Neurotoxicologically Outcomes of Perinatal Chlordiazepoxide Exposure on the Fetal Prefrontal Cortex Cells in Rat Pup
Chlordiazepoxide Effects on Fetal Prefrontal Cortex
Keywords:
Chlordiazepoxide; Prefrontal Cortex; Perinatal Exposure; Neurotoxicity; RatAbstract
Background: Chlordiazepoxide is a benzodiazepine which is widely used as an anxiolytic, sedative and muscle-relaxant and its effects on neurodevelopment is yet to be identified. The aim of the current experimental study was to determine the effects of prenatal exposure to chlordiazepoxide on development of the prefrontal cortex (PFC). Materials and Methods: A total number of 9 pregnant Wister rats that were randomly assigned to three groups receiving standard rat food and drinking water ad libitum (n=3) or chlordiazepoxide (10 mg/kg) (n=3) and an equal volume of vehicle (0.9% NaCl) (n=3) intraperitoneal (i.p.) injection once daily from first to 21st day of gestation, respectively. At the end of the experiment, 14-day-old neonatal rat pups (n=8 per each group) were sacrificed and their PFC cells were extracted. Mitochondria were extracted from the PFC cells and their level of reactive oxygen species (ROS), protein density, Glutathione (GSH) content, mitochondrial membrane potential (MMP), swelling, cytochrome c release and ATP level was identified. We also performed the Nissl staining, DNA fragmentation assay and RNA extraction and real-time polymerase chain reaction (PCR) on PFC cells. Results: We found that isolated mitochondria from rat pups receiving chlordiazepoxide (E), had significantly higher ROS formation (P<0.001), decreased GSH (P<0.001), lower MMP (P<0.001), higher mitochondrial swelling (P<0.001), decreased ATP level (P<0.001), increased cytochrome c release (P<0.001) and higher Bax (P<0.001), p53 (P<0.001), cytochrome c (P<0.001) and caspase 8 mRNAs (P<0.001). The Nissle-stained neurons decreased while the apoptosis significantly increased (P<0.001). Conclusions: The results of this in vivo study provide evidence regarding negative effects of prenatal exposure to chlordiazepoxide on PFC.
References
Iqbal MM, Aneja A, Fremont WP. Effects of chlordiazepoxide (Librium) during pregnancy and lactation. Conn Med. 2003;67(5):259-62.
Dinarvand A, Hashemi M, Dinarvand R, Movassaghi S, Jafarinia M. The Effect of Chlordiazepoxide Consumption on the Hippocampus of Neonatal Rats During Pregnancy. Galen Medical Journal. 2022;11:e2283-e.
https://doi.org/10.31661/gmj.v11i.2283
PMid:36408487 PMCid:PMC9651174
Avnimelech-Gigus N, Feldon J, Tanne Z, Gavish M. The effects of prenatal chlordiazepoxide administration on avoidance behavior and benzodiazepine receptor density in adult albino rats. Eur J Pharmacol. 1986;129(1-2):185-8.
https://doi.org/10.1016/0014-2999(86)90352-3
PMid:3021475
Gavish M, Avnimelech-Gigus N, Feldon J, Myslobodsky M. Prenatal chlordiazepoxide effects on metrazol seizures and benzodiazepine receptors density in adult albino rats. Life Sci. 1985;36(18):1693-8.
https://doi.org/10.1016/0024-3205(85)90550-8
PMid:2984505
Mohammadkhani M, Gholami D, Riazi G. The effects of chronic morphine administration on spatial memory and microtubule dynamicity in male mice's brain. IBRO Neurosci Rep. 2024;16:300-8.
https://doi.org/10.1016/j.ibneur.2024.02.002
PMid:38390235 PMCid:PMC10881431
Armstrong C. ACOG guidelines on psychiatric medication use during pregnancy and lactation. American Family Physician. 2008 Sep 15;78(6):772-8.
Singsai K, Saksit N, Chaikhumwang P. Brain acetylcholinesterase activity and the protective effect of Gac fruit on scopolamine-induced memory impairment in adult zebrafish. IBRO Neurosci Rep. 2024;16:368-72.
https://doi.org/10.1016/j.ibneur.2024.02.004
PMid:38435743 PMCid:PMC10904921
Grigoriadis S, Alibrahim A, Mansfield JK, Sullovey A, Robinson GE. Hypnotic benzodiazepine receptor agonist exposure during pregnancy and the risk of congenital malformations and other adverse pregnancy outcomes: A systematic review and meta-analysis. Acta Psychiatr Scand. 2022;146(4):312-24.
