Elvis Cuevas

RETRACTED: 6-OHDA-induced apoptosis and mitochondrial dysfunction are mediated by early modulation of intracellular signals and interaction of Nrf2 and NF-κB factors

6-Hydroxydopamine (6-OHDA) is a neurotoxin that generates an experimental model of Parkinsons disease in rodents and is commonly employed to induce a lesion in dopaminergic pathways. The characterization of those molecular mechanisms linked to 6-OHDA-induced early toxicity is needed to better understand the cellular events further leading to neurodegeneration. The present work explored how 6-OHDA triggers early downstream signaling pathways that activate neurotoxicity in the rat striatum. Mitochondrial function, caspases-dependent apoptosis, kinases signaling (Akt, ERK 1/2, SAP/JNK and p38) and crosstalk between nuclear factor kappa B (NF-κB) and nuclear factor-erythroid-2-related factor 2 (Nrf2) were evaluated at early times post-lesion. We found that 6-OHDA initiates cell damage via mitochondrial complex I inhibition, cytochrome c and apoptosis-inducing factor (AIF) release, as well as activation of caspases 9 and 3 to induce apoptosis, kinase signaling modulation and NF-κB-mediated inflammatory responses, accompanied by inhibition of antioxidant systems regulated by the Nrf2 pathway. Our results suggest that kinases SAP/JNK and p38 up-regulation may play a role in the early stages of 6-OHDA toxicity to trigger intrinsic pathways for apoptosis and enhanced NF-κB activation. In turn, these cellular events inhibit the activation of cytoprotective mechanisms, thereby leading to a condition of general damage.

Ketamine induces motor neuron toxicity and alters neurogenic and proneural gene expression in zebrafish.

Ketamine, a noncompetitive antagonist of N‐methyl‐d‐aspartate‐type glutamate receptors, is a pediatric anesthetic that has been shown to be neurotoxic in rodents and nonhuman primates when administered during the brain growth spurt. Recently, the zebrafish has become an attractive model for toxicity assays, in part because the predictive capability of the zebrafish model, with respect to chemical effects, compares well with that from mammalian models. In the transgenic (hb9:GFP) embryos used in this study, green fluorescent protein (GFP) is expressed in the motor neurons, facilitating the visualization and analysis of motor neuron development in vivo. In order to determine whether ketamine induces motor neuron toxicity in zebrafish, embryos of these transgenic fish were treated with different concentrations of ketamine (0.5 and 2.0 mm). For ketamine exposures lasting up to 20 h, larvae showed no gross morphological abnormalities. Analysis of GFP‐expressing motor neurons in the live embryos, however, revealed that 2.0 mm ketamine adversely affected motor neuron axon length and decreased cranial and motor neuron populations. Quantitative reverse transcriptase‐polymerase chain reaction analysis demonstrated that ketamine down‐regulated the motor neuron‐inducing zinc finger transcription factor Gli2b and the proneural gene NeuroD even at 0.5 mm concentration, while up‐regulating the expression of the proneural gene Neurogenin1 (Ngn1). Expression of the neurogenic gene, Notch1a, was suppressed, indicating that neuronal precursor generation from uncommitted cells was favored. These results suggest that ketamine is neurotoxic to motor neurons in zebrafish and possibly affects the differentiating/differentiatedneurons rather than neuronal progenitors. Published 2011. This article is a US Government work and is in the public domain in the USA.

S-allyl cysteine protects against 6-hydroxydopamine-induced neurotoxicity in the rat striatum: involvement of Nrf2 transcription factor activation and modulation of signaling kinase cascades.

Pharmacological activation at the basal ganglia of the transcription factor Nrf2, guardian of redox homeostasis, holds a strong promise for the slow progression of Parkinsons disease (PD). However, a potent Nrf2 activator in the brain still must be found. In this study, we have investigated the potential use of the antioxidant compound S-allyl cysteine (SAC) in the activation of Nrf2 in 6-hydoxydopamine (6-OHDA)-intoxicated rats. In the rat striatum, SAC by itself promoted the Nrf2 dissociation of Keap-1, its nuclear translocation, the subsequent association with small MafK protein, and further binding of the Nrf2/MafK complex to ARE sequence, as well as the up-regulation of Nrf2-dependent genes encoding the antioxidant enzymes HO-1, NQO-1, GR, and SOD-1. In vivo and in vitro experiments to identify signaling pathways activated by SAC pointed to Akt as the most likely kinase participating in Nrf2 activation by SAC. In PC12 cells, SAC stimulated the activation of Akt and ERK1/2 and inhibited JNK1/2/3 activation. In the rat striatum, the SAC-induced activation of Nrf2 is likely to contribute to inhibit the toxic effects of 6-OHDA evidenced by phase 2 antioxidant enzymes up-regulation, glutathione recovery, and attenuation of reactive oxygen species (ROS), nitric oxide (NO), and lipid peroxides formation. These early protective effects correlated with the long-term preservation of the cellular redox status, the striatal dopamine (DA) and tyrosine hydroxylase (TH) levels, and the improvement of motor skills. Therefore, this study indicates that, in addition to direct scavenging actions, the activation of Nrf2 by SAC might confer neuroprotective responses through the modulation of kinase signaling pathways in rodent models of PD, and suggests that this antioxidant molecule may have a therapeutic value in this human pathology.

