Ketamine-induced attenuation of reactive oxygen species in zebrafish is prevented by acetyl l-carnitine in vivo

Abstract

Ketamine, an anesthetic, is a non-competitive antagonist of the calcium-permeable N-methyl-d-aspartate (NMDA) receptor. High concentrations of ketamine have been implicated in cardiotoxicity and neurotoxicity. Often, these toxicities are thought to be mediated by reactive oxygen species (ROS). However, findings to the contrary showing ketamine reducing ROS in mammalian cells and neurons in vitro, are emerging. Here, we determined the effects of ketamine on ROS levels in zebrafish larvae in vivo. Based on our earlier studies demonstrating reduction in ATP levels by ketamine, we hypothesized that as a calcium antagonist, ketamine would also prevent ROS generation, which is a by-product of ATP synthesis. To confirm that the detected ROS in a whole organism, such as the zebrafish larva, is specific, we used diphenyleneiodonium (DPI) that blocks ROS production by inhibiting the NADPH Oxidases (NOX). Upon 20 h exposure, DPI (5 and 10 μM) and ketamine at (1 and 2 mM) reduced ROS in the zebrafish larvae in vivo. Using acetyl l-carnitine (ALCAR), a dietary supplement, that induces mitochondrial ATP synthesis, we show elevated ROS generation with increasing ALCAR concentrations. Combined, ketamine and ALCAR counter-balanced ROS generation in the larvae suggesting that ketamine and ALCAR have opposing effects on mitochondrial metabolism, which may be key to maintaining ROS homeostasis in the larvae and affords ALCAR the ability to prevent ketamine toxicity. These results for the first time show ketamine’s antioxidative and ALCAR’s prooxidative effects in a live vertebrate.

GRAPHICAL ABSTRACT

1. Introduction

Ketamine, a pediatric anesthetic, is an antagonist of the calcium permeable N-methyl-d-aspartate (NMDA)-type glutamate receptors [1]. Studies show high concentrations of ketamine to be cardiotoxic and neurotoxic in rodents, non-human primates [2], [reviewed in [3] and zebrafish early life stages [4–11] when treated for a longer duration during early development. How ketamine exerts toxic effects remains a field of continued investigation.

Of late, several studies have shown ketamine’s adverse effects on mitochondrial function and ATP synthesis; for example, high concentrations of ketamine attenuated mitochondrial membrane potential and induced apoptosis in human neural stem cells [12] and led to mitochondrial dysfunction in neurons derived from human induced pluripotent stem cell [13]. In rat primary neurons, ketamine-induced neurotoxicity was concomitant with nitrotyrosine formation [14]. Ketamine induced mitochondrial dysfunction in human lymphocytes and hepatocytes [15,16], while acute ketamine administration impaired mitochondrial function in rat brains [17]. Even subanesthetic concentrations of ketamine altered mitochondrial respiratory chain activity in various regions of rat brains [18]. Furthermore, ketamine suppressed ATP biosynthesis in HepG2 cells [19] and zebrafish embryos [10] potentially through reduction in cellular calcium levels followed by suppressed mitochondrial oxygen metabolism.

In the mitochondria, acetyl l-carnitine (ALCAR) is essential for β-oxidation of fatty acids leading to ATP generation [20]. ALCAR has been shown to prevent mitochondrial injury resulting from oxidative damage to neurons [21]. In ketamine-treated mammalian neurons in vitro, ALCAR’s potential antioxidant effects have been reported [22]. In stressed adult zebrafish, ALCAR treatment (0.1 mg/L with 10 min exposure) for 7 days ameliorated anxiety-like behavior and oxidative damage but showed no effect on the control fish [23]. ALCAR cotreatment at 100 mg/L significantly reduced mortality rates induced by γ-Fe₂CO₃ nanoparticles in 24 h post-fertilization (hpf) embryos [24]. How exactly ketamine and ALCAR influence ROS status in vivo is not known, since ROS is produced as a by-product during ATP synthesis in the mitochondria [25].

