A screen for protective drugs against delayed hypoxic injury

Chun-Ling Sun; Huiliang Zhang; Meng Liu; Wang Wang; C. Michael Crowder

doi:10.1371/journal.pone.0176061

Abstract

Despite longstanding efforts to develop cytoprotective drugs against ischemia/reperfusion (IR) injuries, there remains no effective therapeutics to treat hypoxic injury. The failure of traditional strategies at solving this problem suggests the need for novel and unbiased approaches that can lead to previously unsuspected targets and lead compounds. Towards this end, we report here a unique small molecule screen in the nematode C. elegans for compounds that improve recovery when applied after the hypoxic insult, using a C. elegans strain engineered to have delayed cell non-autonomous death. In a screen of 2000 compounds, six were found to produce significant protection of C. elegans from delayed death. Four of the compounds were tested in an ex vivo mouse heart ischemia/reperfusion model and two, meclocycline and 3-amino-1,2,4-triazole, significantly reduced infarction size. Our work demonstrates the feasibility of this novel C. elegans screen to discover hypoxia protective drugs that are also protective in a mammalian model of hypoxic injury.

Citation: Sun C-L, Zhang H, Liu M, Wang W, Crowder CM (2017) A screen for protective drugs against delayed hypoxic injury. PLoS ONE 12(4): e0176061. https://doi.org/10.1371/journal.pone.0176061

Editor: Guo-Chang Fan, University of Cincinnati College of Medicine, UNITED STATES

Received: January 31, 2017; Accepted: April 4, 2017; Published: April 20, 2017

Copyright: © 2017 Sun et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: The work was supported by National Institute of Neurological Disorders and Stroke [Grants R01 NS045905, R21 NS084360] and by the International Anesthesia Research Society Frontiers in Anesthesia Research Award. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Despite decades of intensive fundamental investigation resulting in numerous clinical trials, no effective treatment has been approved for cytoprotection from ischemia/reperfusion (IR) injury [1–3]. These repeated failures suggest that a full understanding of the mechanisms of IR injury is lacking and that novel, unbiased approaches are needed to make breakthroughs.

A particularly important and vexing question in hypoxic biology is what controls injury after the hypoxic insult and are these factors distinct from those preventing injury before the hypoxic episode. Another fundamental question is the nature of cell non-autonomous injury. How does the injury of one cell promote the death of another nearby or even distant one? This so called “innocent bystander” death is likely to be important in stroke pathology where neurons distant from the ischemic core can undergo delayed death [4]. The difficulty in experimentally manipulating delayed hypoxic cell death and cell non-autonomous death contribute to our incomplete understanding of IR injury.

C. elegans has been developed as a genetic model to identify and study decisive factors of hypoxic injury [5–10]. Forward and reverse genetic screens in C. elegans have identified a substantial number of genes (termed Hyp genes), that promote hypoxic injury and whose reduction-of-function phenotype is resistance to organismal death following hypoxia. Utilizing one of the Hyp gene mutants [8], we have recently developed novel transgenic C. elegans strains where about 3% of non-essential somatic cells were made sensitive to hypoxic injury relative to other cells in the animal. This was accomplished by expressing a wild type version of a Hyp gene in a few cells in the background of the Hyp mutant where all other cells are resistant [11]. We demonstrated that these strains had both delayed cell death and cell non-autonomous death after a hypoxic insult. Thus these strains provide a genetically tractable model to study delayed and cell non-autonomous hypoxic injury and offer tools to screen for genetic changes and chemicals that are protective against delayed hypoxic injury.

Here, we report a screen for drugs to protect from delayed hypoxic organismal death and obtained six compounds that provide reproducibly significant protection in our C. elegans model. Four of the drugs were tested in a mouse perfused-heart model and two were found to be protective, suggesting that the C. elegans model may be generally useful to identify compounds protective in various IR injury paradigms.

Materials and methods

C. elegans strains and culture methods

C. elegans strains were cultured and maintained at 20°C, unless otherwise indicated, on NGM agar with OP50 E. coli food unless otherwise noted [12]. The N2 (Bristol) strain was the standard wild-type strain from the C. elegans Genetics Center (CGC, University of Minnesota). Focal hypoxic injury model strains, rars-1(gc47);gcIs3 and rars-1(gc47);gcSi4 were previously characterized [11]. The mitochondrial UPR reporter strain SJ4100 (zcIs13[hsp-6p::GFP]) was obtained from the CGC.

Hypoxic incubations

Synchronized young adult worms were subjected to hypoxia as described previously except that hypoxic incubation temperature was 26.5°C [9,11]. Briefly, each plate of worms was washed into one 1.5 ml tube with 1 ml of M9 buffer (22 mM KH₂PO₄, 22 mM Na₂HPO₄, 85 mM NaCl, 1 mM MgSO₄). Worms were allowed to settle by gravity, and 900 μl of M9 was removed. The tubes were then placed in the anaerobic chamber (Forma Scientific) at 26.5°C for incubation times ranging from 14–27 hours as indicated. Oxygen tension was always ≤ 0.3%. Following the hypoxic insult unless otherwise noted, worms were placed using glass Pasteur pipettes onto NGM plates spotted with OP50 bacteria and recovered at 20°C for the indicated time. Normoxic incubations were otherwise identical except performed in a 26.5°C room air incubator. To score delayed death in the wild type strain N2, hypoxia was performed for 14–18 hours followed by a 24 hour recovery at 20°C; For gc47;gcIs3 and gc47;gcSi4, 27 hours of hypoxia was used followed by 4 days recovery at 20°C and phenotyping.

Chemical library screen

The Spectrum Collection chemical library from MS-Discovery Inc (Gaylordsville, CT) was supplied through the University of Washington Quellos High Throughput Screening Core. Synchronized young adult gc47; gcIs3 animals were washed off the NGM/OP50 plates with M9 and washed two times with S-medium [12], and re-suspended in S-medium at approximate 800–1000 worms/ml. 3 ml of the worm suspension was transferred into a 3.5 cm petri dish with addition of 20 μl OP50 (100 mg/ml). The petri dish was covered with a breathable sealing film (Alpha) to prevent evaporation and subjected to hypoxia incubation (O₂<0.3%) at 26.5°C for 27 hours. Immediately after hypoxic incubation, worms were robotically dispensed into 384-well plates, and the test compounds were added into each well by the robot. The drug solvent of S-medium with DMSO (0.1%) and doxycycline (10 μM) [13] were added to a few wells on each plate as a solvent/negative and a positive controls, respectively. The worms were then allowed to recover in 384-well plates with the various drug solutions in a normoxic incubator at 20°C for 96 hours. Preliminary screens found the death rate under these conditions to be about 70% with no significant death after a normoxic incubation. Before manual scoring of the hypoxic survival, the 384 plates were vortexed for 2 minutes to stimulate the worms. Survival rates of test compounds better than that of DMSO controls, in at least one of the duplicates of 1^st screen or in one quarter of the wells in a serial dilution in the 2^nd screen, were considered as positive. Animals were scored as dead if immobile and rod-like. For the 1^st screening, compounds were tested at 10 μM in duplicates; for the 2^nd screening, positive compounds in first screening were tested at 14 different concentrations ranging from 0.000031 to 50 μM. For the third screening, 16 positive compounds were tested on NGM agar plates in a serial dilution manner with the final concentration being that of the compound in the agar. The chemicals were purchased from Sigma-Aldrich (St. Louis, MO), with the exception of cypermethrin, which was purchased from Santa Cruz Biotechnology, Inc (Dallas, TX). Drugs were treated either after (post-treatment) or before (pre-treatment) hypoxia. Post-treatment: synchronized young adult worms were recovered from hypoxia on OP50 plates containing indicated compounds. The compound-containing plates were prepared by freshly smearing with compound dissolved in M9 plus 0.4% DMSO (390 μl in total) onto plates 2h before use. Pre-treatment: synchronized young adult worms were incubated on OP50 plates containing indicated compounds for 24 hours at 20°C and washed off before hypoxia incubation.

