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.

Production of bioactive recombinant human fibroblast growth factor 12 using a new transient expression vector in E. coli and its neuroprotective effects.

In recent years, an increasing number of studies have shown that fibroblast growth factor 12 (FGF12) plays important roles in regulating neural development and function. Importantly, changes of FGF12 expression are thought to be related to the pathophysiology of many neurological diseases. However, little research has been performed to explore the protective effect of FGF12 on nerve damage. This study aims to explore its neuroprotective effects using our recombinant humanized FGF12 (rhFGF12). The hFGF12 gene was cloned and ligated into an expression vector to construct a recombinant plasmid pET-3a-hFGF12. Single colonies were screened to obtain high expression engineering strains, and fermentation and purification protocols for rhFGF12 were designed and optimized. The biological activities and related mechanisms of rhFGF12 were investigated by MTT assay using NIH3T3 and PC12 cell lines. The in vitro neurotoxicity model of H2O2-induced oxidative injury in PC12 cells was established to explore the protective effects of rhFGF12. The results indicate that the beneficial effects of rhFGF12 were most likely achieved by promoting cell proliferation and reducing apoptosis. Moreover, a transgenic zebrafish (islet) with strong GFP fluorescence in the motor neurons of the hindbrain was used to establish a central injury model caused by mycophenolate mofetil (MMF). The results suggested that rhFGF12 could ameliorate central injury induced by MMF in zebrafish. In conclusion, we have established an efficient method to express and purify active rhFGF12 using an Escherichia coli expression system. Besides, rhFGF12 plays a protective effect of on nerve damage, and it provides a promising therapeutic approach for nerve injury. KEY POINTS: • Effective expression and purification of bioactive rhFGF12 protein in E. coli. • ERK/MAPK pathway is involved in rhFGF12-stimulated proliferation on PC12 cells. • The rhFGF12 has the neuroprotective effects by inhibiting apoptosis.

Developmental co-exposure of TBBPA and titanium dioxide nanoparticle induced behavioral deficits in larval zebrafish.

Both tetrabromobisphenol A (TBBPA) and titanium dioxide nanoparticle (TiO2 NP) have widespread commercial applications, resulting in their ubiquitous co-presence in the environment and biota. Although environmental chemicals exist as mixtures, toxicity studies are nearly always conducted with single chemicals. Few studies explore potential interactions of different chemical mixtures. In this study, we employ the sensitive developing nerve system in zebrafish to assess the neurotoxicity of TBBPA/TiO2 NP mixtures. Specifically, zebrafish embryos were exposed to solvent control (0.1% DMSO), 2 µM TBBPA, 0.1 mg/L TiO2 NP, and their mixture from 8 to 120 h post fertilization (hpf), and motor/social behavioral assessments were conducted on embryos/larvae at different developmental stages. Our results showed that TBBPA/TiO2 NP single or co-exposures increased spontaneous movement, decreased touch response and swim speed, and affected social behaviors of light/dark preference, shoaling, mirror attack and social contact. In particular, many of these phenotypes were manifested with higher magnitude of changes from the mixture exposure. These behavioral deficits were also accompanied with increased cell death in olfactory region and neuromasts in the lateral line system, increased ROS in gallbladder, pancreas, liver, and intestine, as well as increased lipid peroxidation and decreased ATP levels in whole larval tissue homogenates. Further, genes coding for key cell apoptosis marker and antioxidant enzyme were significantly upregulated by these two chemicals, in particular to their mixture. Interestingly, the co-presence of TBBPA also increased the mean particle size of TiO2 NP in the exposure solutions and the TiO2 NP content in larval tissue. Together, our analysis suggests that TBBPA/TiO2 NP induced behavioral changes may be due to physical accumulation of these two chemicals in the target organs, and TiO2 NP may serve as carriers for increased accumulation of TBBPA. To conclude, we demonstrated that TBBPA/TiO2 NP together cause increased bioaccumulation of TiO2, and heightened responses in behavior, cell apoptosis and oxidative stress. Our findings also highlight the importance of toxicity assessment using chemical mixtures.

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.

Neurodevelopmental toxicity assessments of alkyl phenanthrene and Dechlorane Plus co-exposure in zebrafish.