Working with ketamine and ALCAR, we have reported their counteracting effects in zebrafish (Danio rerio) embryos [8,9,11,26], mostly showing ALCAR preventing ketamine’s adverse effects [4–6,9–11,26], potentially through the mitochondrial bioenergetic pathway involving ATP generation [5,10]. As a follow-up, we explored whether ketamine-induced attenuation in mitochondrial potential and ATP levels in the zebrafish embryos were simultaneous with alternations in the mitochondrial metabolic by-product ROS in vivo and ALCAR modulated the outcome.

2. Materials and methods

2.1. Animals

Adult wild type (WT) zebrafish (Danio rerio, AB strain) were obtained from the Zebrafish International Resource Center (www.zirc.org) (ZIRC, Eugene, OR, USA). Handling and maintenance of zebrafish followed the NIH Guide for the Care and Use of Laboratory Animals and our Institutional Animal Care and Use Committee (IACUC) protocol [10]. Breeding of male to female (ratio of 2:1) was in the in-system breeding tanks. The fish were kept in fish tanks (Aquatic Habitats, FL, USA) at the NCTR/FDA zebrafish facility containing buffered water [(pH 7.5), conductivity 800 μs and dissolved oxygen level set at 7 ppm] at 28.5 °C. The fish were fed daily live brine shrimp and Zeigler dried flake food (Zeiglers, Gardeners, PA, USA). Each 3-liter tank housed eight adult fish. The day-night cycle was maintained at 14:10 h. For in-system breeding, crosses of males and females were set up the previous evening in the tanks with partitions that were taken off the following morning at the time of light onset to stimulate spawning and fertilization. Fertilized eggs were collected, placed in Petri dishes, washed thoroughly with buffered egg water [reverse osmosis water containing 60 mg sea salt (Crystal Sea^®, Aquatic Eco-systems, Inc., Apopka, FL, USA) per liter of water (pH 7.5)]. The pH of 7.5 of the buffered egg water was achieved by adding freshly made sodium bicarbonate solution. The eggs were then allowed to develop in an incubator at 28.5 °C for later use.

2.2. Reagents

Ketamine hydrochloride (10 mg/ml vials) (Product # VINV-KETA-OVED) was purchased from Vedco, Inc. (St. Joseph, MO, USA). Acetyl l-carnitine (ALCAR) (Cat # A6706), diphenyleneiodonium chloride (DPI) (Cat # D2926), 2, 7-dichlorodihydrofluorescein diacetate (H2DCF-DA) (Cat # D6883) and other reagents were purchased from Sigma (St. Louis, MO, USA) unless stated otherwise.

2.3. Treatment of zebrafish embryos with NADPH Oxidase (NOX) inhibitor, diphenyleneiodonium chloride (DPI), ketamine and acetyl l-carnitine (ALCAR)

For treatment with ketamine and ALCAR, we chose 52 hpf embryos based on our previous studies that showed specific response of these embryos to ketamine [4,6], whereas ketamine exposure at the gastrula stage exhibited non-specific effects [9]. The embryos obtained from the eggs fertilized at the exact same time were pooled and divided into the treatment groups. In each treatment group, embryos (n = 15) were placed in one of the wells of the 6-well plates with 5 ml buffered egg water for 20 h (static exposures) exposure to various concentrations of DPI (1–10 μM), ketamine (0.5–2.0 mM) and ALCAR (0.25–1.5 mM). DPI concentrations of 1 μM and 10 μM were chosen as these concentrations have been shown to significantly reduce ROS in mammalian cells [27]. Previously, we have shown that ketamine accumulation in the zebrafish embryos/larvae treated with 2 mM approximated (~8 μM) the lower range of human anesthetic range (plasma concentration) [28] and at this concentration, ketamine induced detectable adverse effects [4,6,7,29]. Although 5 mM ALCAR has been successfully used in PC12 cells to modulate mitochondrial inhibitor 3-nitropropionic acid (3-NPA)-induced toxicity [30], our previous studies showed ALCAR at 1 mM was effective against adverse effects induced by 2 mM ketamine [4–6,10,11] and ALCAR concentrations above 1 mM induced mortality in the zebrafish embryos (our unpublished data). Untreated control groups were examined in parallel. Each experiment was repeated three times.

