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Show Evaluation of the Effects of the Toxicant Carbon Monoxide on Dravet Syndrome Pathophysiology Taylor Simpson, Ashwini Sri Hari Ph.D., Cameron S. Metcalf Ph.D. Department of Pharmacology and Toxicology, College of Pharmacy University of Utah Results Methods 0.03 G6PD activity (mU/ug protein) ns 0.02 0.01 0.01 0.00 ns T+ C O ET +C O W W G6PD activity in forebrain Homogenize • SUDEP is death during or shortly after a seizure unassociated with injury, drowning, or other factors such as status epilepticus • Autopsies have not revealed a toxicological or anatomical cause of SUDEP ✱ 0.02 0.00 C. Sudden Unexpected Death in Epilepsy (SUDEP) D. G6PD activity in forebrain ✱ ns Data CO EXPOSURE Day 1 Treatment groups Sample replicates Plasma carboxyhemoglobin levels (ug/ml) WT+AIR 1 -19.82241274 2 -14.64788732 3 2.039191672 1 -6.625842009 2 -8.769136558 3 3.661971831 1 -0.563380282 2 -0.992039192 3 -4.605021433 1 -4.115125536 Exposure timepoints (in mins) Treatment groups 5 10 20 30 40 45 Avg. exposure (ppm) WT+CO 150 145 170 111 90 0 111.0 HET+CO 160 150 180 121 24 0 105.8 WT+CO Day 2 • There is no literature on the impacts of CO on the Dravet Syndrome population • The aim of this study is to better understand how sub-chronic CO exposure influences Dravet Syndrome pathophysiology. • About 80% of DS patients have a Scn1a gene mutation1, so our model is C57 Black mice with an Scn1aA1783V/WT missense mutation. • If a Dravet patient goes out during a period of high pollution, they could be at high risk WT+CO 223 136 153 172 153 0 139.5 HET+CO 79 130 150 130 109 0 99.7 Day 3 WT+CO 111 157 214 205 117 0 134.0 HET+CO 36 155 174 119 89 0 95.5 Day 4 HET+AIR HET+CO WT+CO 230 139 148 112 45 0 112.3 2 -9.075321494 HET+CO 163 137 277 97 33 0 117.8 3 0.691977955 Table 1. CO exposure paradigm. Age-matched WT and HET mice were exposed to 100-300 parts per million (ppm) CO for 40 minutes daily for 4 consecutive days. Parallelly, control groups (WT+Air and HET+Air) were exposed to room air for 40 minutes daily for 4 consecutive days. Exposure data was analyzed using an unpaired, non-parametric Mann-Whitney test. There was no significant difference in average exposure values between WT+CO and HET+CO groups (p= 0.3166). Detection limit (DL) Below DL Detected Below DL Below DL Detected Table 2. Plasma carboxyhemoglobin (COHb) levels. After the CO exposure paradigm, plasma was collected. Plasma COHb levels were measured using an enzyme-linked immunosorbent assay (ELISA) kit. COHb levels were below the detection limit (see negative values) in most samples due to limited sample availability. Hence, the data was uninterpretable. REFERENCES 1) 2) 3) 4) Ding J, Li X, Tian H, Wang L, Guo B, Wang Y, Li W, Wang F, Sun T. SCN1A Mutation-Beyond Dravet Syndrome: A Systematic Review and Narrative Synthesis. Front Neurol. 2021 Dec 24;12:743726. doi: 10.3389/fneur.2021.743726. PMID: 35002916; PMCID: PMC8739186. Min-Jee Goh, Cassandra E. Deering-Rice, Jacklyn Nguyen, Evalien Duyvesteyn, Alessandro Venosa, Christopher A. Reilly, Cameron S. Metcalf, Breathing dysfunction and alveolar damage in a mouse model of Dravet syndrome bioRxiv 2022.05.20.492889; doi: https://doi.org/10.1101/2022.05.20.492889 Kuo FS, Cleary CM, LoTurco JJ, Chen X, Mulkey DK. Disordered breathing in a mouse model of Dravet syndrome. Elife. 2019 Apr 26;8:e43387. doi: 10.7554/eLife.43387. PMID: 31025941; PMCID: PMC6506208. Chen Z, Yu W, Xu R, Karoly PJ, Maturana MI, Payne DE, Li L, Nurse ES, Freestone DR, Li S, Burkitt AN, Cook MJ, Guo Y, Grayden DB. Ambient air pollution and epileptic seizures: A panel study in Australia. Epilepsia. 2022 Jul;63(7):1682-1692. doi: 10.1111/epi.17253. Epub 2022 Apr 26. PMID: 35395096; PMCID: PMC9543609. 5) Richardson SR, O'Malley GF. Glucose-6-Phosphate Dehydrogenase Deficiency. [Updated 2022 Sep 26]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2024 Jan-. 