Microsomal metabolism and enzyme kinetics of the terpene p-cymene in the common brushtail possum (Trichosurus vulpecula), koala (Phascolarctos cinereus) and rat.

1. p-Cymene is an aromatic monoterpene found in the leaves of Eucalyptus spp. and is ingested in the diet of two marsupial folivores, the brushtail possum (Trichosurus vulpecula) and koala (Phascolarctos cinereus). The metabolism of p-cymene by liver microsomes from the possum, koala and rat (an animal whose diet is not terpene-rich) was examined. 2. The major p-cymene metabolite in each species was cuminyl alcohol, with smaller amounts of other side-chain alcohols being formed. No phenolic metabolites were detected. Possum and koala microsomes further oxidized cuminyl alcohol to cumic acid. 3. Pretreatment with a terpene diet more than doubled the V(max) for cuminyl alcohol formation by possum microsomes. This is similar to a previous finding by our group with 1,8-cineole and indicates that a terpene-containing diet results in induction of the enzymes responsible for terpene metabolism. 4. The rank order of the ability to metabolize p-cymene, measured by intrinsic clearance (Cl(int) = V(max)/K(m) [microl mg protein(-1) min(-1)]) was: terpene-treated possum (128)>control possum (107)>koala (69)>rat (38). 5. The findings support the hypothesis that animals that have adapted to a diet of Eucalypus leaf have a greater capacity than generalist feeders to metabolize dietary terpenes.

Inhibition of the microsomal metabolism of 1,8-cineole in the common brushtail possum (Trichosurus vulpecula) by terpenes and other chemicals.

1. Brushtail possums (Trichosurus vulpecula) ingest large amounts of terpenes in their diet of Eucalyptus leaf. Previously, we showed that dietary terpenes induce the cytochrome P450 enzymes (CYPs) responsible for their metabolism. The present study examined the effects of various CYP inhibitors on the metabolism of 1,8-cineole, the major dietary terpene, by liver microsomes from the possum and rat. 2. Ketoconazole inhibited the major reactions of terpene-induced microsomes in both species: 9-hydroxylation in the possum and 2-hydroxylation in the rat. This suggests the involvement of CYP3A enzymes, although in the possum there was a lack of the expected inhibition by troleandomycin or activation by alpha-naphthoflavone, highlighting the differences between species in CYP forms. Diethyldithiocarbamate also inhibited 9-hydroxylation in the possum, indicating that a CYP2E1-like enzyme contributes to this reaction. 3. Three other dietary terpenes were potent competitive inhibitors of 9-hydroxylation in the possum. K(i) ( micro M) (mean +/- SE, n = 4) were: alpha-pinene, 4.4 +/- 1.1; limonene, 7.8 +/- 2.1; p-cymene, 44.3 +/- 11.2; cuminyl alcohol (a p-cymene metabolite), 6.0 +/- 0.8. It appears likely that p-cymene acts via its metabolite to inhibit 1,8-cineole metabolism. 4. Inhibitory interactions between dietary terpenes, as well as other plant secondary compounds, may impose a significant constraint on foliage consumption in the common brushtail possum, therefore explaining the obligatory generalist nature of this browsing marsupial and other generalist herbivores.

Does excretion of secondary metabolites always involve a measurable metabolic cost? Fate of plant antifeedant salicin in common brushtail possum, Trichosurus vulpecula.

Salicin was administered orally to six brushtail possums by incorporation in food for six days at three dose levels (0.05, 0.5, and 1.5% wet weight), giving mean +/- SD daily intakes of 0.31 +/- 0.09, 2.76 +/- 0.75, and 6.04 +/- 1.12 mmol salicin. Metabolites were identified by mass spectrometry and assayed by HPLC. Salicyl alcohol glucuronide accounted for 56-64% of urinary metabolites over the three doses, salicyluric acid 15-26%, salicin 10-18%, and there were smaller amounts of free (2-4%) and conjugated (0-6%) salicylic acid. beta,2-Dihydroxyphenylpropionic acid was a minor metabolite. The hydrolysis of dietary salicin enabled reconjugation of its aglycone, salicyl alcohol, with a more polar sugar, glucuronic acid, thus enhancing its renal excretion and resulting in little net loss of substrates for conjugation and a low measurable metabolic cost of excretion.

