Abstract
Over the past several years, the Muzaffarpur district of Bihar (India) has witnessed recurrent outbreaks of acute encephalitis illness of unknown etiology, called acute encephalitis syndrome (AES) among young children, especially during the peak-summer season. Pesticide exposure, viral encephalitis, and litchi toxin intake have all been postulated as potential sources of the ailment. However, no conclusive etiology for AES has been identified in the affected children. During recent rounds of the outbreak, metabolic abnormalities have been documented in these children, and a direct correlation was observed between higher environmental temperature during the peak-summer month and AES caseload. The clinical and metabolic profiles of these children suggested the possible involvement of mitochondrial dysfunction during heat stress as one of the several contributory factors leading to multisystem metabolic derangement. The present study observed that mitochondrial function parameters such as cell death, mitochondrial membrane potential, oxidative stress, and mitochondrial pathway-related gene expression in peripheral blood mononuclear cells (PBMCs) isolated from children were affected in peak-summer when compared to post-summer months. Similar observations of mitochondrial function parameters along with impaired bioenergetic parameters were demonstrated in the heat-exposed model of PBMCs isolated from healthy adult individuals. In conclusion, the results suggested that there is an association of transient mitochondrial dysfunction when exposed to sustained heat during the summer months. One may consider mitochondrial dysfunction as one of the important factors leading to an outbreak of AES among the children from affected regions though this needs to be substantiated with further studies.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data availability
The data collected during this study are included in this published article. The data from respondents were collected in paper form. The datasets analyzed during the study are available from the authors on reasonable request.
Abbreviations
- AES :
-
Acute encephalitis syndrome
- PBMCs :
-
Peripheral blood mononuclear cells
- CPK :
-
Creatinine phosphokinase
- LDH :
-
Lactate dehydrogenase
- GC-MS :
-
Gas chromatography mass spectrometry
- MMP :
-
Mitochondrial membrane potential
- TMRM :
-
Tetramethyl rhodamine methyl ester
- ROS :
-
Reactive oxygen species
- DHE :
-
Dihydroethidium
- MitoSOX :
-
Mitochondrial superoxide
- DBPS :
-
Dulbecco’s phosphate-buffered saline media
- RPMI :
-
Roswell Park Memorial Institute
- PI :
-
Propidium iodide
- FITC :
-
Fluorescein isothiocyanate
- PE :
-
Phycoerythrin
- GOI :
-
Gene of interest
- HKG :
-
Housekee** gene
- cDNA :
-
Complementary DNA
- CT :
-
Threshold cycle
- mt-DNA CN :
-
Mitochondrial DNA copy number
- n-DNA CN :
-
Nuclear DNA copy number
- DMSO :
-
Dimethyl sulfoxide
- OCR :
-
Oxygen consumption rate
- DMEM :
-
Dulbecco’s modified Eagle’s medium
- BCA :
-
Bicinchoninic acid
- FCCP :
-
Trifluoromethoxy carbonyl cyanide phenylhydrazone
References
Altintas MM, DiBartolo S, Tadros L, Samelko B, Wasse H (2021) Metabolic changes in peripheral blood mononuclear cells isolated from patients with end stage renal disease. Front Endocrinol (Lausanne) 12:629239. https://doi.org/10.3389/fendo.2021.629239
