The activation of carcinogenic aromatic and heterocyclic amines and benzo[a]pyrene-7,8-diol to intracellular electrophiles by prostaglandin H synthase (COX) is well documented for ovine sources of this enzyme. Here, the arachidonic acid-dependent activation of substrates by human (h)COX-1 and-2 is examined, utilizing recombinant enzymes. The COX-dependent activation of benzidine (BZ), 4-aminobiphenyl, (+)benzo[a]pyrene-7,8-diol, (+)benzo[a]pyrene-7,8-diol, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3-methylimidazo [4,5-f]quinoline (IQ), 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP), and 4,4'-methylenebis(2-chloroaniline) (MOCA) is assessed by means of COX-catalyzed, covalent DNA binding. The hCOX isozymes activated all substrates tested, activation varied from barely detectable for IQ (0.76 and 1.52 pmol bound/mg DNA for COX-1 and -2, respectively) to a high of 65 and 117 pmol bound/mg DNA for COX-1 and -2, respectively, for the activation of MOCA. BZ, which is an excellent peroxidase substrate, did not exhibit high DNA binding levels in hCOX assays and this phenomenon was found to be due to high levels of binding to protein, which effectively competed with the DNA for binding in the assay. The demonstrated ability of the COX enzymes to activate a variety of environmental and dietary carcinogens indicates a potential role for COX in the activation pathway of aromatic and heterocyclic amines and polycyclic hydrocarbons at extra-hepatic sites during early or late stages of carcinogenesis.

2,4,6-Trichlorophenol (TCP) is an environmental contaminant that is toxic, mutagenic, and carcinogenic. We have investigated peroxidase-catalyzed oxidation of TCP as an alternative pathway of TCP bioactivation using horseradish peroxidase (HRP) as a model peroxidase. TCP was shown to function as a reducing substarte for HRP as evidenced by TCP-dependent, HRP-catalyzed reduction of 5-phenyl-4-penten-1-yl hydroperoxide (PPHP) to its corresponding alcohol. In addition, TCP was shown to undergo hydroperoxide (H2O2, ethyl hydroperoxide, or PPHP)-dependent metabolism as evidenced by electronic absorption spectroscopic analysis of reaction mixtures. A single major product was detected by reverse phase HPLC and was identified as 2,6-dichloro-1,4-benzoquinone (2,6-dichloro-2, 5-cyclohexadiene-1,4-dione, CAS no. 697-91-6) on the basis of electronic absorption spectroscopy, mass spectrometry, and cochromatography with synthetic standard. In addition, HRP-catalyzed oxidation of TCP yielded EPR-detectable phenoxyl radical intermediates whose EPR spectrum consisted of a 1:2:1 triplet characterized by proton hyperfine coupling constants aH(3,5) = 2.35 gauss. Mechanisms for the hydroperoxide-dependent, HRP-catalyzed oxidation of TCP are proposed that are consistent with these results.

Coffee drinking has been associated with reduced incidence of colorectal cancer, possibly via chemoprotection/modification of the metabolism of dietary heterocyclic amine carcinogens such as 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine (PhIP) by kahweol and cafestol palmitates (K/C), two components of unfiltered coffee. Using the PhIP-exposed male Fisher F344 rat as a model, K/C have been shown to reduce colonic PhIP-DNA adducts by > 50%. We have used the male F344 rat to investigate the effects of dietary K/C (0.02-0.2% as a 1:1 mixture) on the metabolism of PhIP by N-acetyltransferase- (NAT), sulfotransferase- (SULT), and glutathione-dependent pathways. K/C decreased hepatic NAT-dependent PhIP activation by up to 80% in a dose-dependent manner. Conversely, hepatic glutathione S-transferase (GST) activity/expression increased, e.g., 3-4 fold toward 1-chloro-2,4-dinitrobenzene (total activity), up to 23-fold toward 4-vinylpyridine (rGSTP1), and approximately 7-fold for rGSTA2 protein. These effects had fully developed after 5 days of the test diet and persisted for at least 5 days after withdrawal of K/C. Hepatic glutathione increased two- to threefold and this increase was more short-lived than other changes. K/C did not modify hepatic SULT activity or colon NAT and GST activities. Benzylisothiocyanate and black tea, which have also been shown to reduce the formation of PhIP-DNA adducts in this model, had little effect on hepatic NAT, SULT, GST, or GSH. In primary culture of rat hepatocytes, both kahweol and cafestol palmitates reduced NAT activity by 80%. In summary, the unique potential of K/C to convert rapid acetylators to a slow acetylator phenotype, accompanied by GST induction, might contribute to chemoprevention against cancers associated with heterocyclic amines.

The positive association of decreased risk of colorectal cancer with nonsteroidal antiinflammatory drug (NSAID) use, combined with the observation that cyclooxygenase(COX)-2 is present in a majority of colorectal tumors, has led to the proposed use of isozyme-specific COX inhibitors as preventive agents in polyp and tumor formation in the colon. However, the exact biochemical mechanisms and disease stage at which reduced risk is mediated remain somewhat controversial, in part because of the complex biochemical changes that occur during the progression from aberrant crypt to polyp to tumor. In this study, COX-1 and COX-2 protein expression levels were determined in sets of tumor and normal colon tissue. Changes were characterized in COX-1 and COX-2 expression within individuals, in relation to such factors as sex, tumor grade, and location in the colorectum. COX-1 expression levels were found to be significantly reduced in tumors compared to matched normal tissues (Dunn's method, P < 0.05). Additionally, COX-1 expression was decreased in stage T3 tumors as compared to stage T2 tumors (Student's t-test, P = 0.009). Similar to previous reports, COX-2 protein expression was present in 73% of the tumors studied and appeared to be independent of tumor grade and sex. Interestingly, decreased COX-2 expression correlated with tumor occurrence in rectal mucosa (Wilcoxon two-sample test, P < 0.05). These results warrant further investigation, especially the identification of determinants that would predict which populations would be most responsive to COX-2 inhibition as a means of colorectal cancer chemoprevention.