Long-term use of etomidate disrupts the intestinal homeostasis and nervous system in mice.

Etomidate (ETO) is used as an anesthetic in surgery, but it is being abused in some populations. The damage caused by long-term intake of ETO to intestinal and brain functions is not yet clear, and it remains to be determined whether the drug affects the central nervous system through the gut-brain axis. This study aimed to investigate the neurotoxic and gastrointestinal effects of ETO at doses of 1 mg/kg and 3 mg/kg in mice over 14 consecutive days. The results showed that long-term injection of ETO led to drug resistance in mice, affecting their innate preference for darkness and possibly inducing dependence on ETO. The levels of 5-hydroxytryptamine in the brain, serum, and colon decreased by 37%, 51%, and 42% respectively, while the levels of γ-aminobutyric acid reduced by 38%, 52%, and 41% respectively. H&E staining revealed that ETO reduced goblet cells in the colon and damaged the intestinal barrier. The expression of tight junction-related genes Claudin4 and ZO-1 was downregulated. The intestinal flora changed, the abundance of Akkermansia and Lactobacillus decreased by 33% and 14%, respectively, while Klebsiella increased by 18%. TUNEL results showed that high-dose ETO increased apoptotic cells in the brain. The expression of Claudin1 in the brain was downregulated. Untargeted metabolomics analysis of the colon and brain indicated that ETO caused abnormalities in glycerophospholipid metabolism. Abnormal lipid metabolism might lead to the production or accumulation of lipotoxic metabolites, causing central nervous system diseases. ETO induced changes in the intestinal flora and metabolism, further affecting the central nervous system through the gut-brain axis. The study unveiled the detrimental effects on the brain and gastrointestinal system resulting from long-term intake of ETO, which holds significant implications for comprehending the adverse impact of ETO abuse on human health.

Etomidate is devoid of genotoxicty and mutagenicity in human lymphocytes and in the Salmonella typhimurium/microsomal activation test.

No carcinogenesis or mutagenesis studies have been carried out with etomidate. The current study showed that etomidate has weak cytotoxic potential after 48 h exposure in human lymphocytes and has no hemolytic activity. The weak cytotoxicity seems to be related with redox imbalance of etomidate (40.9 and 81.9 µM) treated lymphocytes. At both etomidate concentrations, a slight decrease of the levels of GSH intracellular content and a significant increase in the amount of carbonylated proteins were observed after 48 h. The contribution of oxidative stress to genetic toxicity was only perceived when the enzyme Fpg was applied in the comet assay. Etomidate (40.9 and 81.9 µM) is a weak generator of oxidative DNA damage in lymphocytes. These damages to DNA probably were repaired, since no DNA strand breaks were detected in the standard alkaline comet assay (in the presence or absence of hepatic S9 microsomal fraction) without Fpg. Also, no micronucleated lymphocytes or carrying chromosomal aberrations were observed. Finally, etomidate (2046.8 and 4093.5 µM) was not mutagenic in the Salmonella/microsome mutagenicity assay, which used four Salmonella typhimurium strains (TA97a, TA98, TA100, and TA102) to detect frameshift and base-substitution mutations. In summary, etomidate is a weak oxidative DNA damaging anesthetic and is devoid of mutagenic properties in eukaryotic and prokaryotic models.

A Novel Strategy to Reverse General Anesthesia by Scavenging with the Acyclic Cucurbit[n]uril-type Molecular Container Calabadion 2.

BACKGROUND: Calabadion 2 is a new drug-encapsulating agent. In this study, the authors aim to assess its utility as an agent to reverse general anesthesia with etomidate and ketamine and facilitate recovery. METHODS: To evaluate the effect of calabadion 2 on anesthesia recovery, the authors studied the response of rats to calabadion 2 after continuous and bolus intravenous etomidate or ketamine and bolus intramuscular ketamine administration. The authors measured electroencephalographic predictors of depth of anesthesia (burst suppression ratio and total electroencephalographic power), functional mobility impairment, blood pressure, and toxicity. RESULTS: Calabadion 2 dose-dependently reverses the effects of ketamine and etomidate on electroencephalographic predictors of depth of anesthesia, as well as drug-induced hypotension, and shortens the time to recovery of righting reflex and functional mobility. Calabadion 2 displayed low cytotoxicity in MTS-3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium-based cell viability and adenylate kinase release cell necrosis assays, did not inhibit the human ether-à-go-go-related channel, and was not mutagenic (Ames test). On the basis of maximum tolerable dose and acceleration of righting reflex recovery, the authors calculated the therapeutic index of calabadion 2 in recovery as 16:1 (95% CI, 10 to 26:1) for the reversal of ketamine and 3:1 (95% CI, 2 to 5:1) for the reversal of etomidate. CONCLUSIONS: Calabadion 2 reverses etomidate and ketamine anesthesia in rats by chemical encapsulation at nontoxic concentrations.

The pituitary adenylate cyclase-activating polypeptide (PACAP) protects adrenal function in septic rats administered etomidate.

