Species Differences in the Metabolism and Toxicity of 4-Ipomeanol
Parkinson, Oliver Thomas
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The studies presented in this dissertation investigate the enzymatic basis for the species differences in target organ toxicity of the pro-toxin, 4-ipomeanol (IPO). <italic>In vitro</italic> studies that monitored the production of a pyrrole-based IPO adduct formed from the reactive ene-dial reactive intermediate, establish a dominant role for CYP4B1 in the <italic>in vitro</italic> bioactivation of IPO in lung and kidney tissues from toxicity-susceptible animal species. Antibodies against CYP4B1 and HET0016, a CYP4-specific chemical inhibitor, eliminated IPO adduct formation in lung microsomes from mice, dogs, rats and rabbits, and in kidney from male mice, indicating that IPO activation in these tissues is catalyzed predominantly, if not exclusively, by CYP4B1. Furthermore, despite the fact that IPO toxicosis is agriculturally relevant due to the relatively frequent occurrence of cattle poisoning, these studies are the first to identify a CYP4B1 ortholog in cow lung and establish a role for this enzyme in IPO-mediated pneumotoxicity in cattle. We also detail the characterization of a <italic>Cyp4b1</italic> null mouse strain and demonstrate that disruption of CYP4B1 expression is protective against IPO poisoning <italic>in vivo</italic>in mice, thereby definitively establishing CYP4B1 as the enzyme responsible for bioactivating IPO. We have shown further that IPO is glucuronidated readily in liver microsomes from all species examined, with the exception of humans. An IPO-glucuronide conjugate was also detected in the urine of IPO-treated mice, the first evidence for UGT-mediated phase II metabolism of IPO beyond that reported for the rat. Cell-based studies with the Ser207 variant of human CYP4B1 demonstrate further that human CYP4B1 in all of its known splice variant forms does not encode a functional protein. Attempts to identify the causative IPO bioactivating enzyme in human liver implicated human CYP1A2, in part, but several other recombinant P450 forms were also capable of catalyzing this reaction. The lack of evidence from <italic>in vitro</italic> studies for a dominant role for human CYP1A2 in the human hepatotoxicity of IPO was complemented by in vivo experiments conducted with transgenic mice that expressed human CYP1A enzymes on a null murine CYP1A2 background. Therefore, the lack of pulmonary toxicity and the prevalent hepatotoxicity observed in humans exposed to IPO is not due to increased rates of IPO activation in human liver or by human liver CYP1A2, but more likely to decreased rates of IPO activation in lung, possibly coupled to a lack of UGT-mediated phase II metabolism of IPO in human liver.
- Medicinal chemistry