Alzeer AH, El-Hazmi MA, Warsy AS, Ansari ZA, Yrkendi MS (1997) Serum enzymes in heat stroke: prognostic implication. Clin Chem 43(7):1182–7
Asthana S, Dixit S, Srivastava A, Kumar A, Singh SP, Tripathi A, Das M (2019) Methylenecyclopropyl glycine, not pesticide exposure as the primary etiological factor underlying hypoglycemic encephalopathy in Muzaffarpur, India. Toxicol Lett 301:34–41. https://doi.org/10.1016/j.toxlet.2018.10.033
Bomer N, Pavez-Giani MG, Grote Beverborg N, Cleland JGF, Van Veldhuisen DJ, Van der Meer P (2022) Micronutrient deficiencies in heart failure: mitochondrial dysfunction as a common pathophysiological mechanism? J Intern Med 291:713–731. https://doi.org/10.1111/joim.13456
Bouchama A, Aziz MA, Mahri SA, Gabere MN, Dlamy MA, Mohammad S, Abbad MA, Hussein M (2017) A model of exposure to extreme environmental heat uncovers the human transcriptome to heat stress. Sci Rep 7(1):9429. https://doi.org/10.1038/s41598-017-09819-5
Bytomski JR, Squire DL (2003) Heat illness in children. Curr Sports Med Rep 2(6):320–324. https://doi.org/10.1249/00149619-200312000-00007
Cartwright SL, McKechnie M, Schmied J, Livernois AM, Mallard BA (2021) Effect of in-vitro heat stress challenge on the function of blood mononuclear cells from dairy cattle ranked as high, average and low immune responders. BMC Vet Res 17:233. https://doi.org/10.1186/s12917-021-02940-8
Chen TH, Koh KY, Lin KM, Chou CK (2022) Mitochondrial dysfunction as an underlying cause of skeletal muscle disorders. Int J Mol Sci 23(21):12926. https://doi.org/10.3390/ijms232112926
Duong A, Evstratova A, Sivitilli A, Hernandez JJ, Gosio J, Wahedi A, Sondheimer N, Wrana JL, Beaulieu JM, Attisano L, Andreazza AC (2021) Characterization of mitochondrial health from human peripheral blood mononuclear cells to cerebral organoids derived from induced pluripotent stem cells. Sci Rep 11(1):4523. https://doi.org/10.1038/s41598-021-84071-6
Ederlé C, Charles AL, Khayath N, Poirot A, Meyer A, Clere-Jehl R, Andres E, De Blay F, Geny B (2019) Mitochondrial function in peripheral blood mononuclear cells (PBMC) is enhanced, together with increased reactive oxygen species, in severe asthmatic patients in exacerbation. J Clin Med 8(10):1613. https://doi.org/10.3390/jcm8101613
Elmore SP, Nishimura Y, Qian T, Herman B, Lemasters JJ (2004) Discrimination of depolarized from polarized mitochondria by confocal fluorescence resonance energy transfer. Arch Biochem Biophys 422(2):145–152. https://doi.org/10.1016/j.abb.2003.12.031
Falk B (1998) Effects of thermal stress during rest and exercise in the paediatric population. Sports Med 25(4):221–240. https://doi.org/10.2165/00007256-199825040-00002
Ghosh S, Basu A (2016) Acute encephalitis syndrome in India: the changing scenario. Ann Neurosci 23(3):131–133. https://doi.org/10.1159/000449177
Gu ZT, Li L, Wu F, Zhao P, Yang H, Liu YS, Geng Y, Zhao M, Su L (2015) Heat stress induced apoptosis is triggered by transcription-independent p53, Ca (2+) dyshomeostasis and the subsequent Bax mitochondrial translocation. Sci Rep 5:11497. https://doi.org/10.1038/srep11497
Heled Y, Moran DS, Mendel L, Laor A, Pras E, Shapiro Y (2004) Human ACE I/D polymorphism is associated with individual differences in exercise heat tolerance. J Appl Physiol 97(1):72–76. https://doi.org/10.1152/japplphysiol.01087.2003
Huang C, Jiao H, Song Z, Zhao J, Wang X, Lin H (2015) Heat stress impairs mitochondria functions and induces oxidative injury in broiler chickens. J Anim Sci 93(5):2144–2153. https://doi.org/10.2527/jas.2014-8739
