Effect of Succinate Dehydrogenase Inhibitors on Mouse Bone Marrow-Derived Macrophages Proliferation, Bioenergetics, and Immune Profile

Abstract

Background: Modern agriculture and food preservation standards require the use of fungicides to treat and prevent fungal contamination. Succinate dehydrogenase inhibitors (SDHIs) are one of the mostly used groups of fungicides applied on many fruits, vegetable, grains, and on the vegetation in public spaces. SDHIs are competitive inhibitors of succinate dehydrogenase (SDH), also known as the mitochondrial respiratory chain (RC) complex II. SDH is a remarkable enzyme that is part of the RC, which represents the core of cellular bioenergetics, and at the same time it catalyzes an essential step in the tricarboxylic acid (TCA) cycle. Moreover, SDH is evolutionary very well conserved across species. Current data shows that mitochondrial bioenergetics and metabolic state are key players in macrophages differentiation and in defining their immune states. Macrophages are highly plastic key players in the innate immunity and participate in the initiation, maintenance, and resolution of inflammation. The role of SDH and the impact of SDH acquired deficiencies on macrophages and the inflammatory process was never assessed. As such we set out to study the effect of the extensively used SDHIs fungicides on the integrity of the immuno-metabolic interplay within the macrophages in off-target species and the potential consequence they may represent on public health. .

Aims: In this study we aim particularly at investigating the effect of several SDHIs on the survival, immune state, anti-oxidant system, and mitochondrial enzymatic activities in a murine model of innate immunity.

Methods: Bone marrow-drived macrophages (BMDM) were extracted from mice and grown in a culture medium containing macrophage colony stimulating factor-1 (MCSF-1) allowing the growth and differentiation of macrophages. Cells were treated with SDHIs in the presence or absence of glucose in the culture medium to assess cell viability using the neutral red spectrophotometric technique. Lipopolysaccharide -activated macrophaes were also exposed to SDHIs to assess the impact on pro-inflammatory genes expression level using RT-qPCR. Moreover, Interleukin-13 treated macrophaes were exposed as well to SDHIs to assess the effect on anti-inflammatory genes expression profile by RT-qPCR. The same technique anti-oxidant enzymes expression level was further asseesed in SDHI treated BMDM by RT-qPCR. Finally, the kinetic activity of specific mitochondrial RC enzymes were assessed by spectrophotometry to detrmine the effect of SDHIs fungicides on SDH activity and determine potential off-targets. . Preliminary results: Bixafen, fluopyram, and boscalid treatment resulted in a reduction in cell viabilty at 48 hours and 72hours. While cyflumetofen reduced cell viability as early as 24 hours. SDHIs treatment of LPS-induced macrophages altered significantly the expression levels of pro-inflammatory genes but not the expression level of anti-oxidant enzymes. Finally, no off-target effect of SDHIs was noted in BMDM mitochondria.