Hypoxic injury scoring

Organismal death was scored as described [11]. Briefly, animals were scored as dead if pharyngeal pumping, spontaneous and evoked movement (touching with a platinum wire) were not observed.

RNAi experiments

Control RNAi strain, L1440 (empty vector) was from the Ahringer C. elegans RNAi library (MRC Gene service) [14] and atfs-1(RNAi) strain was from Vidal ORF-RNAi C. elegans RNAi library [15] (a kind gift from Dr. T. Schedl, Washington University in St. Louis, MO). Bacterial clones containing RNAi plasmids were cultured and induced with 0.1% β-lactose in S-Basal 100 mg/ml ampicillin for 24 hr at 23°C [7]. Worms were synchronized on RNAi plates for 3.5 days (N2) or 4.5 days (animals in a rars-1(gc47) background) until reaching adulthood; worms not reaching adulthood were excluded.

Mitochondrial UPR reporter

SJ4100 (zcIs13[hsp-6::GFP]) was used as a reporter for mitochondrial UPR [16]. L4 animals were incubated on OP50 NGM plates containing indicated drug for 48 hr. The GFP expression were imaged and intensity of fluorescence was determined by ImageJ (NIH).

Langendorff-perfused heart

The Langendorff-perfused mouse heart trials have been performed as described previously, and the protocol was approved by the University of Washington IACUC (Institutional Animal Care and Use Committee) [17,18]. The mice (C57/Bl6, male, 3–4 month old) were heparinized (heparin, 100 U, i.p.) and euthanized (pentobarbital, 270 mg/kg, i.p.) before surgery. After the chest was cleaned with 70% ethyl alcohol, the thoracic cavity was entered and the heart was rapidly removed. The heart was then cannulated via the ascending aorta and perfused in the Langendorff mode with perfusion solution (118 mM NaCl, 25 mM NaHCO₃, 5.3 mM KCl, 2 mM CaCl₂, 1.2 mM MgSO₄, 0.5 mM EDTA) with metabolic substrates (10 mM glucose and 0.5 mM pyruvate), equilibrated with 95% O₂ and 5% CO₂ (pH 7.4) at 37°C. After 30 minutes of baseline perfusion, the perfusion was stopped (no-flow ischemia) for 30 minutes and then resumed for another 60 minutes (reperfusion). The left ventricle (LV) of the heart was separated along the long axis into 6 slices (~1 mm thick each slice) by a heart slicer. The slices were stained by triphenyltetrazolium chloride (TTC) and imaged by a digital camera. The weight of each slice was measured and the LV area and infarct area in each slice was determined by ImageJ software (NIH). The infarct weight was calculated as: (infarct area)/(LV area) x slice weight and the ratio between the total infarct weight and the total LV weight of the 6 slices was expressed as percentage of infarction. To confirm the protective effect of potential candidates, at least two of the trials was carried out by an experimenter blinded to the condition. All compounds were added in the perfusion solutions 30 minutes before the ischemia and throughout the reperfusion period.

Statistics

Two-sided unpaired or paired, as appropriate, t-tests were used for statistical comparisons. Statistics were calculated using GraphPad Prism 6.01 (San Diego, CA, USA) and Excel 2007 (Microsoft, Redmond, WA). Values are expressed as mean ± SD of at least three independent experiments. A P value of ≤ 0.05 was considered significant. EC₅₀ (the half-maximally effective drug concentration) was calculated using GraphPad Prism 6.01 by the equation: Y = Y_min+(Y_max-Y_min)/(1+((x/EC50)^-k)) of at least 5 doses tested where k is the slope and Y_min is the lowest death percentage and Y_max is the highest percentage of death.

Results

Chemical screen identifies six protective compounds against delayed hypoxic organismal death

We have previously established a focal hypoxic injury model [11]. The strains used for this model have selective tissue vulnerability to hypoxic injury. rars-1(gc47);gcIs3 has vulnerable pharyngeal myocytes and rars-1(gc47);gcSi4 has vulnerable GABA neurons. gc47;gcIs3 (pharyngeal myocyte, PM-targeted) was used in our initial chemical screen. To mimic a potential therapeutic given after ischemia, drugs were administered to animals during recovery from hypoxic insult. Following recovery, delayed organismal death was scored (Fig 1A). In the 1^st screen of the chemical library of 2,000 small molecules, 30 strong and 127 mild protective compounds (157 in total) were obtained. 80 of 157 (30 strong plus 50 mild) potential compounds were subjected to a 2^nd screen at multiple drug concentrations. Sixteen compounds (10 of strong compounds and 6 of mild compounds) appeared to have a concentration dependent protection in the 2^nd screen and were subjected to a 3^rd screen (Fig 1B). The 3^rd screen was performed in both gc47;gcIs3 and gc47;gcSi4 (GABA neuron, GN-targeted) and unlike the first and second screen, recovery from hypoxia was on agar plates, which is best validated and most reproducible [11]. Compounds with a statistically significant reduction in organismal death of at least 1.5-fold, as compared to DMSO solvent control, were considered positive. Six compounds were confirmed to be significantly protective against delayed hypoxic injury in both the PM-targeted and GN-targeted strains (Fig 1B and 1C and Fig 2). The half maximal effective concentration (EC₅₀) of the protective compounds ranged from 0.53 to 968 μM (S1 Fig).

Download:

Fig 1. High-throughput screen identifies six compounds protective against delayed hypoxic injuries.

(A) The schematic of the screen to identify hypoxic protective compounds. See the Materials and Methods for more details. (B) Summary of three round of screens tested. (C) Chemical structure of six positive compounds identified.

https://doi.org/10.1371/journal.pone.0176061.g001

Download:

Fig 2. Post-treatment with six compounds protects against delayed focal hypoxic injuries.

(A-B) Delayed organismal death of gc47;gcIs3 (A) and gc47;gcSi4 (B) following hypoxia and recovery with the various drugs and DMSO in NGM plates. Animals were kept on an individual plate for the entire recovery period then scored. Bars represent mean ± SD (n≥3). P values compared to DMSO control; *, p<0.05; **, p<0.01; ***, p<0.001; unpaired two-tailed t-test for mean of the % death. DMSO—dimethyl sulfoxide (solvent negative control, 0.4%); Doxy—doxycycline (positive control, 100 μM) [13]; Meclo—meclocycline (100 μM); Pp—phenazopyridine (50 μM); Cm—cypermethrine (100 μM); Dp—dibutyl phthalate (400 μM); 3-AT—3-amino-1, 2,4-triazole (4000 μM); Om—omeprazole (200 μM). Fold reduction of hypoxic death by drugs compared to DMSO control shown below.

https://doi.org/10.1371/journal.pone.0176061.g002

Compounds protect after but not before against focal hypoxic injury

We next examined the effect of pre-treatment of the protective compounds on delayed hypoxic organismal death. We reasoned that the protective compounds might confer durable protection to the cells not requiring the continued presence of the compounds. However, none of the six compounds showed protection from delayed hypoxic injury when added for the 24 hours preceding hypoxia in either PM-targeted or GN-targeted strains (Fig 3). These results indicate that underlying mechanisms of those compounds are acting specifically after hypoxic insult.

Download:

Fig 3. Effect of pre-treatment of six positive compounds on delayed focal hypoxic death.