Alkyl phenanthrene (A-Phen) and Dechlorane Plus (DP) are ubiquitous environmental pollutants that widely co-exist in the environment. It has been established that both A-Phen and DP elicit neurotoxicity, but the potential interactive toxicity of these contaminants is not well-known. To determine whether a mixture of A-Phen and DP would exhibit interactive effects on neurodevelopment, we co-exposed 3-methylphenanthrene (3-MP), a representative of A-Phen, with DP. Our results illustrated that exposure to 5 or 20 µg/L 3-MP alone or in combination with 60 µg/L DP caused neurobehavioral anomalies in zebrafish. In accordance with the behavioral deficits, 3-MP alone or co-exposed with DP significantly decreased axonal growth of secondary motoneurons, altered intracellular Ca2+ homeostasis and induced cell apoptosis in the muscle of zebrafish. Additionally, 3-MP alone or co-exposed with DP significantly increased reactive oxygen species (ROS) and the mRNA levels of apoptosis-related genes. These findings indicate that 3-MP alone or co-exposed with DP induces neurobehavioral deficits through the combined effects on neuronal connectivity and muscle function. Chemical analysis revealed significant increases in 3-MP and DP bioaccumulation in zebrafish co-exposed with 3-MP and DP. Elevated bioaccumulation resulting from mixture exposure may represent a significant contribution of the synergistic effects observed in combined chemical exposure.

Selective extraction of bisphenol A from water by one-monomer molecularly imprinted magnetic nanoparticles.

One-monomer molecularly imprinted magnetic nanoparticles were prepared as adsorbents for selective extraction of bisphenol A from water in this study. A single bi-functional monomer was adopted for preparation of the molecularly imprinted polymer, avoiding the tedious trial-and-error optimizations as traditional strategy. Moreover, bisphenol F was used as the dummy template for bisphenol A to avoid the interference from residual template molecules. These nanoparticles showed not only large adsorption capacity and good selectivity to the bisphenol A but also outstanding magnetic response performance. Furthermore, they were successfully used as magnetic solid-phase extraction adsorbents of bisphenol A from various water samples, including tap water, river water, and seawater. The developed method was found to be much more efficient, convenient, and economical for selective extraction of bisphenol A compared with the traditional solid-phase extraction. Separation of these nanoparticles can be easily achieved with an external magnetic field, and the optimized adsorption time was only 15 min. The recoveries of bisphenol A in different water samples ranged from 85.38 to 93.75%, with relative standard deviation lower than 7.47%. These results showed that one-monomer molecularly imprinted magnetic nanoparticles had the potential to be popular adsorbents for selective extraction of pollutants from water.

Chronic PFOS Exposure Disrupts Thyroid Structure and Function in Zebrafish.

Perfluorooctane sulfonic acid (PFOS), as a potential endocrine disrupting chemical, is widely detected in the environment, wildlife and human. Currently few studies have documented the effects of chronic PFOS exposure on thyroid in aquatic organisms and the underlying mechanisms are largely unknown. The present study assessed the effect of chronic PFOS exposure on thyroid structure and function using zebrafish model. Zebrafish at 8 h post fertilization (hpf) were exposed to PFOS (250 µg/l) until 120 d post fertilization (dpf). Thyroid hormone (T3 and T4) level, thyroid morphology and thyroid function related gene expression were evaluated in zebrafish at 120 dpf. Our findings demonstrated that chronic PFOS exposure altered thyroid hormone level, thyroid follicular cell structure and thyroid hormone related gene expression, suggesting the validity of zebrafish as an alternative model for PFOS chronic toxicity screening.

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.

Human cardiotoxic drugs delivered by soaking and microinjection induce cardiovascular toxicity in zebrafish.