Zebrafish Model in Drug Safety Assessment

Over the past decade, zebrafish are being increasingly used in assessing the effects of chemical compounds. Especially, the embryos and larvae, due to their microscopically small size and optical transparency, are compatible with multi-well microtiter plates for high throughput screening. Being transparent, they allow for non-invasive visualization of internal organs during early development. The organization of the genome, the genetic pathways controlling signal transduction and the developmental pattern appear to be significantly conserved between zebrafish and humans. Major organ systems including the nervous, cardiovascular, digestive and visual systems of zebrafish are also similar to their mammalian counterparts at the anatomical, physiological and molecular levels. Therefore, zebrafish assays are ideal for evaluating multiple organ toxicities simultaneously that contrast in vitro assays performed on cultured cells or tissue explants and organ slices. Although research on zebrafish as a model system began a few decades ago, later studies on zebrafish developmental biology and developmental genetics resulted in the characterization of a large number of genes involved in vertebrate development and biological pathways thus establishing zebrafish as a relevant human disease model for research. Recently, zebrafish have become an attractive vertebrate model for pharmaceutical and toxicological studies. We have outlined in this review some of the toxicological screens and tools that used zebrafish early life stages, and the efforts made to validate zebrafish assays against mammalian drug screens.

Acetyl l-carnitine protects motor neurons and Rohon-Beard sensory neurons against ketamine-induced neurotoxicity in zebrafish embryos

Ketamine, a non-competitive antagonist of N-methyl-D-aspartate (NMDA) type glutamate receptors is commonly used as a pediatric anesthetic. Multiple studies have shown ketamine to be neurotoxic, particularly when administered during the brain growth spurt. Previously, we have shown that ketamine is detrimental to motor neuron development in the zebrafish embryos. Here, using both wild type (WT) and transgenic (hb9:GFP) zebrafish embryos, we demonstrate that ketamine is neurotoxic to both motor and sensory neurons. Drug absorption studies showed that in the WT embryos, ketamine accumulation was approximately 0.4% of the original dose added to the exposure medium. The transgenic embryos express green fluorescent protein (GFP) localized in the motor neurons making them ideal for evaluating motor neuron development and toxicities in vivo. The hb9:GFP zebrafish embryos (28 h post fertilization) treated with 2 mM ketamine for 20 h demonstrated significant reductions in spinal motor neuron numbers, while co-treatment with acetyl L-carnitine proved to be neuroprotective. In whole mount immunohistochemical studies using WT embryos, a similar effect was observed for the primary sensory neurons. In the ketamine-treated WT embryos, the number of primary sensory Rohon-Beard (RB) neurons was significantly reduced compared to that in controls. However, acetyl L-carnitine co-treatment prevented ketamine-induced adverse effects on the RB neurons. These results suggest that acetyl L-carnitine protects both motor and sensory neurons from ketamine-induced neurotoxicity.

Ketamine attenuates cytochrome p450 aromatase gene expression and estradiol-17β levels in zebrafish early life stages

Ketamine, a dissociative anesthetic, is a noncompetitive antagonist of N‐methyl‐D‐aspartate‐type glutamate receptors. In rodents and non‐human primates as well as in zebrafish embryos, ketamine has been shown to be neurotoxic. In cyclic female rats, ketamine has been shown to decrease serum estradiol‐17β (E2) levels. E2 plays critical roles in neurodevelopment and neuroprotection. Cytochrome p450 (CYP) aromatase catalyzes E2 synthesis from androgens. Although ketamine down‐regulates a number of CYP enzymes in rodents, its effect on the CYP aromatase (CYP19) is not known. Zebrafish have been used as a model system for examining mechanisms underlying drug effects. Here, using wild‐type (WT) zebrafish (Danio rerio) embryos, we demonstrate that ketamine significantly reduced E2 levels compared with the control. However, the testosterone level was elevated in ketamine‐treated embryos. These results are concordant with data from mammalian studies. Ketamine also attenuated the expression of the ovary form of CYP aromatase (cyp19a1a) at the transcriptional level but not the brain form of aromatase, cyp19a1b. Exogenous E2 potently induced the expression of cyp19a1b and vtg 1, both validated biomarkers of estrogenicity and endocrine disruption, but not cyp19a1a expression. Attenuation of activated ERK/MAPK levels, reportedly responsible for reduced human cyp19 transcription, was also observed in ketamine‐treated embryos. These results suggest that reduced E2 levels in ketamine‐treated embryos may have resulted from the suppression of cyp19a1a transcription. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

l-Carnitine rescues ketamine-induced attenuated heart rate and MAPK (ERK) activity in zebrafish embryos

