Carcinogenesis is a multistage process consisting of initiation, promotion, and progression. Protection against both the initiation and tumor promotion stages of carcinogenesis is one of the important findings obtained with phenolic antioxidants such as teas and rosemary (1,2).
Topical application of a methanol extract of rosemary, carnosol, or ursolic acid to mouse skin inhibited the covalent binding of benzo(a)pyrene [B(a)P] to epidermal DNA and inhibited tumor initiation by B(a)P and 7,12-dimethyl-benz(a)anthracene (DMBA) (19). Dietary intake of 0.5-1% crude rosemary extract by rats for 2-3 weeks before administration of DMBA reduced mammary gland tumor incidence and inhibited in vivo binding of DMBA metabolites to mammary epithelial cell DNA (41-43).
Many chemoprotective agents act through induction of Phase II detoxifying enzymes such as glutathione S-transferase (GST), NAD(P)H:qui-none reductase (QR), or UDP-glucuronosyltransferase (UGT) (44,45). In rats fed rosemary as 0.25-1.0% of their diet, the activities of liver GST and QR enzymes were induced 3-4-fold (46). Similarly, rats fed various (water or dichloromethane) extracts of rosemary at 0.5% of their diet for 2 weeks resulted in stimulation of GST, QR, and UGT enzyme activities (20,21). Surprisingly, in contrast to the results reported with ethanolic extracts in human cells (described below), the water extract of rosemary also induced the Phase I, cytochrome P450 (CYP450) enzymes. This difference in effect on CYP450 enzymes may be due to the composition of the water (rich in rosmarinic acid) and ethanolic (rich in carnosol and carnosic acid) extracts. Alternatively, it may be due to a species difference between rat and human CYP450 metabolism or to the generation of secondary metabolites in vivo with different effects from the intact molecules administered in in vitro studies.
Topical application of a methanol extract of rosemary, carnosol, or ursolic acid to mouse skin resulted in strong inhibitory effects on 12-O-
tetradecanoylphorbol-13-acetate (TPA)-induced inflammation, ornithine decarboxylase (ODC) activity, and tumor promotion, as well as on arach-idonic-acid-induced inflammation (19). Anti-tumor-promoting activity in mouse skin has also been reported for betulinic acid and oleanolic acid (17,18).
The antimutagenic (47) and antigenotoxic effects of rosemary components may be partly mediated by their antioxidant properties in scavenging free radicals involved in DNA oxidation. Indeed, carnosic acid, carnosol, rosma-rinic acid, and ursolic acid effectively inhibited DNA strand breakage induced by the Fenton reaction, suggesting effective scavenging of the hydroxyl radical (48), and an ethanolic extract of rosemary showed antigenotoxic effects against DNA damage induced by H2O2 in CaCo-2 colon cancer cells and in hamster lung cells V79 (49).
Modulation of metabolic enzymes involved in activation and detoxification of carcinogens is an important mechanism in chemoprotection. An ethanolic extract of rosemary extract, carnosol and carnosic acid, inhibited DNA adduct formation by the lung carcinogen B(a)P in human bronchial (BEAS-2B) cells through inhibition of the activity of the CYP450 enzyme CYP1A1, involved in the activation of B(a)P to its DNA-binding epoxide, benzo(a)pyrene-( + )-anti-7,8-dihydrodiol-9,10-epoxide (anti-BPDE) (50). Furthermore, carnosol (3 ||M) induced expression of the Phase II enzyme glutathione S-transferase k (GST k) involved in the detoxification of the proximate carcinogenic metabolite of B(a)P. Therefore, in human bronchial cells rosemary extract acts by a dual mechanism involving inhibition of Phase I-activating enzymes and induction of Phase II-detoxifyig enzymes. Similarly, in human liver cells expressing CYP1A2 or CYP3A4, rosemary extract inhibited the formation of DNA adducts by the epoxide of the mycotoxin aflatoxin B1 (AFB1) through inhibiting the enzyme activity of CYP1A2 and CYP3A4 (51). In the human liver, it is not clear how aflatoxin epoxide (AFBO) is detoxified, as none of the GSTs have a strong affinity for AFBO; therefore, inhibition of CYP450 metabolism may be the dominant mechanism of protection against AFB1-induced genotoxicity.
The enzyme NADPH:quinone reductase (QR) is important for detoxification of highly reactive quinone intermediates and has been associated with anticarcinogenic activity (45). Many chemoprotective agents induce QR by a transcriptional activation mechanism acting through upstream promoter sequences (52). Indeed, carnosol induced QR mRNA levels in human bronchial cells (50). Quinone reductase activity has been conveniently studied in the mouse hepatoma cell line Hepa 1c1c7 (53). Rosemary components effectively induce QR in these cells (54). In our studies, we found that QR activity was induced twofold by 1.0 ||M carnosol compared to 0.2 ||M sulforaphane, a component of broccoli known to be a potent inducer of QR.
Cellular mechanisms relevant to tumor promotion are affected by rosemary components. Growth inhibitory and differentiation effects of carnosic acid (2.5-10 |M) were shown in human leukemic cells (55). Carnosic acid inhibited cell proliferation through interference with cell cycle progression without induction of apoptotic or necrotic cell death. Low concentrations of carnosic acid significantly potentiated the action of the differentiation agents all-trans retinoic acid and 1,25-dihydroxyvitamin D3. In contrast, in another study with carnosol and leukemic cell lines, carnosol induced apoptotic cell death and downregulated Bcl-2 (56).
Nitric oxide (NO) is a small, short-lived molecule that is synthesized from L-arginine by NO synthase (NOS) and released from cells in response to a number of homeostatic and pathological stimuli (57). NO is involved in diverse physiological and pathological processes such as vasodilation, neurotransmission, inflammation and the immune response, platelet inhibition, cellular signalling, and free radical (peroxynitrite)-induced cytotoxicity and can be regulated by dietary factors (58,59). The inducible form of NOS (iNOS) is upregulated under inflammatory conditions and in response to cytokines, resulting in a relatively high and sustained level of NO production. Overproduction of NO may lead to production of damaging reactive nitrogen species such as nitrate, nitrite, peroxynitrite, and 3-nitrotyrosine with cytotoxic and genotoxic consequences (60,61). Carnosol has been shown to inhibit lipopolysaccharide and interferon-gamma induced NO production in activated mouse macrophages in a concentration-related manner (2-10 |M) (48,62). The mechanism involves inhibition of iNOS mRNA and protein expression by blocking activation of the transcription factor NF-kB through interference with the signal-induced phosphorylation of its inhibitor, IkB (48). The NF-kB family of transcription factors regulates the expression of many genes involved in immune and inflammatory responses. Therefore, inhibition of NF-kB activation provides a possible mechanism for the anti-inflammatory and anti-tumor-promoting action of carnosol.
The lipoxygenase pathways of arachidonic acid metabolism produce reactive oxygen species, which may play a role in inflammation and tumor promotion. Rosemary extracts carnosol and ursolic acid inhibited soybean 15-lipoxygenase activity (31). Since soybean lipoxygenase bears many similarities to the mammalian lipoxygenase enzyme (63), these results further suggest that rosemary components have the potential to inhibit lipoxygenase enzymes, which is another potential mechanism for the anti-tumor-promoting activity of rosemary components.
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