Yongyong Guo

Disruption of endocrine function in in vitro H295R cell-based and in in vivo assay in zebrafish by 2,4-dichlorophenol

Chlorophenols in the aquatic environment have been of concern due to their potential effects on human and wildlife. In the present study, the endocrine disrupting effects of 2,4-dichlorophenol (2,4-DCP) were investigated in vitro and in vivo. In the in vitro assay, H295R human adrenocortical carcinoma cells were used to determine the potential effects of 2,4-DCP on steroidogenesis. Exposure to 0, 0.1, 0.3 or 1.0 mg 2,4-DCP/L resulted in less production of 17β-estradiol (E2) and alterations in transcript expressions of genes involved in steroidogenesis, including cytochrome P450 (CYP11A, CYP17, CYP19), 3βHSD, 17βHSD and StAR. In the in vivo study, effects of 0, 0.03, 0.1 or 0.3 mg 2,4-DCP/L on concentrations of steroid hormones in plasma of adult zebrafish (Danio rerio) were measured and expression of mRNA of selected genes in hypothalamic-pituitary-gonadal (HPG) axis and liver were determined. Exposure of zebrafish to 2,4-DCP resulted in lesser concentrations of E2 accompanied by down-regulation of CYP19A mRNA in the females. In males, exposure to 2,4-DCP resulted in greater concentrations of testosterone (T) and E2 along with greater mRNA expression of CYP17 and CYP19A. The mRNA expression of prostaglandin synthase (Ptgs2) gene, which regulates ovulation, was down-regulated in females, but up-regulated in males. The hepatic estrogenic receptor (ERα and ERβ) and vitellogenin (VTG1 and VTG3) mRNAs were up-regulated in both females and males. The average number of eggs spawned was significantly less upon exposure to 2,4-DCP. Exposure of adult zebrafish to 2,4-DCP resulted in lesser rates of hatching of eggs. The results demonstrated that 2,4-DCP modulates transcription of steroidogenetic genes in both H295R cells and in the zebrafish HPG-axis and disrupts steroidogenesis, which in turn, can cause adverse effects on reproduction in fish.

Effect of titanium dioxide nanoparticles on the bioavailability, metabolism, and toxicity of pentachlorophenol in zebrafish larvae

This study investigated the influence of titanium dioxide nanoparticles (n-TiO2) on the bioavailability, metabolism, and toxicity of pentachlorophenol (PCP) in fish. Zebrafish (Danio rerio) embryos or larvae (2-h post-fertilization) were exposed to PCP (0, 3, 10, and 30 μg/L) alone or in combination with n-TiO2 (0.1mg/L) until 6 days post-fertilization. Results showed that n-TiO2 treatment alone did not induce lipid peroxidation, DNA damage, as well as the generation of reactive oxygen species (ROS) in the larvae. As compared with PCP treatment, the co-exposure of PCP and n-TiO2 enhanced the induction of ROS generation, eventually leading to lipid peroxidation and DNA damage. The nuclear factor erythroid 2-related factor 2 gene transcriptions were significantly upregulated in both PCP treatment alone and in combination with n-TiO2. Chemical analysis and histological examination showed that n-TiO2 adsorb PCP, and n-TiO2 are taken up by developing zebrafish larvae; however, PCP content was not enhanced in the presence of n-TiO2, but the metabolism of PCP to tetrachlorohydroquinone was enhanced in larvae. The results indicate that n-TiO2 enhanced the metabolism of PCP and caused oxidative damage and developmental toxicity, suggesting that NPs can influence the fate and toxicity of associated organic pollutants in the aquatic environment.

Developmental exposure to the organophosphorus flame retardant tris(1,3-dichloro-2-propyl) phosphate: Estrogenic activity, endocrine disruption and reproductive effects on zebrafish

Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) is an organophosphate flame retardant that is detectable in the environment and biota, prompting concern over its risk to wildlife and human health. Our objective was to investigate whether long-term exposure to low concentrations of TDCPP can affect fish reproduction. Zebrafish embryos were exposed to low concentrations (0, 4, 20 and 100μg/L) of TDCPP from 2h post-fertilization until sexual maturation. Exposure to TDCPP significantly increased plasma estradiol and testosterone levels in females, but had no effect in males. TDCPP exposure also caused a significant reduction in fecundity as indicated by decreased egg production. Real-time PCR was performed to examine selected genes in the hypothalamic-pituitary-gonadal (HPG) axis and liver. Principle component analysis (PCA) showed that sex hormone levels and fecundity were related to the mRNA level of several genes in the HPG axis. Furthermore, hepatic vitellogenin (vtg1 and vtg3) expression was upregulated in both females and males, suggesting TDCPP has estrogenic activity. Histological examination revealed promotion of oocyte maturation in the females, but retardation of spermiation in males. Reduced egg quality (e.g., egg diameter) and increased malformation rates were observed in the F1 generation. Chemical analysis showed significant levels of TDCPP and its metabolite bis(1,3-dichloro-2-propyl) phosphate in the gonads of males and females. In conclusion, long-term exposure to low concentrations of TDCPP impairs fish reproduction.

