Polystyrene nanoplastics induced size-dependent developmental and neurobehavioral toxicities in embryonic and juvenile zebrafish.

Because of widespread environmental contamination, there is growing concern that nanoplastics may pose a risk to humans and the environment. Due to their small particle size, nanoplastics may cross the blood-nerve barrier and distribute within the nervous system. The present study systematically investigated the uptake/distribution and developmental/neurobehavioral toxicities of different sizes (80, 200, and 500 nm) of polystyrene nanoplastics (PS) in embryonic and juvenile zebrafish. The results indicate that all three sizes of PS could cross the chorion, adsorb by the yolk, and distribute into the intestinal tract, eye, brain, and dorsal trunk of zebrafish, but with different patterns. The organ distribution and observed developmental and neurobehavioral effects varied as a function of PS size. Although all PS exposures induced cell death and inflammation at the cellular level, only exposures to the larger PS resulted in oxidative stress. Meanwhile, exposure to the 80 nm PS increased the expression of neural and optical-specific mRNAs. Collectively, these studies indicate that early life-stage exposures to PS adversely affect zebrafish neurodevelopment and that the observed toxicities are influenced by particle size.

miRNA-660-3p inhibits malignancy in glioblastoma via negative regulation of APOC1-TGFß2 signaling pathway.

Glioblastoma as the most common and aggressive central nervous system tumor in adults. Its prognosis and therapeutic outcome are poor due to the limited understanding of its molecular mechanism. Apolipoprotein C-1 (APOC1) as a member of the apolipoprotein family that acts as a tumor promoter in various cancers. MicroRNA (miRNA) can silence gene expression and suppress tumor progression. However, the role of APOC1 and its upstream miRNA has not been explored in glioblastoma. Two glioblastoma cell lines (U87 and U251) were used to explore the role of APOC1 and its upstream miRNA-660-3p in glioblastoma tumorigenesis in vitro. Cells with APOC1/miRNA-660-3p overexpression or knockdown were assessed for their proliferation, migration, and invasion in vitro, and tumorigenesis in vivo. Gene and protein expression was assessed by qRT-PCR and western blot, respectively. Cell proliferation was assessed by the MTT assay and the EdU and Ki67 staining. Cell migration and invasion were assessed by the transwell assay. Tumorigenesis in vivo was assessed in U87 cells with a xenograft mouse model. APOC1 was overexpressed in glioblastoma compared with normal peritumoral tissue and was inversely related to patient prognosis. APOC1 overexpression promotes cell proliferation, migration, and invasion in vitro. APOC1 inhibition reduced tumor growth in vivo. miRNA-660-3p inhibits tumorigenesis by directly targeting APOC1. Mechanistically, APOC1 drives the malignancy of glioblastoma by activating the TGFß2 signaling pathway. miRNA-660-3p suppresses tumorigenesis by targeting APOC1. Therefore, miRNA-660-3p/APOC1 axis can serve as potential intervention targets in managing glioblastoma progression.

6PPD and its metabolite 6PPDQ induce different developmental toxicities and phenotypes in embryonic zebrafish.

The automobile tire antioxidant N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) and its quinone metabolite 6PPDQ have recently received much attention for their acute aquatic toxicity. The present study investigated the mechanistic developmental toxicity of 6PPD and 6PPDQ in embryonic zebrafish. Neither compound induced significant mortality but significantly decreased spontaneous embryo movement and heart rate. Both compounds induced malformations with different phenotypes; the 6PPD-exposed larvae manifested a myopia-like phenotype with a convex eyeball and fusion vessels, while the 6PPDQ-exposed embryonic zebrafish manifested enlarged intestine and blood-coagulated gut, activated neutrophils, and overexpressed enteric neurons. mRNA-Seq and quantitative real-time PCR assays showed that 6PPD- and 6PPDQ-induced distinct differential gene expression aligned with their toxic phenotype. 6PPD activated the retinoic acid metabolic gene cyp26a, but 6PPDQ activated adaptive cellular response to xenobiotics gene cyp1a. 6PPD suppressed the gene expression of the eye involved in retinoic acid metabolism, phototransduction, photoreceptor function and visual perception. In contrast, 6PPDQ perturbed genes involved in inward rectifier K+ and voltage-gated ion channels activities, K+ import across the plasma membrane, iron ion binding, and intestinal immune network for IgA production. The current study advances the present understanding the reason of why many fish species are so adversely impacted by 6PPD and 6PPDQ.

