Materials and Methods


(+)limonene, (-)limonene, limonene oxide, menthol, alpha-terpineol and terpinene-4-ol were purchased from Sigma Chemical Co. (Poole, Dorset. UK). Radiolabelled [14C]lauric acid was obtained from the Radiochemical centre (Amersham, Bucks. UK). All other chemicals were purchased from standard commercial sources and were of reagent grade.

Animals and Treatment

Three groups of fifteen male Wistar rats (140 - 220g body weight; University of Surrey breeders) were pretreated by gavage once daily for three consecutive days with each of the cyclic monoterpenes. Each compound was administered to five animals at a dose level of 1.5g/kg body weight. The compounds were administered as a solution in corn oil in a total volume of 5ml/kg body weight; control rats were given a similar volume of corn oil. All animals were killed at the start of the fourth day, i.e. 24hr. after the last dose by cervical fracture and the livers immediately removed.

Preparation of subcellular fractions

The livers after removal were washed in ice-cold homogenized medium (1.15% KCl). They were weighed, scissor-minced and homogenized in 1.15%KCl using a Potter- Elvehjem glass-Teflon homogenizer. The liver homogenate was adjusted so that 1ml=250mg of liver by the addition of 1.15%KCl. Homogenates were prepared by centrifugation at 10,000rpm for 20min. in a J2-21 Beckman centrifuge. The supernatant was stored at -80°C until required, without loss of original activity. The microsomes were prepared by centrifugation of the thawed samples at 45,000rpm for 60min. (60Ti rotor in a Beckman LK65 refrigerated ultracentrifuge) to obtain the microsomal pellet which was resuspended in the original volume of homogenizing medium prior to use.


Protein determination

The protein concentration was determined by Lowry et al. (1951) using bovine serum albumin as standard.

Cytochrome P450

Cytochrome P450 content was quantified in the microsomal fraction by the method of Omura and Sato (1964) using a difference absorption coefficient (450-490nm) of 91mM-1cm-1 for the sodium dithionite-reduced CO adduct.

Lauric acid hydroxylase

Lauric acid hydroxylase was measured using [14C]lauric acid essentially as described by Parker and Orton (1980). The incubation medium was applied to Merck silica gel TLC plates. The plates were developed in a hexane:diethylether:acetic acid system (70 : 28 : 1) by volume. The plates were scanned and the radioactivity quantified using a Berthold TLC plate scanner. The results presented are the combined w and w-1 hydroxylations of lauric acid, since the TLC method does not separate these two metabolites.

p-nitrophenol hydroxylase

The hydroxylation of p-nitrophenol was followed essentially by the method of Reinke and Mayer (1985) as modified by McCoy and Koop (1988). After a 3min. incubation of the reactants at 37°C in a shaking water bath, the reaction was initiated by the addition of 0.1ml of 10mM NADPH which was terminated after a further 10min. by the addition of 0.5ml. of ice-cold 0.6N percloric acid. Centrifugation at 300rpm for 10min. precipitated the protein. 0.1ml of 10M NaOH was added to 1ml of supernatant and the absorbance read at 536nm.

Erythromycin N-demethylase

The demethylation of erythromycin is followed by measuring the formaldehyde formed from the methyl group (Wrighton et al., 1985). Incubation was carried out for 10min. at 37°C in a shaking water bath after initiation of the reaction with 0.1ml of 10mM NADPH. The reaction was terminated by the addition of 0.5ml of ice-cold 12.5% (w/v) trichloroacetic acid. The tubes were then centrifuged at 3000rpm for 10min to remove the protein. 1ml of the supernatant was added to 1ml of Nash reagent. The tubes were heated in a water bath at 50°C for 30min. and after cooling the absorbance was read at 412nm.

Ethoxyresorufin O-deethylase

The O-deethylation of ethoxyresorufin was determined by the method of Burke and Mayer (1974), using the difference in fluorescent properties of ethoxyresorufin (excitation wavelength=456nm, emission wavelength=510nm) and the product resorufin (excitation wavelength=560nm, emission wavelength=586nm). The fluorimeter was set at excitation wavelength=510nm with a slit=10 and an emission wavelength=586nm with a slit=2.5.

Pentoxyresorufin O-depentylase

The method used is essentially that of Lubet et al. (1985). Principally, the same as the assay for ethoxyresorufin. The only difference is the amount of substrate used.

Cyanide-insensitive palmitoyl coenzyme A oxidation

The method of Bronfman et al., (1979) was adapted as follows. A reaction cocktail consisting of 75µM-coenzyme A (CoA), 180µM-FAD, 555µM-NAD+, 141mM-nicotinamide, 4.2mM-dithiothreitol, 3mM-potassium cyanide and 0.225mg BSA/ml in 60mM-Tris-HCl (pH 8.3) was freshly prepared and kept on ice. In addition 1% (v/v) Triton X-100 in 60mM-Tris-HCl (pH 8.3) and 7.65mM-palmitoyl CoA in 60mM-Tris-HCl (pH 8.3) were also prepared and kept on ice. The assay was carried out at 340nm at 37°C using a Konton Uvikon 860 spectrophotometer. Samples of whole homogenates were diluted 1:1 with 1% Triton X-100 and kept at 37°C. Reference and sample cuvettes were set up containing 2.0ml of the reaction cocktail, 0.94ml of 60mM-Tris-HCl buffer (pH 8.3) and 40µl of the diluted whole homogenate. The cuvettes were then placed in the spectrophotometer and a baseline recorded. 20µl of 7.5mM Palmitoyl CoA was then added to the sample cuvette and the change in extintion recorded for at least 5min. The activity was then calculated using a molar absorption coefficient of 6.22 x 10-3 mM-1 cm-1 NADH at 340nm. The homogenate was used for protein determination by the method of Lowry et al., (1951).

Statistical analysis
Statistical data evaluation was performed using the Student's t-test.

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