2.4. Detection of reactive oxygen species (ROS) and embryo imaging

ROS in zebrafish embryos/larvae were measured by fluorescence microscopy using the cell membrane-permeable compound, 2’, 7’-di-chlorodihydrofluorescein diacetate (H2DCF-DA) fluorescent dye, a permanent ROS marker [31], essentially following a method used for zebrafish embryos [32]. H2DCF-DA is deacetylated by cellular esterases to non-fluorescent 2’,7’-dichlorfluorescein (DCFH). In response to cellular ROS production, DCFH is converted to the green fluorescent di-chlorofluorescein (DCF), the fluorescent intensity of which can be visualized and quantified using fluorescence microscopy. In brief, embryos/larvae were stained with H2DCF-DA for 30 min in dark at 28.5° C. The fluorescence of the whole embryo/larva was visualized using a DP2 BSW microscope (Olympus, Tokyo, Japan) through the green channel and images were acquired. Fluorescence was measured using the DP2 BSW microscope digital camera software. Data for each group (n = 15) were used to calculate the mean and standard deviation (SD). Data acquisition and analysis were blind to the treatments. The statistical significance of ROS fluorescence intensities was determined by one-way ANOVA (Sigma Stat) using Holm-Sidak pairwise multiple comparison post-hoc analysis. Statistical significance was set at P < 0.05 (*) and P < 001 (**).

3. Results

3.1. The NADPH Oxidase (NOX) inhibitor, diphenyleneiodonium chloride (DPI), reduces ROS

We hypothesized that calcium induced mitochondrial respiration would be hampered by inhibition of calcium-permeable NMDA receptors by ketamine, whereas ALCAR would increase mitochondrial respiration via activating the l-type calcium channels [33] and through fatty acid oxidation [34]. With mitochondrial respiration, ATP is generated along with ROS as a by-product [35]. Based on this background information (Fig. 1A), we set out to measure ROS in zebrafish larvae in vivo. We exposed 52 hpf embryos to DPI, an inhibitor of NADPH oxidase (NOX), for 20 h. In the 72 hpf larvae, ROS levels decreased with increasing DPI concentrations (Fig. 1 B, C) suggesting that DPI reduced mitochondrial respiration. Data analyses revealed significant changes in ROS levels among the treatment groups [F (3, 55) = 376.103, P < 0.001]. Compared to control, significant decrease in ROS by DPI occurred at 5 μM (P < 0.05) and 10 μM concentrations (P < 0.001).

Fig. 1.

Effect of the NADPH Oxidase (NOX) inhibitor, diphenyleneiodonium chloride (DPI) on ROS in live zebrafish embryos. (A) Schematic diagram shows generation of ROS, a by-product of ATP synthesis in the mitochondria, as a downstream event of calcium signaling that may be inhibited by ketamine, a calcium antagonist and induced by ALCAR, an activator of L-type calcium channels. (B) Representative images of ROS levels in the 72 hpf larvae after 20 h of exposure to DPI, detected in vivo, are shown. Zebrafish embryos at 52 hpf were treated for 20 h with various concentrations of DPI; (C) Relative ROS fluorescence intensities of the experimental groups are presented as mean ± SD. Asterisks (* P < 0.05; ** P < 0.001) indicate a statistically significant difference with control.

3.2. Ketamine-induced reduction in ROS is concentration-dependent

Previously, we have shown that 2 mM ketamine treatment for 20 h can be both cardiotoxic and neurotoxic in zebrafish early life stages [4–8,36]. To determine whether ketamine affects ROS levels, 52 hpf embryos were exposed to 0.5, 1.0 and 2.0 mM ketamine for 20 h. Data analyses revealed significant changes in ROS levels among the treatment groups [F (3, 55) = 52.215, P < 0.001]. Post-exposure, 72 hpf larvae showed ketamine-induced attenuation of ROS levels at 1 mM (P < 0.05) and 2 mM concentrations (P < 0.001) compared to control (Fig. 2A, B). These results suggested that ketamine could reduce ROS levels in vivo.

