Hypothermia Suppresses Uncoupling of Oxidative- Phosphorylation after Neonatal Cerebral Hypoxia-Ischemia

Hypothermia is the best available therapy for neonatal hypoxia ischemia (HI) brain injury, but its primary mechanisms remain uncertain. We hypothesize that HI induces, whereas hypothermia represses, uncoupling of oxidative phosphorylation (OXPHOS), an increase of the cerebral metabolic rate of oxygen (CMRO2) despite reduction of the mitochondrial energy output. We used a multiparametric photoacoustic microscopy (PAM) system to compare the effects of HI and post HI hypothermic treatment on CMRO2 in awake 10 day old (P10) mice. Here we show that hypoxia (10% O2) elevated CMRO2, but the addition of unilateral carotid artery ligation suppressed CMRO2 and sparked a rapid overshoot of post HI CMRO2 in the ipsilateral cerebral cortex for at least 2 hours. The post HI surge of CMRO2 was linked to an increase of mitochondrial oxygen consumption and superoxide outburst, despite reduction of the mitochondrial membrane potential. Notably, post HI hypothermia blocked the surge of superoxide and CMRO2, primarily by limiting oxygen extraction fraction (OEF), leading to better preservation of adenosine triphosphate (ATP), creatine (Cr) and N acetylaspartate (NAA) after HI. Mice that did not receive hypothermia exhibited ~80% reduction of CMRO2 at 24 h post HI, coupled to a large cortical infarction. These results suggest that mitigation of post HI uncoupling of OXPHOS is an early and/or pivotal effect of hypothermia. Further, optical measurement of CMRO2 may be a sensitive and noninvasive method to monitor brain damage in hypoxic ischemic encephalopathy (HIE).

Coupling/Uncoupling Reversibility in Isolated Mitochondria from Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae uses fermentation as the preferred pathway to obtain ATP and requires the respiratory chain to re-oxidize the NADH needed for activity of Glyceraldehyde-3-phosphate. This process is favored by uncoupling of oxidative phosphorylation (OxPhos), which is at least partially controlled by the mitochondrial unspecific pore (ScMUC). When mitochondrial ATP synthesis is needed as in the diauxic phase or during mating, a large rise in Ca2+ concentration ([Ca2+]) closes ScMUC, coupling OxPhos. In addition, ScMUC opening/closing is mediated by the ATP/ADP ratio, which indicates cellular energy needs. Here, opening and closing of ScMUC was evaluated in isolated mitochondria from S. cerevisiae at different incubation times and in the presence of different ATP/ADP ratios or varying [Ca2+]. Measurements of the rate of O2 consumption, mitochondrial swelling, transmembrane potential and ROS generation were conducted. It was observed that ScMUC opening was reversible, a high ATP/ADP ratio promoted opening and [Ca2+] closed ScMUC even after several minutes of incubation in the open state. In the absence of ATP synthesis, closure of ScMUC resulted in an increase in ROS.

Amperometric Cytosensor for Studying Mitochondrial Interferences Induced by Plasticizers Bisphenol B and Bisphenol A

The widespread presence of plasticizers Bisphenol B (BPB) and Bisphenol A (BPA) in food contact materials, medical equipment, and common household products is a toxicological risk factor for health due to internal exposure after environmental dietary exposure. This work describes the use of an amperometric cytosensor (i.e., a whole cell-based amperometric biosensoristic device) for studying mitochondrial interferences of BPA and BPB (5–100 µg/mL) in the yeast Saccharomyces cerevisiae model following long-term (24 h) exposure (acute toxicity). Percentage interference (%ρ) on yeast aerobic mitochondrial catabolism was calculated after comparison of aerobic respiration of exposed and control S. cerevisiae cell suspensions. Results suggested the hypothesis of a dose-dependent co-action of two mechanisms, namely uncoupling of oxidative phosphorylation and oxidative stress. These mechanisms respectively matched with opposite effects of hyperstimulation and inhibition of cellular respiration. While uncoupling of oxidative phosphorylation and oxidative stress have been previously described as separate effects from in vitro BPA exposure using other biochemical endpoints and biological systems, effects of BPB on cellular aerobic respiration are here reported for the first time. Results highlighted a similar hyperstimulation effect after exposure to 5 µg/mL BPA and BPB. About a 2-fold higher cellular respiration inhibition potency was observed after exposures to 15, 30, and 100 µg/mL BPB compared to BPA. 2,4-Dinitrophenol (2,4-DNP) was used as model uncoupling agent. A time-dependent mechanism of mitochondrial interference was also highlighted.