Structure-based developmental toxicity and ASD-phenotypes of bisphenol A analogues in embryonic zebrafish.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder that has become more prevalent in recent years. Environmental endocrine disruptor bisphenol A (BPA) has been linked to ASD. BPA analogues (BPs) are structure-modified substitutes widely used as safer alternatives in consumer products, yet few studies have explored the developmental neurotoxicity (DNT) of BPA analogues. In the present study, we used the larval zebrafish model to assess the DNT effects of BPA and its analogues. Our results showed that many BPA analogues are more toxic than BPA in the embryonic zebrafish assay regarding teratogenic effect and mortality, which may partially due to differences in lipophilicity and/or different substitutes of structural function groups such as CF3, benzene, or cyclohexane. At sublethal concentrations, zebrafish embryos exposed to BPA or BPs also displayed reduced prosocial behavior in later larval development, evidenced by increased nearest neighbor distance (NND) and the interindividual distance (IID) in shoaling, which appears to be structurally independent. An in-depth analysis of BPA, bisphenol F (BPF), and bisphenol S (BPS) revealed macrocephaly and ASD-like behavioral deficits resulting from exposures to sublethal concentrations of these chemicals. The ASD-like behavioral deficits were characterized by hyperactivity, increased anxiety-like behavior, and decreased social contact. Mechanistically, accelerated neurogenesis that manifested by increased cell proliferation, the proportion of newborn mature neurons, and the number of neural stem cells in proliferation, as well as upregulated genes related to the K+ channels, may have contributed to the observed ASD-like morphological and behavioral alterations. Our findings indicate that BPF and BPS may also pose significant risks to ASD development in humans and highlight the importance of a comprehensive assessment of DNT effects for all BPA analogues in the future.

Cold stress during the perinatal period leads to developmental and neurobehavioral toxicity in zebrafish larvae.

In nature, cold stress is a core threat to aquatic organisms. But the neurodevelopmental effects of cold stress during the perinatal period on the offspring development were unknown. In the present study, adult zebrafish were cold-stressed at 18 °C for five days before spawning, and then the fertilized eggs were raised at 18, 24, or 28 °C from 0 to 120 h post fertilization (hpf). The resulting embryos and larvae were assessed for developmental and neurobehavioral responses. Our findings showed that embryos raised at 18 °C (Cold+++) suffered hatching failure and death, at 24 °C (Cold++) had decreased hatching, while those raised at 28 °C (Cold+) exhibited no developmental adversity. The neurobehavioral assessment showed that embryos from Cold+ and Cold++ groups displayed decreased motor behaviors, including spontaneous movement at 20-24 hpf, touch response at 48 hpf, and swimming speed at 120 hpf. In addition, cold stress during perinatal stage irreversibly affected larval social behaviors examined during 10-13 days post fertilization (dpf), such as unconsolidated shoaling, increased mirror attacks, and decreased social contacts. Notably, behavioral adversity was more pronounced in larvae from the Cold ++ group than those from the Cold+ group. Mechanistically, cold stress increased cell apoptosis, evidenced by increased acridine orange positive cells at 24 hpf and upregulation of casp8 at 120 hpf, increased oxidative stress (upregulation of cat and nos1) at 120 hpf, delayed motor neuron extension at 72 hpf, and upregulated nrxn2 and rab33a at 120 hpf. Our data indicate that cold stress during the perinatal period impaired neural development in zebrafish larvae, showing high mental health risk. These findings highlight cold stress should be avoided during the perinatal period for both aquatic fish or even humans.

Lifetime exposure to benzophenone-3 at an environmentally relevant concentration leads to female-biased social behavior and cognition deficits in zebrafish.