Jadali Z, Amiri MM, Ravanbakhsh M (2010) Apoptosis of peripheral blood mononuclear cells in patients with sepsis. Indian J Pathol Microbiol 53(4):646–650. https://doi.org/10.4103/0377-4929.72013
Javadov S, Kozlov AV, Camara AKS (2020) Mitochondria in health and diseases. Cells 9(5):1177. https://doi.org/10.3390/cells9051177
John TJ, Das M (2014) Acute encephalitis syndrome in children in Muzaffarpur: hypothesis of toxic origin. Curr Sci 106:1184–1185
Jones N, Piasecka J, Bryant AH, Jones RH, Skibinski DO, Francis NJ, Thornton CA (2015) Bioenergetic analysis of human peripheral blood mononuclear cells. Clin Exp Immunol 182(1):69–80. https://doi.org/10.1111/cei.12662
Kadam S, Ghosh B, Apte KK, Brahme A, Salvi S, Agarwal AR (2017) Metabolic changes in peripheral blood mononuclear cells (PBMCs) of subjects with chronic obstructive pulmonary disease (COPD). Eur Respir J 50:PA3917. https://doi.org/10.1183/1393003.congress-2017.PA3917
Karabatsiakis A, Böck C, Salinas-Manrique J, Kolassa S, Calzia E, Dietrich DE, Kolassa IT (2014) Mitochondrial respiration in peripheral blood mononuclear cells correlates with depressive sub symptoms and severity of major depression. Transl Psychiatry 4(6):397. https://doi.org/10.1038/tp.2014.44
Karamercan MA, Weiss SL, Villarroel JP, Guan Y, Werlin E, Figueredo R, Becker LB, Sims C (2013) Can peripheral blood mononuclear cells be used as a proxy for mitochondrial dysfunction in vital organs during hemorrhagic shock and resuscitation? Shock 40(6):476–484. https://doi.org/10.1097/shk.0000000000000026
Li H, Fukuda S, Hasegawa Y, Kobayashi H, Purevsuren J, Mushimoto Y, Yamaguchi S (2010) Effect of heat stress and bezafibrate on mitochondrial β-oxidation: comparison between cultured cells from normal and mitochondrial fatty acid oxidation disorder children using in vitro probe acylcarnitine profiling assay. Brain Dev 32:362–370. https://doi.org/10.1016/j.braindev.2009.06.001
Liu CT, Brooks GA (2012) Mild heat stress induces mitochondrial biogenesis in C2C12 myotubes. J Appl Physiol 112(3):354–361. https://doi.org/10.1152/japplphysiol.00989
Liu X, Zhang Z, Li D, Lei M, Li Q, Liu X, Zhang P (2021) DNM1L-Related mitochondrial fission defects presenting as encephalopathy: A case report and literature review. Front Pediatr 9:626657. https://doi.org/10.3389/fped.2021.626657
Marcuello A, González-Alonso J, Calbet JA, Damsgaard R, López-Pérez MJ, Díez-Sánchez C (2005) Skeletal muscle mitochondrial DNA content in exercising humans. J Appl Physiol 99(4):1372–1377. https://doi.org/10.1152/japplphysiol.00289.2005
McGlynn ML, Schnitzler H, Shute R, Ruby B, Slivka D (2021) The acute effects of exercise and temperature on regional mtDNA. Int J Environ Res Public Health 18(12):6382. https://doi.org/10.3390/ijerph18126382
Meddeb R, Dache ZAA, Thezenas S, Otandault A, Tanos R, Pastor B, Sanchez C, Azzi J, Tousch G, Azan S, Mollevi C, Adenis A, El Messaoudi S, Blache P, Thierry AR (2019) Quantifying circulating cell-free DNA in humans. Sci Rep 9(1):5220. https://doi.org/10.1038/s41598-019-41593-4
Mosallaei M, Ehtesham N, Rahimirad S, Saghi M, Vatandoost N, Khosravi S (2022) PBMCs: a new source of diagnostic and prognostic biomarkers. Arch Physiol Biochem 128(4):1081–1087. https://doi.org/10.1080/13813455.2020.1752257