Thyroid endocrine system disruption by pentachlorophenol: An in vitro and in vivo assay

The present study aimed to evaluate the disruption caused to the thyroid endocrine system by pentachlorophenol (PCP) using in vitro and in vivo assays. In the in vitro assay, rat pituitary GH3 cells were exposed to 0, 0.1, 0.3, and 1.0 μM PCP. PCP exposure significantly downregulated basal and triiodothyronine (T3)-induced Dio 1 transcription, indicating the antagonistic activity of PCP in vitro. In the in vivo assay, zebrafish embryos were exposed to 0, 1, 3, and 10 μg/L of PCP until 14 days post-fertilization. PCP exposure resulted in decreased thyroxine (T4) levels, but elevated contents of whole-body T3. PCP exposure significantly upregulated the mRNA expression of genes along hypothalamic-pituitary-thyroid (HPT) axis, including those encoding thyroid-stimulating hormone, sodium/iodide symporter, thyroglobulin, Dio 1 and Dio 2, alpha and beta thyroid hormone receptor, and uridinediphosphate-glucuronosyl-transferase. PCP exposure did not influence the transcription of the transthyretin (TTR) gene. The results indicate that PCP potentially disrupts the thyroid endocrine system both in vitro and in vivo.

The synthetic progestin megestrol acetate adversely affects zebrafish reproduction

Synthetic progestins contaminate the aquatic ecosystem, and may cause adverse health effects on aquatic organisms. Megestrol acetate (MTA) is present in the aquatic environment, but its possible effects on fish reproduction are unknown. In the present study, we investigated the endocrine disruption and impact of MTA on fish reproduction. After a pre-exposure period of 14 days, reproductively mature zebrafish (Danio rerio) (F0) were exposed to MTA at environmental concentrations (33, 100, 333, and 666 ng/L) for 21 days. Egg production was decreased in F0 fish exposed to MTA, with a significant decrease at 666 ng/L. The exposure significantly decreased the circulating concentrations of estradiol (E2) and testosterone (T) in female fish or 11-keto testosterone (11-KT) in male fish. MTA exposure significantly downregulated the transcription of certain genes along the hypothalamic-pituitary-gonadal (HPG) axis. MTA did not affect early embryonic development or hatching success in the F1 generation. The present study showed that MTA is a potent endocrine disruptor in fish, and short-term exposure to MTA could significantly affect reproduction in fish and negatively impact the fish population.

Enhanced Bioconcentration of Bisphenol A in the Presence of Nano-TiO2 Can Lead to Adverse Reproductive Outcomes in Zebrafish

Titanium dioxide nanoparticles (n-TiO2) and bisphenol A (BPA) are widespread environmental contaminants in the aquatic environment. We hypothesized that n-TiO2 may adsorb BPA, and thus modify its bioavailability and toxicity to aquatic organisms. In this study, the bioavailability and toxicity of BPA (0, 2, 20, 200 μg/L) was investigated in the presence of n-TiO2 (100 μg/L). The n-TiO2 sorbed BPA and the resulting nanoparticles were taken up by zebrafish, where they translocated to the liver, brain, and gonad tissues. Increased tissue burdens of both BPA and n-TiO2 were observed following coexposure, and they also caused a reduction in plasma concentrations of estradiol (E2), testosterone (T), follicle-stimulating hormone (FSH), and luteinizing hormone (LH). Plasma vitellogenin (VTG) concentrations were significantly increased in males and females upon exposure to BPA. Histological examination of the ovary and testes did not show obvious morphological alterations; however, inhibition of egg production was noted in the presence of n-TiO2. The results indicated that n-TiO2 acts as a carrier of BPA and enhances its bioconcentration in zebrafish, leading to endocrine disruption and impairment of reproduction.

Characterization of a bystander effect induced by the endocrine-disrupting chemical 6-propyl-2-thiouracil in zebrafish embryos

This study was conducted to evaluate possible bystander effects induced by the model chemical 6-propyl-2-thiouracil (PTU) on melanin synthesis. Zebrafish (Danio rerio) embryos were treated with PTU by either microinjection exposure, via waterborne exposure or indirectly through bystander exposure. Melanin content, related mRNA and protein expression were examined at the end of exposure (36 h post-fertilization). Direct exposure to PTU decreased the melanin content, up-regulated mRNA expressions of oculocutaneous albinism type 2 (OCA2), tyrosinase (TYR), dopachrometautomerase (DCT), tyrosinase-related protein 1 (TYRP1) and silver (SILV), and increased the protein expressions of TYR and SILV. Bystander exposure also up-regulated mRNA and protein expressions of TYR and SILV but increased melanin contents. Correlation analysis demonstrated that mRNA expressions of OCA2, TYR, DCT, TYRP1, SILV and protein expressions of TYR and SILV in bystander exposure groups were positively correlated with corresponding expressions in microinjection exposure groups. The results might have environmental implications and highlight the need to consider the bystander effects when assessing potential risks of endocrine-disrupting chemicals.