Benzophenone-3 (BP3) is an organic UV filter widely used in the commercial formulations of various personal care products. It has been detected ubiquitously in the environment and human tissues. Recently, BP3-induced neurotoxicity has been identified as the main health risk to humans and aquatic organisms. However, most research has been focused on embryonic development, and few studies explore chronic lifetime exposure. In the present study, we evaluated the neurotoxicity of lifetime exposure to an environmentally relevant concentration of BP3 in zebrafish. Our findings revealed that continuous BP3 exposure at 10 µg/L (0.04 µM) from 6 h post fertilization (hpf) to adulthood at 5 months led to female-biased social behavioral deficits and learning and memory impairment. These neurobehavioral effects were characterized by decreased prosocial activities in the social preference test and mirror biting assay, and reduced learning and memory in a T-maze test. Furthermore, these effects were accompanied by female-specific decreases in brain weight and brain dopamine concentration, female-biased decrease of neurogenesis in the telencephalon as well as female-specific increases in apoptotic cells and expression levels of genes and proteins related to the apoptosis pathway in the brain. Our results suggest that BP3-induced social behavior and learning/memory deficits are correlated to the cell loss in the telencephalon region of the zebrafish brain.

Characterization of Developmental Neurobehavioral Toxicity in a Zebrafish MPTP-Induced Model: A Novel Mechanism Involving Anemia.

Zebrafish represent an economical alternative to rodents for developmental neurotoxicity (DNT) testing. Mechanistic understanding is the key to successfully translating zebrafish findings to humans. In the present study, we used a well-known dopaminergic (DA) neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) as a model chemical to uncover the molecular pathways for observed DNT effects. To enhance the specificity of potential molecular targets, we restricted our exposure to a concentration that is nonteratogenic yet exhibits high DNT effects and an exposure window sensitive to MPTP. Our DNT assessment based on a battery of motor and social behavioral tests revealed an effective concentration of 1 µM and a sensitive window of 48-96 h postfertilization (hpf) for MPTP-induced hypoactivity. It is worth noting that this hypoactivity persisted into later larval development until 28 dpf. We observed increased cell apoptosis, oxidative stress, and decreased ATP levels in larvae immediately after exposure at 96 hpf. Significant reductions of DA neurons were found in the retina at 72, 96, and 120 hpf. No visible deformity was found in motoneurons at 72, 96, and 120 hpf. Transcriptome analysis uncovered a novel pathway manifested by significant upregulation of genes enriched with erythropoiesis. Sensitive window exposure of MPTP and other DA neurotoxins rotenone and paraquat exhibited a concentration-dependent effect on transcriptional changes of embryonic hemoglobins and anemia. Given that anemia is a significant risk factor for Parkinson's disease and MPTP is known to cause parkinsonism in humans, we concluded that anemia resulting from dysregulation of primitive erythropoiesis during embryonic development might serve as a common mechanism underlying DA neurotoxin-induced DNT effects between zebrafish and humans.

Transient MPTP exposure at a sensitive developmental window altered gut microbiome and led to male-biased motor and social behavioral deficits in adult zebrafish.

Zebrafish is an economical alternative model for developmental neurotoxicity (DNT) testing. DNT studies in zebrafish have been focused on acute effects; few studies explore enduring neurotoxicity in adults. More recently, gut microbiome has emerged as an important modulator between chemical exposure and neurotoxicity, rendering its necessity to be included in DNT testing. The present study used a well-known dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) as a model chemical to explore long-lasting neurotoxicity in adults after transient exposure during early development. We demonstrated that transient MPTP exposure at 1 µM during a sensitive developmental window of 48-96 h post-fertilization (hpf) altered gut microbiome and led to male-biased locomotion and behavioral deficits in adult fish. The locomotion deficit was manifested as hypoactivity observed in adult males under light conditions or specifically the reduction of fast swim bouts. The social behavioral deficits were characterized by the reduced number of times fish crossed the mirror zone in the mirror response assay and the reduced percent time fish spent at the area proximal to conspecific fish shoal in the social preference test. Gut microbiome analysis revealed that transient MPTP exposure during early development might render fish more susceptible to the colonization of the pathogenic Vibrio. In conclusion, our study revealed that transient MPTP exposure during early development could lead to long-lasting neurotoxicity in adult fish and cause altered gut microbiome composition in both larval and adult fish.