BACKGROUND: Both hyperinflammation during sepsis and etomidate can suppress adrenal function. In this study, we explored whether treatment with pituitary adenylate cyclase-activating polypeptide (PACAP) relieves adrenal suppression in cecal ligation and puncture (CLP)-induced septic rats. MATERIALS AND METHODS: Female Sprague-Dawley rats were randomly divided into five groups (n=7 per group), including the sham group, sepsis group (CLP group), sepsis and etomidate group (CLP+ETO group), PACAP group, and etomidate alone group (ETO group). Rats were sacrificed on the third day of sepsis, and blood and adrenal gland samples were obtained for further testing. RESULTS: The PACAP reduced the apoptosis rate of adrenal cells and peripheral lymphocytes, improving adrenal function, inhibiting the secretion of interferon gamma (IFN-γ) from peripheral lymphocytes, and slightly relieving the suppression of the adrenal function induced by the injection of etomidate in sepsis. CONCLUSION: In septic conditions, the PACAP protects the adrenal gland by regulating peripheral inflammation, which slightly relieves the toxic effects of etomidate on adrenal function.

Adrenocortical endocrine disruption.

The adrenal has been neglected in endocrine disruption regulatory testing strategy. The adrenal is a vital organ, adrenocortical insufficiency is recognised in life threatening "adrenal crises" and Addison's disease, and the consequences of off-target toxicological inhibition of adrenocortical steroidogenesis is well recognised in clinical medicine, where drugs such as aminoglutethimide and etomidate killed patients via unrecognised inhibition of adrenocortical steroidogenic enzymes (e.g. CYP11B1) along the cortisol and aldosterone pathways. The consequences of adrenocortical dysfunction during early development are also recognised in the congenital salt wasting and adrenogenital syndromes presenting neonatally, yet despite a remit to focus on developmental and reproductive toxicity mechanisms of endocrine disruption by many regulatory agencies (USEPA EDSTAC; REACH) the assessment of adrenocortical function has largely been ignored. Further, every step in the adrenocortical steroidogenic pathway (ACTH receptor, StAR, CYP's 11A1, 17, 21, 11B1, 11B2, and 3-hydroxysteroid dehydrogenase Δ4,5 isomerase) is known to be a potential target with multiple examples of chemicals inhibiting these targets. Many of these chemicals have been detected in human and wildlife tissues. This raises the question of whether exposure to low level environmental chemicals may be affecting adrenocortical function. This review examines the omission of adrenocortical testing in the current regulatory frameworks; the characteristics that make the adrenal cortex particularly vulnerable to toxic insult; chemicals and their toxicological targets within the adrenocortical steroidogenic pathways; the typical manifestations of adrenocortical toxicity (e.g. human iatrogenically induced pharmacotoxicological adrenal insufficiency, manifestations in typical mammalian regulatory general toxicology studies, manifestations in wildlife) and models of adrenocortical functional assessment. The utility of the in vivo ACTH challenge test to prove adrenocortical competency, and the H295R cell line to examine molecular mechanisms of steroidogenic pathway toxicity, are discussed. Finally, because of the central role of the adrenal in the physiologically adaptive stress response, the distinguishing features of stress, compared with adrenocortical toxicity, are discussed with reference to the evidence required to claim that adrenal hypertrophy results from stress rather than adrenocortical enzyme inhibition which is a serious adverse toxicological finding. This article is part of a special issue entitled 'Endocrine disruptors and steroids'.

Effects of Etomidate on the Steroidogenesis of Rat Immature Leydig Cells.

BACKGROUND: Etomidate is a rapid hypnotic intravenous anesthetic agent. The major side effect of etomidate is the reduced plasma concentration of corticosteroids, leading to the abnormal reaction of adrenals. Cortisol and testosterone biosynthesis has similar biosynthetic pathway, and shares several common steroidogenic enzymes, such as P450 side chain cleavage enzyme (CYP11A1) and 3ß-hydroxysteroid dehydrogenase 1 (HSD3B1). The effect of etomidate on Leydig cell steroidogenesis during the cell maturation process is not well established. METHODOLOGY: Immature Leydig cells isolated from 35 day-old rats were cultured with 30 µM etomidate for 3 hours in combination with LH, 8Br-cAMP, 25R-OH-cholesterol, pregnenolone, progesterone, androstenedione, testosterone and dihydrotestosterone, respectively. The concentrations of 5α-androstanediol and testosterone in the media were measured by radioimmunoassay. Leydig cells were cultured with various concentrations of etomidate (0.3-30 µM) for 3 hours, and total RNAs were extracted. Q-PCR was used to measure the mRNA levels of following genes: Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Srd5a1, and Akr1c14. The testis mitochondria and microsomes from 35-day-old rat testes were prepared and used to detect the direct action of etomidate on CYP11A1 and HSD3B1 activity. RESULTS AND CONCLUSIONS: In intact Leydig cells, 30 µM etomidate significantly inhibited androgen synthesis. Further studies showed that etomidate also inhibited the LH- stimulated androgen production. On purified testicular mitochondria and ER fractions, etomidate competitively inhibited both CYP11A1 and HSD3B1 activities, with the half maximal inhibitory concentration (IC50) values of 12.62 and 2.75 µM, respectively. In addition, etomidate inhibited steroidogenesis-related gene expression. At about 0.3 µM, etomidate significantly inhibited the expression of Akr1C14. At the higher concentration (30 µM), it also reduced the expression levels of Cyp11a1, Hsd17b3 and Srd5a1. In conclusion, etomidate directly inhibits the activities of CYP11A1 and HSD3B1, and the expression levels of Cyp11a1 and Hsd17b3, leading to the lower production of androgen by Leydig cells.