https://doi.org/10.1111/acps.13441
PMid:35488412
Hartz SC, Heinonen OP, Shapiro S, Siskind V, Slone D. Antenatal exposure to meprobamate and chlordiazepoxide in relation to malformations, mental development, and childhood mortality. N Engl J Med. 1975;292(14):726-8.
https://doi.org/10.1056/NEJM197504032921405
PMid:1113782
Milkovich L, van den Berg BJ. Effects of prenatal meprobamate and chlordiazepoxide hydrochloride on human embryonic and fetal development. N Engl J Med. 1974;291(24):1268-71.
https://doi.org/10.1056/NEJM197412122912402
PMid:4431433
Bellantuono C, Tofani S, Di Sciascio G, Santone G. Benzodiazepine exposure in pregnancy and risk of major malformations: a critical overview. Gen Hosp Psychiatry. 2013;35(1):3-8.
https://doi.org/10.1016/j.genhosppsych.2012.09.003
PMid:23044244
Paxinos G, Watson C. The rat brain in stereotaxic coordinates 2nd ed. Academic: New York, NY, USA; 1986.
Ghazi-Khansari M, Mohammadi-Bardbori A, Hosseini MJ. Using Janus green B to study paraquat toxicity in rat liver mitochondria: role of ACE inhibitors (thiol and nonthiol ACEi). Ann N Y Acad Sci. 2006;1090:98-107.
https://doi.org/10.1196/annals.1378.010
PMid:17384251
Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248-54.
https://doi.org/10.1016/0003-2697(76)90527-3
PMid:942051
Fortunato F, Deng X, Gates LK, McClain CJ, Bimmler D, Graf R et al. Pancreatic response to endotoxin after chronic alcohol exposure: switch from apoptosis to necrosis? Am J Physiol Gastrointest Liver Physiol. 2006;290(2):G232-41.
https://doi.org/10.1152/ajpgi.00040.2005
PMid:15976389
Gao X, Zheng CY, Yang L, Tang XC, Zhang HY. Huperzine A protects isolated rat brain mitochondria against beta-amyloid peptide. Free Radic Biol Med. 2009;46(11):1454-62.
https://doi.org/10.1016/j.freeradbiomed.2009.02.028
PMid:19272446
Sadegh C, Schreck RP. The spectroscopic determination of aqueous sulfite using Ellman's reagent. MURJ. 2003;8:39-43.
Hosseini MJ, Shaki F, Ghazi-Khansari M, Pourahmad J. Toxicity of vanadium on isolated rat liver mitochondria: a new mechanistic approach. Metallomics. 2013;5(2):152-66.
https://doi.org/10.1039/c2mt20198d
PMid:23306434
Zhao Y, Ye L, Liu H, Xia Q, Zhang Y, Yang X et al. Vanadium compounds induced mitochondria permeability transition pore (PTP) opening related to oxidative stress. J Inorg Biochem. 2010;104(4):371-8.
https://doi.org/10.1016/j.jinorgbio.2009.11.007
PMid:20015552
Tafreshi N, Hosseinkhani S, Sadeghizadeh M, Sadeghi M, Ranjbar B, Naderi-Manesh H. The influence of insertion of a critical residue (Arg356) in structure and bioluminescence spectra of firefly luciferase. J Biol Chem. 2007;282(12):8641-7.
https://doi.org/10.1074/jbc.M609271200
PMid:17197704
Faghani M, Ejlali F, Sharifi ZN, Molladoost H, Movassaghi S. The Neuroprotective Effect of Atorvastatin on Apoptosis of Hippocampus Following Transient Global Ischemia/Reperfusion. Galen Medical Journal. 2016;5(2):82-9.
https://doi.org/10.31661/gmj.v5i2.656
Laviola G, de Acetis L, Bignami G, Alleva E. Prenatal oxazepam enhances mouse maternal aggression in the offspring, without modifying acute chlordiazepoxide effects. Neurotoxicol Teratol. 1991;13(1):75-81.
https://doi.org/10.1016/0892-0362(91)90030-Z
PMid:1646381
Chandrasekaran K, Stoll J, Giordano T, Atack JR, Matocha MF, Brady DR et al. Differential expression of cytochrome oxidase (COX) genes in different regions of monkey brain. J Neurosci Res. 1992;32(3):415-23.
https://doi.org/10.1002/jnr.490320313
PMid:1279190
Ansari MA, Scheff SW. Oxidative stress in the progression of Alzheimer disease in the frontal cortex. J Neuropathol Exp Neurol. 2010;69(2):155-67.
https://doi.org/10.1097/NEN.0b013e3181cb5af4
PMid:20084018 PMCid:PMC2826839
Gawryluk JW, Wang JF, Andreazza AC, Shao L, Young LT. Decreased levels of glutathione, the major brain antioxidant, in post-mortem prefrontal cortex from patients with psychiatric disorders. Int J Neuropsychopharmacol. 2011;14(1):123-30.