(A-B) Delayed organismal death of gc47;gcIs3 (A) and gc47;gcSi4 (B) following hypoxia and recovery. Bars represent mean ± SD (n = 3). None of the values were significantly different from the DMSO negative control at the p < 0.05 threshold. Abbreviations and doses were as described in Fig 2. Fold change in hypoxic death by drugs compared to DMSO control shown below.

https://doi.org/10.1371/journal.pone.0176061.g003

To explore in more detail the timing requirements for improved recovery, we exposed the hypoxic-injured worms to meclocycline at various times during their recovery. Treatment with meclocycline for at least 2 days of the four-day recovery period was required for significant protection and the treatment could be delayed until day 3 and 4 of recovery and still impart a protective effect (Fig 4). These results indicate that meclocycline treatment can improve recovery with a surprisingly delayed time course, up to two days after the initial hypoxic injury.

Download:

Fig 4. Time course of meclocycline treatment for protection from delayed hypoxic injury.

(A) Schematic diagram of meclocycline (Drug) treatment (d1, d2, d3, d4 is the number of days of recovery following hypoxia). Animals were transferred daily to fresh plates containing meclocycline (100 μM) and DMSO (0.15%), as a solvent control. (B) Delayed organismal death of gc47;gcIs3 following hypoxia with exposure to meclocycline at the indicated recovery intervals. Bars represent mean ± SD (n = 3). P values compared to DMSO control; *, p<0.05 (unpaired two-tailed t-test for mean death).

https://doi.org/10.1371/journal.pone.0176061.g004

We next asked whether the compounds were protective in the wild type genetic background where no cells have been genetically modified to increase or decrease their sensitivity to hypoxia relative to their neighbors, thereby modeling global hypoxic injury. None of the compounds protected the wild type strain N2 when added after a short hypoxic exposure that results in a similar level and time course of delayed death as is seen in the gc47;gcIs3 and gc47;gcSi4 (S2A and S2B Fig). Notably, phenazopyridine (Pp) appeared to be additive with hypoxia producing injury in the wild type strain suggesting a toxic effect. However, none of the drugs showed toxicity of the adult animals in the absence of hypoxia (S3 Fig).

Likewise, when added before hypoxia, all but one of the compounds failed to protect the wild type strain (S2C and S2D Fig). In contrast, meclocycline as well as its congener doxycycline, when added before hypoxia, were strongly protective in wild type animals (S2C and S2D Fig). Doxycycline and tetracycline were also protective in the focal hypoxic model strains when added after the hypoxic injury (Fig 2A and 2B and S4 Fig). Given that these drugs are antibiotics, we considered the possibility that meclocycline and its congeners might be killing or altering the bacteria in some way that indirectly protects C. elegans from hypoxia. To test this hypothesis, we utilized heat-killed bacteria. Meclocycline, as well as doxycycline and tetracycline, were hypoxia protective when applied with heat-killed bacteria (S4 Fig), indicating that killing or altering the function of the bacteria is not required for protective effect of meclocycline and its congeners.

mtUPR activation is required for hypoxic protection by meclocycline

Doxycycline protects C. elegans in both the global and focal hypoxia injury models via activating mitochondrial unfolded protein response (mtUPR) [13]. We examined whether meclocycline might also protect by activating the mtUPR. Meclocycline treatment induced robust expression of hsp-6::GFP, which is a mitochondrial UPR reporter (Fig 5A and 5B). atfs-1 is a transcription factor central to mtUPR activation [19]. RNAi knockdown of atfs-1 fully suppressed the protection by meclocycline against delayed hypoxic injury (Fig 5C), indicating that the normal function of the mtUPR is required for the hypoxia protection by meclocycline.

Download:

Fig 5. Activation of mtUPR by meclocycline is hypoxia protective.

(A, B) Activation of hsp-6::GFP by doxycycline, meclocycline, and tetracycline. Two-day exposure of adult animals on plates containing DMSO (0.15%); Doxy, doxycycline (100 μM); Meclo, meclocycline (100 μM); Tetra, tetracycline, (100 μM). (B) Quantitation of hsp-6::GFP activation. Fluorescence intensity in arbitrary units/μm² was scored. Data represent mean ± SD (n = 10). P values compared to DMSO control; ***P<0.001 (unpaired two-tailed t-test). (C) Effect of atfs-1 RNAi on meclocycline-mediated delayed hypoxic injuries. Delayed organismal death of gc47;gcIs3 following hypoxia and recovery with the various drugs and DMSO on empty vector (L4440) or atfs-1(RNAi) plates. Bars represent mean ± SD (n = 4). p-values: black, compared to DMSO control; white, atfs-1(RNAi) compared to L4440 control for the same drug; n.s., not significant; *, p<0.05; **, p<0.01; ***, p<0.001 (paired two-tailed t-test for mean of the % Death). DMSO—dimethyl sulfoxide (solvent negative control, 0.15%); Doxy—doxycycline (positive control, 100 μM); Meclo—meclocycline (100 μM); Tetra—tetracycline (100 μM).

https://doi.org/10.1371/journal.pone.0176061.g005

Meclocycline and 3-AT protect against IR injury in a Langendorff-perfused heart

To test whether the protective compounds can protect against IR-mediated hypoxic injury in mammals, we applied the drugs to a Langendorff-perfused mouse heart model. Both the meclocycline- and 3-amino-1,2,4-triazole-perfused hearts were significantly resistant to IR-mediated infarction in comparison with DMSO solvent-perfused controls (Fig 6). By contrast, phenazopyridine-perfused hearts showed no protection and dibutyl phthalate-perfusion was toxic to the heart (data now shown). Cypermethrin and Omeprazole were not tested.

Download:

Fig 6. Cardioprotection by two compounds identified from the screen.

(A) Examples of representative left ventricular cross-section images (left to right, from bottom to top). DMSO (0.03%), a solvent control; Meclo, meclocycline (20 μM); 3-AT, 3-amino-1,2,4-triazole (600 μM). Bars = 1 mm. (B) Quantitation of infarct size. Data represent mean ± SD (n = 6). P values are by unpaired two-tailed t-test compared to DMSO control; **, p<0.01; for meclocycline, p = 0.0023; for 3-amino-1,2,4-triazole, p = 0.0019.

https://doi.org/10.1371/journal.pone.0176061.g006

Discussion

The aim of the current study was to develop a new potentially high throughput screening model for compounds that can improve recovery from hypoxic injury when added after the hypoxic insult. A second goal was to target specifically cell non-autonomous death, which to our knowledge has not been the focus of previous screens, in order to maximize the likelihood of identifying novel therapeutic leads. The potential for novel compounds is particularly valuable here given that a large number of drugs screens for hypoxia protection have been reported and presumably many more have been performed by the pharmaceutical industry without resulting in an approved therapeutic. Using a C. elegans strain that dies from a delayed cell non-autonomous death, we screened a library of 2,000 chemicals and identified six protective compounds with structural and pharmacological diversity.

Interestingly, none of the six compounds provided protection of the wild type strain when added after the hypoxic insult. In the wild type strain, we believe that cells are more or less similarly sensitive to hypoxia and therefore most closely model a global hypoxic injury paradigm. Whereas in the engineered strain used for the screen, a small number of cells are deliberately sensitized to hypoxic injury so that the primary mode of cell death must be by a secondary, non-autonomous mechanism. Thus, the ability of these six compounds to protect the engineered strain but not the wild type strain when added after the hypoxic injury demonstrates that the mechanisms underlying delayed cell non-autonomous hypoxic death and delayed global hypoxic death are distinct. Also, none of the six compounds protected the engineered strain when present only before the hypoxic insult. However, one of the six compounds, meclocycline, did strongly protect the wild type strain when added only before the hypoxic insult. Doxycycline similarly strongly protected the wild type strain when added before hypoxia. These results show, not surprisingly, that the mechanisms that prevent hypoxic injury are not identical with those that improve recovery after the injury. However, the efficacy of meclocycline and doxycycline both before and after hypoxia in the wild type strain suggests overlap of some aspects of the mechanisms operant in the two scenarios. Given the divergent protective properties and structures of the compounds, a more detailed discussion of the bioactivities of those compound warrants some discussion.

