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Broad applications and exposure to the fungicide maneb can lead to toxicity in non-target organisms. Maneb is also associated with neurogenerative diseases such as Parkinson's disease (PD). The objectives of this study were to determine the acute toxicity of maneb to zebrafish by measuring mitochondrial bioenergetics, locomotor activity, and the expression of genes related to the oxidative damage response, as well as those related to dopamine signaling due to its association with PD. Zebrafish embryos at 6 h post-fertilization (hpf) were exposed to either solvent control (0.1% DMSO, v/v), or one dose of 0.1, 0.5, 1.0 and 10.0 µM maneb for 96 h. Maneb was moderately toxic to zebrafish embryos, and had a 96-h LC50 value of 4.29 μM (~ 1.14 mg/L). Maneb induced a dose-dependent increase in mortality, decreased hatching rate, and increased notochord deformity rate at both 1.0 and 10.0 µM after 72 and 96 h. Total body length was also significantly reduced with 1.0 µM maneb. A 50–60% decrease in mean basal oxygen consumption rate was also observed in embryos following a 24 hpf exposure to 10.0 µM maneb but oligomycin-induced ATP production and FCCP-induced maximum respiration remained unaffected. No change was detected in the expression levels of genes associated with oxidative stress (sod1 and sod2), nor those related to dopamine synthesis (th1), dopamine transporter (dat), dopamine receptors (drd1, drd2a, drd3, and drd4b). Thus, modifying the expression of these transcripts may not be a mechanism for maneb-induced developmental toxicity in zebrafish. To assess the potential for neurotoxicity, a dark photokinesis assay was conducted in larvae following 7 d exposure to 0.1, 0.5 and 1.0 μM maneb. Larvae exposed to 0.5 and 1.0 μM maneb showed signs related to hypoactivity, and this reduced activity is hypothesized to be associated with notochord defects as this deformity was prevalent at higher concentrations of maneb. Overall, these data demonstrate that maneb negatively affects embryonic development (i.e. notochord development), affects basal oxygen consumption rates of embryos, and induces hypoactivity in larval fish. This study improves understanding regarding the developmental neurotoxicity of the fungicide maneb to zebrafish.

Synthetic phenolic antioxidant 2,4-di-tert-butylphenol (2,4-DTBP) has gained growing concerns due to relatively high concentrations in aquatic ecosystems. There are, however, significant knowledge gaps regarding its potential toxicity to aquatic organisms. In this study, zebrafish (Danio rerio) larvae were exposed to 0.01, 0.1, or 1 μM 2,4-DTBP for 6 d. Transcriptomic analysis of larvae revealed that biological processes related to anti-inflammatory function of macrophage M2 lineage were inhibited by 0.01 μM 2,4-DTBP. Decreases of transcripts related to the IL1B-MYD88-NF-κB pathway (i.e., il1b, il1rl1, myd88, irak4, irak1, traf6, ikbkg, nfkbia, nfkb) and protein levels of NF-κB in larvae intestine confirmed anti-inflammatory effects of 2,4-DTBP. Subsequently, larvae exposed to 2,4-DTBP were challenged with E. coli and showed higher survival rate, suggesting sustained activation of inflammation via LPS can be attenuated by 2,4-DTBP. Moreover, histological examination revealed that intestine barrier was compromised and there was an imbalance of intestine macrophage homeostasis. Food intake was also reduced following exposure to 0.1 and 1 μM 2,4-DTBP. In addition, a risk assessment revealed that 2,4-DTBP in surface water pose low to high ecological risks to aquatic organisms. Taken together, exposure to environmentally relevant concentrations of 2,4-DTBP could negatively affect immune response in zebrafish and may elicit ecological risk in fish population.

