Succinate-driven methyl thiazolyl tetrazolium (MTT) reduction is coupled to mitochondrial electron transport chain (ETC) activity: comparative evidence from subcellular fractionation in the lobster cockroach (Nauphoeta cinerea) and zebrafish (Danio rerio).

The tetrazolium-based MTT assay is widely used to assess cellular metabolic activity, yet the subcellular origin and mechanistic basis of MTT reduction remain incompletely defined. Here, we investigated the localization and biochemical determinants of MTT reductive activity using subcellular fractionation of lobster cockroach Nauphoeta cinerea head tissue and zebrafish Danio rerio brain, combined with substrate specificity, protein-dependence analysis, and mitochondrial electron transport chain (ETC) inhibition. Succinate-driven MTT reduction was predominantly localized to mitochondrial fractions (P2 and P3), accounting for ~60-65% of total activity in both species, while post-mitochondrial supernatants exhibited minimal activity. MTT reduction increased linearly with protein concentration across all fractions (R2 = 0.95-0.99), with mitochondrial fractions displaying the highest protein-normalized reductive capacity. MTT reduction was highly sensitive to malonate and significantly inhibited by antimycin A and sodium azide, confirming dependence on Complex II-mediated electron entry and downstream ETC activity, whereas rotenone produced only partial inhibition, consistent with bypass of Complex I under succinate-driven conditions. Collectively, these findings demonstrate that MTT reduction is strongly dependent on substrate selection and experimental conditions. Under succinate-driven conditions, MTT reduction reflects mitochondrial ETC activity, with minimal non-mitochondrial contribution. This work provides a mechanistic framework that clarifies the intracellular origin of MTT reduction and supports its use as a substrate-dependent indicator of mitochondrial function in bioenergetic and toxicological studies. The consistent findings obtained in insect and fish tissues further support the conservation of the underlying mechanisms across phylogenetically distinct species and reinforce the value of alternative experimental models for investigating mitochondrial bioenergetics.