Fig. 2.

Concentration-dependent effect of ketamine on ROS in zebrafish larvae. Zebrafish embryos at 52 hpf were treated for 20 h with ketamine (0.5, 1.0 and 2.0 mM). Untreated embryos were used as control. (A) Representative images of post-treatment larvae show ROS levels in vivo; (B) Relative ROS fluorescence intensities of the experimental groups are presented as mean ± SD. Asterisks (* P < 0.05; ** P < 0.001) indicate a statistically significant difference with control.

3.3. ALCAR-induced ROS generation is concentration-dependent

Both prooxidative and antioxidative effects of l-carnitine have been reported in mammals [34,37,38]. We tested the effects of ALCAR on ROS generation in the zebrafish embryos. Embryos at 52 hpf were treated with various concentrations of ALCAR (0.25, 0.5, 1.0 and 1.5 mM) for 20 h. Data analyses showed that ALCAR induced significant changes in ROS levels [F (4, 69) = 4790.862, P < 0.001]. Post-treatment, the 72 hpf larvae showed a gradual increase in ROS levels with increasing ALCAR concentrations compared to control (Fig. 3 A, B). These results suggest that ALCAR induced ROS generation potentially by increasing mitochondrial respiration.

Fig. 3.

Concentration-dependent effect of ALCAR on ROS in zebrafish larvae. Zebrafish embryos at 52 hpf were treated for 20 h with ALCAR (0.25, 0.5, 1.0 and 1.5 mM). Untreated embryos were used as Control. (A) Representative images of post-treatment larvae show ROS levels in vivo; (B) Relative ROS fluorescence intensities of the experimental groups are presented as mean ± SD. Asterisks (* P < 0.05; ** P < 0.001) indicate statistically significant differences.

3.4. ALCAR prevents ketamine-induced reduction of ROS

Based on the observations that ketamine reduced ROS levels while ALCAR induced ROS generation, we explored the status of ROS when zebrafish embryos were exposed to both the compounds. We have shown in our previous studies that ketamine’s neurotoxic and cardiotoxic effects at 2 mM, which results in an internal exposure of ^~8 μM [28], is prevented by cotreatment with 1 mM ALCAR [4–6,9–11,26]. In 52 hpf embryos, a 20 h coexposure with ketamine (2 mM) and ALCAR (1 mM) continued for 20 h. Data analyses revealed significant changes in ROS levels among the treatment groups [F (3, 55) = 15881.038, P < 0.001]. ALCAR and ketamine cotreatment resulted in significantly increased ROS compared to control (P < 0.001) and ketamine (P < 0.05), although the level was significantly lower than in the 1 mM ALCAR-treated group (P < 0.001) (Fig. 4A, B). ALCAR-induced ROS levels were significantly higher than the control (P < 0.001) and ketamine-treated larvae (P < 0.001). Ketamine significantly reduced ROS compared to control (P < 0.05). These results suggest that ketamine and ALCAR have contrasting effects on ROS generation, which may be critical to ALCAR’s ameliorative effects on ketamine toxicity through maintaining ROS homeostasis.

Fig. 4.

ALCAR counteracts ketamine-induced attenuation of ROS in zebrafish larvae. Zebrafish embryos at 52 hpf were treated for 20 h with 2 mM ketamine with or without 1 mM ALCAR. (A) Representative images of post-treatment larvae show ROS levels in vivo; (B) Relative ROS fluorescence intensities of the experimental groups are presented as mean ± SD. Asterisks (* P < 0.05; ** P < 0.001) indicate statistically significant differences.

4. Discussion

The goal of this study was to determine how ketamine and ALCAR affect ROS generation in vivo. ROS are natural by-products generated during normal metabolism in the mitochondria [39]. In vitro, ketamine-induced ROS generation was shown in rat neurons in culture [22], and in neurons differentiated from human embryonic stem cells [40]. Ketamine-induced neurodegeneration in neonatal rats followed by long-term cognitive deficits is thought to be mediated by ROS [41]. In contrast, ketamine is shown to be an antioxidant in mouse hippocampi [42], macrophages [43] and rat lungs [44]. The reason for such conflicting results may be attributed to the difference in ROS detection between in vitro and in vivo conditions. The zebrafish embryo model is a system in which the ultimate biological readout of drug effects on a whole organism can be visualized in vivo.