Developmental titanium dioxide nanoparticle exposure induces oxidative stress and neurobehavioral changes in zebrafish.

The widespread commercial application of titanium dioxide nanoparticles (TiO2 NPs) leads to ubiquitous presence of TiO2 NPs in the aquatic environment, which highlights the necessity to determine their potential adverse effects on aquatic organisms. The developing nerve system is particularly susceptible to environment perturbation. However, few studies have explored the developmental neurobehavioral toxicity of TiO2 NPs, especially at smaller particle size ranges (≤20 nm) that have relatively longer retention time in the water column. In this study, zebrafish embryos were exposed to non-teratogenic concentrations of 0.1 and 1 mg/L TiO2 NPs (average size of 14-20 nm) from 8 to 108 h post-fertilization (hpf) followed by various assessments at different time points up to 12 days post-fertilization (dpf). Our findings revealed that 1 mg/L TiO2 NPs perturbed the motor and social behaviors in larval zebrafish. These behavioral changes were characterized by decreased swimming speed in a locomotor response test at 5 dpf, increased travel distance in a flash stimulus test at 5 dpf, increased preference to the light zone in a light/dark preference test at 10 dpf, and increased mirror attack and percent time spent in the mirror zone in a mirror stimulus response assay at 12 dpf. Mechanistic examinations at 5 dpf revealed elevated cell apoptosis and oxidative stress. Cell apoptosis was characterized by increased acridine orange (AO) positive cells in the olfactory region and neuromasts of the lateral line system. Oxidative stress was characterized by increased lipid peroxidation, increased ROS production, and upregulated catalase (cat) gene expression. In addition, TiO2 NP exposure also upregulated genes associated with the developmental nervous system such as the growth associated protein 43 (gap43) and proliferating cell nuclear antigen (pcna). Our results suggest that the neurobehavioral changes in larvae exposed to 1 mg/L TiO2 NPs during early development may result from cell apoptosis and oxidative stress induced neuronal damages.

Early life stage transient aristolochic acid exposure induces behavioral hyperactivity but not nephrotoxicity in larval zebrafish.

Aristolochic acids (AA) are nitrophenanthrene carboxylic acids found in plants of the Aristolochiaceae family. Humans are exposed to AA by deliberately taking herbal medicines or unintentionally as a result of environmental contamination. AA is notorious for its nephrotoxicity, however, fewer studies explore potential neurotoxicity associated with AA exposure. The developing nervous system is vulnerable to xenobiotics, and pregnant women exposed to AA may put their fetuses at risk. In the present study, we used the embryonic zebrafish model to evaluate the developmental neurotoxicity associated with AA exposure. At non-teratogenic concentrations (≤ 4 µM), continuous AA exposure from 8 to 120 hours post fertilization (hpf) resulted in larval hyperactivity that was characterized by increased moving distance, elevated activity and faster swimming speeds in several behavioral assays. Further analysis revealed that 8-24 hpf is the most sensitive exposure window for AA-induced hyperactivity. AA exposures specifically increased motor neuron proliferation, increased apoptosis in the eye, and resulted in cellular oxidative stress. In addition, AA exposures increased larval eye size and perturbed the expression of vision genes. Our study, for the first time, demonstrates that AA is neurotoxic to the developmental zebrafish with a sensitive window distinct from its well-documented nephrotoxicity.

Environmental relevant concentrations of benzophenone-3 induced developmental neurotoxicity in zebrafish.