With the younger onset age of female lower genital tract diseases, there are increasing demands for protecting organ and tissue structures to preserve fertility and, therefore, effective fertility-sparing treatments that cause minimal normal tissue damage and less adverse reactions are urgently needed.This study is aimed at reviewing information and achieving consensus on recommendations on the clinical applications of aminolevulinic acid-based photodynamic therapy (ALA-PDT) in female lower genital tract diseases.Members of the expert panel held online and in-person meetings to discuss and revise drafts created by the steering committee based on the literature review and the clinical experiences of the expert panel. Opinions of the experts were transcribed and discussed in detail to ensure that the consensus statement best reflects the current advances in the field and the experts' view.After numerous rounds of meetings, experts unanimously agreed on the importance of ALA-PDT in the treatment of cervical squamous intraepithelial lesions (SIL), vaginal SIL, vulvar SIL, vulvar lichen sclerosus (VLS), and condyloma acuminatumon (CA). Experts also reached consensus on the recommended treatment regimen and treatment methods.This consensus aimed to provide practical basis and guidance for the clinical applications of ALA-PDT in female lower genital tract diseases in China. Of note, this is the only expert consensus prepared by board-certified specialists in gynecology and obstetrics in China. More evidence-based clinical studies should be made to update and expand the current recommendations.

Benzotriazole (BT) and its associated derivatives are used ubiquitously in industrial processes, and can be detected in indoor temperature coolants and in chemicals designed to inhibit corrosion. This chemical has been widely detected in aquatic environments and shows some degree of environmental persistence. Evidence has shown that BT exposure can negatively affect endocrine systems and can result in neurotoxicity in fish. However, no study has examined whether this chemical exhibits hepatotoxicity in fish, and if so, what are the underlying mechanism associated with the damage. To address this knowledge gap, we measured the liver proteome of adult male Chinese rare minnow (Gobiocypris rarus) exposed to either 0.05, 0.5, or 5 mg/L BT for 28 days. Overall, 17 proteins were induced and 9 were reduced in abundance following BT treatment (ratio > 1.5, p < 0.05). Pathway analysis revealed that cellular processes affected by BT included xenobiotic clearance, oxidative stress response, apoptosis, and translation. Moreover, transcripts related to these toxic pathways were also significantly affected by BT. In addition, rare minnows exposed to BT showed signs of hypertrophy of hepatocytes, nuclei pyknosis, and higher levels of cellular vacuolization compared to the controls, thus these early proteomic responses in the liver may be related to pathology (i.e. adverse outcome pathway). Our data demonstrate that BT dysregulates molecular responses in the liver and tissue pathology indicative of damage. This study provides new insight into BT hepatotoxicity in Chinese rare minnow.

Sertraline (SER) is one of the most prevalent antidepressants detected in aquatic environments, but its impact on fish behavior and growth remain poorly understood. As such, behavior and growth were assessed in yellow catfish (Tachysurus fulvidraco) following SER exposure. SER induced shoaling, reduced food consumption and growth, and increased cannibalism at environmentally relevant concentrations. To ascertain toxicity mechanisms, acetylcholinesterase (AChE) activity and transcripts related to growth and feeding were measured. AChE activity was increased in fish exposed to 10 and 100 μg/L SER. Transcript levels of neuropeptide Y, somatostatin, growth hormone, and insulin growth factor 1 were reduced in the brain following SER exposure. RNA-seq conducted in brain and liver revealed that gene networks associated with feeding and growth (i.e. leptin expression networks in the brain and insulin signaling pathways in the liver) were altered, proposed to be associated with the decreased food intake and growth. The brain also accumulated SER, which may relate to neurobehavioral responses. Lastly, the main metabolite of SER, norsertraline, was detected in the liver, and may also relate to toxicity. This study uncovers mechanisms and key events proposed to lead to impaired behavior and growth after exposure to some antidepressants.

Sertraline (SER) is one of the most frequently detected antidepressant drugs in aquatic environments. However, knowledge regarding SER-induced behavioral alterations in fish is insufficient, as well as the mechanisms underlying SER-induced toxicity. The present study aimed to determine behavioral and molecular responses in larval fish following SER exposure with a focus on its mode of action. Zebrafish embryos (~6 h-post-fertilization, hpf) were exposed to one of three concentrations of SER (1, 10, 100 μg/L) for 6 days, respectively. Evaluated parameters included development, behavior, transcripts related to serotonin signaling, serotonin levels, and acetylcholinesterase activity. Accelerated hatching of zebrafish embryos was observed for those fish exposed to 100 μg/L SER at 54 hpf. Locomotor activity (e.g. distance moved and mobile cumulative duration) was significantly reduced in larval zebrafish following exposure to 10 and 100 μg/L SER. Conversely, larval fish showed increased dark-avoidance after exposure to 1–100 μg/L SER. Of the measured transcripts related to serotonin signaling, only serotonin transporter (serta) and serotonin receptor 2c (5-ht2c) mRNA levels were increased in fish in response to 10 μg/L SER treatment. However, serotonin levels were unaltered in larvae exposed to SER. There were no differences among groups in acetylcholinesterase activity at any concentration tested. Taking together, the results evidenced that exposure to SER alters behavioral responses in early-staged zebrafish, which may be related to the abnormal expression of 5-ht2c. This study elucidates molecular responses to SER and characterizes targets that may be sensitive to antidepressant pharmaceuticals in larval fish.

