Advertisement

Neurotoxicity Research

, Volume 34, Issue 1, pp 121–136 | Cite as

2,4 Dinitrophenol Attenuates Mitochondrial Dysfunction and Improves Neurobehavioral Outcomes Postanoxia in Neonatal Rats

  • Puneet K. Samaiya
  • Gopeshwar Narayan
  • Ashok Kumar
  • Sairam Krishnamurthy
ORIGINAL ARTICLE
  • 119 Downloads

Abstract

Following anoxia, a rapid and marked mitochondrial-linked cell death occurs in the cerebral cortex of newborn rats which leads to insult advancement within a couple of days and causes lifelong neurobehavioral abnormalities. The present study investigated the role of 2,4 dinitrophenol (2,4 DNP) in three doses, i.e.,1, 2.5, and 5 mg/kg on anoxia-induced time-dependent mitochondrial dysfunction and associated neurobehavioral outcome using a well-established global model of anoxia. Briefly, rat pups of 30-h age (P2) were subjected to two episodes of anoxia (10 min each) at 24 h of the time interval in an enclosed chamber supplied with 100% N2 and immersed in a water bath (35–37 °C) to avoid hypothermia. Results demonstrated that the uncoupler 2,4 DNP, in the dose 2.5 and 5 mg/kg injected i.p. within 5 min after second anoxic episode significantly (P < 0.05) preserved mitochondrial function on day 7 preferentially by maintaining mitochondrial membrane potential (MMP) and inhibiting mitochondrial permeability transition (MPT) pore. Further, 2,4 DNP preserved mitochondrial function by improving different states of mitochondrial respiration (s2, s3, s4, s5), respiratory control ratio (RCR), antioxidant enzyme system like superoxide dismutase (SOD) and catalase (CAT), and mitochondrial complex enzymes (I, II, IV, V) after anoxia. Furthermore, a marked decrease in the levels of expression of cytochrome C (cyt C) and pro-apoptotic (Bcl-2 family) and apoptotic (caspase-9/3) proteins was observed on day 7 indicating that the treatment with 2,4 DNP prevented mitochondrial dysfunction and further insult progression (day 1 to day 7). Moreover, 2,4 DNP decreased the apoptotic cell death on day 7 and overall improved the neurobehavioral outcomes like reflex latency and hanging latency which suggests its role in treating neonatal anoxia.

Keywords

Anoxia Mitochondrial function 2,4 Dinitrophenol Neurobehavior Apoptosis Animal model 

Abbreviations

2,4 DNP

2,4 Dinitrophenol

NO

Nitric oxide

UPCs

Mitochondrial uncouplers

MMP`

Mitochondrial membrane potential

MPT

Mitochondrial permeability transition

RCR

Respiratory control ratio

ETC

Electron transport chain

LPO

Lipid peroxidation

SOD

Superoxide dismutase

CAT

Catalase

Cyt C

Cytochrome C

GABA

Gamma-aminobutyric acid

CNS

Central nervous system

EAA

Excitatory amino acid

ATP

Adenosine triphosphate

ADP

Adenosine diphosphate

NADH

Nicotinamide adenine dinucleotide

N2

Nitrogen

ROS

Reactive oxygen species

RNS

Reactive nitrogen species

Bcl-2

B cell lymphoma 2

Bax

Bcl-2-associated X protein

Ca2+

Calcium ion

s

State

P

Pyruvate

M

Malate

i.p.

Intraperitoneal

TMRM

Tetramethylrhodamine methyl ester

HEPES

(4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid)

EGTA

(Ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid)

DMSO

Dimethyl sulfoxide

SDH

Succinate dehydrogenase

SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

PVDF

Polyvinylidene fluoride

FITC

Fluorescein isothiocyanate

PBS

Phosphate buffer saline

ECL

Chemiluminescence

PI

Propidium iodide

ANT

Adenosine nucleotide translocase

ER

Endoplasmic reticulum

MAMs

Mitochondria-associated ER membranes

NBT

Nitro blue tetrazolium

dfz

Diformazan

MDA

Malondialdehyde

ANOVA

Analysis of variance

Notes

Funding Information

SK is thankful to Department of Biotechnology (DBT), New Delhi, India, for assistance in terms of research grant [102/IFD/SAN/4654/2011-2012].