Owiredu S, Ranganathan A, Greenwood JC, Piel S, Janowska JI, Eckmann DM, Kelly M, Ehinger JK, Kilbaugh TJ, Jang DH (2020) In vitro comparison of hydroxocobalamin (B12a) and the mitochondrial directed therapy by a succinate prodrug in a cellular model of cyanide poisoning. Toxicol Rep 7(1263–1271):2214–7500. https://doi.org/10.1016/j.toxrep.2020.09.002
Paital B, Chainy GB (2014) Effects of temperature on complexes I and II mediated respiration, ROS generation and oxidative stress status in isolated gill mitochondria of the mud crab Scylla serrata. J Therm Biol 41:104–111. https://doi.org/10.1016/j.jtherbio.2014.02.013
Puente-Maestu L, Lázaro A, Tejedor A, Camaño S, Fuentes M, Cuervo M, Navarro BO, Agustí A (2011) Effects of exercise on mitochondrial DNA content in skeletal muscle of patients with COPD. Thorax 66(2):121–127. https://doi.org/10.1136/thx.2010.153031
Qian L, Song X, Ren H, Gong J, Cheng S (2004) Mitochondrial mechanism of heat stress-induced injury in rat cardiomyocyte. Cell Stress Chaperones 9(3):281. https://doi.org/10.1379/csc-20r.1
Ren MQ, Kazman JB, Abraham PA, Atias-Varon D, Heled Y, Deuster PA (2019) Gene expression profiling of humans under exertional heat stress: Comparisons between persons with and without exertional heat stroke. J Therm Biol 85:102423. https://doi.org/10.1016/j.jtherbio.2019.102423
Sahni GS (2012) Recurring epidemics of acute encephalopathy in children in Muzaffarpur, Bihar. Indian Pediatr 49(6):502–503
Samuel PP, Muniaraj M, Thenmozhi V, Tyagi BK (2013) Entomo-virological study of a suspected Japanese encephalitis outbreak in Muzaffarpur district, Bihar, India. Indian J Med Res 137:991–992
Shen K, Pender CL, Bar-Ziv R, Zhang H, Wickham K, Willey E, Durieux J, Ahmad Q, Dillin A (2022) Mitochondria as cellular and organismal signaling hubs. Annu Rev Cell Dev Biol 38:179–218. https://doi.org/10.1146/annurev-cellbio-120420-015303
Shrivastava A, Kumar A, Thomas JD, Laserson KF, Bhushan G, Carter MD, Chhabra M, Mittal V, Khare S, Sejvar JJ, Dwivedi M, Isenberg SL, Johnson R, Pirkle JL, Sharer JD, Hall PL, Yadav R, Velayudhan A, Papanna M, Singh P, Somashekar D, Pradhan A, Goel K, Pandey R, Kumar M, Kumar S, Chakrabarti A, Sivaperumal P, Kumar AR, Schier JG, Chang A, Graham LA, Mathews TP, Johnson D, Valentin L, Caldwell KL, Jarrett JM, Harden LA, Takeoka GR, Tong S, Queen K, Paden C, Whitney A, Haberling DL, Singh R, Singh RS, Earhart KC, Dhariwal AC, Chauhan LS, Venkatesh S, Srikantiah P (2017) Association of acute toxic encephalopathy with litchi consumption in an outbreak in Muzaffarpur, India, 2014: a case-control study. Lancet Glob Health 5(4):e458–e466. https://doi.org/10.1016/S2214-109X(17)30035-9
Singh AK, Shahi SK, Kumar B, Das MK (2022) Is ambient weather a risk factor for acute encephalopathy outbreaks in children in Muzaffarpur, Bihar, India? Insight from a 9-year analysis. J Trop Pediatr 68(1):fmab111. https://doi.org/10.1093/tropej/fmab111
Singh AK, Jhalani M, Shahi SK, Christopher R, Kumar B, Das MK (2023) Acute encephalopathy in children from Muzaffarpur, Bihar, India, and the potential role of ambient heat stress-induced mitochondrial dysfunction. Cureus 15(4):e37073. https://doi.org/10.7759/cureus.37073
Slimen IB, Najar T, Ghram A, Dabbebi H, Ben Mrad M, Abdrabbah M (2014) Reactive oxygen species, heat stress and oxidative-induced mitochondrial damage. A review. Int J Hyperthermia 30(7):513–523. https://doi.org/10.3109/02656736.2014.971446