Proteomics plays a significant role in discerning the effects of chemical exposures in animal taxa. Multi-omics applications have become more pervasive in toxicology, however questions remain about whether proteomics is being utilized by the community to its full potential - are we placing too much stock in transcriptomics and other omics approaches for developing adverse outcome pathways? Proteins are more relevant than transcripts because they are direct mediators of the resulting phenotype. There is also rarely perfect stoichiometry between transcript and protein abundance and transcript abundance may not accurately predict physiologic response. Proteins direct all levels of phenotype: structural proteins dictate physical form, enzymes catalyze biochemical reactions, and proteins act as signaling proteins, antibodies, transporters, ion pumps, and transcription factors to control gene expression. Molecular initiating events (MIEs) of AOPs predominantly occur at the level of the protein (e.g. ligand-receptor binding) and proteomics can elucidate novel MIEs and mapping KEs in AOPs. This critical review highlights the need for proteomics in multi-omics studies in environmental toxicology and outlines steps required for inclusion and wider acceptance in chemical risk assessment. We also present case studies of multi-omics approaches that utilize proteomics and discuss some of the challenges and opportunities for proteomics in comparative ecotoxicology. Our intention is not to minimize the importance of other omics technologies, as each has strengths and limitations, but rather to encourage researchers to consider proteomics-based methods in multi-omics studies and AOP development.

Advances in proteomics have greatly improved chemical toxicity assessment and predictions of adverse outcomes in organisms. Ecotoxicoproteomics has been employed to elucidate biological pathways affected by chemicals and provide data that can be incorporated into adverse outcome pathways (AOP) to better define the ecological risk of emerging pollutants. Microplastics (MPs) and plastic additives have raised global concern due to their widespread use in aquatic environments, bioaccumulation in tissues, and toxic effects in aquatic organisms. Despite showing sublethal toxicity in many cases, mechanisms underlying these emerging pollutants are underexplored. In this review, adverse effects and recent ecotoxicoproteomic studies of MPs and typical additives (i.e. plasticizers, flame retardants, antioxidants, and UV stabilizers) in aquatic organisms are summarized. Proteomics data show that MPs adversely affect ingestion and reproduction via disrupting pathways related to energy metabolism, stress-related defense, and cytoskeletal dynamics. Biological processes including lipid metabolism, energy homeostasis, skeletal development, neurotransmitter signaling, and immune response are modulated by additives and induce developmental malformations in fish embryos/larvae. Furthermore, plastic additives also exert reproductive toxicity, hepatotoxicity, and neurotoxicity in invertebrates (e.g. mussel, abalone, and oyster) and fish by disrupting detoxification/oxidative stress, hormonal modulation, signal transduction, and apoptosis. Additional studies are needed to complement the omic knowledge of chemical additives that are not well documented (e.g. UV stabilizers) for improving understanding into toxic mechanisms and for characterizing ecological risk linked to plastic contaminants.

Fipronil is a phenylpyrazole pesticide that is used in both residential and agricultural applications. Fipronil is detected in run-off and water systems that are near areas in which the pesticide has been applied. The pesticide acts to antagonize gamma aminobutyric acid receptors, leading to over-excitation in the central nervous system. Fipronil has relatively high toxicity to fish, but the mechanisms underlying the toxicity are not well understood in embryonic stages. Zebrafish embryos were exposed to a single concentration of fipronil for 48 h at ∼3-4 h-post-fertilization. Following a 7-day depuration phase, transcriptome and behavioral analyses were conducted. Transcriptomics identified neural processes as those differentially expressed with different doses of fipronil (0.2 µg, 200 µg and 2 mg fipronil/L). Gene networks associated with astrocyte differentiation, myelination, neural tube development, brain stem response, innervation, nerve regeneration, astrocyte differentiation, among other pathways were altered with exposure. In addition, miRNA-related events are disrupted by fipronil exposure and genes associated with primary or pri-miRNA processing were increased in larval fish exposed to the pesticide. These data present putative mechanisms associated with neurological impacts at later ages of zebrafish. This is important because it is not clear how early exposure to pesticides like fipronil affect central nervous system function and organisms later in life.