Benzophenone-3 (BP3 or oxybenzone) is an organic UV filter that has been widely used in personal care products. Its frequent detection in the environment and humans as well as its structural similarity to estradiol have prompted most research focus on its endocrine effect. However, these effects are usually associated with concentrations 10-100 fold higher than its environmental relevant concentrations. Few studies explore its adverse effects at environmental relevant concentrations. In the present study, we evaluated the developmental neurotoxic (DNT) effects of low concentration BP3 exposure during a sensitive developmental window in zebrafish. Our findings revealed that BP3 exposure at 10 µg/L (0.04 µM) during 6-24 h post fertilization (hpf) led to various DNT effects such as increased spontaneous movement at 21 and 24 hpf, decreased touch response at 27 hpf, heightened hyperactivity in locomotor response at 5 day post fertilization (dpf), decreased shoaling behavior at 11 dpf and decreased mirror attacks at 12 dpf. These effects were accompanied with decreased axonal growth at 27 hpf, decreased cell proliferation and increased cell apoptosis in the head region of larval zebrafish immediately after BP3 exposure at 24 hpf, and increased expression of retinoid X receptor gene rxrgb at 5 dpf. Interestingly, rxrgb knockdown through morpholino injection largely restored most of BP3-induced DNT effects, axonal growth delay, cell proliferation and cell apoptosis, suggesting that BP3-induced DNT effects are likely mediated through the Rxrgb receptor. In considering with recent findings on the endocrine effects of BP3, we conclude that BP3 at environmental relevant concentrations has limited estrogenic effect, but is neurotoxic to developing embryos in zebrafish.

Cryopreservation disrupts lipid rafts and heat shock proteins in yellow catfish sperm.

Lipid rafts and associated membrane proteins (flotillin, caveolin) play important roles in cell signaling and sperm fertilization while heat shock proteins (Hsp) ensure properly protein folding to fulfill their physiological functions. The markedly reduced fertility in thawed sperm after cryopreservation could result from disrupted membrane lipid rafts and these proteins. To explore the effect of sperm cryopreservation on lipid rafts and heat shock proteins, we compared lipid raft integrity, and the expression levels of lipid raft associated proteins (Flot-1, Flot-2, Cav-1) as well as heat shock proteins (Hsp90, Hsp70) in fresh and thawed sperm cryopreserved under different scenarios in yellow catfish. We found higher lipid raft integrity, higher protein expression levels of Flot-1, Flot-2, Cav-1, Hsp90, and Hsp70 in fresh sperm samples than in thawed sperm samples, in thawed sperm samples cryopreserved with optimal cooling rate than those cryopreserved with sub-optimal cooling rate, and in thawed sperm samples cryopreserved with extenders supplemented with cholesterol than those supplemented with methyl-ß-cyclodextrin (for cholesterol removal). Our findings indicate that lipid raft integrity, and expression levels of Flot-1, Flot-2, Cav-1, Hsp90, and Hsp70 are clearly associated with sperm quality, and together they may play a cumulative role in reduced fertility associated with thawed sperm in aquatic species.

Maternal exposure to low dose BDE209 and Pb mixture induced neurobehavioral anomalies in C57BL/6 male offspring.

Polybrominated diphenyl ethers (PBDEs) and lead (Pb) are common pollutants that co-exist in the environment. These chemicals may be associated with autism spectrum disorder (ASD), yet direct evidence is lacking. More importantly, how co-exposure of these chemicals might affect ASD has never been explored. For assessing the relationship between PBDE/Pb exposure and ASD, pregnant C57BL/6 J female mice were exposed to BDE209 (0.12 ng/day), Pb (1.2 ng/day), or a BDE209/Pb mixture from gestational day (GD) 9.5 to postnatal day (PND) 21 using ALZET osmotic pumps. Polyinosinic-polycytidylic acid (poly I:C) was included as a positive control, as its single dose injection (20 mg/kg.bw; i.p.) at mid-pregnancy (GD 12.5) produces ASD-like behaviors in mouse offspring. These ASD-like phenotypes include decreased preference for social novelty, increased marble burying behavior, and learning impairment. Similar to the poly I:C control, perinatal exposure to Pb or BDE209/Pb mixture elicited increased marble burying and learning impairment, but it had no effect on sociability. Consistent with these behavioral anomalies, Pb and BDE209/Pb co-exposure as well as poly I:C exposure increased the production of pro-inflammation cytokines interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 10 (IL-10), tumor necrosis factor α (TNFα), interferon γ (IFNγ), and interleukin 17 A (IL-17 A) in the serum, and decreased neuronal cells in the CA1 and CA3 subregions of the hippocampus. The majority of these changes in the BDE209/Pb mixture group were due to the effect of Pb rather than BDE209. However, BDE209/Pb co-exposure elicited a synergistic increase in the production of IL-4, IL-6, TNFα, IFNγ, and IL-17A in the serum. BDE209 exposure alone also significantly affected spatial learning and increased the production of IL-10, TNFα, and IL-17 A in the serum of male offspring. Our work demonstrates that perinatal exposure to a low dose of Pb or the BDE209/Pb mixture, although it did not induce typical ASD-like symptoms, elicited restricted, repetitive patterns of behavior and affected learning in male offspring. In addition, the synergistic increase in the systemic inflammatory response in the BDE209/Pb co-exposure group underscores the importance of evaluating chemical mixtures in disease onset.

