Developmental Phenotypic and Transcriptomic Effects of Exposure to Nanomolar Levels of 4-Nonylphenol, Triclosan, and Triclocarban in Zebrafish (Danio rerio).

Triclosan, triclocarban and 4-nonylphenol are all chemicals of emerging concern found in a wide variety of consumer products that have exhibited a wide range of endocrine-disrupting effects and are present in increasing amounts in groundwater worldwide. Results of the present study indicate that exposure to these chemicals at critical developmental periods, whether long-term or short-term in duration, leads to significant mortality, morphologic, behavioral and transcriptomic effects in zebrafish (Danio rerio). These effects range from total mortality with either long- or short-term exposure at 100 and 1000 nM of triclosan, to abnormalities in uninflated swim bladder seen with long-term exposure to triclocarban and short-term exposure to 4-nonylphenol, and cardiac edema seen with short-term 4-nonylphenol exposure. Additionally, a significant number of genes involved in neurological and cardiovascular development were differentially expressed after the exposures, as well as lipid metabolism genes and metabolic pathways after exposure to each chemical. Such changes in behavior, gene expression, and pathway abnormalities caused by these three known endocrine disruptors have the potential to impact not only the local ecosystem, but human health as well.

Developmental phenotypic and transcriptomic effects of exposure to nanomolar levels of metformin in zebrafish.

Metformin is found in the majority of lakes and streams in the United States, leading to widespread environmental exposure. Results of the present study indicate that extended duration metformin exposure at critical developmental periods leads to decreased survival rates in zebrafish (danio rerio), an NIH approved human model. Significant abnormalities are seen with extended duration metformin exposure from 4 h post fertilization up to 5 days post fertilization, although short term metformin exposure for 24 h at 4-5 days post fertilization did not lead to any significant abnormalities. Both extended and short term duration did however have an impact on locomotor activity of zebrafish, and several genes involved in neurological and cardiovascular development were differentially expressed after exposure to metformin. The changes seen in behavior, gene expression and morphological abnormalities caused by metformin exposure should be examined further in future studies in order to assess their potential human health implications as metformin prescriptions continue to increase worldwide.

Detection of endocrine disrupting chemicals in Danio rerio and Daphnia pulex: Step-one, behavioral screen.

Anthropogenic surface and ground water contamination by chemicals is a global problem, and there is an urgent need to develop tools to identify and elucidate biological effects. Contaminants of emerging concern (CECs) are not typically monitored or regulated and those with known or suspected endocrine disrupting potential have been termed endocrine disrupting chemicals (EDCs). Many CECs are known to be neurotoxic (e.g., insecticides) and many are incompletely characterized. Behavioral responses can identify chemicals with neuroactive properties, which can be relevant to EDC mechanisms (e.g., neuroendocrine disturbances). Two freshwater species, Daphnia pulex and Danio rerio, were evaluated for swimming behavior alterations resulting from 24-hr exposure to 9 CECs: triclosan, triclocarban, chlorpyrifos, dieldrin, 4-nonylphenol, bisphenol-A, atrazine, metformin, and estrone. This is the first step in the development of a bioassay for detecting estrogenic and/or anti-androgenic activity with the goal to evaluate complex mixtures of uncharacterized contaminants in water samples. The second step, described in a subsequent report, examines transcriptome alterations following chemical exposure. Significant differences in the swimming behavior response and sensitivity were found across chemicals within a species and across species for a given chemical in this unique optical bioassay system. In the concentration ranges studied, significant behavioral alterations were detected for 6 of 9 CECs for D. pulex and 4 of 9 CECs for D. rerio. These results underscore the utility of this bioassay to identify behavioral effects of sublethal concentrations of CECs before exploration of transcriptomic alterations for EDC detection.

The phenotypic and transcriptomic effects of developmental exposure to nanomolar levels of estrone and bisphenol A in zebrafish.