Cypermethrin, a synthetic pyrethroid, is commonly used as an insecticide. Cypermethrin is a fast-acting neurotoxin in insects and also induces dopaminergic neurodegeneration in rats [20,21]. Excessive exposure of cypermethrin in humans can cause nausea, headache, muscle weakness, salivation, shortness of breath and seizures [22]. It has been documented that cypermethrin can cause oxidative stress, DNA damage and inhibition of gap junctions, so it is therefore classified as a possible human carcinogen [22–24]. Whether cypermethrin protects from delayed hypoxic injury through any of these known actions remains to be investigated.

Dibutyl phthalate is a commonly used plasticizer and also used as an additive to adhesives or printing inks. Exposure of phthalates may cause reproductive and developmental defect [25]. However, how phthalates modulate biological pathways is not understood.

Phenazopyridine is a chemical generally prescribed for its local analgesic effects on the urinary tract; it is rapidly excreted by the kidneys directly into the urine [26]. However, the underlying mechanism of phenazopyridine action is not well known. 2,3,6-triaminopyridine, a metabolite of phenazopyridine, causes muscle necrosis and renal damage in rats and promotes generation of superoxide radical and hydrogen peroxide [27,28]. Whether phenazopyridine inhibits delayed hypoxic injury through its metabolite, 2,3,6-triaminopyridine, by regulating generation of reactive oxygen species remains to be determined.

Omeprazole is a proton pump inhibitor which blocks the release of stomach acid [29]. In addition, cellular proton levels are strictly controlled and crucial for normal physiology, such as mitochondrial homeostasis [30,31]. How a proton pump, like omeprazole, regulates delayed hypoxic injury is of interest.

3-amino-1,2,4,-triazole is characterized as a catalase inhibitor [32] and has been tested for its role in heat shock (hyperthemia)-mediated cardiac injury; however the results were controversial [33,34]. 3-amino-1,2,4,-triazole has also been shown to be neuroprotective in an ischemic brain model [35]. Our data show that 3-AT is also protective against ischemia/reperfusion injury in mouse heart (Fig 6). Although ROS has been thought to be a primary cause of IR injury, findings in this study suggest that increased ROS levels by 3-AT and perhaps phenazopyridine may protect from IR mediated heart infarction. Alternatively, an unknown mechanism underlies their protective actions and further investigations are warranted.

Meclocycline is a derivative of a tetracycline antibiotic, which blocks bacterial protein synthesis through inhibiting the 30S bacterial ribosomal subunit. In this study, we have demonstrated that meclocycline protects from hypoxic injury in C. elegans and IR-injury in mouse heart. Tetracycline and its derivatives, such as doxycycline and minocycline, have been shown to protect ischemia/reperfusion injuries in various tissues, including neuronal, liver and myocardium cells, where roles of tetracyclines in anti-inflammation and inhibition of mitochondrial permeability transition for protection against IR injury have been proposed [36–40]. In C. elegans, we go on to show that meclocycline at the protective concentrations activates the mtUPR and that the hypoxia protection requires the function of the mtUPR transcriptional activator gene atfs-1 (Figs 2 and 5). Another tetracycline-type antibiotic, doxycycline has been found to be hypoxia protective in C. elegans and induce the mtUPR, both by an atfs-1-dependent mechanism [13,19], therefore arguing that mtUPR activation has therapeutic potential. Activation of the mtUPR by cco-1(RNAi) is also protective from hypoxic injury in C. elegans [13,19]. Tetracycline is also protective from focal hypoxic injury (Fig 5 and S4 Fig). Additional members of this class of drugs may also be protective against cardiac IR injury, and doxycycline, tetracycline, and any other compounds in this family should be tested.

Statins are a class of cholesterol lowing agents that act by inhibiting HMG-CoA reductase, which is the rate limiting enzyme for mevalonate pathway of cholesterol biosynthesis [41]. Activation of the mtUPR is protective against the toxic effects of mevalonate inhibition by statins in C. elegans [42]. In a chemical screen, Rauthan et al. identified a tetracycline antibiotic, methacycline, as a potent inducer of mtUPR; however, methacycline exhibits only moderate, if any, protection against statins [43]. Methacycline would be another tetracycline-family member that should be tested in both C. elegans and in mammalian IR models.

Gou et al. have developed a cell-based IR assay for high-throughput drug screens and have identified 37 protective compounds, of which 4 showed cardioprotection in the Langendorff- perfused rat heart [44]. The 37 compounds are completely distinct from the 6 identified in the current study from the same chemical library. As opposed to the Gou et al screen, an essential feature of our screen was that the compounds were applied after the hypoxic insult. Additionally, unlike the Gou et al screen, our screen was performed in a strain where the death of the animal is due to secondary cell non-autonomous hypoxic injury. These two factors may explain the distinct set of protective compounds isolated in the two screens.

Future studies will include screening additional compounds for other novel hits as well as testing in other IR paradigms such as rodent stroke models. Collectively, our findings demonstrate the feasibility and value of our C. elegans model for identifying compounds that can improve recovery from hypoxic injury when added after the insult.

Supporting information

S1 Fig. EC50 of six protective compounds.

(A-F) Dose response curve of protective compounds against hypoxic organismal death in gc47;gcIs3. (G) Summary of EC₅₀, k and R square of the compounds tested, which were calculated using GraphPad Prism 6.01 (San Diego, CA, USA). Data represent mean ± SD.

https://doi.org/10.1371/journal.pone.0176061.s001

(TIF)

S2 Fig. Effect of six protective compounds against global hypoxic injury.

(A-B) Post-treatment. Wildtype N2 animals were treated with 14 (A) or 16 (B) hr hypoxia followed by 1-day recovery on indicated compound-containing plates. Organismal death scored. (C-D) Pre-treatment. Animals were pre-treated with compounds 24h before hypoxia. Organismal death scored 1 day after a 14 (A and C) or 16 (B and D) hr hypoxia. Bars represent mean ± SD (n = 3). Abbreviations were as described in Fig 2. P values compared to DMSO control; ***, p<0.001; paired two-tailed t-test for mean of the % death.

https://doi.org/10.1371/journal.pone.0176061.s002

(TIF)

S3 Fig. Test for toxic effects of positive compounds.

(A, B) Delayed organismal death of gc47;gcIs3 (A) and gc47;gcSi4 (B) following 27h normoxia at 26.5°C and 4 days recovery (n = 1). Abbreviations and doses were as described in Fig 2.

https://doi.org/10.1371/journal.pone.0176061.s003

(TIF)

S4 Fig. Protective effect of tetracyclines is independent of bacterial survival.

(A, B) gc47;gcIs3 (A) or gc47;gcSi4 (B) were fed before and after hypoxia with either live or heat-killed OP50. Delayed organismal death (death) scored by 4-day recovery following hypoxia. Bars represent mean ± SD (n = 3). P values are by unpaired two-tailed t-test compared to DMSO control; *, p<0.05; n.s., not significant. DMSO (0.15%) as a solvent control; Doxy—doxycycline (100 μM); Meclo—meclocycline (100 μM); Tetra—tetracycline (100 μM).

https://doi.org/10.1371/journal.pone.0176061.s004

(TIF)

Acknowledgments

We thank Barbara Scott for her technical contributions to the work. We also grateful to Timothy J Martins and James Annis (Quellos High Throughput Screening Core, UW) for the assistance of high-throughput screen. Strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440).