Developmental bisphenol A exposure impairs sperm function and reproduction in zebrafish.

The developmental and reproductive toxicity of bisphenol A (BPA) has been demonstrated in a variety of model systems. Zebrafish (Danio rerio) were waterborne-exposed to BPA during three different developmental stages: embryonic period:6 h post fertilization (hpf) to 5 months post fertilization (mpf); larval period: 6 days post fertilization (dpf) to 5 mpf; and sexually mature period: 3 mpf to 5 mpf. Evaluations included F0 adult growth, reproduction parameters, and F1 offspring development. BPA exposure did not affect zebrafish growth in any of exposure groups. Testis weight was decreased only following the 6 hpf to 5 mpf 0.001 µM BPA exposure. The lowest effect level indicated by a reduction in sperm volume, density, motility, and velocity across a range of exposure durations was 0.001 µM, with all but sperm density significant for the longest exposure duration, which was also the only significant endpoint for the lowest exposure concentration in the 3-5 mpf exposure group. Nonmonotonic concentration-response curves were noted for all F0 reproductive endpoints for at least one of the two longest exposure durations. For the F1 offspring of fish exposed from 6 hpf to 5 mpf, malformations and mortality were increased following 0.001 µM BPA exposure, while egg production and fertilization were reduced in higher concentration treatment groups. Overall, BPA exposure during three different developmental periods impaired zebrafish reproductive development, with most significance changes found in the lowest concentration treatment groups. Genetic impacts on gamete development may underlie the secondary effects of reduced fertilization rate, embryonic mortality, and malformations.

Subchronic perfluorooctanesulfonate (PFOS) exposure induces elevated mutant frequency in an in vivo λ transgenic medaka mutation assay.

Perfluorooctanesulfonate (PFOS) has been widely detected in the environment, wildlife and humans, but few studies have ever examined its mutagenic effect in vivo. In the present study, we use a transgenic fish model, the λ transgenic medaka, to evaluate the potential mutagenicity of PFOS in vivo following a subchronic exposure of 30 days. The mutant frequency of cII target gene was 3.46 × 10-5 in liver tissue from control fish, which increased by 1.4-fold to 4.86 × 10-5 in fish exposed to 6.7 µg/L PFOS, 1.55-fold to 5.36 × 10-5 in fish exposed to 27.6 µg/L PFOS, and 2.02-fold to 6.99 × 10-5 in fish exposed to 87.6 µg/L PFOS. This dose-dependent increase of mutant frequency was also accompanied with mutational spectrum changes associated with PFOS exposure. In particular, PFOS-induced mutation was characterized by +1 frameshift mutations, which increased from 0% in control fish to 13.2% in fish exposed to 27.6 µg/L PFOS and 14.6% in fish exposed to 87.6 µg/L PFOS. Our findings provide the first evidence of PFOS's mutagenicity in an aquatic model system. Given the fact that most conventional mutagenic assays were negative for PFOS, we propose that PFOS-induced mutation in liver tissue of λ transgenic medaka may be mediated through compromised liver function.