Benzotriazole ultraviolet stabilizers (BUVSs) are widely used additives in industrial materials and personal care products that protect products from ultraviolet damage. Due to their high production volume and potential to bioaccumulate, BUVSs are an environmental pollutant of concern. In this study, juvenile zebrafish (Danio rerio) were exposed to 4 BUVSs (UV-234, UV-326, UV-329, and UV-P) at 10 and 100 μg/L for 28 d. BUVSs induced hepatic vacuolization and nuclei pyknosis in the liver following 100 μg/L UV-234 and UV-329 exposure. Transcriptomic analysis in the liver uncovered pathways related to inflammation that were affected by BUVSs. Based upon these data, we measured the expression levels of 9 genes involved in AHR-IL17/IL22 pathway in zebrafish larvae exposed to each BUVSs at one dose of either 10 or 100 μg/L for 6 days in a second set experiment. Transcript levels of interleukins il17a and il22 were decreased, while il6 mRNA was increased with exposure to UV-234, UV-329, and UV-P. No change to targeted transcripts was observed with UV-326 treatments. Moreover, cyp1a1 and ahr2 levels were increased in larvae treated with 100 μg/L UV-329 or UV-P. Consistent with expression data, protein abundance of IL22 was decreased by 29% with exposure to 100 μg/L UV-P. Taken together, these results demonstrate that exposure to different benzotriazole congeners may be associated with immunotoxicity in zebrafish through the AHR-IL17/IL22 pathway, and this may be associated with hepatic damage with prolonged exposures. This study provides new insight into unique pathways perturbed by specific BUVSs congeners.

Carbamazepine (CBZ), an anticonvulsant and mood-stabilizing pharmaceutical, is a widespread contaminant in aquatic environments. In this study, the effects of chronic exposure to environmentally relevant CBZ concentrations were investigated in freshwater clams Corbicula fluminea. Adult C. fluminea were exposed to 0.5, 5, and 50 μg/L of CBZ for 30 days, after which siphoning behavior (filtration rates), biomarker levels, and heat shock protein expression were measured. The filtration rates were significantly decreased (p

Benzotriazole (BT), an anticorrosive agent, is widely used in industrial applications and household dish-washing agents. Despite its reported toxicity to aquatic organisms, little is known about its endocrine disrupting effects. In this study, adult Chinese rare minnows (Gobiocypris rarus) were exposed to 0.05, 0.5, and 5 mg L-1 BT for 28 d. The pathological damage in liver was associated with hypertrophy of the hepatocytes, nuclei pyknosis and vacuolization at 5 mg L-1 groups. Additionally, the degeneration of the ovary and the stimulation of spermatogenesis were observed at 5 mg L-1 groups. The plasma 17 beta-estradiol level was significantly increased in the males but decreased in the females at 5 mg L-1 (p < 0.05). In the brain, the up-regulation of CYP19B, GnRHs, and LH beta mRNA was detected across all doses (p < 0.05). In the gonad, the transcriptional levels of StAR, CYP11A, 3 beta HSD, CYP17, 17 beta HSD, and CYP19A were generally decreased in the males at 5 mg L-1 (p < 0.05), whereas these genes, except for 3 beta HSD, were significantly increased in females at all concentrations (p < 0.05). Moreover, the expression level of VTG in the livers from all exposure groups was significantly increased compared with controls (p < 0.05). Taken together, our results indicate that BT could adversely affect the rare minnows in a sex-dependent manner. (C) 2014 Elsevier Ltd. All rights reserved.