6) Jian Cheng, Hong Su, Jian Song, Xu Wang, Short-term effect of air pollution on childhood epilepsy in eastern China: A space–time-stratified case-crossover and pooled analysis, Environment International, Volume 170, 2022, 107591, ISSN 0160-4120 7) Images created with BioRender ET +A IR ir T+ A W Figure 1. Glucose-6-phosphate dehydrogenase (G6PD) enzyme activity. After the CO exposure paradigm, forebrain and brainstem tissue were collected. G6PD activity (milli units/microgram protein (mU/ug protein)) in samples was measured using a colorimetric assay kit. Mean G6PD activity in the brainstem (A, B). Mean G6PD activity in the forebrain (C, D). Data are represented as mean ± standard deviation (SD). Data was analyzed using a one-way ANOVA with Sidak’s multiple comparison test (A, C), and using an unpaired, parametric, Student’s t-test (B, D). ns: not significant; *p<0.05. N = 3-4 mice/group. Key Conclusions • Basal forebrain G6PD activity is lower in HETs compared to WTs • Brainstem G6PD activity in HETs is more susceptible to CO exposure compared to WTs • G6PD activity appears to vary in different brain regions in HETs Detected Below DL 0.01 0.00 ET +A IR H ET +C O The Bradford protein assay measures how much protein there is in a tissue sample H • ir The carboxyhemoglobin assay measures how much carbon monoxide is in the blood from inhalation 0.02 H • High air pollution events are associated with a higher risk for epilepsy-associated hospitalizations • Carbon monoxide (CO) is a highly common pollutant inhaled by humans • CO has been shown to have a significant impact on respiratory and nervous system health, and prolonged exposure often leads to brain injury • 0.00 T+ C O Pollution’s Effect on Epilepsy The glucose-6-phosphate dehydrogenase assay measures the amount of G6PD enzyme activity in tissue or blood Bradford Protein Assay W • Carboxyhemoglobin T+ A G6PD Assay 0.01 W • During seizures, many patients experience changes in respiration such as irregular breathing patterns and depressed respiratory processes • Dravet Syndrome patients are at high risk for SUDEP, likely as a result of respiration depression in the ictal and post-ictal periods ns 0.02 ✱ 0.03 G6PD activity (mU/ug protein) G6PD activity (mU/ug protein) 0.03 Respiration Introduction ns T+ A • Dravet Syndrome is a rare type of childhood epilepsy and has a high mortality rate • It is resistant to current anti-convulsant drugs • It is a genetic form of epilepsy caused by de novo mutations in the Scn1a gene • Seizures have variable severity and may be influenced by age • Associated with a much higher risk for status epilepticus and sudden unexpected death in epilepsy (SUDEP) 0.03 G6PD activity in brainstem T+ C O Dravet Syndrome (DS) • Whole body plethysmography is a lung function test Measures pressure changes in the chamber to assess respiration volume and changes ns ir Background • A. ET +A IR H ET +C O • 100 ppm – 300 ppm • 40 minutes per day for 4 consecutive days • Tissues collected on day 5, 18-24 hours after exposure B. ns H Whole Body Plethysmography H CO Exposure G6PD activity (mU/ug protein) Does sub-chronic CO exposure impact brain metabolism and respiration in Dravet Syndrome mice? G6PD activity in brainstem W Research Question Future Research • • • • Plasma COHb studies need to be repeated with sufficient sample volume G6PD activity assay should be repeated to validate results The exact mechanism(s) by which CO exposure might alter G6PD activity in HETs needs to be explored further Future studies may assess other pollutants such as particulate matter, ozone, and dust particles from the Great Salt Lake Acknowledgments • Thank you to the ACCESS Scholars program and the Science Research Initiative for supporting my involvement in this research. • Thank you to the following supporting labs: The ADD Lab, Rice Lab, Reilly Lab, and the Center for Human Toxicology (CHT) |