TBBPA chronic exposure produces sex-specific neurobehavioral and social interaction changes in adult zebrafish.

The toxicity of tetrabromobisphenol A (TBBPA) has been extensively studied because of its high production volume. TBBPA is toxic to aquatic fish based on acute high concentration exposure tests, and few studies have assessed the behavioral effects of low concentration chronic TBBPA exposures in aquatic organisms. The present study defined the developmental and neurobehavioral effects associated with exposure of zebrafish to 0, 5 and 50nM TBBPA during 1-120days post-fertilization (dpf) following by detoxification for four months before the behaviors assessment. These low concentration TBBPA exposures were not associated with malformations and did not alter sex ratio, but resulted in reduced zebrafish body weight and length. Adult behavioral assays indicated that TBBPA exposed males had significantly higher average swim speeds and spent significantly more time in high speed darting mode and less time in medium cruising mode compared to control males. In an adult photomotor response assay, TBBPA exposure was associated with hyperactivity in male fish. Female zebrafish responses in these assays followed a similar trend, but the magnitude of TBBPA effects was generally smaller than in males. Social interaction evaluated using a mirror attack test showed that 50nM TBBPA exposed males had heightened aggression. Females exposed to 50nM TBBPA spent more time in the vicinity of the mirror, but did not show increased aggression toward the mirror compared to unexposed control fish. Overall, the hyperactivity and social behavior deficits ascribed here to chronic TBBPA exposure was most profound in males. Our findings indicate that TBBPA can cause developmental and neurobehavioral deficits, and may pose significant health risk to humans.

In vivo DNA mismatch repair measurement in zebrafish embryos and its use in screening of environmental carcinogens.

Impairment of DNA mismatch repair (MMR) function leads to the development and progression of certain cancers. Many environmental contaminants can target DNA MMR system. Currently, measurement of MMR activity is limited to in vitro or in vivo methods at the cell line level, and reports on measurement of MMR activity at the live organism level are lacking. Here, we report an efficient method to measure DNA MMR activity in zebrafish embryos. A G-T mismatch was introduced into enhanced green fluorescent protein (EGFP) gene. Repair of the G-T mismatch to G-C in the heteroduplex plasmid generates a functional EGFP expression. The heteroduplex plasmid and a similarly constructed homoduplex plasmid were injected in parallel into the same batch of embryos at 1-cell stage and EGFP expression in EGFP positive embryos was quantified at 24 h after injection. MMR efficiency was calculated as the total fluorescence intensity of embryos injected with the heteroduplex construct divided by that of embryos injected with the homoduplex construct. Our results showed 73% reduction of MMR activity in embryos derived from MMR-deficient mlh1 mutant fish (positive control) when compared with embryos from MMR-competent wild type AB line fish, indicating feasibility of in vivo MMR activity measurement in zebrafish embryos. We further applied this novel assay for measurement of MMR efficiency in embryos exposed to environmental chemicals such as cadmium chloride (CdCl2), benzo[a]pyrene (BaP), and perfluorooctanesulphonic acid (PFOS) from 6 hpf to 24 hpf. We observed significant reductions of MMR efficiency in embryos exposed to 0.1 µM CdCl2 (52%) and 0.5 µM BaP (34%), but no effect in embryos exposed to PFOS. Our study for the first time provides a model system for in vivo measurement of DNA MMR activity at the organism level, which has important implications in risk assessment of various environmental carcinogens.

Reproductive toxicity of low level bisphenol A exposures in a two-generation zebrafish assay: Evidence of male-specific effects.

