Endocrine Abstracts

JOINT3292

Background: Bacterial sepsis is a serious threat to homeostasis and is the most common cause of mortality in non-coronary intensive care units (ICUs). It is characterized by compromised function of several vital organs, including the adrenal glands. However, little is known about the mechanisms involved. Recently, it has been reported that iron overload and ferroptosis might be involved in sepsis-induced multiorgan damage. Our previous results indicated that the human adrenocortical cell line, NCI-H295R, is sensitive to ferroptosis induced by inhibiting glutathione peroxidase 4 (GPX4) and can be enhanced by active steroidogenesis. However, whether primary adrenal cells are also sensitive to ferroptosis and whether bacterial LPS can modulate this process remains unexplored until now.

Objectives: The main objective of this study was to investigate whether primary adrenal cells are susceptible to ferroptosis induction by known ferroptosis inducers or in septic conditions in vitro in 3D culture.

Methods: Primary adrenal cells were isolated from mouse adrenal glands and cultivated as 3D spheroids using AggreWell microwell plates and low attachment conditions. The sensitivity of 3D spheroids was tested by pharmacological inhibition of GPX4 using RSL3 and incubation with bacterial LPS. Spheroid diameter, cell viability (FDA/PI staining), lipid peroxidation (C11-BODIPY), and necrosis induction (LDH release) were measured 24 h thereafter.

Results: Our study demonstrated that administration of RSL3 negatively affected the morphology and size of primary adrenal spheroids, which were associated with enhanced lipid peroxidation and necrosis induction. These changes were mitigated by ferroptosis inhibitor, ferrostatin-1. Our preliminary data based on spheroids isolated from mice with adrenocortical cell-specific deletion of GPX4 support these results. However, no significant increase in lipid peroxidation or LDH release could be observed after LPS treatment.

Conclusions: In summary, our findings suggest that primary adrenal cells are susceptible to ferroptosis induction through direct inhibition of GPX4 but not bacterial LPS. Further experiments, including the detailed characterization of spheroids, especially those isolated from GPX4-deficient adrenal cells, are ongoing. Moreover, a more thorough molecular investigation of LPS effects on primary mouse spheroids is necessary to elucidate its potential involvement in adrenal necrosis.