The blood concentration of 1,8-cineole and its metabolites was measured in six male brushtail possums while they voluntarily fed on diets laced with varying concentrations of cineole for 3 d. On the third day, blood samples were collected during and after each bout of feeding for 3 hr. Blood cineole was measured by using headspace solid-phase microextraction (SPME), while cineole metabolites were measured by liquid-liquid extraction followed by gas chromatography-mass spectroscopy. Feeding patterns were measured by continual recording of residual food weight and time. Cineole absorption was rapid, resulting in a peak blood concentration at the end of each feeding bout. The blood concentration of cineole did not exceed a critical value (51.8 +/- 14.1 micromol/l) regardless of the concentration in the diet. Food and, therefore, cineole intake was regulated. The amount of food ingested in the first feeding bout decreased from 236 +/- 52 g on the control diet to 36 +/- 20 g on the 4% cineole diet. The amount of cineole ingested in the first bout (1.18 +/- 1.10 g) was the same regardless of the dietary concentration and was controlled by the size of the meal. Total food eaten during the 7-hr feeding session decreased by 64% from 368 +/- 94 g (control diet) to 131 +/- 52 g (4% diet). Total cineole intake increased from 2.47 +/- 0.60 g (1% diet) to 5.05 +/- 2.41 g (4% diet). Cineole metabolites accumulated throughout the sampling period and were generally still rising at the end of blood sampling period. Blood levels of metabolites were at least 10-fold higher than cineole levels. The immediate control of feeding seems to be regulated by blood levels of cineole, whereas metabolites are likely to be more important in regulating the chronic ingestion of cineole.

1,8-Cineole (cineole) is a Eucalyptus leaf toxin that defends against predation by herbivores such as the brushtail possum (Trichosurus vulpecula). The aim of the current study was to characterize the pharmacokinetics of cineole in the possum to improve understanding about how possums can avoid cineole toxicity when eating a Eucalyptus diet. Nine male possums were trapped in the wild and acclimated to captivity; a subcutaneous port was then implanted for venous blood sampling. Cineole was administered intravenously (10 and 15 mg kg(-1)) via a lateral tail vein and orally (30, 100 and 300 mg kg(-1)) by gavage, and blood concentrations of cineole and its metabolites were determined by gas chromatography. Cineole had a large terminal volume of distribution (V(z) = 27 l kg(-1)) and a high clearance (43 ml min(-1) kg(-1)), equal to hepatic blood flow. The terminal half-life was approximately 7 h. Oral bioavailability was low (F = 0.05) after low doses, but increased tenfold with dose, probably due to saturable first-pass metabolism. These findings indicate that when possums feed on a cineole diet, they eat until the cineole consumed is sufficient to saturate pre-systemic metabolism, leading to a rapid rise in bioavailability and cineole blood levels, and a cessation of the feeding bout. This is the first report on the pharmacokinetics of a dietary toxin in a wild herbivore, and provides insights into the interactions between the blood concentration of a plant secondary metabolite and the browsing behaviour of a herbivore.

No abstract

The common brushtail possum (Trichosurus vulpecula) is a generalist herbivore whose diet includes Eucalyptus leaves that are well defended by plant secondary metabolites (PSM) such as the terpene 1,8-cineole (cineole). We accustomed possums to a terpene-free diet, then challenged them with the addition of 2% cineole to the diet. Initially, there was a 50% reduction in total overnight food consumption associated with a marked decrease in the mass of the major feeding bout. After nine nights, however, cineole tolerance had developed as total food consumption had returned to the control amount. Compared to the control diet, the cineole diet was eaten in a larger number of smaller bouts, which were also eaten at a slower rate. The experiment was repeated with animals that had been accustomed to day-time feeding to take blood samples during feeding sessions. Feeding variables and blood concentration data for cineole were compared on the first and seventh day of the cineole diet. Although the total food consumed increased 2.5-fold after 7 days of the cineole diet, there was no increase in average blood cineole concentration, measured as the area under the concentration-time curve. This indicates that induction of liver enzymes resulted in greater pre-systemic metabolism of cineole and reduced bioavailability. The maximum tolerated blood concentration of cineole also increased, suggesting some adaptation of the central nervous system to the cineole aversive effects. This appears to be the first report in a vertebrate herbivore that consumption of a dietary PSM leads to metabolism induction and that this contributes to development of tolerance to the PSM. Overall, herbivores adapt to newly encountered dietary PSMs by immediate changes in feeding behavior followed by development of increased metabolism of PSM and probably diminished cellular responsiveness to effects.

The resistance of Eucalyptus to browsing mammals has been related to the level and type of formylated phloroglucinol compounds (FPCs) present in the leaf. The antifeedant activity of FPCs appears to depend on their aldehyde groups, but little else is known of their mode of action. We have sought to elucidate this further by examining the biological reactivity and disposition of jensenone, a model FPC. Neither jensenone nor any metabolites were detected in urine or feces of marsupial brushtail or ringtail possums that had ingested up to 725 mg x kg(-0.75). When jensenone was incubated in rat gastrointestinal segments in vitro, it rapidly disappeared. Jensenone also reacted rapidly with glutathione, cysteine, glycine, ethanolamine, and trypsin, and more slowly with acetylcysteine and albumin. Sideroxylonal, a more complex FPC, exhibited the same reactivity. Torquatone, a related compound that lacks both aldehyde groups and antifeedant activity, was unreactive. Mass spectroscopic analysis indicated that the adducts were Schiff bases formed between the aldehyde groups of FPCs and amine groups of the conjugating molecules. Successive adducts were formed with the two aldehyde groups of jensenone, and the four groups of sideroxylonal. The jensenone bis-glutathione adduct appeared to cyclize to the disulfide form. These findings suggest that the antifeedant effects of FPCs are due to their facile binding to amine groups on critical molecules in the gastrointestinal tract, leading to a loss of metabolic function. The consequent toxic reaction, probably involving chemical mediators such as 5-hydroxytryptamine (5HT), may cause colic, nausea, and a general malaise, resulting in anorexia.