Cardiovascular toxicity is a major challenge for the pharmaceutical industry and predictive screening models to identify and eliminate pharmaceuticals with the potential to cause cardiovascular toxicity in humans are urgently needed. In this study, taking advantage of the transparency of larval zebrafish, Danio rerio, we assessed cardiovascular toxicity of seven known human cardiotoxic drugs (aspirin, clomipramine hydrochloride, cyclophosphamide, nimodipine, quinidine, terfenadine and verapamil hydrochloride) and two non-cardiovascular toxicity drugs (gentamicin sulphate and tetracycline hydrochloride) in zebrafish using six specific phenotypic endpoints: heart rate, heart rhythm, pericardial edema, circulation, hemorrhage and thrombosis. All the tested drugs were delivered into zebrafish by direct soaking and yolk sac microinjection, respectively, and cardiovascular toxicity was quantitatively or qualitatively assessed at 4 and 24 h post drug treatment. The results showed that aspirin accelerated the zebrafish heart rate (tachycardia), whereas clomipramine hydrochloride, cyclophosphamide, nimodipine, quinidine, terfenadine and verapamil hydrochloride induced bradycardia. Quinidine and terfenadine also caused atrioventricular (AV) block. Nimodipine treatment resulted in atrial arrest with much slower but regular ventricular heart beating. All the tested human cardiotoxic drugs also induced pericardial edema and circulatory disturbance in zebrafish. There was no sign of cardiovascular toxicity in zebrafish treated with non-cardiotoxic drugs gentamicin sulphate and tetracycline hydrochloride. The overall prediction success rate for cardiotoxic drugs and non-cardiotoxic drugs in zebrafish were 100% (9/9) as compared with human results, suggesting that zebrafish is an excellent animal model for rapid in vivo cardiovascular toxicity screening. The procedures we developed in this report for assessing cardiovascular toxicity in zebrafish were suitable for drugs delivered by either soaking or microinjection.

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.

Cooling rate optimization for zebrafish sperm cryopreservation using a cryomicroscope coupled with SYBR14/PI dual staining.

The Zebrafish has gained increased popularity as an aquatic model species in various research fields, and its widespread use has led to numerous mutant strains and transgenic lines. This creates the need to store these important genetic materials as frozen gametes. Sperm cryopreservation in zebrafish has been shown to yield very low post-thaw survival and many protocols suffer from great variability and poor reproducibility. The present study was intended to develop a freezing protocol that can be reliably used to cryopreserve zebrafish sperm with high post-thaw survival. In particular, our study focused on cooling protocol optimization with the aid of cryomicroscopy. Specifically, sperm suspended in 8% DMSO or 4% MeOH were first incubated with live/dead fluorescent dyes (SYBR14/PI) and then cooled at various rates from 4°C to different intermediate stopping temperatures such as -10, -20, -30 and -80°C before rewarming to 35°C at the rate of 100°C/min. %PI-positive (dead) cells were monitored throughout the cooling process and this screening yielded an optimal rate of 25°C/min for this initial phase of freezing. We then tested the optimal cooling rate for the second phase of freezing from various intermediate stopping temperatures to -80°C before plunging into liquid nitrogen. Our finding yielded an optimal intermediate stopping temperature of -30°C and an optimal rate of 5°C/min for this second phase of freezing. When we further applied this two-step cooling protocol to the conventional controlled-rate freezer, the average post-thaw motility measured by CASA was 46.8 ± 6.40% across 11 males, indicating high post-thaw survival and consistent results among different individuals. Our study indicates that cryomiscroscopy is a powerful tool to devise the optimal cooling conditions for species with sperm that are very sensitive to cryodamage.

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.

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.

The importance of mitochondrial metabolic activity and mitochondrial DNA replication during oocyte maturation in vitro on oocyte quality and subsequent embryo developmental competence.

Mitochondrial metabolic capacity and DNA replication have both been shown to affect oocyte quality, but it is unclear which one is more critical. In this study, immature oocytes were treated with FCCP or ddC to independently inhibit the respective mitochondrial metabolic capacity or DNA replication of oocytes during in vitro maturation. To differentiate their roles, we evaluated various parameters related to oocyte maturation (germinal vesicle break down and nuclear maturation), quality (spindle formation, chromosome alignment, and mitochondrial distribution pattern), fertilization capability, and subsequent embryo developmental competence (blastocyst formation and cell number of blastocyst). Inhibition of mitochondrial metabolic capacity with FCCP resulted in a reduced percent of oocytes with nuclear maturation; normal spindle formation and chromosome alignment; evenly distributed mitochondria; and an ability to form blastocysts. Inhibition of mtDNA replication with ddC has no detectable effect on oocyte maturation and mitochondrial distribution, although high-dose ddC increased the percent of oocytes showing abnormal spindle formation and chromosome alignment. ddC did, however, reduce blastocyst formation significantly. Neither FCCP nor ddC exposure had an effect on the rate of fertilization. These findings suggest that the effects associated with lower mitochondrial DNA copy number do not coincide with the effects seen with reduced mitochondrial metabolic activity in oocytes. Inhibiting mitochondrial metabolic activity during oocyte maturation has a negative impact on oocyte maturation and subsequent embryo developmental competence. A reduction in mitochondrial DNA copy number, on the other hand, mainly affects embryonic development potential, but has little effect on oocyte maturation and in vitro fertilization.

