP314, MRP4, P-gp, EcR and Vtg in the acute exposure (up to 96 h) were also determined. Results showed that the molting frequency, days to the first brood and days to the first egg production of D. magna were affected under the 50 µg/L concentration of APAP in the chronic exposure test. In the acute test, the transcriptional expression of HR96 was up-regulated under APAP exposure for 24 and 48 h. Similar performances were also observed in the expression of CYP360A8, CYP314, MRP4 and P-gp. However, with exposure time extended to 96 h, the induction of HR96 decreased or even reversed in some cases. It may indicate that the defense system in Daphnia is activated for a short time of exposure or becomes adaptive after longer term of exposure. APAP exposure also affected reproduction-related genes expression, which was related to the exposure time and concentration of APAP. In summary, APAP significantly affected the expression of genes associated with detoxification metabolism and altered some physiological parameters in D. magna.

Responses of the Nrf2/Keap1 signaling pathway in Mugilogobius abei (M. abei) exposed to environmentally relevant concentration aspirin.

Aspirin (ASA) is a widely used non-steroidal anti-inflammatory drug. Its high detection frequency in various waterborne and environmental residues has drawn wide attention. Limited information were provided for the effects of aspirin exposure on oxidative stress signaling pathway in fish, which is closely related to pathological and immunological process of fish. In this study, a small fish - Mugilogobius abei (M. abei) distributing widely in aquatic ecosystems in southern China, was employed as testing organism and the key genes of the detoxification metabolism were cloned for the first time. The responses of Nrf2/Keap1 signaling pathway were investigated under the environmentally relevant concentration aspirin exposure (0.5 µg L-1, 5 µg L-1, and 50 µg L-1) for 24 h, 72 h, and 168 h then. The transcriptional expression of the key genes (Nrf2, Keap1, GCLC, GPx, GST, SOD, CAT, Trx2, and TrxR) as well as the changes of the related enzymatic activities (GPx, GST, SOD, and CAT) and GSH and MDA content were also determined. Results showed that Nrf2 and Keap1 gene expression displayed a negative correlation to some extent under ASA exposure, the transcriptional expressions of the downstream related genes (GCLC, GST, SOD, CAT, Trx2, and TrxR) in Nrf2/Keap1 signaling pathway showed inhibition at 24 h but induction at 72 h and 168 h. At the protein level, ASA exposure can improve the antioxidant capacity by increasing GSH synthesis and enzymatic activity of GPx, GST, SOD, and CAT to reduce the degree of lipid peroxidation. We proposed that ASA exposure may interfere with the redox balance in M. abei at an early stage but sub-chronic ASA exposure can activate the Nrf2 signaling pathway to improve the antioxidant capacity of M. abei.

Effects of norfloxacin nicotinate on the early life stage of zebrafish (Danio rerio): Developmental toxicity, oxidative stress and immunotoxicity.

Norfloxacin nicotinate (NOR-N), an adduct of norfloxacin (NOR) and nicotinic acid, has been widely used for replacing NOR in animal husbandry and fishery industry. Nowadays, increasing evidences showed that NOR could pose toxic effects on fish and other aquatic organisms, but as its adduct, whether NOR-N could cause adverse effects on aquatic organisms is still unclear. To evaluate the toxic effects of NOR-N on the early life stage of zebrafish, we determined the changes in embryonic development (hatching rate, body length, malformation rate and mortality), antioxidant enzyme (superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (Gpx)) activities, malondialdehyde (MDA) content and gene expression levels related to antioxidant enzymes (Cu/Zn-sod, Mn-sod, CAT and Gpx) and innate immune system (tumor necrosis factor α (TNFα), interferon (IFN), Interleukin-1 beta (IL-1ß), IL-8, CXCL-clc, CC-chemokine, lysozyme (Lzy) and complement factors (C3)) after embryonic exposure to NOR-N till 96 hpf. The results showed that NOR-N exposure could decreased the hatching rate and body length, and increased abnormality and mortality as concentration-dependent during embryonic development process. NOR-N induced oxidative stress in zebrafish larvae through increasing the contents of MDA and the activities of SOD, CAT and Gpx, as well as the mRNA levels of genes related to these antioxidant enzymes. Moreover, the expression of TNFα, IFN, IL-1ß, IL-8, CXCL-clc, CC-chemokine, Lzy and C3 genes were significantly up-regulated after exposure to high concentration (5 and/or 25 mg/L) of NOR-N till 96 hpf, indicating that the innate immune system in zebrafish larvae was disturbed by NOR-N. Overall, our results suggested that NOR-N caused development toxicity, oxidative stress and immunotoxicity on the early life stage of zebrafish. Thus, widespread application of NOR-N might pose potential ecotoxicological risk on aquatic ecosystems.

Simvastatin affect the expression of detoxification-related genes and enzymes in Daphnia magna and alter its life history parameters.