Microsomal metabolism of the terpene 1,8-cineole in the common brushtail possum (Trichosurus vulpecula), koala (Phascolarctos cinereus), rat and human.

1. This study reports on the pathways of metabolism and enzyme kinetics of the Eucalyptus terpene, 1,8-cineole, by liver microsomes from the brushtail possum (Trichosurus vulpecula) and koala (Phascolarctos cinereus) (animals that normally include this terpene in their diet), rat and human. 2. The rank order of the ability to metabolize 1,8-cineole with respect to overall 1,8-cineole intrinsic clearance (CL'int = Vmax/Kmax in microl mg protein(-1) min(-1)) was koala (188) > possum (181)>> rat (28) > human (12). This order supports the hypothesis that adaptation to a Eucalyptus diet involves enhanced metabolism of terpenes. 3. The metabolism of 1,8-cineole was also studied in the liver from brushtail possum pretreated with a mixture of terpenes, which have previously been shown to induce cytochrome P450 enzymes. Rats were pretreated with the same mixture of terpenes or phenobarbitone. 4. Terpene pretreatment more than doubled the CL'int of 1,8-cineole by brushtail possum liver microsomes (from 180 to 394 microl mg protein(-1) min(-1)) and increased rat CL'int by nearly 10-fold (from 28 to 259 microl mg protein(-1) min(-1)), but still less than the induced possum value. However, phenobarbitone had the greatest inducing effect, increasing the rat CL'int to 1,825 microl mg protein(-1) min(-1). 5. A regioselective preference of oxidation was evident between adapted and non-adapted species. In rat and human oxidation was preferred at the aliphatic ring carbons over methyl substituents. In possum, many of the available carbons were utilized, however metabolism at methyl substituents was preferred. In the koala, oxidation occurred primarily at the methyl substituents.

Plant secondary metabolites as mammalian feeding deterrents: separating the effects of the taste of salicin from its post-ingestive consequences in the common brushtail possum (Trichosurus vulpecula).

The effect of the phenolic glycoside, salicin, on food intake of the common brushtail possum (Trichosurus vulpecula) was studied in a series of feeding experiments. Increasing the concentration of salicin in a diet of fruits and cereals led to significant reductions of food intake in the short term (6 days). After prolonged (20 days) exposure to salicin, food intake (19 g kg(-0.75) day(-1)) was still reduced relative to controls (31 g kg(-0.75) day(-1)) but not reduced to the same extent as in the short-term experiments. Nonetheless, over these 20 days, common brushtail possums regulated their intake of salicin so as not to exceed a threshold limit of 1.9 +/- 0.1 g kg(-0.75) day(-1). Manipulative experiments sought to determine whether this threshold intake was in response to pre-ingestive factors (taste) or the post-ingestive consequences of ingesting salicin. Dietary salicin (0.17-5.0% DM) had no significant effect on nitrogen balance or urea metabolism and injection of a specific serotonin receptor antagonist, ondansetron, did not lead to increases in salicin intake as has been found for some other plant secondary metabolites. Similarly, administration of 1.3 g salicin by gavage had no significant effect on the subsequent intake of salicin compared to controls that were gavaged with water. We concluded that pre-ingestive factors were responsible for common brushtail possums limiting their intake of salicin-rich diets rather than any measurable post-ingestive consequence of feeding.

Characterisation of tolbutamide hydroxylase activity in the common brushtail possum, (Trichosurus vulpecula) and koala (Phascolarctos cinereus): inhibition by the eucalyptus terpene 1,8-cineole.