Estrone and BPA are two endocrine disrupting chemicals (EDCs) that are predicted to be less potent than estrogens such as 17ß-estradiol and 17α-ethinylestradiol. Human exposure concentrations to estrone and BPA can be as low as nanomolar levels. However, very few toxicological studies have focused on the nanomolar-dose effects. Low level of EDCs can potentially cause non-monotonic responses. In addition, exposures at different developmental stages can lead to different health outcomes. To identify the nanomolar-dose effects of estrone and BPA, we used zebrafish modeling to study the phenotypic and transcriptomic responses after extended duration exposure from 0 to 5 days post-fertilization (dpf) and short-term exposure at days 4-5 post fertilization. We found that non-monotonic transcriptomic responses occurred after extended duration exposures at 1 nM of estrone or BPA. At this level, estrone also caused hypoactivity locomotive behavior in zebrafish. After both extended duration and short-term exposures, BPA led to more apparent phenotypic responses, i.e. skeletal abnormalities and locomotion changes, and more significant transcriptomic responses than estrone exposure. After short-term exposure, BPA at concentrations equal or above 100 nM affected locomotive behavior and changed the expression of both estrogenic and non-estrogenic genes that are linked to neurological diseases. These data provide gaps of mechanisms between neurological genes expression and associated phenotypic response due to estrone or BPA exposures. This study also provides insights for assessing the acceptable concentration of BPA and estrone in aquatic environments.

Toxic effects of combined stressors on Daphnia pulex: Interactions between diazinon, 4-nonylphenol, and wastewater effluent.

Contaminant exposure in aqueous systems typically involves complex chemical mixtures. Given the large number of compounds present in the environment, it is critical to identify hazardous chemical interactions rapidly. The present study utilized a prototype for a novel high-throughput assay to quantify behavioral changes over time to identify chemical interactions that affect toxicity. The independent and combined effects of 2 chemicals, diazinon (an insecticide) and 4-nonylphenol (a detergent metabolite), on the swimming behavior of the freshwater crustacean Daphnia pulex were examined. Cumulative distance and change in direction were measured repeatedly via optical tracking over 90 min. Exposure to low concentrations of diazinon (0.125-2 µM) or 4-nonylphenol (0.25-4 µM) elicited significant concentration- and time-dependent effects on swimming behavior. Exposure to 0.5 µM 4-nonylphenol alone did not significantly alter mean cumulative distance but did elicit a small, significant increase in mean angle, the measure of change in direction. When 0.5 µM 4-nonylphenol was used in combination with diazinon (0.125-0.5 µM), it augmented the adverse impact of diazinon on the swimming behavior of Daphnia. Additionally, enhanced sensitivity to diazinon was observed in animals exposed to treated wastewater effluent for 24 h prior to a diazinon challenge. The present experiments demonstrate that exposure to 4-nonylphenol and complex chemical mixtures (e.g., treated wastewater) can enhance the toxicity of exposure to the insecticide diazinon.

Optical bioassay for measuring sublethal toxicity of insecticides in Daphnia pulex.

Many emerging contaminants tend to be biologically active at very low concentrations, occur in water as part of complex mixtures, and impact biota in ways that are not detected using traditional toxicity tests (e.g., median lethal concentration). To evaluate emerging contaminants, the authors developed a method for detecting sublethal behavioral effects by quantifying the swimming behavior of Daphnia pulex, a model organism for studying aquatic toxicity. This optical tracking technique is capable of measuring many swimming parameters, 2 of which-cumulative distance and angular change-are presented. To validate this technique, 2 prototypical compounds that exhibit different modes of action as well as corresponding insecticides that are commonly found in surface waters were investigated. The acetylcholinesterase (AChE) inhibitor physostigmine was used as the prototypical compound for the large number of AChE inhibitor insecticides (e.g., chlorpyrifos). Nicotine was used as the prototypical compound for neonicotinoid insecticides (e.g., imidacloprid). Results demonstrate that this assay is capable of detecting sublethal behavioral effects that are concentration-dependent and that insecticides with the same mode of action yield similar results. The method can easily be scaled up to serve as a high-throughput screening tool to detect sublethal toxic effects of a variety of chemicals. This method is likely to aid in enhancing the current understanding of emerging contaminants and to serve as a novel water-quality screening tool.

Postnatal inorganic lead exposure reduces midbrain dopaminergic impulse flow and decreases dopamine D1 receptor sensitivity in nucleus accumbens neurons.