Author Contributions

Conceptualization: CS CC.
Formal analysis: CS CC.
Funding acquisition: CC.
Investigation: CS ML CC.
Methodology: CS ML HZ.
Project administration: CS CC.
Resources: WW CC.
Supervision: CC.
Validation: CC.
Visualization: CC.
Writing – original draft: CS CC.
Writing – review & editing: CS WW CC.

References

1. Kalogeris T, Baines CP, Krenz M, Korthuis RJ (2012) Cell biology of ischemia/reperfusion injury. Int Rev Cell Mol Biol 298: 229–317. pmid:22878108
- View Article
- PubMed/NCBI
- Google Scholar
2. O'Collins VE, Macleod MR, Donnan GA, Horky LL, van der Worp BH, Howells DW (2006) 1,026 experimental treatments in acute stroke. Ann Neurol 59: 467–477. pmid:16453316
- View Article
- PubMed/NCBI
- Google Scholar
3. Gerczuk PZ, Kloner RA (2012) An update on cardioprotection: a review of the latest adjunctive therapies to limit myocardial infarction size in clinical trials. J Am Coll Cardiol 59: 969–978. pmid:22402067
- View Article
- PubMed/NCBI
- Google Scholar
4. Moskowitz MA, Lo EH, Iadecola C (2010) The science of stroke: mechanisms in search of treatments. Neuron 67: 181–198. pmid:20670828
- View Article
- PubMed/NCBI
- Google Scholar
5. Miller DL, Roth MB (2009) C. Elegans Are Protected from Lethal Hypoxia by an Embryonic Diapause. Current Biology 19: 1233–1237. pmid:19576771
- View Article
- PubMed/NCBI
- Google Scholar
6. Menuz V, Howell KS, Gentina S, Epstein S, Riezman I, Fornallaz-Mulhauser M, et al. (2009) Protection of C. elegans from anoxia by HYL-2 ceramide synthase. Science 324: 381–384. pmid:19372430
- View Article
- PubMed/NCBI
- Google Scholar
7. Mabon ME, Mao X, Jiao Y, Scott BA, Crowder CM (2009) Systematic identification of gene activities promoting hypoxic death. Genetics 181: 483–496. pmid:19047414
- View Article
- PubMed/NCBI
- Google Scholar
8. Anderson LL, Mao X, Scott BA, Crowder CM (2009) Survival from hypoxia in C. elegans by inactivation of aminoacyl-tRNA synthetases. Science 323: 630–633. pmid:19179530
- View Article
- PubMed/NCBI
- Google Scholar
9. Scott BA, Avidan MS, Crowder CM (2002) Regulation of hypoxic death in C. elegans by the insulin/IGF receptor homolog DAF-2. Science 296: 2388–2391. pmid:12065745
- View Article
- PubMed/NCBI
- Google Scholar
10. Wojtovich AP, DiStefano P, Sherman T, Brookes PS, Nehrke K (2012) Mitochondrial ATP-sensitive potassium channel activity and hypoxic preconditioning are independent of an inwardly rectifying potassium channel subunit in Caenorhabditis elegans. FEBS Lett 586: 428–434. pmid:22281198
- View Article
- PubMed/NCBI
- Google Scholar
11. Sun CL, Kim E, Crowder CM (2014) Delayed innocent bystander cell death following hypoxia in Caenorhabditis elegans. Cell Death Differ 21: 557–567. pmid:24317200
- View Article
- PubMed/NCBI
- Google Scholar
12. Brenner S (1974) The genetics of Caenorhabditis elegans. Genetics 77: 71–94. pmid:4366476
- View Article
- PubMed/NCBI
- Google Scholar
13. Kaufman DM, Crowder CM (2015) Mitochondrial Proteostatic Collapse Leads to Hypoxic Injury. Curr Biol 25: 2171–2176. pmid:26234215
- View Article
- PubMed/NCBI
- Google Scholar
14. Kamath RS, Fraser AG, Dong Y, Poulin G, Durbin R, Gotta M, et al. (2003) Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421: 231–237. pmid:12529635
- View Article
- PubMed/NCBI
- Google Scholar
15. Rual JF, Ceron J, Koreth J, Hao T, Nicot AS, Hirozane-Kishikawa T, et al. (2004) Toward improving Caenorhabditis elegans phenome mapping with an ORFeome-based RNAi library. Genome Res 14: 2162–2168. pmid:15489339
- View Article
- PubMed/NCBI
- Google Scholar
16. Yoneda T, Benedetti C, Urano F, Clark SG, Harding HP, Ron D (2004) Compartment-specific perturbation of protein handling activates genes encoding mitochondrial chaperones. J Cell Sci 117: 4055–4066. pmid:15280428
- View Article
- PubMed/NCBI
- Google Scholar
17. Gong G, Wang W (2013) Confocal imaging of single mitochondrial superoxide flashes in intact heart or in vivo. J Vis Exp: e50818. pmid:24300235
- View Article
- PubMed/NCBI
- Google Scholar
18. Yu Q, Lee CF, Wang W, Karamanlidis G, Kuroda J, Matsushima S, et al. (2014) Elimination of NADPH oxidase activity promotes reductive stress and sensitizes the heart to ischemic injury. J Am Heart Assoc 3: e000555. pmid:24470522
- View Article
- PubMed/NCBI
- Google Scholar
19. Nargund AM, Pellegrino MW, Fiorese CJ, Baker BM, Haynes CM (2012) Mitochondrial import efficiency of ATFS-1 regulates mitochondrial UPR activation. Science 337: 587–590. pmid:22700657
- View Article
- PubMed/NCBI
- Google Scholar
20. Hu JX, Li YF, Li J, Pan C, He Z, Dong HY, et al. (2013) Toxic effects of cypermethrin on the male reproductive system: with emphasis on the androgen receptor. J Appl Toxicol 33: 576–585. pmid:22147539
- View Article
- PubMed/NCBI
- Google Scholar
21. Singh AK, Tiwari MN, Upadhyay G, Patel DK, Singh D, Prakash O, et al. (2012) Long term exposure to cypermethrin induces nigrostriatal dopaminergic neurodegeneration in adult rats: postnatal exposure enhances the susceptibility during adulthood. Neurobiol Aging 33: 404–415. pmid:20371137
- View Article
- PubMed/NCBI
- Google Scholar
22. Singh AK, Tiwari MN, Prakash O, Singh MP (2012) A current review of cypermethrin-induced neurotoxicity and nigrostriatal dopaminergic neurodegeneration. Curr Neuropharmacol 10: 64–71. pmid:22942879
- View Article
- PubMed/NCBI
- Google Scholar
23. Amer SM, Aboul-ela EI (1985) Cytogenetic effects of pesticides. III. Induction of micronuclei in mouse bone marrow by the insecticides cypermethrin and rotenone. Mutat Res 155: 135–142. pmid:3974628
- View Article
- PubMed/NCBI
- Google Scholar
24. Tateno C, Ito S, Tanaka M, Yoshitake A (1993) Effects of pyrethroid insecticides on gap junctional intercellular communications in Balb/c3T3 cells by dye-transfer assay. Cell Biol Toxicol 9: 215–221. pmid:8299001
- View Article
- PubMed/NCBI
- Google Scholar
25. Lyche JL, Gutleb AC, Bergman A, Eriksen GS, Murk AJ, Ropstad E, et al. (2009) Reproductive and developmental toxicity of phthalates. J Toxicol Environ Health B Crit Rev 12: 225–249. pmid:20183522
- View Article
- PubMed/NCBI
- Google Scholar
26. Thomas BH, Whitehouse LW, Solomonraj G, Paul CJ (1990) Excretion of phenazopyridine and its metabolites in the urine of humans, rats, mice, and guinea pigs. J Pharm Sci 79: 321–325. pmid:2352143
- View Article
- PubMed/NCBI
- Google Scholar
27. Munday R, Manns E (1998) 2,3,6-triaminopyridine, a metabolite of the urinary tract analgesic phenazopyridine, causes muscle necrosis and renal damage in rats. J Appl Toxicol 18: 161–165. pmid:9570699
- View Article
- PubMed/NCBI
- Google Scholar
28. Munday R, Fowke EA (1994) Generation of superoxide radical and hydrogen peroxide by 2,3,6-triaminopyridine, a metabolite of the urinary tract analgesic phenazopyridine. Free Radic Res 21: 67–73. pmid:7921165
- View Article
- PubMed/NCBI
- Google Scholar
29. Kosma CI, Lambropoulou DA, Albanis TA (2016) Analysis, occurrence, fate and risks of proton pump inhibitors, their metabolites and transformation products in aquatic environment: A review. Sci Total Environ 569–570: 732–750. pmid:27380396
- View Article
- PubMed/NCBI
- Google Scholar
30. Divakaruni AS, Brand MD (2011) The regulation and physiology of mitochondrial proton leak. Physiology (Bethesda) 26: 192–205.
- View Article
- Google Scholar
31. Sena LA, Chandel NS (2012) Physiological roles of mitochondrial reactive oxygen species. Mol Cell 48: 158–167. pmid:23102266
- View Article
- PubMed/NCBI
- Google Scholar
32. Margoliash E, Novogrodsky A (1958) A study of the inhibition of catalase by 3-amino-1:2:4:-triazole. Biochem J 68: 468–475. pmid:13522646
- View Article
- PubMed/NCBI
- Google Scholar
33. Kingma JG Jr., Simard D, Rouleau JR, Tanguay RM, Currie RW (1996) Effect of 3-aminotriazole on hyperthermia-mediated cardioprotection in rabbits. Am J Physiol 270: H1165–1171. pmid:8967353
- View Article
- PubMed/NCBI
- Google Scholar
34. Auyeung Y, Sievers RE, Weng D, Barbosa V, Wolfe CL (1995) Catalase inhibition with 3-amino-1,2,4-triazole does not abolish infarct size reduction in heat-shocked rats. Circulation 92: 3318–3322. pmid:7586320
- View Article
- PubMed/NCBI
- Google Scholar
35. Nistico R, Piccirilli S, Cucchiaroni ML, Armogida M, Guatteo E, Giampa C, et al. (2008) Neuroprotective effect of hydrogen peroxide on an in vitro model of brain ischaemia. Br J Pharmacol 153: 1022–1029. pmid:18223675
- View Article
- PubMed/NCBI
- Google Scholar
36. Schwartz J, Holmuhamedov E, Zhang X, Lovelace GL, Smith CD, Lemasters JJ (2013) Minocycline and doxycycline, but not other tetracycline-derived compounds, protect liver cells from chemical hypoxia and ischemia/reperfusion injury by inhibition of the mitochondrial calcium uniporter. Toxicol Appl Pharmacol 273: 172–179. pmid:24012766
- View Article
- PubMed/NCBI
- Google Scholar
37. Yrjanheikki J, Tikka T, Keinanen R, Goldsteins G, Chan PH, Koistinaho J (1999) A tetracycline derivative, minocycline, reduces inflammation and protects against focal cerebral ischemia with a wide therapeutic window. Proc Natl Acad Sci U S A 96: 13496–13500. pmid:10557349
- View Article
- PubMed/NCBI
- Google Scholar
38. Kagawa N, Senbonmatsu TA, Satoh K, Ichihara K, Yamagata N, Hatano O, et al. (2005) Tetracycline protects myocardium against ischemic injury. Front Biosci 10: 608–619. pmid:15569603
- View Article
- PubMed/NCBI
- Google Scholar
39. Pires PW, Rogers CT, McClain JL, Garver HS, Fink GD, Dorrance AM (2011) Doxycycline, a matrix metalloprotease inhibitor, reduces vascular remodeling and damage after cerebral ischemia in stroke-prone spontaneously hypertensive rats. Am J Physiol Heart Circ Physiol 301: H87–97. pmid:21551278
- View Article
- PubMed/NCBI
- Google Scholar
40. Theruvath TP, Zhong Z, Pediaditakis P, Ramshesh VK, Currin RT, Tikunov A, et al. (2008) Minocycline and N-methyl-4-isoleucine cyclosporin (NIM811) mitigate storage/reperfusion injury after rat liver transplantation through suppression of the mitochondrial permeability transition. Hepatology 47: 236–246. pmid:18023036
- View Article
- PubMed/NCBI
- Google Scholar
41. Morck C, Olsen L, Kurth C, Persson A, Storm NJ, Svensson E, et al. (2009) Statins inhibit protein lipidation and induce the unfolded protein response in the non-sterol producing nematode Caenorhabditis elegans. Proc Natl Acad Sci U S A 106: 18285–18290. pmid:19826081
- View Article
- PubMed/NCBI
- Google Scholar
42. Rauthan M, Ranji P, Aguilera Pradenas N, Pitot C, Pilon M (2013) The mitochondrial unfolded protein response activator ATFS-1 protects cells from inhibition of the mevalonate pathway. Proc Natl Acad Sci U S A 110: 5981–5986. pmid:23530189
- View Article
- PubMed/NCBI
- Google Scholar
43. Rauthan M, Pilon M (2015) A chemical screen to identify inducers of the mitochondrial unfolded protein response in C. elegans. Worm 4: e1096490. pmid:27123370
- View Article
- PubMed/NCBI
- Google Scholar
44. Guo S, Olm-Shipman A, Walters A, Urciuoli WR, Devito S, Nadtochiy SM, et al. (2012) A cell-based phenotypic assay to identify cardioprotective agents. Circ Res 110: 948–957. pmid:22394516
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Kalogeris T, Baines CP, Krenz M, Korthuis RJ (2012) Cell biology of ischemia/reperfusion injury. Int Rev Cell Mol Biol 298: 229–317. pmid:22878108
View Article
PubMed/NCBI
Google Scholar