To determine whether the detected ROS is specific, here, we show that DPI, an NADPH Oxidase (NOX) inhibitor, which blocks cellular ROS without affecting ATP generation [45], reduces ROS levels concentration-dependently in the zebrafish larvae. During early development, significant levels of ROS in the intestine and liver of zebrafish larvae were observed [46,47], possibly due to the rapidly developing digestive system, where mitochondrial metabolic activity must be pronounced at the time, relative to other parts of the body. The current study revealed that ketamine reduced ROS, a by-product of ATP synthesis [35]. Although different from a report that ketamine increases ROS in 24 hpf zebrafish embryos after the blastulae (2–3 hpf with a hollow sphere of undifferentiated cells without any organs/organ rudiments, especially the liver where major drug metabolism occurs) were exposed to ketamine for only 20 min [48], the current data, on embryos with functional organs with a capacity to metabolize drugs as opposed to the 24 hpf embryos, are consistent with our earlier findings that 2 mM ketamine reduces ATP synthesis along with mitochondrial potential [10] and are concordant with reports that ketamine reduced ATP levels in mouse brain [49] and rapidly reduced ROS in mouse hippocampus [42]. In ketamine-induced cardiotoxicity [2] and neurotoxicity [reviewed in [3] in mammals, whether ROS play any role in such adverse outcomes in vivo is not known. ROS detection in vivo can only be performed in lower vertebrates, such as the transparent zebrafish larvae. In these larvae, the drug effect would be more predictive since a whole organism, following exposure, can fine tune all the signals through its homeostatic control mechanisms before manifesting a phenotype, as opposed to cultured cells in vitro.

ROS is produced downstream of calcium. ALCAR’s ability to activate l-type calcium channels [33] could potentially compensate for ketamine’s antagonism of calcium-permeable NMDA receptors in preventing ketamine toxicities [5,6,10], and ketamine-induced reduction in ATP levels [10]. In this context, our current data show that ALCAR concentration-dependently induced ROS generation, a downstream event of calcium signaling. More importantly, we show that ketamine reduced ALCAR-triggered ROS generation. Previously, we have shown that cotreatment with ketamine and ALCAR prevented a variety of ketamine toxicities in the zebrafish early life stages [4–6,9–11,26]. In stressed adult zebrafish, although ALCAR treatment (O.1 mg/L with 10 min exposure) for 7 days ameliorated oxidative damage [23] and ALCAR co-treatment at 100 mg/L significantly reduced mortality rates induced by γ-Fe₂0₃ nanoparticles in 24 hpf embryos [24], in our studies ALCAR concentrations at 1 mM was efficacious against ketamine toxicity [10,11,50]. It would have been conventional to speculate that ketamine toxicities might occur due to elevated ROS. However, our current study proves otherwise, and is consistent with findings that ALCAR increases mitochondrial ROS in rat liver by enhancing mitochondrial function [37,38]. From this in vivo study, we hypothesize that ketamine toxicities in these embryos at the specific age are due to mitochondrial suppression resulting in lower metabolic ROS production. ALCAR improves mitochondrial function thus counteracting ketamine’s suppressive effects. In the current study, ROS may not be the cause of ketamine toxicity, but reduced ROS is an indication that mitochondrial function is below normal physiological levels and ALCAR helps restore mitochondrial function thereby causing homeostasis of physiologic ROS.

In conclusion, we show that in vivo, ketamine impairs ROS generation potentially suppressing mitochondrial function. This reduction in ROS is overcome by ALCAR co-treatment possibly by restoration of mitochondrial function that may be key to its ability in mitigating ketamine toxicity, we have reported earlier [4–6,9–11,26]. This study, for the first time, provides evidence that ketamine and ALCAR have opposite effects on ROS generation in vivo in a live vertebrate.