Biotransformation of terpenoids in rats : studies on the pulmonary mixed function oxidase systems

Abstract

Metabolic fate of natural products in mammals has been of interest since the beginning of this century. Terpenoids, one of the largest groups of naturally occurring compounds, are components of many commonly used food additives, cosmetics, etc. In vivo metabolism of several of these compounds has been reported earlier. However, there are only a few reports on the details of the enzyme systems involved in their metabolism, although the liver has been implicated as a possible site of metabolism. Since the logical target tissue for volatile compounds is the lung, it is reasonable to expect metabolism of terpenoids in pulmonary tissue as well. However, involvement of pulmonary tissue in terpenoid metabolism has not been documented before. The present study encompasses different aspects of terpenoid metabolism in the mammalian system. Acyclic monoterpene alcohols are widely distributed in nature, and living systems are frequently exposed to them. Since only scanty information exists on their mode of metabolism in mammals, studies on the biotransformation of representative acyclic monoterpene alcohols in rats were undertaken. Studies carried out with geraniol revealed the formation of 8 hydroxygeraniol, 8 carboxygeraniol, Hildebrandt’s acid, geranic acid, and 3 hydroxycitronellic acid. The products identified from the urine extract of linalool fed rats were 8 hydroxylinalool and 8 carboxylinalool. Prolonged administration of these terpenoids by the aerosol route resulted in the induction of microsomal enzymes in both lungs and liver. However, the effects on hepatic microsomal enzymes were more pronounced following administration of the terpenoid by gastric intubation. A comparative study of liver and lung microsomal enzymes was carried out in detail. Microscopic examination of geraniol treated tissues showed pathological changes in both lung and liver at the end of pretreatment by inhalation and ingestion for ten days, respectively. Electron microscopic studies of the exposed hepatic tissue also indicated morphological changes. The present study, for the first time, clearly demonstrated that rat pulmonary microsomes effectively transform acyclic monoterpene alcohols to their respective ( )-hydroxylated products. The microsomal terpene hydroxylase was shown to be induced by pretreating rats with naphthoflavone ( NF) and not by phenobarbital (PB). This observation suggested the involvement of a cytochrome P 448-mediated mixed function oxidase in the hydroxylation reaction. Inhibitor studies further supported the involvement of a P 448 system. Rat kidney microsomes exhibited lower hydroxylase activity than lung microsomes. The NF-treated microsomal enzyme system was also capable of hydroxylating other acyclic monoterpene alcohols such as linalool, citronellol, and nerol. The NF-induced pulmonary microsomal hydroxylase was solubilized, resolved, and partially purified into its components-cytochrome P 448 and NADPH-cytochrome c (P 448) reductase. With partially purified cytochrome P 448 and reductase, hydroxylase activity was reconstituted in the presence of dilauroylphosphatidylcholine. Some properties of the NADPH-cytochrome c (P 448) reductase were studied to compare it with other biological systems. The in vivo metabolism of the pharmaceutically important monoterpene ether 1,8 cineole resulted in the formation of its 2 and 3 hydroxy derivatives, along with 1,8 dihydroxy 10 carbomethoxy p menthane. Administration of this terpenoid resulted in a significant increase in hepatic cytochrome P 450 levels when given by aerosol. Pulmonary geraniol hydroxylase was marginally enhanced, and cytochrome P 450 levels in the lung remained unchanged. The acyclic sesquiterpene alcohol nerolidol bears structural similarity to the monoterpene alcohol linalool. Since linalool metabolism was studied in rats earlier, it was of interest to establish the metabolic fate of nerolidol. Metabolites from the urine of nerolidol treated rats showed the presence of 3,7,11 trimethyl 3,11 diol 1,6 dodecatriene and 3,7,11 trimethyl 3,7,11 trihydroxydodec 1 ene, the structures of which were confirmed by NMR spectroscopy. In conclusion, the role of the pulmonary mixed function oxidase system in the biotransformation of acyclic monoterpene alcohols has been emphasized, and the significance of their metabolic pathways has been discussed in relation to different life forms.