[2] View Article

[3] PubMed/NCBI

[4] Google Scholar

[ref2] 2. O'Collins VE, Macleod MR, Donnan GA, Horky LL, van der Worp BH, Howells DW (2006) 1,026 experimental treatments in acute stroke. Ann Neurol 59: 467–477. pmid:16453316
View Article
PubMed/NCBI
Google Scholar

[6] View Article

[7] PubMed/NCBI

[8] Google Scholar

[ref3] 3. Gerczuk PZ, Kloner RA (2012) An update on cardioprotection: a review of the latest adjunctive therapies to limit myocardial infarction size in clinical trials. J Am Coll Cardiol 59: 969–978. pmid:22402067
View Article
PubMed/NCBI
Google Scholar

[10] View Article

[11] PubMed/NCBI

[12] Google Scholar

[ref4] 4. Moskowitz MA, Lo EH, Iadecola C (2010) The science of stroke: mechanisms in search of treatments. Neuron 67: 181–198. pmid:20670828
View Article
PubMed/NCBI
Google Scholar

[14] View Article

[15] PubMed/NCBI

[16] Google Scholar

[ref5] 5. Miller DL, Roth MB (2009) C. Elegans Are Protected from Lethal Hypoxia by an Embryonic Diapause. Current Biology 19: 1233–1237. pmid:19576771
View Article
PubMed/NCBI
Google Scholar

[18] View Article

[19] PubMed/NCBI

[20] Google Scholar

[ref6] 6. Menuz V, Howell KS, Gentina S, Epstein S, Riezman I, Fornallaz-Mulhauser M, et al. (2009) Protection of C. elegans from anoxia by HYL-2 ceramide synthase. Science 324: 381–384. pmid:19372430
View Article
PubMed/NCBI
Google Scholar