Compliance with Ethical Standards

The experimental procedures were approved by the Institutional Animal Ethical Committee of BHU (Protocol No. Dean/11-12/CAEC/328). All experiments were performed as per guidelines of laboratory animal care (National Research Council US Committee for the Update of the Guide for the Care and Use of Laboratory Animals 2011) guidelines.

Conflict of Interest

The authors declare that they have no conflict of interest.

References

  1. Beers RF, Sizer IW (1952) A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J Biol Chem 195(1):133–140PubMedGoogle Scholar
  2. Bel FV, Groenendaal F (2016) Drugs for neuroprotection after birth asphyxia: pharmacologic adjuncts to hypothermia. Semin Perinatol 40(3):152–159.  https://doi.org/10.1053/j.semperi.2015.12.003 CrossRefPubMedGoogle Scholar
  3. Berman SB, Hastings TG (1999) Dopamine oxidation alters mitochondrial respiration and induces permeability transition in brain mitochondria. J Neurochem 73(3):1127–1137CrossRefPubMedGoogle Scholar
  4. Boardman WW (1935) Rapidly developing cataracts after dinitrophenol. Calif West Med 43(2):118–119Google Scholar
  5. Borutaite V, Toleikis A, Brown GC (2013) In the eye of the storm: mitochondrial damage during heart and brain ischaemia. FEBS J 280:4999–5014CrossRefPubMedGoogle Scholar
  6. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72(1-2):248–254.  https://doi.org/10.1016/0003-2697(76)90527-3 CrossRefPubMedGoogle Scholar
  7. Cerio FGD, Lara-Celador I, Alvarez A, Hilario E (2013) Neuroprotective therapies after perinatal hypoxic-ischemic brain injury. Brain Sci 3:191–214CrossRefPubMedPubMedCentralGoogle Scholar
  8. Cheng G, Kong RH, Zhang LM, Zhang JN (2012) Mitochondria in traumatic brain injury and mitochondrial-targeted multipotential therapeutic strategies. Br J Pharmacol 167(4):699–719.  https://doi.org/10.1111/j.1476-5381.2012.02025.x CrossRefPubMedPubMedCentralGoogle Scholar
  9. Chowdhury I, Tharakan B, Bhat GK (2008) Caspases—an update. Comparative Biochemistry and Physiology Part B: Biochem Mol Biol 151:10–27CrossRefGoogle Scholar
  10. Diano S, Matthews RT, Patrylo P, Yang L, Beal MF, Barnstable CJ, Horvath TL (2003) Uncoupling protein 2 prevents neuronal death including that occurring during seizures: a mechanism for preconditioning. Endocrinol 144(11):5014–5021.  https://doi.org/10.1210/en.2003-0667 CrossRefGoogle Scholar
  11. Ennis SR, Keep RF (2006) Effects of 2,4-dinitrophenol on ischemia-induced blood-brain barrier disruption. Acta Neurochir Suppl 96:295–298.  https://doi.org/10.1007/3-211-30714-1_63 CrossRefPubMedGoogle Scholar
  12. Erpapazoglou Z, Mouton-Liger F, Corti O (2017) From dysfunctional endoplasmic reticulum-mitochondria coupling to neurodegeneration. Neurochem Int 17:30081–30085.  https://doi.org/10.1016/j.neuint.2017.03.021. Google Scholar
  13. Fan L-W, Lin S, Pang Y, Lei M, Zhang F, Rhodes PG, Cai Z (2005) Hypoxia-ischemia induced neurological dysfunction and brain injury in the neonatal rat. Behav Brain Res 165(1):80–90.  https://doi.org/10.1016/j.bbr.2005.06.033 CrossRefPubMedGoogle Scholar
  14. Feng J, Lu S, Ding Y, Zheng M, Wang X (2016) Homocysteine activates T cells by enhancing endoplasmic reticulum-mitochondria coupling and increasing mitochondrial respiration. Protein Cell 7(6):391–402.  https://doi.org/10.1007/s13238-016-0245-x CrossRefPubMedPubMedCentralGoogle Scholar