Ketamine, an antagonist of the N-methyl-D-aspartate (NMDA)-type glutamate receptors, is a pediatric anesthetic. Ketamine has been shown to be neurotoxic and cardiotoxic in mammals. Here, we show that after 2 h of exposure, 5 mM ketamine significantly reduced heart rate in 26 h old zebrafish embryos. In 52 h old embryos, 1 mM ketamine was effective after 2 h and 0.5 mM ketamine at 20 h of exposure. Ketamine also induced significant reductions in activated MAPK (ERK) levels. Treatment of the embryos with the ERK inhibitor, PD 98059, also significantly reduced heart rate whereas the p38/SAPK inhibitor, SB203580, was ineffective. Ketamine is known to inhibit lipolysis and a decrease of ATP content in the heart. Co-treatment with l-carnitine that enhances fatty acid metabolism effectively rescued ketamine-induced attenuated heart rate and ERK activity. These findings demonstrate that l-carnitine counteracts ketamines negative effects on heart rate and ERK activity in zebrafish embryos.

Developmental toxicity assay using high content screening of zebrafish embryos

Typically, time‐consuming standard toxicological assays using the zebrafish (Danio rerio) embryo model evaluate mortality and teratogenicity after exposure during the first 2 days post‐fertilization. Here we describe an automated image‐based high content screening (HCS) assay to identify the teratogenic/embryotoxic potential of compounds in zebrafish embryos in vivo. Automated image acquisition was performed using a high content microscope system. Further automated analysis of embryo length, as a statistically quantifiable endpoint of toxicity, was performed on images post‐acquisition. The biological effects of ethanol, nicotine, ketamine, caffeine, dimethyl sulfoxide and temperature on zebrafish embryos were assessed. This automated developmental toxicity assay, based on a growth‐retardation endpoint should be suitable for evaluating the effects of potential teratogens and developmental toxicants in a high throughput manner. This approach can significantly expedite the screening of potential teratogens and developmental toxicants, thereby improving the current risk assessment process by decreasing analysis time and required resources. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

Nicotine alters the expression of molecular markers of endocrine disruption in zebrafish.

Nicotine, a drug of abuse, has been reported to have many adverse effects on the developing nervous system. In rodents, chronic nicotine exposure inhibits estrogen-mediated neuroprotection against cerebral ischemia in females suggesting that nicotine could disrupt endocrine targets. Zebrafish have been used as a model system for examining mechanisms underlying nicotinic effects on neuronal development. Here, using zebrafish embryos, we demonstrate that nicotine alters the expression of the validated endocrine disruption (ED) biomarkers, vitellogenin (vtg 1 and vtg 2) and cytochrome p450 aromatase (cyp19a1a and cyp19a1b) at the transcriptional level. Increased expression of three of these molecular markers (vtg 1, vtg 2 and cyp19a1b) in response to 17β-estradiol (E2) was more pronounced in 48hpf (hours post-fertilization) embryos than in the 24hpf embryos. While 24hpf embryos were non-responsive in this regard to 25μM nicotine, a similar exposure of the 48hpf embryos for 24h significantly down-regulated the expression of all four ED biomarker genes indicating that nicotines anti-estrogenic effects are detectable in the 48hpf zebrafish embryos. These results provide direct molecular evidence that nicotine is an endocrine disruptor in zebrafish.

On the in vivo early toxic properties of Aβ25–35 peptide in the rat hippocampus: Involvement of the Receptor-for-Advanced Glycation-End-Products and changes in gene expression

Amyloid-beta peptide (Aβ) deposition is assumed to play a pathogenic role in the brain of Alzheimers disease patients. To date, the precise mechanisms underlying Aβ toxicity are not fully understood. A recent hypothesis suggesting that the Receptor-for-Advanced-Glycation-End-Products (RAGE)-a trans-membrane protein signaling for oxidative stress-is involved in Aβ toxicity is gaining attention. Early Aβ toxicity could indeed help to explain the deleterious events further produced by this molecule in the brain. In this work, we evaluated the pattern of early expression of RAGE in the toxic model induced by Aß₂₅₋₃₅ in rat CA1 region. Intrahippocampal injections of Aβ₂₅₋₃₅ in rats increased the RAGE expression at 24 h post-injection; this event was accompanied by increased components of RAGE downstream signaling in hippocampal cells, such as enhanced expression of the pro-apoptotic factor NF-κB, increased nitric oxide production, LDH leakage, mitochondrial dysfunction, increased TNF-α expression, antioxidant genes down-regulation, and augmented neurodegeneration. Our findings support an active role of RAGE during the early stages of Aβ₂₅₋₃₅ toxicity in the hippocampus.