Lead treatment via drinking water for 3 to 6 weeks at 250 ppm was found to significantly decrease the number of spontaneously active dopamine (DA) neurons in both the substantia nigra and ventral tegmental area that were recorded using standard extracellular electrophysiological recording techniques. Lead exposure did not affect the discharge rate or discharge pattern of these DA neurons. No significant decrease in the number of tyrosine hydroxylase immunopositive cells was detected in lead-treated animals relative to controls even though the length of lead exposure was extended beyond that of the electrophysiological studies. The significant lead-induced decrease in spontaneously active cells observed in the electrophysiological studies was, therefore, not due to cell death. An acute drug challenge with the DA receptor agonist apomorphine at a dose known to hyperpolarize midbrain DA neurons (50 mug/kg i.v.) was used to determine whether hyperpolarization would normalize the number of spontaneously active DA neurons. The results suggest that depolarization inactivation was most likely not the cause for this lead effect. The D(1) receptor agonist SKF-38393 [1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol] was iontophoretically applied to type I nucleus accumbens (Nacb) neurons. The results demonstrated that type I Nacb neurons have a significantly lower basal discharge rate in lead-treated animals relative to controls and that the Nacb DA D(1) receptors were significantly less sensitive to SKF-38393 in the lead-treated animals. Therefore, lead exposure decreases DA neuron impulse flow presynaptically and decreases DA D(1) receptor sensitivity postsynaptically in the nucleus accumbens.

Inorganic lead exposure in the rat activates striatal cFOS expression at lower blood levels and inhibits amphetamine-induced cFOS expression at higher blood levels.

The impact of inorganic lead exposure on dopamine (DA) neurotransmission in the basal ganglia was examined. Amphetamine (AMPH)-induced cFOS immunoreactivity (cFOS-IR) in the striatum was determined after a 3-week exposure to lead acetate (0, 50, or 250 ppm). On the 21st day of lead exposure, rats were challenged with AMPH (4 mg/kg i.p.) or saline vehicle (Veh) and were assayed for presence of cFOS-IR. In the untreated control (Con) group, AMPH challenge (Con/AMPH) increased cFOS-IR expression by approximately 35-fold over Veh challenge (Con/Veh) (P < 0.01). In the Pb50/Veh group, cFOS-IR expression was approximately 7-fold greater than in the Con/Veh group (P < 0.05). Given that there was negligible cFOS-IR expression in the Con/Veh group, this indicates that the Pb50 exposure induced cFOS expression. The increase in cFOS-IR in the Pb50/AMPH was also significant (P < 0.01), but it was not different from the Con/AMPH (P > 0.20). Neither the Pb250/Veh group nor the Pb250/AMPH group had a significant increase in cFOS-IR relative to Con/Veh (P > 0.20). These results indicate that chronic 50 ppm lead exposure induced a low but statistically significantly level of cFOS gene activation and that it did not affect the AMPH-induced cFOS activation. However, chronic 250 ppm lead exposure inhibited AMPH-induced activation of cFOS in the striatum by about 89%. Therefore, lead is capable of both activating cFOS expression at low levels of exposure (mean blood lead level 21.6 +/- 1.9 microg/dl) and inhibiting AMPH-induced cFOS expression at higher levels of exposure (mean blood lead level 47.4 +/- 2.6 microg/dl).

Protein kinase C does not mediate the inhibitory action of lead on vitamin D3-dependent production of osteocalcin in osteoblastic bone cells.

The level of osteocalcin in serum is lower in lead-intoxicated children than in their normal counterparts. To explain this clinical observation, we investigated the mechanism of action of lead on vitamin D3-dependent osteocalcin production. Lead (5-20 microM) blocked the stimulating effects of vitamin D3 on osteocalcin production in cultured rat osteosarcoma cells (ROS 17/2.8). It is often suggested that activation of protein kinase C (PKC) is a critical mediator of the toxic actions of lead. Treatment of ROS cells with Gö6976, an inhibitor of PKC alpha and beta isozymes, produced similar effects as lead on vitamin D3-dependent osteocalcin production, while activation of PKC by phorbol-12-myristate-13-acetate (TPA) did not reverse or mimic this effect of lead. Thus activation of PKC is not consistent with the actions of lead on vitamin D3-dependent osteocalcin production. Measurement of PKC enzyme activity showed that 10 microM lead treatment does not activate or inhibit the activity of PKC in ROS cells. Western blot analysis indicated that lead treatment does not translocate PKC alpha, beta, or zeta from cytosol to membrane. Therefore, we concluded that PKC does not mediate the cellular toxicity of lead on vitamin D3-dependent osteocalcin production.