Bisphenol A (BPA), a high-volume chemical used to make polycarbonate plastic and epoxy resins, is a ubiquitous contaminant in environment and human body. To investigate the reproductive effects of long-term exposure to low concentrations of BPA, a two-generation study was conducted using the aquatic model species of zebrafish. Our findings revealed that exposure to 1nM (0.228µg/L) BPA for continuous two generations resulted in female-biased sex ratio in both F1 and F2 adult population, decreased sperm density, and decreased sperm quality as measured by motility, velocity, ATP content and lipid peroxidation in F1 and F2 males. Females were less sensitive to BPA exposures than males as no adverse effects were found in female gonads or gametes. Delayed hatching at 48hpf and increased malformation and mortality were found in the offspring from BPA exposed F2, but not F1 parents. Most importantly, the adverse effect on larval development and survival from BPA exposed F2 parents was paternal-specific, resulting mainly from BPA exposed males. Subsequent transcription analysis of F2 male gonads revealed dysregulated mitochondrial biogenesis and significant activation of non-canonical Wnt/planar cell polarity and Wnt/Calcium signaling pathways. Gene expression analysis of larvae from BPA exposed F2 parents showed significant reduced expression of DNA methyltransferases such as dnmt1, dnmt3, and dnmt5. In conclusion, low level BPA exposures for continuous two generations not only affects sex ratio and sperm quantity/quality in F1 and F2 adults, reproductive success in offspring from F2 parents, but also perturbs various molecular pathways potentially contributing to these BPA induced male-specific reproductive defects.

Upregulation of uncoupling protein Ucp2 through acute cold exposure increases post-thaw sperm quality in zebrafish.

Oxidative stress plays an important role in sperm damage during cryopreservation. Mild mitochondrial uncoupling has been shown to reduce excessive reactive oxygen species (ROS) and thus mitigate oxidative stress. Uncoupling protein (Ucp2) regulates mitochondrial uncoupling and can be induced by temperature fluctuation. In the present study, we explored a novel approach of acute cold exposure on Ucp2 activation and its association with oxidative damage and post-thaw sperm quality in zebrafish. Our study revealed that acute cold exposure of zebrafish at 18 °C for 24 h led to significant increase of ucp2 mRNA and Ucp2 protein in zebrafish fresh sperm as well as thawed sperm after cryopreservation. Although cold exposure had no effect on fresh sperm quality except for decreasing lipid peroxidation, sperm collected from cold-exposed zebrafish exhibited higher resistance to cryodamage, which was demonstrated by increased post-thaw motility, decreased lipid peroxidation, increased ATP production, and ultimately increased fertilization success. However, except for reduced lipid peroxidation, we did not observe any significant ROS reduction associated with increased Ucp2 activation in cold-exposed group, suggesting mechanisms other than mitochondrial uncoupling could have contributed to cold exposure associated benefits in post-thaw sperm survival. Nevertheless, our findings indicate that acute cold exposure prior to sperm cryopreservation is beneficial for post-thaw sperm survival in zebrafish, and this novel approach may be used to improve post-thaw sperm quality for other aquatic species.

Inhibition of ROS production through mitochondria-targeted antioxidant and mitochondrial uncoupling increases post-thaw sperm viability in yellow catfish.

Reactive oxygen species (ROS) are one of the main causes for decreased viability in cryopreserved sperm. Many studies have reported the beneficial effect of antioxidant supplements in freezing media for post-thaw sperm quality. In the present study, we explored two new approaches of ROS inhibition in sperm cryopreservation of yellow catfish, namely mitochondrial-targeted antioxidant and metabolic modulator targeting mitochondrial uncoupling pathways. Our study revealed that addition of MitoQ, a compound designed to deliver ubiquinone into mitochondria, significantly decreased ROS production, as well as lipid peroxidation, and increased post-thaw viability. Similarly, sperm incubated with 2,4-dinitrophenol (DNP), a chemical protonophore that induces mitochondrial uncoupling, also had reduced ROS production, as well as lipid peroxidation, and increased post-thaw sperm viability. Conversely, activation of uncoupling protein (UCP2) by 4-hydroxynonenal (HNE) neither reduced ROS production nor increased post-thaw sperm viability. Our findings indicate that ROS inhibition through mitochondrial-targeted antioxidant or mild mitochondrial uncoupling is beneficial for sperm cryopreservation in yellow catfish. Our study provides novel methods to mitigate oxidative stress induced damage in cryopreserved sperm for future applications.