  15. Fiske CH, Subbarow Y (1925) The colorimetric determination of phosphorus. J biol Chem 66:375–400Google Scholar
  16. Forkink M, Manjeri GR, Liemburg-Apers DC, Nibbeling E, Blanchard M, Wojtala A, Smeitink JA, Wieckowski MR, Willems PH, Koopman WJ (2014) Mitochondrial hyperpolarization during chronic complex I inhibition is sustained by low activity of complex II, III, IV and V. Biochim Biophys Acta 1837(8):1247–1256.  https://doi.org/10.1016/j.bbabio.2014.04.008 CrossRefPubMedGoogle Scholar
  17. Gilmer LK, Ansari MA, Roberts KN, Scheff SW (2010) Age-related changes in mitochondrial respiration and oxidative damage in the cerebral cortex of the Fischer 344 rat. Mech Ageing Dev 131(2):133–143.  https://doi.org/10.1016/j.mad.2009.12.011 CrossRefPubMedPubMedCentralGoogle Scholar
  18. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR (1982) Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids. Anal Biochem 126(1):131–138.  https://doi.org/10.1016/0003-2697(82)90118-X CrossRefPubMedGoogle Scholar
  19. Griffiths DE, Houghton RL (1974) Studies on energy-linked reactions: modified mitochondrial ATPase of oligomycin-resistant mutants of Saccharomyces cerevisiae. Eur J Biochem/ FEBS 46(1):157–167.  https://doi.org/10.1111/j.1432-1033.1974.tb03608.x CrossRefGoogle Scholar
  20. Grundlingh J, Dargan PI, El-Zanfaly M, Wood DM (2011) 2,4-Dinitrophenol (DNP): a weight loss agent with significant acute toxicity and risk of death. J Med Toxicol 7(3):205–212.  https://doi.org/10.1007/s13181-011-0162-6 CrossRefPubMedPubMedCentralGoogle Scholar
  21. Harper JA, Dickinson K, Brand MD (2001) Mitochondrial uncoupling as a target for drug development for the treatment of obesity. Obes Rev 2:255–265CrossRefPubMedGoogle Scholar
  22. Hermans RHM, Hunter DE, McGivern RF, Cain CD, Longo LD (1992) Behavioral sequelae in young rats of acute intermittent antenatal hypoxia. Neurotoxicol Teratol 14(2):119–129.  https://doi.org/10.1016/0892-0362(92)90060-N CrossRefPubMedGoogle Scholar
  23. Huang SG (2002) Development of a high throughput screening assay for mitochondrial membrane potential in living cells. J Biomol Screen 7(4):383–389.  https://doi.org/10.1177/108705710200700411 CrossRefPubMedGoogle Scholar
  24. Kakkar P, Das B, Viswanathan PN (1984) A modified spectrophotometric assay of superoxide dismutase. Indian J Biochem Biophys 21:130–132PubMedGoogle Scholar
  25. Kenwood BM, Weaver JL, Bajwa A, Poon IK, Byrne FL, Murrow BA, Calderone JA, Huang L, Divakaruni AS, Tomsig JL, Okabe K, Lo RH, Cameron Coleman G, Columbus L, Yan Z, Saucerman JJ, Smith JS, Holmes JW, Lynch KR, Ravichandran KS, Uchiyama S, Santos WL, Rogers GW, Okusa MD, Bayliss DA, Hoehn KL (2014) Identification of a novel mitochondrial uncoupler that does not depolarize the plasma membrane. Mol Metabol 3(2):114–123.  https://doi.org/10.1016/j.molmet.2013.11.005 CrossRefGoogle Scholar
  26. Koch RA, Lee RC, Tainter ML (1935) Dinitrophenol on liver function. Calif West Med 43(5):337–339Google Scholar
  27. Korde AS, Pettigrew LC, Craddock SD, Maragos WF (2005) The mitochondrial uncoupler 2,4-dinitrophenol attenuates tissue damage and improves mitochondrial homeostasis following transient focal cerebral ischemia. J Neurochem 94(6):1676–1684.  https://doi.org/10.1111/j.1471-4159.2005.03328.x CrossRefPubMedGoogle Scholar