The developmental neurotoxicity of polybrominated diphenyl ethers: Effect of DE-71 on dopamine in zebrafish larvae.

The potential neurotoxicity of polybrominated diphenyl ethers (PBDEs) is still a great concern. In the present study, the authors investigated whether exposure to PBDEs could affect the neurotransmitter system and cause developmental neurotoxicity in zebrafish. Zebrafish embryos (2 h postfertilization) were exposed to different concentrations of the PBDE mixture DE-71 (0-100 μg/L). The larvae were harvested at 120 h postfertilization, and the impact on dopaminergic signaling was investigated. The results revealed significant reductions in content of whole-body dopamine and its metabolite, dihydroxyphenylacetic acid, in DE-71-exposed larvae. The transcription of genes involved in the development of dopaminergic neurons (e.g., manf, bdnf, and nr4a2b) was significantly downregulated upon exposure to DE-71. Also, DE-71 resulted in a significant decrease of tyrosine hydroxylase and dopamine transporter protein levels in dopaminergic neurons. The expression level of tyrosine hydroxylase in forebrain neurons was assessed by whole-mount immunofluorescence, and the results further demonstrated that the tyrosine hydroxylase protein expression level was reduced in dopaminergic neurons. In addition to these molecular changes, the authors observed reduced locomotor activity in DE-71-exposed larvae. Taken together, the results of the present study demonstrate that acute exposure to PBDEs can affect dopaminergic signaling by disrupting the synthesis and transportation of dopamine in zebrafish, thereby disrupting normal neurodevelopment. In accord with its experimental findings, the present study extends knowledge of the mechanisms governing PBDE-induced developmental neurotoxicity.

The progestin levonorgestrel affects sex differentiation in zebrafish at environmentally relevant concentrations.

Synthetic progestins have become widespread environmental contaminants and may cause adverse effects on fish. In the present study, we investigated the effects of levonorgestrel (LNG) on sex differentiation in zebrafish (Danio rerio). Embryos were exposed to LNG at environmentally relevant concentrations (0, 1, 10, 33, and 100ng/L) and allowed to develop until sexual maturity. Histological examination at 63 days post fertilization (dpf) caused complete sex reversal and 100% males were observed in the 10, 33 and 100ng/L treatments; gross morphological and histological examination of gonads at 142dpf further confirmed 100% males at these exposure concentrations. The results indicate androgenic activity of LNG, and masculinization during zebrafish gonadal differentiation. The mRNA expression levels of genes involved in fish sex differentiation and gonadal development were examined at 28 and 42dpf. Down-regulation of the mRNA expression of aromatase (e.g., cyp19a1a, cyp19a1b), the forkhead transcription factor gene L2 (foxl2) and the Fushi tarazu factor-1d (nr5a1b) were observed. In contrast, transcription of the doublesex and mab-3-related transcription factor 1 (dmrt1) gene was up-regulated. Androgen receptor (ar) mRNA expression was significantly down-regulated at 28 and 42dpf. Co-exposure to flutamide (an androgen antagonist) and LNG, led to a decrease in the sex inversion potency of LNG. Our study has demonstrated that environmentally relevant concentrations of LNG could alter sex differentiation and gonadal development in zebrafish. Our results also suggest a potentially high ecological risk of LNG to fish populations in LNG-contaminated aquatic environments.

Effect of combined exposure to lead and decabromodiphenyl ether on neurodevelopment of zebrafish larvae.

The effect of combined exposure to decabromodiphenyl ether (BDE-209) and lead (Pb) on neurodevelopment of zebrafish (Danio rerio) larvae was investigated. Zebrafish embryos were exposed to Pb (0, 5, 10, 20 µg/L) and BDE-209 (0, 50, 100, 200 µg/L), either alone or in combination (Mix1: 5 + 50 µg/L, Mix2: 10 + 100 µg/L, Mix3: 20 + 200 µg/L) for up to 144 h post-fertilization. Growth of secondary motoneuron axons and expression of genes related to central nervous system development was significantly inhibited in Mix3 co-exposure group. A significant increase in reactive oxygen species (ROS), lipid peroxidation, DNA damage, and perturbation of the antioxidant system was detected in the Mix3 group compared to single-toxicant treatments or control. Depressed locomotor activity was recorded in the Mix2 and Mix3 groups. Addition of N-acetyl cysteine to Mix3 eliminated excessive ROS, and protected against lipid peroxidation, DNA damage, and locomotor dysfunction. Pb uptake was increased in the presence of BDE-209, but BDE-209 bioconcentration and the ability to metabolize BDE-209 were decreased in the presence of Pb. These results suggest that BDE-209 and Pb have a synergistic disruptive effect on neurodevelopment in zebrafish larvae by enhanced generation of ROS, which is a major factor that contributes to developmental neurotoxicity.