Plant constituents such as terpenes are major constituents of the essential oil in Eucalyptus sp. 1,8-Cineole and p-cymene (Terpenes present in high amounts in Eucalyptus leaves) are potential substrates for the CYP family of enzymes. We have investigated tolbutamide hydroxylase as a probe substrate reaction in both koala and terpene pretreated and control brushtail possum liver microsomes and examined inhibition of this reaction by Eucalyptus terpenes. The specific activity determined for tolbutamide hydroxylase in the terpene treated brushtails was significantly higher than that for the control animals (1865+/-334 nmol/mg microsomal protein per min versus 895+/-27 nmol/mg microsomal protein per min). The activity determined in koala microsomes was 8159+/-370 nmol/mg microsomal protein per min. Vmax values and Km values for the terpene treated possum, control, possum and koala were 1932-2225 nmol/mg microsomal protein per min and 0.80 0.81 mM; 1406-1484 nmol/mg microsomal protein per min and 0.87-0.92 mM and 5895-6403 nmol/mg microsomal protein per min and 0.067-0.071 mM, respectively. Terpenes were examined as potential inhibitors of tolbutamide hydroxylase activity. 1,8-Cineole was found to be a competitive inhibitor for the enzyme responsible for tolbutamide hydroxylation (Ki 15 microM) in the possum. In koala liver microsomes stimulation of tolbutamide hydroxylase activity was observed when concentrations of cineole were increased. Therefore, although inhibition was observed, the type of inhibition could not be determined.

Induction of xenobiotic metabolising enzymes in the common brushtail possum, Trichosurus vulpecula, by Eucalyptus terpenes.

The mixed function oxidase (MFO) activity and content was studied in the liver of common brushtail possums fed for 10 days on a diet containing a mixture of terpenes found in Eucalyptus leaves on which possums commonly browse. The MFOs were compared to the MFOs in possums fed a control diet of fruits and cereals only. The terpenes chosen were 1,8-cineole, p-cymene, alpha-pinene and limonene. The selected terpenes caused induction of P450 enzymes, as shown by a 53% higher cytochrome P450 content and a 45% increase in aminopyrine demethylase activity in the test animals. Aniline hydroxylase activity was significantly increased, with levels of 2.95 and 1.43 nmol min(-1) mg(-1) microsomal protein in the test and control animals, respectively. There was also a significant increase in androstenedione 16alpha-hydroxylase activity in the test group, 0.85 as compared to 0.50 nmol mg(-1) min(-1) in the control group. Western blot studies using human CYP2E1 and rat CYP2C11 and CYP2C6 antibodies gave CYP2E, CYP2C11 and CYP2C6 immunoreactive bands of greater intensity in the test animals as compared to the control group. This study has shown experimentally that dietary terpenes cause enzyme induction in folivorous marsupials. It also confirms the importance of knowledge of diet when studying xenobiotic metabolising enzymes, particularly in wild animals such as the brushtail possum.

Administration of a 5HT3 receptor antagonist increases the intake of diets containing Eucalyptus secondary metabolites by marsupials.

The effect of a naturally occurring plant phenolic constituent (the acylphloroglucinol derivative, jensenone, derived from Eucalyptus jensenii) on the food intake of two folivorous marsupials, the common ringtail (Pseudocheirus peregrinus) and the common brushtail possum (Trichosurus vulpecula) was studied. When fed diets containing varying concentrations of jensenone, both species regulated their intake of jensenone so as not to exceed a ceiling intake. This ceiling was about twice as high for common ringtails as for common brushtails from northern Australia. Southern populations of common ringtails showed greatly reduced capacities to tolerate jensenone. When common brushtails were injected (0.5 mg.kg-0.75 body mass) with ondansetron (a selective antagonist of serotonin 5HT3 receptors), they ate significantly more jensenone than animals injected with physiological saline. The same pattern was observed when common ringtails were fed diets containing both jensenone and ondansetron (0.0035 mg.g-1 wet mass of diet). Ondansetron injection had no effect on food intake when the food did not contain jensenone while the addition of higher doses of ondansetron to diets of common ringtails very slightly reduced food intakes of a non-jensenone diet. When common brushtails were given 50 mg of jensenone by gastric lavage, their average subsequent intake of dietary jensenone matched the difference between the daily threshold and the dose given, although the response of individuals was highly variable. Lavage with water alone had no effect on subsequent jensenone intake compared with the pre-dose period. We interpret these results as evidence that the antifeedant effects of jensenone and related compounds are partly mediated by serotonin action on 5HT3 receptors most likely via "nausea" to condition a food aversion.