Assessing protein responses to endocrine disrupting chemicals is critical for understanding the mechanisms of chemical action and for the assessment of hazards. In this study, the response of the liver proteome of male rare minnows (Gobiocypris rarus) treated with 17β-estradiol (E2) and females treated with 17α-methyltestosterone (MT) were analyzed. A total of 23 and 24 proteins were identified with differential expression in response to E2 and MT, respectively. Pyruvate carboxylase (PC) was the only common differentially expressed protein in both males and females after E2- and MT-treatments. The mRNA as well as the protein levels of PC were significantly down-regulated compared with that of the controls (p

a b s t r a c t Dechlorane Plus (DP), an alternative to decabromodiphenyl ether (BDE-209), is a widely used polychlo- rinated flame retardant that is frequently detected in aquatic ecosystems. While the mechanisms of toxicity of BDE-209 have been well documented, less is known about the toxicity of DP. In this study, juvenile Chinese sturgeon (Acipenser sinensis) were treated with DP at doses of 1, 10, and 100 mg/kg wet weight for 14 days via a single intraperitoneal injection (i.p.). After 14 days, liver proteomes of juvenile Chinese sturgeon were analyzed using two-dimensional electrophoresis (2-DE) coupled matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). A total of 39 protein spots were significantly altered in abundance (>2-fold) and of these proteins, 27 were suc- cessfully identified. Proteins related to the stress response that included heat shock cognate protein 70 and T-complex protein 1 were significantly increased and decreased in abundance, respectively. More- over, Ras-related protein Rab-6B and GDP dissociation inhibitor 2, proteins that are involved in small G-protein signal cascades, were decreased in abundance 2- to 5-fold. Annexin A4, which is associated with Ca 2+ signaling pathways, was also markedly decreased by 2-fold in the liver. Pathway analysis of differentially regulated proteins revealed that DP interfered with metabolism and was associated with proteins related to apoptosis and cell differentiation. Based upon protein responses, we suggest that DP has effects on the generalized stress response, small G-protein signal cascades, Ca 2+ signaling pathway, and metabolic process, and may induce apoptosis in the liver. This study offers novel mechanistic insight into the protein responses induced in the liver with DP, an increasingly used and understudied flame retardant.

Molecular initiating events and downstream transcriptional/proteomic responses provide valuable information for adverse outcome pathways, which can be used predict the effects of chemicals on physiological systems. There has been a paucity of research that addresses sex-specific expression profiling in toxicology and due to cost, time, and logistic considerations, sex as a variable has not been widely considered. In response to this deficiency, federal agencies in the United States, Canada, and Europe have highlighted the importance of including sex as a variable in scientific investigations. Using case studies from both aquatic and mammalian toxicology, we report that there can be less than ~ 20–25% consensus in how the transcriptome and proteome of each sex responds to chemicals. Chemicals that have been shown to elicit sex-specific responses in the transcriptome or proteome include pharmaceuticals, anti-fouling agents, anticorrosive agents, and fungicides, among others. Sex-specific responses in the transcriptome and proteome are not isolated to whole animals, as investigations demonstrate that primary cell cultures isolated from each sex responds differently to toxicants. This signifies that sex is important, even in cell lines. Sex has significant implications for predictive toxicology, and both male and female data are required to improve robustness of adverse outcome pathways. Biological significance Clinical toxicology recognizes that sex is an important variable, as pharmacokinetics (ADME; absorption, distribution, metabolism, and excretion) can differ between females and males. However, few studies in toxicology have explored the implication of sex in relation to the transcriptome and proteome of whole organisms. High-throughput molecular approaches are becoming more frequently applied in toxicity screens (e.g. pre-clinical experiments, fish embryos, cell lines, synthetic tissues) and such data are expected to build upon reporter-based cell assays (e.g. receptor activation, enzyme inhibition) used in toxicant screening programs (i.e. Tox21, ToxCast, REACH). Thus, computational models can more accurately predict the diversity of adverse effects that can occur from chemical exposure within the biological system. Our studies and those synthesized from the literature suggest that the transcriptome and proteome of females and males respond quite differentially to chemicals. This has significant implications for predicting adverse effects in one sex when using molecular data generated in the other sex. While molecular initiating events are not expected to differ dramatically between females and males (i.e. an estrogen binds estrogen receptors in both sexes), it is important to acknowledge that the downstream transcriptomic and proteomic responses can differ based upon the presence/absence of co-regulators and inherent sex-specific variability in regulation of transcriptional and translational machinery. Transcriptomic and proteomic studies also reveal that cell processes affected by chemicals can differ due to sex, and this can undoubtedly lead to sex-specific physiological responses.