Incipient resistance to an effective pesticide results from genetic adaptation and the canalization of gene expression

The resistance of bacteria, disease vectors, and pest species to chemical controls has vast ecological, economic, and societal costs. In most cases, resistance is only detected after spreading throughout an entire population. Detecting resistance in its incipient stages, by comparison, provides time to implement preventative strategies. Incipient resistance can be detected by coupling standard toxicology assays with large-scale gene expression experiments. We apply this approach to a system where an invasive parasite, sea lamprey (Petromyzon marinus), has been treated with the highly-effective pesticide 3-trifluoromethyl-4-nitrophenol (TFM) for 60 years. Toxicological experiments revealed that lamprey from treated populations did not have higher survival to TFM exposure than lamprey from their native range, demonstrating that full-fledged resistance has not yet evolved. In contrast, we find hundreds of genes differentially expressed in response to TFM in the population with the longest history of exposure, many of which relate to TFM’s primary mode of action, the uncoupling of oxidative phosphorylation and subsequent depletion of ATP. Three genes critical to oxidative phosphorylation, ATP5PB, PLCB1, and NDUFA9, were nearly fixed for alternative alleles in comparisons of SNPs between native and treated populations (FST > 5 SD from the mean). ATP5PB encodes subunit b of ATP synthase and an additional subunit, ATP5F1B, was canalized for high expression in treated populations, but remained plastic in response to TFM treatment in individuals from the native range. These combined genomic and transcriptomic results demonstrate that an adaptive, genetic response to TFM is driving incipient resistance in a damaging pest species.

Interaction of Potent Mitochondrial Uncouplers with Thiol-Containing Antioxidants

It is generally considered that reactive oxygen species (ROS) are involved in the development of numerous pathologies. The level of ROS can be altered via the uncoupling of oxidative phosphorylation by using protonophores causing mitochondrial membrane depolarization. Here, we report that the uncoupling activity of potent protonophores, such as carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), and fluazinam, can be abrogated by the addition of thiol-containing antioxidants to isolated mitochondria. In particular, N-acetylcysteine, glutathione, cysteine, and dithiothreitol removed both a decrease in the mitochondrial membrane potential and an increase in the respiration rate that is caused by FCCP. The thiols also reduced the electrical current that is induced by FCCP and CCCP across planar bilayer lipid membranes. Thus, when speculating on the mechanistic roles of ROS level modulation by mitochondrial uncoupling based on the antioxidant reversing certain FCCP and CCCP effects on cellular processes, one should take into account the ability of these protonophoric uncouplers to directly interact with the thiol-containing antioxidants.

Correlation of MRI with the Neuropathologic Changes in Two Cats with Bromethalin Intoxication

ABSTRACT Two cats were presented with multifocal neurological signs. One cat’s signs progressed over 2 wk; the other cat progressed over 5 days. Examinations were consistent with a process involving the prosencephalon, vestibular system, and general proprioceptive/upper motor neuron systems. MRI of the brain and cervical spinal cord reveal widespread T2 hyperintensity of the white matter. Affected areas included the cerebrum, cerebral peduncles, corticospinal tracts of the pons and medulla, and the cerebellum. T2 hyperintensity was present in all funiculi of the spinal cord. Diffusion-weighted imaging (DWI) and apparent diffusion coefficient (ADC) maps were consistent with cytotoxic or intramyelinic edema. Differential diagnosis included toxic or metabolic/degenerative leukoencephalopathies. Necropsies revealed widespread spongy degeneration of the central nervous system white matter. Toxicologic assays of liver specimens revealed desmethylbromethalin, a metabolite of bromethalin. Bromethalin is a rodenticide that causes uncoupling of oxidative phosphorylation. Antemortem diagnosis is challenging. DWI and ADC maps were instrumental in narrowing the differential diagnosis and raised the index of suspicion for bromethalin. Bromethalin intoxication should be considered in all animals with a progressive course of multifocal neurologic deficits. MRI, specifically, DWI and ADC maps, may serve as a biomarker of cytotoxic or intramyelinic edema associated with spongiform leukoencephalomyelopathy.

Melatonin and Its Metabolites Ameliorate UVR-Induced Mitochondrial Oxidative Stress in Human MNT-1 Melanoma Cells

Melatonin (Mel) is the major biologically active molecule secreted by the pineal gland. Mel and its metabolites, 6-hydroxymelatonin (6(OH)Mel) and 5-methoxytryptamine (5-MT), possess a variety of functions, including the scavenging of free radicals and the induction of protective or reparative mechanisms in the cell. Their amphiphilic character allows them to cross cellular membranes and reach subcellular organelles, including the mitochondria. Herein, the action of Mel, 6(OH)Mel, and 5-MT in human MNT-1 melanoma cells against ultraviolet B (UVB) radiation was investigated. The dose of 50 mJ/cm2 caused a significant reduction of cell viability up to 48%, while investigated compounds counteracted this deleterious effect. UVB exposure increased catalase activity and led to a simultaneous Ca++ influx (16%), while tested compounds prevented these disturbances. Additional analysis focused on mitochondrial respiration performed in isolated mitochondria from the liver of BALB/cJ mice where Mel, 6(OH)Mel, and 5-MT significantly enhanced the oxidative phosphorylation at the dose of 10−6 M with lower effects seen at 10−9 or 10−4 M. In conclusion, Mel, 6(OH)Mel and 5-MT protect MNT-1 cells, which express melatonin receptors (MT1 and MT2) against UVB-induced oxidative stress and mitochondrial dysfunction, including the uncoupling of oxidative phosphorylation.