Introduction


It has been of some concern for sometime that food additives used with the intention to improve the quality of food and contaminants from packaging material migrants such as the phthalate diesters may have adverse effects on both man and animals. These fears surfaced mainly through research carried out on experimental animals. Diesters of phthalic acid are extensively used as plasticisers in various plastic formulations, including those used in food packaging.

The acute toxicity of the phthalate esters is low (Krauskopf, 1973; Shibko and Blumenthal, 1973) but chronic administration to rats leads to liver enlargement, changes in hepatic enzyme activity and in the case of di-n-butyl and di-(2-ethylhexyl)-phthalate(DEHP), to testicular atrophy (Carpenter et al., 1953; Lake et al., 1975; Cater et al., 1976; Gray et al., 1977; Brown et al., 1978). There is evidence that DEHP does produce liver tumours in rats and mice (Cohen and Grasso , 1981; Reddy and Lalwani, 1983) through a non-genotoxic mechanism for they give negative results in standard mutagenicity tests and there is no evidence of covalent binding to DNA (Reddy and Lalwani, 1983).

They have a similar mechanism of action to the hypolipidaemic drugs such as clofibrate by lowering serum triglyceride which has the indirect effect of causing a temporary increase in lipid deposition in the liver which then produces a pronounced proliferation of peroxisomes.

The acyclic monoterpenes, citral and linolool have been shown to cause peroxisome proliferation in the liver of rats ( Jackson et al., 1987; Roffey et al. , 1990) but only citral was found to induce cytochrome P450IVA1 (Roffey et al. , 1990).

There is no evidence suggesting a similar mechanism of action for the cyclic monoterpenes. Some of the toxic effects of the cyclic monoterpenes has been reviewed in the literature survey. Twenty-eight cyclic terpenes were studied by COMPACT (Computer Optimised Molecular Parametric Analysis for Chemical Toxicity) computer graphic analysis and none was likely to be a substrate of either the P450 I or P450 IIE1 isoenzymes (Lewis et al. , 1992), on the other hand their COMPACT - 3D data does make them possible substrate for the P450IIB, P450III and P450IV isoforms.

Based on these findings, six cyclic monoterpenes were selected for the present study in order to examine the above hypothesis further by the use of biochemical toxicological assays to find out which of the P450 isoforms are induced, what are the implications and how does it compare with the COMPACT studies, and if the results could be used in a structural / activity relationship in order to forcast the activity of other cyclic monoterpenes in the future. If the selected compounds could be identified as inducers of specific isoforms of the cytochrome P450 family, then the mode of action of the compound could be envisaged from the known properties of the specific isoform induced. For example, P450I has the ability to oxygenate molecules in conformationally hindered (bay-region) positions, thereby resulting in their activation (Ioannides and Parke, 1990). The enzyme is also induced by the inducers interacting with the Ah receptor, a steroid-like regulatory receptor (Nebert and Jones, 1989) which forms a receptor- inducer complex that migrates into the nucleus where it results in the production of new mRNA for cytochrome P450, for the Ah receptor, protein kinase c, a UDP-glucuronyl transferase, and other enzymes.

The optimum spatial conformation of the active site of P450I and the binding site of the Ah receptor have been shown to be very similar (Lewis et al., 1986), therefore, inducers of cytochrome P450I should also be potential substrates of this isoform.

P450IIE1 is a cytochrome P450 normally present in the high spin state, so that it can activate molecular oxygen in the absence of substrate, and hence generate superoxide anions and reactive oxygen species (ROS). Substrates of P450IIE1 are difficult to oxidatively metabolise, and are thought to undergo non-enzymic oxygenation by the enzymically-generated ROS ( Lewis et al., 1992).

P450IIB is generally known for detoxication. P450IV exhibits a high preference for hydroxylation of the terminal (w - n) methylene groups (literature survey), of saturated and unsaturated fatty acids, including derivatives such as the prostaglandins, thromboxanes and prostacyclins (Kupfer, 1980). They are also associated with hepatic peroxisome proliferation (Sharma et al., 1988).

P450III is involved in the activation of the dihydrodiol derivatives of polycyclic aromatic hydrocarbons to the ultimate mutagens and carcinogens (Shimada et al., 1989). Generally it is known to have affinity for large, nonplanar substrates such as the macrolide antibiotics, and is known to metabolise cyclosporin, ergotamine derivatives and some steroids. The assay mrethods for these selected P450 isoforms were used to investigate the inductive properties of the selected cyclic monoterpenes.