Effects of monoterpenoids on in vivo DMBA-DNA adduct formation and on phase I hepatic metabolizing enzymes.
We have previously demonstrated the anticarcinogenic effects of monocyclic monoterpenes such as limonene when given during the initiation phase of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary cancer in Wistar-Furth (WF) rats. Here we investigated the possible mechanisms for this chemoprevention activity including limonene's effects on DMBA-DNA adduct formation and hepatic metabolism of DMBA. Twenty-four hours after carcinogen administration, there were approximately 50% of the total DMBA-DNA adducts found in control animals formed in the liver, spleen, kidney and lung of limonene-fed animals. While circulating levels of DMBA and/or its metabolites were not different in control and limonene-fed rats, there was a 2.3-fold increase in DMBA and/or DMBA-derived metabolites in the urine of the limonene-fed animals. Studies of the effects of limonene and sobrerol, a hydroxylated monocyclic monoterpenoid with increased chemoprevention activity, on phase I metabolizing enzymes revealed that these terpenoids modulated cytochrome P450 (CYP) and epoxide hydratase (EH) activity. The 5% limonene diet increased total CYP to the same extent as phenobarbital (PB) treatment when compared to control, while 1% sobrerol (isoeffective in chemoprevention to 5% limonene) did not. However, both 5% limonene and 1% sobrerol diets greatly increased the levels of microsomal EH protein and associated hydrating activities towards benzo[a]pyrene 4,5-oxide when compared to control and PB treatment. These changes also modified the rate and regioselectivity of in vitro microsomal DMBA metabolism when compared to PB treatment or control. Identification of the specific isoforms of CYP induced by these terpenoids was performed using antibodies to CYP isozymes in Western blot analysis and inhibition studies of microsomal DMBA metabolism. Five per cent limonene was more effective than 1% sobrerol at increasing the levels of members of the CYP2B and 2C families but was equally effective at increasing EH. Furthermore, both terpenoid diets caused increased formation of the proximate carcinogen, DMBA 3,4-dihydrodiol. While these terpene-induced changes in hepatic CYP and EH do not explain the anticarcinogenic mechanism of these chemopreventive agents, or the ability of limonene systemically to reduce DMBA-DNA binding, they do reveal novel and selective induction mechanisms of hepatic enzymes.
Department of Human Oncology, University of Wisconsin-Madison 53792.
T H
TH
Maltzman
M
M
Christou
M N
MN
Gould
C R
CR
Jefcoate
D015127:9,10-Dimethyl-1,2-benzanthracene / Q000494:pharmacology*; D000818:Animals; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D053138:Cyclohexenes; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis; D004247:DNA / Q000187:drug effects*; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004851:Epoxide Hydrolases / Q000032:analysis; D005260:Female; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D000077222:Limonene; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D010634:Phenobarbital / Q000494:pharmacology; D051381:Rats; D011920:Rats, Inbred WF; D013729:Terpenes / Q000494:pharmacology*
D053138:Cyclohexenes; D013729:Terpenes; D015127:9,10-Dimethyl-1,2-benzanthracene; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D000077222:Limonene; C003316:sobrerol; D004851:Epoxide Hydrolases; D010634:Phenobarbital