Impaired mitochondrial function in murine oocytes is associated with controlled ovarian hyperstimulation and in vitro maturation.

The present study was designed to determine whether controlled ovarian hyperstimulation (COH) and in vitro maturation (IVM), two common clinical procedures in human IVF treatment, have an impact on mitochondrial DNA (mtDNA) copy number and mitochondrial function in oocytes. Matured mouse oocytes recovered following COH, IVM and natural cycles (NC), which simulated those treatments in human clinic IVF treatment. The copies of mtDNA, the activity of mitochondria as determined by inner mitochondrial membrane potential and oocyte adenosine trisphosphate (ATP) content, pattern of mitochondrial distribution, reactive oxygen species (ROS) levels and the integrity of the cytoskeleton were evaluated in oocytes. Significant differences were detected between COH and NC groups in all measures, except the pattern of mitochondrial distribution and ROS levels. There were also significant differences detected between IVM and NC treatment groups in the copies of mitochondrial DNA, the level of ROS and the integrity of the cytoskeleton in oocytes. In conclusion, the results of this investigation indicate that non-physiological COH and IVM treatments inhibit mtDNA replication, alter mitochondrial function and increase the percentage of abnormal cytoskeleton and ROS production. Damage related to the mitochondria may partly explain the low efficiency of IVF and high rate of embryonic loss associated with these clinical procedures.

Polybrominated diphenyl ethers (PBDEs) in human serum from Southeast China.

Polybrominated diphenyl ethers (PBDEs) have been used extensively in electrical and electronic products, but little is known about the exposure level in the electrical appliance factories workers and nearby local residents. In this study, we assessed body burdens of PBDEs in 194 Chinese injection workers from electrical appliance factories and also measured 205 blood samples from catering workers, leather factory workers, umbilical cord and infertile men for comparison. Twelve PBDE congeners in serum samples were measured by GC-MS. The highest concentration for total PBDEs was found in injection workers, which is positively correlated to employment duration. BDE-209 was the most dominant congener followed by BDE-47, 28 and 99. We also found the presence of all twelve PBDEs in cord blood, suggesting an evidence of fetal exposure. Concentration of BDE-47 was particularly higher in serum samples from infertile men in comparison with that of catering workers and leather factory workers.

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.

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.

Frozen-thawed rhesus sperm retain normal morphology and highly progressive motility but exhibit sharply reduced efficiency in penetrating cervical mucus and hyaluronic acid gel.

The preservation of the genetic diversity of captive populations of rhesus monkeys is critical to the future of biomedical research. Cryopreservation of rhesus macaque sperm is relatively simple to perform, yields high post-thaw motility, and theoretically, provides via artificial insemination (AI) a way to easily transfer genetics among colonies of animals. In the interest of optimizing semen cryopreservation methods for use with vaginal AI, we evaluated the ability of frozen-thawed rhesus sperm to penetrate periovulatory cervical mucus (CM). Motile sperm concentration of pre-freeze ("fresh") and post-thawed ("thawed") samples from five different males were normalized for both computer assisted sperm motion analysis and CM penetration experiments. Sperm samples were deposited into slide chambers containing CM or gel composed of hyaluronic acid (HA) as a surrogate for CM and numbers of sperm were recorded as they entered a video field a preset distance from the sperm suspension-CM (or HA) interface. Fresh and thawed sperm were dried on glass slides, "Pap"-stained, and assessed for changes in head dimensions and head and flagellar shape. While retaining better than 80% of fresh sperm progressive motility, thawed sperm from the same ejaculate retained on average only 18.6% of the CM penetration ability. Experiments using HA gel yielded similar results only with reduced experimental error and thus improved detection of treatment differences. Neither the percentage of abnormal forms nor head dimensions differed between fresh and thawed sperm. While findings suggests that sperm-CM interaction is a prominent factor in previous failures of vaginal AI with cryopreserved macaque sperm, neither sperm motility nor morphology appears to account for changes in the ability of cryopreserved sperm to penetrate CM. Our data points to a previously unidentified manifestation of cryodamage which may have implications for assessment of sperm function beyond the cervix and across mammalian species.