Staroscik A (2004) Calculator for determining the number of copies of a template. URI Genomics & Sequencing Center, 19:2012. Available online at: https://cels.uri.edu/gsc/cndna.html
Steinberg GR, Hardie DG (2023) New insights into activation and function of the AMPK. Nat Rev Mol Cell Biol 24(4):255–327. https://doi.org/10.1038/s41580-022-00547-x
Von Schulze AT, Geiger PC (2022) Heat and mitochondrial bioenergetics. Curr Opin in Phys 27:2468–8673. https://doi.org/10.1016/j.cophys.2022.100553
Wang Z, Cai F, Chen X, Luo M, Hu L, Lu Y (2013) The role of mitochondria-derived reactive oxygen species in hyperthermia-induced platelet apoptosis. PloS one 8(9):e75044. https://doi.org/10.1371/journal.pone.0075044
Wang Q, Zou MH (2018) Measurement of reactive oxygen species (ROS) and mitochondrial ROS in AMPK knockout mice blood vessels. Methods Mol Biol (Clifton, N.J.) 1732:507–517. https://doi.org/10.1007/978-1-4939-7598-3_32
White MG, Saleh O, Nonner D, Barrett EF, Moraes CT, Barrett JN (2012) Mitochondrial dysfunction induced by heat stress in cultured rat CNS neurons. J Neurophysiol 108(8):2203–2214. https://doi.org/10.1152/jn.00638.2011
**e JH, Li YY, ** J (2020) The essential functions of mitochondrial dynamics in immune cells. Cell Mol Immunol 17:712–721. https://doi.org/10.1038/s41423-020-0480-1
Zorov DB, Juhaszova M, Sollott SJ (2014) Mitochondrial reactive oxygen species (ROS) and ROS-induced ROS release. Physiol Rev 94(3):909–950. https://doi.org/10.1152/physrev.00026.2013
Acknowledgements
We sincerely acknowledge the children and their parents for their participation in the study and contribution of information and samples for analysis. We appreciate the efforts by the field research team: Dharmesh Kumar, Upendra Rajak, Manish Kumar, and Rohit Chaudhury and the facilitation from Dr Sridhar Srikantiah, and Neeraj Kumar for conduct of the study.
Funding
This work was supported by a grant from Indian council of Medical Research (ICMR), grant number GAP0242 (VIR/12/2020/ECD-1).
Author information
Authors and Affiliations
Contributions
Conceptualization: Manoja K. Das, Aastha Mishra, and Arun K. Singh; data curation: Kanika Singh, Swati Kumari, and Manzoor Ali; formal analysis: Kanika Singh, Swati Kumari, and Manzoor Ali; methodology: Kanika Singh, Swati Kumari, and Manzoor Ali; resources: Aastha Mishra and Arun K. Singh; software: Aastha Mishra; writing—original draft: Kanika Singh and Aastha Mishra; writing—review and editing: Kanika Singh, Swati Kumari, Manzoor Ali, Manoja K. Das, Aastha Mishra, and Arun K. Singh.
Corresponding authors
Ethics declarations
Ethics approval and consent to participate
Ethical approval was granted by the Institutional Human Ethics Certificate Board of CSIR-Institute of Genomics and Integrative Biology, Delhi, India (ref no. CSIR-IGIB/IHEC/2022–23/01) and All India Institute of Medical Sciences, Jodhpur, India (ref no. AIIMS/IEC/2019–20/040).
Competing interests
The authors declare no competing interests.
Additional information
Aastha Mishra and Arun K. Singh share the senior authorship.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Singh, K., Kumari, S., Ali, M. et al. Association of transient mitochondrial functional impairment with acute heat exposure in children from Muzaffarpur region of Bihar, India. Int J Biometeorol 67, 1975–1989 (2023). https://doi.org/10.1007/s00484-023-02555-8
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00484-023-02555-8