An In Vivo Assay for Elucidating the Importance of Cytochromes P450 for the Ability of a Wild Mammalian Herbivore (Neotoma lepida) to Consume Toxic Plants

Abstract

An in vivo assay using the cytochrome P450 (P450) suicide inhibitor 1-aminobenzotriazole (ABT) and 24 hour food intake was developed to determine the relative importance of P450s in two populations of Neotoma lepida with respect to biotransformation of plant secondary compounds (PSCs) in the animals’ natural diets. The efficacy of ABT as a P450 inhibitor was first validated using hypnotic state assays with and without pretreatment with ABT. Pretreatment with 100mg/kg ABT by gavage increased hexobarbital sleep times 3–4 fold in both populations, showing effective inhibition of P450s in woodrats. Next, the Great Basin population was fed a terpene-rich juniper diet, and the Mojave population was fed a phenolic-rich creosote diet, with rabbit chow serving as the control diet in each group. Treatment with ABT inhibited food intake in the Great Basin population fed the juniper diet to a greater extent (35%) than the Great Basin population fed the control diet (19%) or the Mojave population fed the creosote diet (16%). The food intake of the Mojave population fed the control diet was not significantly inhibited by ABT. The findings suggest that the biotransformation of terpenes in juniper relies more heavily on P450s than that of phenolics in creosote. This assay provides an inexpensive and non-invasive method to explore the relative importance of P450s in the biotransformation strategies of wild mammalian herbivores.

Dengue viruses cleave STING in humans but not in nonhuman primates, their presumed natural reservoir

“…The fact that only a small handful of mammals encode an RG at position 78/79, out of the entire database, may in part explain why modeling dengue viruses in animals has been so difficult. We next wished to determine if STING is in fact cleaved by dengue in these rodent species, since all three already serve as animal models for biological research ( Keane et al, 2014 ; Nathaniel et al, 2013 ; Shimoyama et al, 2016 ; Campbell et al, 2016 ; Skopec et al, 2013 ). We synthesized HA-tagged STING genes for the rodent species discussed in Figure 6A , as well as an additional rodent (13-lined ground squirrel) which does not have the ‘RG’ motif at STING 78/79, as a negative control.…”

“…Our work suggests the identity of three such species: the naked mole rat, the common chinchilla, and the desert woodrat. All three of these small rodents are already used as animal models in biomedical research, and the genomes of all three have been sequenced ( Keane et al, 2014 ; Nathaniel et al, 2013 ; Shimoyama et al, 2016 ; Campbell et al, 2016 ; Skopec et al, 2013 ). These species could be superior to STING knockout mice, in that the STING pathway would be intact and the cleavage of STING by the virus would be naturally modeled rather than just bypassed.…”

Role of cytochrome P450 2B sequence variation and gene copy number in facilitating dietary specialization in mammalian herbivores

“…To further our understanding of the mechanisms underlying dietary specialization in herbivorous mammals, we have been conducting studies of cytochrome P450 enzymes, particularly those within the subfamily 2B (CYP2B), in specialist and generalist woodrats (genus Neotoma ). Our previous work indicates that cytochrome P450 detoxification enzymes are critical in the ability of woodrats to ingest juniper (Skopec, Malenke, Halpert, & Dearing, ). Junipers are high in terpenes, a class of PSMs known for their toxicity to the central nervous system (Waidyanatha et al., ).…”

Trio‐binned genomes of the woodrats Neotoma bryanti and Neotoma lepida reveal novel gene islands and rapid copy number evolution of xenobiotic metabolizing genes

“…To better understand the genomic basic of adaptation to different toxic diets, we interrogated the genetic architecture and possible expansion dynamics of several xenobiotic metabolizing gene families, with a particular focus on the cytochromes P450 (henceforth P450) 2A, 2B, and 3A genes. The P450 enzymes produced by these genes are critical in the biotransformation of toxins like those present in creosote bush and juniper (Huo et al, 2017; Magnanou et al, 2009; Shah et al, 2016; Skopec et al, 2013; Wilderman et al, 2014). Novel dietary niches involving toxic food are hypothesized to exert strong selection pressure for herbivore defence against potential toxic effects (Dearing et al, 2005), and previous work in these woodrats and other herbivorous mammals suggests that the evolution of gene copies, specifically those in P450 subfamily 2B, play an important role in dietary adaptation (Kitanovic et al, 2018; Magnanou et al, 2009; Malenke et al, 2012).…”