The results presented in this project show that the cyclic monoterpenes tested have a profound influence on the activity of pentoxyresorufin O-depentylase. Pentoxyresorufin O-depentylase is a selectively catalysed reaction of phenobarbital inducible cytochrome P450IIB1. This isoform is minor constitutive but highly inducible (Ioannides and Parke, 1990). Although P450IIB1 directs the overall oxidative metabolism of chemicals towards subsequent conjugation and detoxication, however, there are a few instances where induction leads to toxicity such as the activation of cyclophosphasmide, bromobenzene, carbon tetrachloride and aflatoxin. In the case of aflatoxin, the epoxide formed binds to N7 of guanine on DNA and RNA to form an adduct, which may be repaired or could lead to mutations. If the epoxides become substrates of epoxide hydroxylase then dihydrodiols will form eventually leading to the formation of aldehydes, which will then form a schiff base with amines on proteins.
From the results, it can be said that menthol and limonene oxide are highly potent inducers of P450IIB1. On one hand, they will be very useful in stimulating the detoxication of other chemicals. The other question which needs to be addressed is, what is their fate, since they will automatically become substrates for this increased enzyme activity? Will they follow the path of the aflatoxin so that although they help in the detoxication of other chemicals, they end up as toxic entities? Because menthol is an alcohol, c-hydroxylation of one of the carbons adjacent to the hydroxyl group will turn it into a dihydrodiol without the intervention of epoxide hydrolase. This could break up and form an an aldehyde which forms a schiff's base with proteins. Limonene oxide is an epoxide which when acted upon by epoxide hydrolase could end up the same way or bind to DNA and RNA on the N7 of guanine. Alpha-terpineol could easily form an epoxide and follow the same path.
What is interesting is the enantiomers of limonene. At P<0.05 (-)limonene induces P450IIB1 but not the (+) isomer. The (+) isomer instead increases the activity of P450IIE1 as compared to the (-) isomer. A close examination of figure 5 shows that the inductive capacity of the compounds for P450IIB1 is inversely related to their effect on P450IIE. Menthol being the highest for P450IIB1 and the lowest for P450IIE whilst (+)limonene is the lowest for P450IIB1 and the highest for P450IIE. The other compounds also follow this relative pattern. Apart from limonene, the other compounds are racemic. It will be interesting to find out how the enantiomers of the other compounds will behave in this assay system. The possible metabolic fate of d-limonene is shown in appendix C. There is some species differences but a good model for man is the guinea pig, since they both excrete (M-IV) as the major metabolite in the urine. Male rats have an abundance of x2u - globulin which reversibly binds d-limonene-1,2-oxide, leading to x2u - globulin nephropathy (literature survey). There is a very high significant difference in cyanide-insensitive palmitoyl-CoA oxidation which is an indication of peroxisome proliferation, from the administration of alpha-terpineol.
There is no significant difference in the activity of lauric acid hydroxylase, therefore, it can be said that alpha-terpineol may behave similarly to linalool by causing the induction of peroxisome enzymes but not cytochrome P450IVA1. The same dose was used for all the compounds but terpinene-4-ol killed all the rats, although it is included in the group LD50 of monoterpenes. The LD50 for this particular compound needs to be established with the appropriate acute toxicity studies.
In the case of accessing (+)limonene's induction of P450IIE by measuring the activity of p-nitrophenol hydroxylase, it might be possible that a type II error has been made in this case i.e. accepting the hypothesis that there is no difference between the means at P<0.05 when actually there might be a difference. There is a difference at P<0.1 which is not acceptable as a cut off point. The large variability as shown by the standard deviation may have just put the lower end of the test sample into the highr end of the control population. This experiment needs to be re-examined because this is the data for the repeat experiment. The first experiment which is not published showed a significant difference between the control and test.
In the present studies the compounds were administered in corn oil as the vehicle. Food flavours are normally added to food and many of these substances interact with components of food or other chemical substances in food. For example, the prenylation of coumarins can lead to furano or pyrano coumarins. A linear furano-coumarin like psoralen and xanthotoxin are phototoxic. Prenylation of isoflavonoids can form compounds like retonone which is an insecticide and fish poison. Prenalation of tryptophan can lead to the formation of indole alkaloids.