[22] View Article

[23] PubMed/NCBI

[24] Google Scholar

[ref7] 7. Mabon ME, Mao X, Jiao Y, Scott BA, Crowder CM (2009) Systematic identification of gene activities promoting hypoxic death. Genetics 181: 483–496. pmid:19047414
View Article
PubMed/NCBI
Google Scholar

[26] View Article

[27] PubMed/NCBI

[28] Google Scholar

[ref8] 8. Anderson LL, Mao X, Scott BA, Crowder CM (2009) Survival from hypoxia in C. elegans by inactivation of aminoacyl-tRNA synthetases. Science 323: 630–633. pmid:19179530
View Article
PubMed/NCBI
Google Scholar

[30] View Article

[31] PubMed/NCBI

[32] Google Scholar

[ref9] 9. Scott BA, Avidan MS, Crowder CM (2002) Regulation of hypoxic death in C. elegans by the insulin/IGF receptor homolog DAF-2. Science 296: 2388–2391. pmid:12065745
View Article
PubMed/NCBI
Google Scholar

[34] View Article

[35] PubMed/NCBI

[36] Google Scholar

[ref10] 10. Wojtovich AP, DiStefano P, Sherman T, Brookes PS, Nehrke K (2012) Mitochondrial ATP-sensitive potassium channel activity and hypoxic preconditioning are independent of an inwardly rectifying potassium channel subunit in Caenorhabditis elegans. FEBS Lett 586: 428–434. pmid:22281198
View Article
PubMed/NCBI
Google Scholar

[38] View Article

[39] PubMed/NCBI

[40] Google Scholar

[ref11] 11. Sun CL, Kim E, Crowder CM (2014) Delayed innocent bystander cell death following hypoxia in Caenorhabditis elegans. Cell Death Differ 21: 557–567. pmid:24317200
View Article
PubMed/NCBI
Google Scholar

[42] View Article

[43] PubMed/NCBI

[44] Google Scholar

[ref12] 12. Brenner S (1974) The genetics of Caenorhabditis elegans. Genetics 77: 71–94. pmid:4366476
View Article
PubMed/NCBI
Google Scholar

[46] View Article

[47] PubMed/NCBI

[48] Google Scholar

[ref13] 13. Kaufman DM, Crowder CM (2015) Mitochondrial Proteostatic Collapse Leads to Hypoxic Injury. Curr Biol 25: 2171–2176. pmid:26234215
View Article
PubMed/NCBI
Google Scholar

[50] View Article

[51] PubMed/NCBI

[52] Google Scholar

[ref14] 14. Kamath RS, Fraser AG, Dong Y, Poulin G, Durbin R, Gotta M, et al. (2003) Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421: 231–237. pmid:12529635
View Article
PubMed/NCBI
Google Scholar

[54] View Article

[55] PubMed/NCBI

[56] Google Scholar

[ref15] 15. Rual JF, Ceron J, Koreth J, Hao T, Nicot AS, Hirozane-Kishikawa T, et al. (2004) Toward improving Caenorhabditis elegans phenome mapping with an ORFeome-based RNAi library. Genome Res 14: 2162–2168. pmid:15489339
View Article
PubMed/NCBI
Google Scholar

[58] View Article

[59] PubMed/NCBI

[60] Google Scholar

[ref16] 16. Yoneda T, Benedetti C, Urano F, Clark SG, Harding HP, Ron D (2004) Compartment-specific perturbation of protein handling activates genes encoding mitochondrial chaperones. J Cell Sci 117: 4055–4066. pmid:15280428
View Article
PubMed/NCBI
Google Scholar

[62] View Article

[63] PubMed/NCBI

[64] Google Scholar

[ref17] 17. Gong G, Wang W (2013) Confocal imaging of single mitochondrial superoxide flashes in intact heart or in vivo. J Vis Exp: e50818. pmid:24300235
View Article
PubMed/NCBI
Google Scholar

[66] View Article

[67] PubMed/NCBI

[68] Google Scholar

[ref18] 18. Yu Q, Lee CF, Wang W, Karamanlidis G, Kuroda J, Matsushima S, et al. (2014) Elimination of NADPH oxidase activity promotes reductive stress and sensitizes the heart to ischemic injury. J Am Heart Assoc 3: e000555. pmid:24470522
View Article
PubMed/NCBI
Google Scholar

[70] View Article

[71] PubMed/NCBI

[72] Google Scholar

[ref19] 19. Nargund AM, Pellegrino MW, Fiorese CJ, Baker BM, Haynes CM (2012) Mitochondrial import efficiency of ATFS-1 regulates mitochondrial UPR activation. Science 337: 587–590. pmid:22700657
View Article
PubMed/NCBI
Google Scholar

[74] View Article

[75] PubMed/NCBI

[76] Google Scholar

[ref20] 20. Hu JX, Li YF, Li J, Pan C, He Z, Dong HY, et al. (2013) Toxic effects of cypermethrin on the male reproductive system: with emphasis on the androgen receptor. J Appl Toxicol 33: 576–585. pmid:22147539
View Article
PubMed/NCBI
Google Scholar

[78] View Article

[79] PubMed/NCBI

[80] Google Scholar

[ref21] 21. Singh AK, Tiwari MN, Upadhyay G, Patel DK, Singh D, Prakash O, et al. (2012) Long term exposure to cypermethrin induces nigrostriatal dopaminergic neurodegeneration in adult rats: postnatal exposure enhances the susceptibility during adulthood. Neurobiol Aging 33: 404–415. pmid:20371137
View Article
PubMed/NCBI
Google Scholar

[82] View Article

[83] PubMed/NCBI

[84] Google Scholar

[ref22] 22. Singh AK, Tiwari MN, Prakash O, Singh MP (2012) A current review of cypermethrin-induced neurotoxicity and nigrostriatal dopaminergic neurodegeneration. Curr Neuropharmacol 10: 64–71. pmid:22942879
View Article
PubMed/NCBI
Google Scholar

[86] View Article

[87] PubMed/NCBI

[88] Google Scholar

[ref23] 23. Amer SM, Aboul-ela EI (1985) Cytogenetic effects of pesticides. III. Induction of micronuclei in mouse bone marrow by the insecticides cypermethrin and rotenone. Mutat Res 155: 135–142. pmid:3974628
View Article
PubMed/NCBI
Google Scholar

[90] View Article

[91] PubMed/NCBI

[92] Google Scholar

[ref24] 24. Tateno C, Ito S, Tanaka M, Yoshitake A (1993) Effects of pyrethroid insecticides on gap junctional intercellular communications in Balb/c3T3 cells by dye-transfer assay. Cell Biol Toxicol 9: 215–221. pmid:8299001
View Article
PubMed/NCBI
Google Scholar

[94] View Article

[95] PubMed/NCBI

[96] Google Scholar

[ref25] 25. Lyche JL, Gutleb AC, Bergman A, Eriksen GS, Murk AJ, Ropstad E, et al. (2009) Reproductive and developmental toxicity of phthalates. J Toxicol Environ Health B Crit Rev 12: 225–249. pmid:20183522
View Article
PubMed/NCBI
Google Scholar

[98] View Article

[99] PubMed/NCBI

[100] Google Scholar

[ref26] 26. Thomas BH, Whitehouse LW, Solomonraj G, Paul CJ (1990) Excretion of phenazopyridine and its metabolites in the urine of humans, rats, mice, and guinea pigs. J Pharm Sci 79: 321–325. pmid:2352143
View Article
PubMed/NCBI
Google Scholar

[102] View Article

[103] PubMed/NCBI

[104] Google Scholar

[ref27] 27. Munday R, Manns E (1998) 2,3,6-triaminopyridine, a metabolite of the urinary tract analgesic phenazopyridine, causes muscle necrosis and renal damage in rats. J Appl Toxicol 18: 161–165. pmid:9570699
View Article
PubMed/NCBI
Google Scholar