  28. Liu D, Chan SL, de Souza-Pinto NC, Slevin JR, Wersto RP, Zhan M, Mustafa K, de Cabo R, Mattson MP (2006) Mitochondrial UCP4 mediates an adaptive shift in energy metabolism and increases the resistance of neurons to metabolic and oxidative stress. NeuroMolecular Med 8(3):389–413.  https://doi.org/10.1385/NMM:8:3:389 CrossRefPubMedGoogle Scholar
  29. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193(1):265–275PubMedGoogle Scholar
  30. Lozinsky OV, Lushchak OV, Storey JM, Storey KB, Lushchak VI (2013) The mitochondrial uncoupler 2,4-dinitrophenol attenuates sodium nitroprusside-induced toxicity in Drosophila melanogaster: potential involvement of free radicals. Comp Biochem Physiol C Toxicol Pharmacol 158(4):244–252.  https://doi.org/10.1016/j.cbpc.2013.09.002 CrossRefPubMedGoogle Scholar
  31. Mehta SL, Manhas N, Raghubir R (2007) Molecular targets in cerebral ischemia for developing novel therapeutics. Brain Res Rev 54:34–66CrossRefPubMedGoogle Scholar
  32. Old SL, Johnson MA (1989) Methods of microphotometric assay of succinate dehydrogenase and cytochrome c oxidase activities for use on human skeletal muscle. Histochem J 21(9-10):545–555.  https://doi.org/10.1007/BF01753355 CrossRefPubMedGoogle Scholar
  33. Pandya JD, Pauly JR, Nukala VN, Sebastian AH, Day KM, Korde AS, Maragos WF, Hall ED, Sullivan PG (2007) Post-injury administration of mitochondrial uncouplers increases tissue sparing and improves behavioral outcome following traumatic brain injury in rodents. J Neurotrauma 24(5):798–811.  https://doi.org/10.1089/neu.2006.3673 CrossRefPubMedGoogle Scholar
  34. Patel SP, Sullivan PG, Pandya JD, Rabchevsky AG (2009) Differential effects of the mitochondrial uncoupling agent, 2,4-dinitrophenol, or the nitroxide antioxidant, Tempol, on synaptic or nonsynaptic mitochondria after spinal cord injury. J Neurosci Res 87(1):130–140.  https://doi.org/10.1002/jnr.21814 CrossRefPubMedPubMedCentralGoogle Scholar
  35. Perez-Pinzon MA, Xu GP, Born J, Lorenzo J, Busto R, Rosenthal M, Sick TJ (1999) Cytochrome C is released from mitochondria into the cytosol after cerebral anoxia or ischemia. J Cereb Blood Flow Metab 19(1):39–43.  https://doi.org/10.1097/00004647-199901000-00004 CrossRefPubMedGoogle Scholar
  36. Petrenko AY, Cherkashina DV, Somov AY, Tkacheva EN, Semenchenko OA, Lebedinsky AS, Fuller BJ (2010) Reversible mitochondrial uncoupling in the cold phase during liver preservation/reperfusion reduces oxidative injury in the rat model. Cryobiology 60(3):293–300.  https://doi.org/10.1016/j.cryobiol.2010.02.001 CrossRefPubMedGoogle Scholar
  37. Pizzo P, Pozzan T (2007) Mitochondria–endoplasmic reticulum choreography: structure and signaling dynamics. Trends Cell Biol 17(10):511–517.  https://doi.org/10.1016/j.tcb.2007.07.011 CrossRefPubMedGoogle Scholar
  38. Puka-Sundvall M, Wallin C, Gilland E, Hallin U, Wang X, Sandberg M, Karlsson J, Blomgren K, Hagberg H (2000) Impairment of mitochondrial respiration after cerebral hypoxia–ischemia in immature rats: relationship to activation of caspase-3 and neuronal injury. Dev Brain Res 125:43–50CrossRefGoogle Scholar
  39. Rice JE III, Vannucci RC, Brierley JB (1981) The influence of immaturity on hypoxic-ischemic brain damage in the rat. Ann Neurol 9(2):131–141.  https://doi.org/10.1002/ana.410090206 CrossRefPubMedGoogle Scholar
