Adaptive metabolic strategies employed by Eucalyptus leaf-eating marsupials : the role of cytochrome P450

It has long been hypothesised that the major drug-metabolising enzymes, the cytochrome P450s (CYPs), play a key role for both plants and animals in the 'evolutionary arms race'. Herbivorous mammals are said to have evolved highly developed xenobiotic metabolising systems that enable them to metabolise large quantities of dietary plant secondary metabolites (PSMs). In spite of this, there is a paucity of data on the interactions between plants and mammals with respect to CYPs. This study aimed to address the validity of the hypothesis that animals including significant quantities of potentially toxic PSMs in their diet, have necessarily developed highly specialised enzymatic systems, enabling them to metabolise these compounds. Trees belonging to the genus, Eucalyptus, are well defended from herbivory as they contain significant levels of PSMs, yet some Australian marsupials, such as the common brushtail possum (Trichosurus vulpecula) and koala (Phascolarctos cinereus), include significant proportions of Eucalyptus leaves in their diet. Therefore, the relationship between Eucalyptus PSMs and the common brushtail possum was the model chosen to test the above-mentioned hypothesis. The effects of Eucalyptus terpenes on CYP activity and content in the liver of this generalist Eucalyptus browser were initially investigated. Possums were fed for ten days on a basal diet with or without a mixture of the four terpenes: 1,8-cineole, p-cymene, limonene and a-pinene. Total hepatic CYP content, as well as activity usually attributed to specific isoforms (CYP2E1 and CYP2C11), was significantly increased in hepatic microsomes from terpene-treated possums. Western blot studies also showed immunoreactive bands of greater intensity for human and rat CYP2E, 2C11 and CYP2C6 in microsomes from terpene-treated animals compared to untreated possums, suggesting that the isoforms induced were similar to human and rat CYP2E and CYP2C. The in vitro hepatic metabolism of 1,8-cineole and p-cymene was then investigated in the specialist Eucalyptus feeder, the koala and the generalist brushtail possum, as well as in non-adapted Eucalyptus feeders, rats and humans. The metabolites were identified using gas chromatography and mass spectrometry and the pathways of metabolism found in vitro for the possum and koala correlated well with those observed in vivo. The rank order of the ability to metabolise 1,8-cineole with respect to the overall 1,8-cineole intrinsic clearance (CL`\\(_{int}\\), = V\\(_{max}\\)/Km) was found to be: terpenetreated possum > koala ‚Äöv¢‚Ä¢ control possum >> rat > human. A similar pattern was observed for in vitro p-cymene metabolism. In addition, the CL`\\(_{int}\\) of 1,8-cineole by microsomes from terpene-treated possums was more than double to that found in untreated possums, providing direct evidence in support of the hypothesis that adaptation to a Eucalyptus diet involves enhanced metabolism of terpenes. Rats treated with the same terpenes as possums also showed a greater oxidative capacity of 1,8-cineole, with the CL`\\(_{int}\\) increasing nearly tenfold, however this was still less than the induced possum value. The nature of CYP isoforms involved in 1,8-cineole metabolism in both possums and rats showed similarities to CYP3A, with the selective CYP3A inhibitor, ketoconazole, reducing the formation of 1,8-cineole metabolites to around 25% of control activity in both species. In the possum, piperonyl butoxide, a general CYP inhibitor, was also found to potently inhibit 1,8-cineole metabolism, while diethyldithiocarbamate, known to be selective for CYP2El, exhibited moderate inhibition in this species. p-Cymene, limonene, ˜í¬±-pinene and cuminyl alcohol were all found to potentiy inhibit the in vitro metabolism of 1,8-cineole, with metabolite formation reduced by up to 86%. This provides direct evidence to support the hypothesis that PSMs belonging to the same class are metabolised by common CYP isozymes and, as a result, this may be a major factor contributing to the obligatory generalist nature of many mammalian herbivores. By including a variety of plant foods in their diet, mammalian herbivores avoid saturation of these metabolic pathways. A single-dose of disulfiram, the precursor to diethyldithiocarbamate, had no effect of 1,8-cineole blood levels. However, evidence of the impressive inducing nature of 1,8-cineole was discovered. 1,8-Cineole blood levels were significantly reduced in possums one week after the animals were given the same dose (50 mg/kg) of this terpene. 1,8-Cineole was absorbed rapidly, with peak blood levels occurring at approximately 20 min. These results highlight the adaptive nature of the enzymatic system in this Eucalyptus leaf-eating marsupial.