Mechanism-based inactivation of hepatic ethoxyresorufin O-dealkylation activity by naturally occurring coumarins.

Several naturally occurring coumarins contained in the human diet have been found to be effective inhibitors and inactivators of murine hepatic ethoxyresorufin O-dealkylase (EROD) and pentoxyresorufin O-dealkylase in vitro [Cai, Y., Bennett, D., Nair, R.V., Ceska, O., Ashwood-Smith, M., and DiGiovanni, J. (1993) Chem. Res. Toxicol. 6, 872-879]. In the present study, these same coumarins decreased the content of cytochrome P450 (P450) in either 3-methylcholanthrene (MC)- or phenobarbital-induced murine hepatic microsomes but did not have a major effect on heme content. Detailed in vitro studies with [14C]coriandrin, which selectively inhibits and inactivates P450 1A1-mediated EROD activity, demonstrated that it covalently bound, in a preferential manner, to hepatic microsomal protein from MC-pretreated mice. A linear relationship was observed between covalent binding and loss of EROD activity. The inclusion of electrophile trapping agents in the incubations significantly inhibited the covalent binding of [14C]coriandrin to microsomal protein. In addition, the covalent binding of [14C]coriandrin was decreased 46% by 7,8-benzoflavone (7,8-BF), 58% by a monoclonal antibody with specificity toward MC-induced form(s) of P450, and 60% by ethoxyresorufin, implicating the bioactivation of coriandrin by P450 1A1. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [14C]coriandrin-bound microsomal protein from MC-pretreated mice showed that [14C]coriandrin bound covalently to a protein with an approximate molecular mass of 49 kDa. Again, addition of 7,8-BF or polyclonal antibody against P450 1A1 reduced the covalent binding of [14C]coriandrin to this specific protein band. Interestingly, coriandrin was also found to be a potent inhibitor and inactivator of purified human P450 1A1. These results demonstrate that certain coumarins to which humans are exposed in the diet are bioactivated by P450 1A1 to reactive intermediates that subsequently form covalent adducts with the apoprotein, effectively destroying enzyme activity. Thus, certain naturally occurring coumarins may have a significant effect on human health.

Inhibition and inactivation of murine hepatic ethoxy- and pentoxyresorufin O-dealkylase by naturally occurring coumarins.

The present study was designed to evaluate the effects of a series of natural coumarins on ethoxyresorufin O-dealkylase (EROD) and pentoxyresorufin O-dealkylase (PROD) activities in vitro using hepatic tissues from SENCAR mice. Fifteen different coumarins were examined for potential modulating activities. Several naturally occurring coumarins, found in the human diet, were effective inhibitors of hepatic EROD activity in vitro, including coriandrin, bergamottin, isoimperatorin, and ostruthin. Notably, coriandrin and bergamottin were approximately as potent as 7,8-benzoflavone, a relatively selective inhibitor of cytochrome P450 1A1. Several naturally occurring coumarins were also potent inhibitors of hepatic PROD activity, including imperatorin, bergamottin, isopimpinellin, and angelicin. Kinetic studies of the type of inhibition revealed that these compounds inhibited hepatic EROD and PROD activity by a variety of modes rather than by a uniform one. Furthermore, experiments using a two-stage incubation assay revealed that coriandrin, imperatorin, ostruthin, and several other natural coumarins inactivated hepatic EROD activity (i.e., predominantly cytochrome P450 1A1-mediated) and that isopimpinellin inactivated hepatic PROD activity (i.e., predominantly cytochrome P450 2B1-mediated). Finally, the results indicate that some coumarins had selective inhibitory effects for EROD vs PROD and preliminary analyses suggested a possible structural basis for the observed differences. The current data suggest that certain naturally occurring coumarins, to which humans are exposed in the diet, are potent modulators of cytochrome P450. Furthermore, these compounds may be capable of influencing the metabolic activation of other xenobiotics, including chemical carcinogens.

The photobiological activity of 5-geranoxypsoralen and its photoproducts.

5-geranoxypsoralen (Bergamottin) does not photosensitize bacteria or a bacterial virus. It does, however, photosensitize mammalian cells in tissue culture. Irradiation with either black light (300-400 nm) or fluorescent ceiling lights produced at least four photobiologically active degradation products, the chemical nature of which still remains to be elucidated. Prolonged exposure to black light resulted in the formation of inactive molecule(s).

Photobiological properties of a novel, naturally occurring furoisocoumarin, coriandrin.

The photobiological properties of a novel, naturally occurring furoisocoumarin isolated from coriander and named coriandrin are described. Photosensitized lethal and mutagenic effects in bacteria indicate that it is more active than psoralen. It is a weak frameshift mutagen in the dark. Mammalian cells in tissue culture are photosensitized more actively with coriandrin than with psoralen even though preliminary evidence from interrupted radiation experiments and DNA analysis suggest that coriandrin does not form DNA interstrand crosslinks. Sister chromatid exchanges were induced with a unit dose of 1.1 x 10(-2) with coriandrin; the value for psoralen is 3 x 10(-3). Coriandrin appears to be metabolized more rapidly than furocoumarins by liver mixed function oxidases. Skin photosensitizing activity is very weak compared with psoralen, a surprising observation considering its potency in biological test systems.

Oxypeucedanin, a Major Furocoumarin in Parsley, Petroselinum crispum.

Fresh parsley leaves and roots were analyzed by HPLC and photobiological assay for photoactive furocoumarins. Oxypeucedanin ( 7), not previously reported from parsley, was found to be the major component (70-100 ppm wet weight). Although only moderately photoactive, its high concentration in parsley may be partially responsible for contact photodermatitis. Other photoactive compounds, namely 5-MOP ( 2), 8-MOP ( 3), psoralen ( 1), isopimpinellin ( 4) and imperatorin ( 5) were also present and quantified.

Detection of furocoumarins in plants and plant products with an ultrasensitive biological photoassay employing a DNA-repair-deficient bacterium.

The application of an ultrasensitive photobiological assay which detects photosensitizing furocoumarins with sensitivities as high as 1 × 10(-11) g is discussed in relation to these molecules as phytoalexins. Examples of the utilization of this technique, verified by both HPLC and TLC, are the analyses of healthy and diseased celery and carrots, dry seeds, plant extracts and oils, and whole plants and leaves. The usefulness of this method in following the metabolic detoxification of furocoumarins is also illustrated. The extreme sensitivity of the test has permitted the detection, for the first time, of both 5-methoxypsoralen and 8-methoxypsoralen in fresh carrot roots.