[106] View Article

[107] PubMed/NCBI

[108] Google Scholar

[ref28] 28. Munday R, Fowke EA (1994) Generation of superoxide radical and hydrogen peroxide by 2,3,6-triaminopyridine, a metabolite of the urinary tract analgesic phenazopyridine. Free Radic Res 21: 67–73. pmid:7921165
View Article
PubMed/NCBI
Google Scholar

[110] View Article

[111] PubMed/NCBI

[112] Google Scholar

[ref29] 29. Kosma CI, Lambropoulou DA, Albanis TA (2016) Analysis, occurrence, fate and risks of proton pump inhibitors, their metabolites and transformation products in aquatic environment: A review. Sci Total Environ 569–570: 732–750. pmid:27380396
View Article
PubMed/NCBI
Google Scholar

[114] View Article

[115] PubMed/NCBI

[116] Google Scholar

[ref30] 30. Divakaruni AS, Brand MD (2011) The regulation and physiology of mitochondrial proton leak. Physiology (Bethesda) 26: 192–205.
View Article
Google Scholar

[118] View Article

[119] Google Scholar

[ref31] 31. Sena LA, Chandel NS (2012) Physiological roles of mitochondrial reactive oxygen species. Mol Cell 48: 158–167. pmid:23102266
View Article
PubMed/NCBI
Google Scholar

[121] View Article

[122] PubMed/NCBI

[123] Google Scholar

[ref32] 32. Margoliash E, Novogrodsky A (1958) A study of the inhibition of catalase by 3-amino-1:2:4:-triazole. Biochem J 68: 468–475. pmid:13522646
View Article
PubMed/NCBI
Google Scholar

[125] View Article

[126] PubMed/NCBI

[127] Google Scholar

[ref33] 33. Kingma JG Jr., Simard D, Rouleau JR, Tanguay RM, Currie RW (1996) Effect of 3-aminotriazole on hyperthermia-mediated cardioprotection in rabbits. Am J Physiol 270: H1165–1171. pmid:8967353
View Article
PubMed/NCBI
Google Scholar

[129] View Article

[130] PubMed/NCBI

[131] Google Scholar

[ref34] 34. Auyeung Y, Sievers RE, Weng D, Barbosa V, Wolfe CL (1995) Catalase inhibition with 3-amino-1,2,4-triazole does not abolish infarct size reduction in heat-shocked rats. Circulation 92: 3318–3322. pmid:7586320
View Article
PubMed/NCBI
Google Scholar

[133] View Article

[134] PubMed/NCBI

[135] Google Scholar

[ref35] 35. Nistico R, Piccirilli S, Cucchiaroni ML, Armogida M, Guatteo E, Giampa C, et al. (2008) Neuroprotective effect of hydrogen peroxide on an in vitro model of brain ischaemia. Br J Pharmacol 153: 1022–1029. pmid:18223675
View Article
PubMed/NCBI
Google Scholar

[137] View Article

[138] PubMed/NCBI

[139] Google Scholar

[ref36] 36. Schwartz J, Holmuhamedov E, Zhang X, Lovelace GL, Smith CD, Lemasters JJ (2013) Minocycline and doxycycline, but not other tetracycline-derived compounds, protect liver cells from chemical hypoxia and ischemia/reperfusion injury by inhibition of the mitochondrial calcium uniporter. Toxicol Appl Pharmacol 273: 172–179. pmid:24012766
View Article
PubMed/NCBI
Google Scholar

[141] View Article

[142] PubMed/NCBI

[143] Google Scholar

[ref37] 37. Yrjanheikki J, Tikka T, Keinanen R, Goldsteins G, Chan PH, Koistinaho J (1999) A tetracycline derivative, minocycline, reduces inflammation and protects against focal cerebral ischemia with a wide therapeutic window. Proc Natl Acad Sci U S A 96: 13496–13500. pmid:10557349
View Article
PubMed/NCBI
Google Scholar

[145] View Article

[146] PubMed/NCBI

[147] Google Scholar

[ref38] 38. Kagawa N, Senbonmatsu TA, Satoh K, Ichihara K, Yamagata N, Hatano O, et al. (2005) Tetracycline protects myocardium against ischemic injury. Front Biosci 10: 608–619. pmid:15569603
View Article
PubMed/NCBI
Google Scholar

[149] View Article

[150] PubMed/NCBI

[151] Google Scholar

[ref39] 39. Pires PW, Rogers CT, McClain JL, Garver HS, Fink GD, Dorrance AM (2011) Doxycycline, a matrix metalloprotease inhibitor, reduces vascular remodeling and damage after cerebral ischemia in stroke-prone spontaneously hypertensive rats. Am J Physiol Heart Circ Physiol 301: H87–97. pmid:21551278
View Article
PubMed/NCBI
Google Scholar

[153] View Article

[154] PubMed/NCBI

[155] Google Scholar

[ref40] 40. Theruvath TP, Zhong Z, Pediaditakis P, Ramshesh VK, Currin RT, Tikunov A, et al. (2008) Minocycline and N-methyl-4-isoleucine cyclosporin (NIM811) mitigate storage/reperfusion injury after rat liver transplantation through suppression of the mitochondrial permeability transition. Hepatology 47: 236–246. pmid:18023036
View Article
PubMed/NCBI
Google Scholar

[157] View Article

[158] PubMed/NCBI

[159] Google Scholar

[ref41] 41. Morck C, Olsen L, Kurth C, Persson A, Storm NJ, Svensson E, et al. (2009) Statins inhibit protein lipidation and induce the unfolded protein response in the non-sterol producing nematode Caenorhabditis elegans. Proc Natl Acad Sci U S A 106: 18285–18290. pmid:19826081
View Article
PubMed/NCBI
Google Scholar

[161] View Article

[162] PubMed/NCBI

[163] Google Scholar

[ref42] 42. Rauthan M, Ranji P, Aguilera Pradenas N, Pitot C, Pilon M (2013) The mitochondrial unfolded protein response activator ATFS-1 protects cells from inhibition of the mevalonate pathway. Proc Natl Acad Sci U S A 110: 5981–5986. pmid:23530189
View Article
PubMed/NCBI
Google Scholar

[165] View Article

[166] PubMed/NCBI

[167] Google Scholar

[ref43] 43. Rauthan M, Pilon M (2015) A chemical screen to identify inducers of the mitochondrial unfolded protein response in C. elegans. Worm 4: e1096490. pmid:27123370
View Article
PubMed/NCBI
Google Scholar

[169] View Article

[170] PubMed/NCBI

[171] Google Scholar

[ref44] 44. Guo S, Olm-Shipman A, Walters A, Urciuoli WR, Devito S, Nadtochiy SM, et al. (2012) A cell-based phenotypic assay to identify cardioprotective agents. Circ Res 110: 948–957. pmid:22394516
View Article
PubMed/NCBI
Google Scholar

[173] View Article

[174] PubMed/NCBI

[175] Google Scholar

Figures

Abstract

Introduction

Materials and methods

C. elegans strains and culture methods

Hypoxic incubations

Chemical library screen

Hypoxic injury scoring

RNAi experiments

Mitochondrial UPR reporter

Langendorff-perfused heart

Statistics

Results

Chemical screen identifies six protective compounds against delayed hypoxic organismal death

Compounds protect after but not before against focal hypoxic injury

mtUPR activation is required for hypoxic protection by meclocycline

Meclocycline and 3-AT protect against IR injury in a Langendorff-perfused heart

Discussion

Supporting information

S1 Fig. EC50 of six protective compounds.

S2 Fig. Effect of six protective compounds against global hypoxic injury.

S3 Fig. Test for toxic effects of positive compounds.

S4 Fig. Protective effect of tetracyclines is independent of bacterial survival.

Acknowledgments

Author Contributions

References