Evidence from animal as well as epidemiological studies has shown that DHA and other ω-3 PUFAs possess cancer chemopreventive effects, yet earlier studies have shown that ω-3 PUFAs generate more DNA damage, such as Acr-dG, than ω-6 PUFAs or saturated fatty acids (13
). A possible explanation that we propose and study here is that specific DNA damage may contribute to the initiation of DHA induced apoptosis and cell cycle arrest, and thus protect the cells against carcinogenesis. Several other possible mechanisms have been suggested for the apoptotic effects of DHA and other ω-3 PUFAs, including prostaglandin metabolism by cyclooxgenase and lipooxygenase, nuclear receptors such as peroxisome prolifirator-activated receptors and PUFAs’ membrane altering effects (6
). Protein modification by enals and other PUFA oxidative products has also been shown to play a role in ω-3 PUFAs induced apoptosis (42
). It is conceivable that more than one mechanisms are likely involved in ω-3 PUFAs induced apoptosis.
Although extensively studied, the mutagenic potential of Acr-dG is uncertain due to conflicting results (17
). Other related cyclic DNA adducts from ω-6 PUFAs, specifically HNE-dG and the etheno adducts, have been shown to be mutagenic (47
). Because of its relatively high levels of modification induced by ω-3 PUFAs, Acr-dG formation in cellular DNA could be one of the triggers in apoptosis. Here we investigated whether Acr-dG and 8-oxo-dG, two major in vivo
DNA lesions induced by DHA and other ω-3 PUFAs, play a role in this process. This study showed that DHA is a potent inducer of apoptosis in HT-29 cells compared with AA and LA. We also found that the apoptotic responses in cells treated with various concentrations of DHA, AA or LA are in parallel with Acr-dG formation, but not with 8-oxo-dG, in DNA. Furthermore, we demonstrated that BSA can block the apoptosis in cells treated with DHA with simultaneous decrease in Acr-dG formation, by trapping Acr to form protein carbonyls before it reacts with nuclear DNA. Additionally, our results showed that incubation of Acr induced apoptosis and Acr-dG formation in HT-29 cells. Together, the results established a close correlation between apoptosis and Acr-dG formation in HT-29 cells, suggesting a potential role of Acr-dG in the apoptosis induced by DHA.
Our data showed the same patterns of dose-response in apoptosis induction and Acr-dG formation in cells treated with DHA, suggesting that the apoptotic response could be initiated only when Acr-dG adduct levels in cells reach a threshold of around 300nmol Acr-dG/mol dG, which coincide with the data from the Acr treatment experiments where the apoptosis was initiated when Acr-dG levels reached and passed around 500nmol Acr-dG/mol dG. The lack of dose-response in Acr-dG formation with the lower concentrations of DHA () may be explained by efficient repair of Acr-dG. Acr-dG can be readily removed through the nucleotide excision repair (NER)-mediated pathway similar to that for HNE-dG (52
). DNA repair can act as genome ‘caretakers’ to repair and remove DNA damages, thus maintain the genomic integrity. However, new evidence shows that they can also act as ‘gatekeepers’ reacting to DNA damage by themselves or through interacting with other molecules to initiate cell cycle arrest and apoptosis pathways when certain DNA damage reaches a threshold level (26
). NER has been shown to be involved in transcription-coupled DNA damage sensing and signaling, whereas mismatch repair in DNA replication-related signaling recognition (24
Our data suggest that when the Acr-dG and perhaps DNA strand breaks induced by DHA reached a certain threshold level beyond the capability of the NER and other pathways, cells may undergo cell cycle arrest or initiate the apoptosis signaling pathways. More studies are needed in order to determine the roles of the NER repair in DHA induced apoptosis. Nevertheless, our results suggest that NER repair and NER engaged transcription-coupled DNA damage signaling pathway may be involved in the apoptotic response to the Acr-dG formation and other possible oxidative DNA damage caused by DHA. In addition, the apoptosis induced by DHA is likely via a p53-independent pathway because HT-29 cells express mutant p53. Our preliminary experiments with HCT116 and p53 knocked out HCT116 cells also showed that there is no difference in apoptosis responses from these two cell lines treated with DHA (data not shown).
It should be noted that the concentrations of fatty acids used in this study are considerably higher than the in vivo
intracellular free fatty acid concentration generally reported (<10µM) (56
). However, the concentrations may still be relevant with the consideration of the following factors: 1) once in the cell, most fatty acids non-covalently bind to various fatty acid binding proteins, they are enzymatically converted into fatty acyl-CoA-thioesters (FA-CoA). FA-CoA level is highly variable and dependent on the cell examined. For example, in liver FA-CoA can range from 110~152µM (56
); 2) the serum lipid level is relatively high as the average free fatty acids concentration in the post-absorptive stage is reported to be 0.7mM, and this level can be much higher during the absorptive phase following ingestion of a meal rich in fat. Other factors including age, gender, diet and genetic background can also affect the expression and regulation of genes involved in fatty acid metabolism and, therefore, its concentrations (57
); and 3) DHA in the culture media may be oxidized and its oxidative products can readily react with proteins in the media resulting in the reduced effective intracellular DHA concentration.
It was interesting to observe that the addition of BSA to the cell culture media led to decrease of DNA adduct formation and apoptosis, since BSA and other fatty acid binding proteins have been widely used to facilitate fatty acid absorption and transportation by cells. Our results suggest that because BSA and other fatty acid binding proteins can readily react with certain reactive oxidative species, the oxidative effects of PUFAs on DNA and other proteins in cells may be altered. Caution needs to be exercised to interpret the data on oxidation when BSA and other fatty acid binding proteins were used.
A recent study showed that the accumulation of 8-oxo-dG in nuclear and mitochondrial DNAs can lead to a buildup of the mismatch repair mediated DNA single strand breaks, that trigger two distinctive apoptotic pathways (58
). While our study showed that Acr-dG may play an important role in apoptosis induced by DHA, it is possible that 8-oxo-dG and maybe other types of DNA damage including the etheno adducts in cells treated with DHA could contribute to DNA strand breakage due to effective repair. It is therefore reasonable to assume that a variety of DNA damage could collectively be responsible for DHA-induced apoptosis. Nonetheless, our studies demonstrated a clear correlation between Acr-dG formation and apoptotic response in human colon HT-29 cells, and thus shed light on a potential role of Acr-dG adduct formation in the protective effect of DHA against tumorigenesis.