Integrated Toxicokinetics Underlie Selective Sequestration of Cardiotonic Steroids in an Asian Snake Rhabdophis...

The sequestration of dietary toxins as defensive compounds requires coordinated physiological processes, including metabolism, uptake into defensive organs and excretion. However, these processes are often studied separately, which can obscure how dietary toxins are ultimately selected and retained for defence. The Asian natricine snake Rhabdophis tigrinus acquires chemical defence by preying on toads and storing toad-derived bufadienolides (BDs), a class of cardiotonic steroids, in specialized nuchal glands. Whether this sequestration primarily reflects physicochemical retention or compound-specific physiological handling remains unclear. Here, we experimentally administered three cardiotonic steroids: bufalin, a naturally encountered BD, and two non-dietary cardenolides, digitoxigenin and ouabain. Using LC/MS and UV analyses, we quantified nuchal gland accumulation, excretion and chemical modification. Bufalin was extensively converted into multiple derivatives and accumulated efficiently in the nuchal glands. Digitoxigenin showed limited modification and high excretion, with lower mean accumulation than bufalin but no significant difference, whereas ouabain showed limited modification, high excretion and significantly lower accumulation than bufalin. Ouabain, the most polar compound, showed the lowest accumulation; however, the contrasting handling of bufalin and digitoxigenin-two structurally similar aglycones that differ in lactone-ring type-indicates that polarity alone cannot explain sequestration selectivity. Instead, coordinated differences in metabolism, accumulation and excretion reveal fine-scale discrimination among cardiotonic steroids. These results show that toxin sequestration in R. tigrinus is an integrated toxicokinetic process rather than simple retention, providing mechanistic insight into how vertebrate predators convert dietary toxins into chemical defences.