  40. Rosenberg AA, Parks JK, Murdaugh E, Parker WD Jr (1989) Mitochondrial function after asphyxia in newborn lambs. Stroke 20(5):674–679.  https://doi.org/10.1161/01.STR.20.5.674 CrossRefPubMedGoogle Scholar
  41. Samaiya PK, Krishnamurthy S (2015) Characterization of mitochondrial bioenergetics in neonatal anoxic model of rats. J Bioenerg Biomembr 47(3):217–222.  https://doi.org/10.1007/s10863-015-9603-2 CrossRefPubMedGoogle Scholar
  42. Samaiya PK, Narayan G, Kumar A, Krishnamurthy S (2016) Neonatal anoxia leads to time dependent progression of mitochondrial linked apoptosis in rat cortex and associated long term sensorimotor deficits. Int J Dev Neurosci 52:55–65.  https://doi.org/10.1016/j.ijdevneu.2016.05.005 CrossRefPubMedGoogle Scholar
  43. Schlagowski AI, Singh F, Charles AL, Gali Ramamoorthy T, Favret F, Piquard F, Geny B, Zoll J (2014) Mitochondrial uncoupling reduces exercise capacity despite several skeletal muscle metabolic adaptations. J Appl Physiol (1985) 116:364–375CrossRefGoogle Scholar
  44. Shapiro BL, Feigal RJ, Lam LF (1979) Mitrochondrial NADH dehydrogenase in cystic fibrosis. Proc Natl Acad Sci U S A 76(6):2979–2983.  https://doi.org/10.1073/pnas.76.6.2979 CrossRefPubMedPubMedCentralGoogle Scholar
  45. Storrie B, Amadden E (1990) Isolation of subcellular organelles. Methods Enzymol 182:203–225.  https://doi.org/10.1016/0076-6879(90)82018-W CrossRefPubMedGoogle Scholar
  46. Sunderman F, Marzouk A, Hopfer S, Zaharia O, Reid M (1985) Increased lipid peroxidation in tissues of nickel chloride-treated rats. Ann Clin Lab Sci 15:229–236PubMedGoogle Scholar
  47. Tainter ML, Cutting WC, Stockton AB (1934) Use of dinitrophenol in nutritional disorders: a critical survey of clinical results. Am J Public Health Nations Health 24:1045–1053CrossRefPubMedPubMedCentralGoogle Scholar
  48. Tedeschi H, Harris DL (1958) Some observations on the photometric estimation of mitochondrial volume. Biochim Biophys Acta 28(2):392–402.  https://doi.org/10.1016/0006-3002(58)90487-6 CrossRefPubMedGoogle Scholar
  49. Utkina-Sosunova IV, Niatsetskaya ZV, Sosunov SA, Ratner VI, Matsiukevich D, Ten VS (2013) Nelfinavir inhibits intra-mitochondrial calcium influx and protects brain against hypoxic-ischemic injury in neonatal mice. PLoS One 8(4):e62448.  https://doi.org/10.1371/journal.pone.0062448 CrossRefPubMedPubMedCentralGoogle Scholar
  50. Wassink G, Gunn ER, Drury PP, Bennet L, Gunn AJ (2014) The mechanisms and treatment of asphyxial encephalopathy. Front Neurosci 8:40CrossRefPubMedPubMedCentralGoogle Scholar
  51. Zhang Y, Marcillat O, Giulivi C, Ernster L, Davies KJ (1990) The oxidative inactivation of mitochondrial electron transport chain components and ATPase. J Biol Chem 265:16330–16336PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Puneet K. Samaiya
    • 1
  • Gopeshwar Narayan
    • 2
  • Ashok Kumar
    • 3
  • Sairam Krishnamurthy
    • 1
  1. 1.Neurotherapeutics Laboratory, Department of Pharmaceutical Engineering and TechnologyIndian Institute of Technology (Banaras Hindu University)VaranasiIndia
  2. 2.Department of Molecular and Human GeneticsBanaras Hindu UniversityVaranasiIndia
  3. 3.Department of Pediatrics, Institute of Medical SciencesBanaras Hindu UniversityVaranasiIndia

Personalised recommendations