Simvastatin (SV), as an hypocholesterolaemic drug, has been detected in various aquatic environment. However, limited information is available on the effects of SV on freshwater invertebrates. In the present study, we investigated the toxic effects of SV on Daphnia. magna (D. magna) through measuring the physiological changes (e.g., survival, growth rate, and reproduction) in a 21-d chronic toxicity test We also determined the expression of seven detoxification and reproduction-related genes (i.e. HR96, P-gp, CYP360A8, GST, CYP314, EcR and Vtg) and several enzymes (i.e. APND, ERND, GST and CAT) in a acute test (24 h). Results showed that high concentration (e.g. 50 µg L-1) of SV for short time exposure (e.g. 24 h) significantly induced the expression of HR96 and P-gp (e.g. up to 2.5 folds)and enzymes (e.g. increasing 4.0 folds for ERND and GST activity) in D. magna.. The long-term chronic exposure (21 days) may cause the changes of life history parameters such as decreasing total egg production number per individual and intrinsic growth rates etc. SV may act as a potential endocrine disruptor to D. magna and the reproduction parameters were more sensitive endpoints than the survival and growth for evaluating SV exposure.

Assessment of oxidative stress of paracetamol to Daphnia magna via determination of Nrf1 and genes related to antioxidant system.

Paracetamol (APAP) is one of the most widely used anti-inflammatory and analgesic drugs in human being health care and has been universally detected in various aquatic environments. However, its potential adverse effects and toxic mechanisms on freshwater invertebrates still remain unclear. In the present study, the effects of APAP on the expressions of Nrf1 and the antioxidant related genes including GCLC, GST, GPX, CAT, TRX, TrxR and Prx1 in Daphnia magna (D. magna) were evaluated after 24, 48 and 96 h, and the changes of GPX, GST and CAT enzyme activities, as well as the GSH and MDA content under APAP exposure for 48 h were also determined. Results showed that paracetamol affected the expressions of Nrf1 and antioxidant related genes in D. magna, which were related to the exposure time and concentration of APAP. Nrf1 was inhibited at 48 h, but induced at 96 h under the APAP exposure, being about two fold of the control in 5.0 µg/L. CAT were significantly induced in all treatments. But Prx decreased in an concentration-dependent manner in all treatments. In comparison with the mRNA expression, antioxidant enzymes activity displayed less changes in D. magna. Overall, APAP exposure altered the expression of Nrf1 and genes related to antioxidant system and disturbed the redox homeostasis of D. magna.

Effects of simvastatin on the PXR signaling pathway and the liver histology in Mugilogobius abei.

Simvastatin is one of the most commonly cholesterol-lowering prescribed drugs all over the world. With the increase of consumption of these pharmaceuticals and subsequent their discharge into the aquatic environment in recent years, they are present at detectable levels in most sewage effluents. Unfortunately, limited information is provided about their potential impacts on aquatic organisms, especially on the detoxification-related metabolism in fish. In the present study, one local native benthic fish (Mugilogobius abei) in southern China was employed as test species and exposed to SV (0.5 µg L-1, 5 µg L-1, 50 µg L-1 and 500 µg L-1) for 72 h. The transcriptional expression of nucleus transcriptional factor pregnane X receptor (PXR) and its downstream targeted genes including multixenobiotics resistance protein or permeability glycoprotein (P-gp), cytochrome 1A (CYP1A), cytochrome P450 3A (CYP3A), glutathione-S-transferase (GST) and the expression of associated microRNA such as miR-27, miR-34 and miR-148 in Mugilogobius abei were investigated. Result showed that the expressions of P-gp, CYP 1A, CYP 3A, GST and PXR were induced to some extend under simvastatin exposure for 72 h. A positive correlation was observed between PXR and CYP1A, CYP3A and P-gp. While for microRNA, a negative relationship was found between miR-34a and CYP3A, CYP1A. The expression of miR-148a was significantly induced under the exposure of SV (50 µg L-1), which was positive related to the transcriptional expression of PXR. For enzyme activity, erythromycin N-demethylase (ERND) significantly increased at 24 h and the activity of catalase (CAT) and superoxide dismutase (SOD) exhibited different trends. CAT was slightly inhibited at 24 h exposure but SOD was significantly induced in high concentration. Glutathione-S-transferase (GST) activity was significant inhibited after 72 h exposure. The reductive small molecule glutathione (GSH) content showed obvious decrease, while the quantity of malondialdehyde (MDA) increased significantly in high concentrations of SV exposure. GSH and MDA showed a typical negative correlation to some degree. Moreover, simvastatin caused histological changes in the liver tissues of M. abei, especially the size of adipocyte significantly decreased. The present study indicated that environmentally relevant concentration SV may affect the PXR signaling pathway in M. abei and pose potential ecological risks to non-target organisms like fish.