Implications
(i) Drug-drug interactions
(ii) Carcinogenesis
(iii) Cytotoxicity
Focus
(i) Inhibition of specific activities of cytochromes P450 (CYP1A, 2A6, 2C9, 2D6, 2E1, 3A4)
(ii) Induction/repression of CYP 1A
Models
(i) CYP activities in human liver microsomes
(ii) Hepatoma cell line HepG2 (alternative: primary rat hepatocytes)
Why effects on CYP enzymes?
Phase I biotransformation is frequently the decisive step in obtaining beneficial vs adverse effects of a substance. Cytochromes P450 (CYP) is a superfamily of enzymes responsible for catalysis of reactions, usually hydroxylation, turning a hydrophobic compound into a more water soluble substance which can be excreted. CYP enzymes are present predominantly in liver but some, particularly CYP 1A, are found in extrahepatic tissues as well. Inhibition of CYP enzymes may cause adverse effects by changing pharmacological profile of a simultaneously applied drug. Same result is observed if expression of a particular CYP enzyme is changed by the compound/extract under investigation. CYP enzymes, and CYP 1A in particular, were implicated in carcinogenesis because of metabolising procarcinogens, e.g. polycyclic aromatic hydrocarbons, into carcinogenic substances. Similarly, harmless compounds may be metabolised into cytotoxic agents causing cell damage and necrosis/apoptosis.
Assessment of interaction with CYP enzymes
Test:
(i) Effect of the test substance/extract/phytopreparation on activities liver CYP family from microsomes
Aim: To find out whether substances/extracts inhibit specific activities of CYP enzymes in human liver microsomes.
Procedure: The following activities will be used: CYP 1A, 7-ethoxyresorufin de-ethylation; CYP 2A6, coumarin hydroxylation; CYP 2C9, diclofenac 4´-hydroxylation; CYP 2D6, AMMC hydroxylation; CYP2E1, nitrophenol hydroxylation; CYP 3A4, testosterone 6b-hydroxylation. Inhibition will be compared to known inhibitors of corresponding activities.
Induction/repression of CYP 1A in living cells
Tests:
(i) Effect on expression of CYP in HepG2
(ii) Effect on expression of CYP in primary rat hepatocytes
Aim: To test whether substances/extracts affect expression of CYP 1A.
Procedure: Human hepatoma cell line HepG2 expressing inducible CYP1A1 will be treated with known inducers for comparison. Expression of CYP 1A will be evaluated by Western blotting and specific activity measured directly in whole cells. Rat primary hepatocytes expressing inducible CYP 1A2 will serve as an alternative model.