https://doi.org/10.1017/S1461145710000805
PMid:20633320
Lenaz G. The mitochondrial production of reactive oxygen species: mechanisms and implications in human pathology. IUBMB Life. 2001;52(3-5):159-64.
https://doi.org/10.1080/15216540152845957
PMid:11798028
Andreazza AC, Shao L, Wang JF, Young LT. Mitochondrial complex I activity and oxidative damage to mitochondrial proteins in the prefrontal cortex of patients with bipolar disorder. Arch Gen Psychiatry. 2010;67(4):360-8.
https://doi.org/10.1001/archgenpsychiatry.2010.22
PMid:20368511
Sakamuru S, Attene-Ramos MS, Xia M. Mitochondrial Membrane Potential Assay. High-Throughput Screening Assays in Toxicology. 2016:17-22.
https://doi.org/10.1007/978-1-4939-6346-1_2
PMid:27518619 PMCid:PMC5375165
Yuan J, Yankner BA. Apoptosis in the nervous system. Nature. 2000;407(6805):802-9.
https://doi.org/10.1038/35037739
PMid:11048732
Dong Y, Zhang W, Lai B, Luan WJ, Zhu YH, Zhao BQ et al. Two free radical pathways mediate chemical hypoxia-induced glutamate release in synaptosomes from the prefrontal cortex. Biochim Biophys Acta. 2012;1823(2):493-504.
https://doi.org/10.1016/j.bbamcr.2011.10.004
PMid:22057390
Jarskog LF, Gilmore JH, Glantz LA, Gable KL, German TT, Tong RI et al. Caspase-3 activation in rat frontal cortex following treatment with typical and atypical antipsychotics. Neuropsychopharmacology. 2007;32(1):95-102.
https://doi.org/10.1038/sj.npp.1301074
PMid:16641945
Hara Y, Yuk F, Puri R, Janssen WG, Rapp PR, Morrison JH. Presynaptic mitochondrial morphology in monkey prefrontal cortex correlates with working memory and is improved with estrogen treatment. Proc Natl Acad Sci U S A. 2014;111(1):486-91.
https://doi.org/10.1073/pnas.1311310110
PMid:24297907 PMCid:PMC3890848
Kar AN, Sun CY, Reichard K, Gervasi NM, Pickel J, Nakazawa K et al. Dysregulation of the axonal trafficking of nuclear-encoded mitochondrial mRNA alters neuronal mitochondrial activity and mouse behavior. Dev Neurobiol. 2014;74(3):333-50.
https://doi.org/10.1002/dneu.22141
PMid:24151253 PMCid:PMC4133933
Reichel JM, Nissel S, Rogel-Salazar G, Mederer A, Kafer K, Bedenk BT et al. Distinct behavioral consequences of short-term and prolonged GABAergic depletion in prefrontal cortex and dorsal hippocampus. Front Behav Neurosci. 2014;8:452.
https://doi.org/10.3389/fnbeh.2014.00452
PMid:25628548 PMCid:PMC4292780
Petrides M, Tomaiuolo F, Yeterian EH, Pandya DN. The prefrontal cortex: comparative architectonic organization in the human and the macaque monkey brains. Cortex. 2012;48(1):46-57.
https://doi.org/10.1016/j.cortex.2011.07.002
PMid:21872854
Yoon JH, Grandelis A, Maddock RJ. Dorsolateral Prefrontal Cortex GABA Concentration in Humans Predicts Working Memory Load Processing Capacity. J Neurosci. 2016;36(46):11788-94.
https://doi.org/10.1523/JNEUROSCI.1970-16.2016
PMid:27852785 PMCid:PMC5125231
Perlman SB, Almeida JR, Kronhaus DM, Versace A, Labarbara EJ, Klein CR et al. Amygdala activity and prefrontal cortex-amygdala effective connectivity to emerging emotional faces distinguish remitted and depressed mood states in bipolar disorder. Bipolar Disord. 2012;14(2):162-74.
https://doi.org/10.1111/j.1399-5618.2012.00999.x
PMid:22420592 PMCid:PMC3703524
Swartz JR, Carrasco M, Wiggins JL, Thomason ME, Monk CS. Age-related changes in the structure and function of prefrontal cortex-amygdala circuitry in children and adolescents: a multi-modal imaging approach. Neuroimage. 2014;86:212-20.
https://doi.org/10.1016/j.neuroimage.2013.08.018
PMid:23959199 PMCid:PMC3947283
Published
How to Cite
Issue
Section
License
Copyright (c) 2025 Galen Medical Journal
This work is licensed under a Creative Commons Attribution 4.0 International License.
