Curcumin was an effective inhibitor of iron toxicity in T51B cells. It prevented iron-induced ROS generation and signaling of iron to several oxidative stress responsive pathways. Based on its ability to bind iron, and on similar protective effects of iron chelating drugs such as desferoxamine, we predicted initially that curcumin would significantly reduce iron loading of the cells. This was supported by a recent report in the literature that curcumin lowered iron levels in mice (27
). However, we found that curcumin did not block iron uptake in T51B cells, even though we could demonstrate high affinity iron binding. In addition, the potency of curcumin as a protective agent in several assays was independent of the iron concentration. These observations indicate curcumin reduces the toxicity of intracellular iron by mechanisms other than preventing iron loading or depleting iron stores.
It remains possible that a direct interaction with iron contributes to curcumin’s effects, however. We did see evidence of a slight inhibition of the calcein-iron interaction by curcumin ( and ). Other support comes from comparing the effects of curcumin to those of Vitamin E, another antioxidant that does not bind iron. We observed little effect of 50 μM vitamin E on ROS generation by iron, but found an IC50 for jnk activation near 10 μM. We interpret this to mean that vitamin E acts most potently at some step downstream from ROS generation by iron but prior to jnk activation. In contrast, curcumin inhibited both ROS generation and jnk activation with an IC50 between 5 and 10 μM. This indicates curcumin interacts directly with some component of the ROS generating reaction, quenching at the earliest possible step.
Optimal protection against iron toxicity is predicted from an agent that (i) binds iron, (ii) prevents redox cycling of iron, and (iii) independently quenches free radicals formed by iron (48
). Since iron chemistry is biologically critical, however, an agent that is too effective may itself be toxic. With these criteria in mind, three properties of curcumin contribute to effective neutralization of iron toxicity. First, curcumin is a hydrophobic molecule that freely enters cells. This was demonstrated in our study by curcumin’s ability to block the rapid generation of ROS and activation of oxidative stress pathways caused by iron and 8-hydroxyquinoline. Second, curcumin binds iron with relatively low affinity (near 1 μM) compared to biological transport and storage proteins (27
). Because of this, curcumin-bound iron retains its bioavailability and may be readily transferred to ferritin. One curcumin molecule may undergo many cycles of binding iron, releasing it to ferritin, and then binding more iron. Third, curcumin has potent antioxidant and free radical quenching activity (12
). Others have also shown the redox activity of iron is significantly reduced by curcumin (49
). Many small molecule iron ligands, including citrate and ascorbate, do not quench the redox activity of bound iron, and may even increase it by catalyzing redox cycling. Conversely, iron is not redox active when bound to transferrin, ferritin, or chelating drugs such as desferoxamine. Curcumin may share this important iron detoxifying property, as suggested by our results using the H2DCFDA ROS assay. We speculate that curcumin’s cell permeability, moderate affinity for iron, and unique antioxidant properties all contribute to prevention of iron toxicity.
Curcumin may inhibit other biochemical targets in T51B cells. A number of curcumin-sensitive enzymes and pathways have been identified in other systems (12
). These include: (i) cyclooxygenase (COX)-1 and -2, (ii) inducible nitric oxide synthase (iNOS), (iii) AP-1 and NF-kappaB transcription factors, (iv) growth factor receptors, and (v) thioredoxin reductase (51
). Reported pro-oxidant activity (52
) and induction of phase II drug metabolizing enzymes (55
) represent additional mechanisms in some cell types. The importance of specific enzymatic targets for inhibition of some cellular processes is underscored by the finding that curcumin analogs lacking antioxidant activity were still effective inhibitors of NF-kappaB and AP-1 activation in screening assays (58
). These reports suggest that curcumin may have effects downstream of iron in our experiments. Indeed, the available evidence suggests that curcumin acts by multiple mechanisms (17
). We propose that an additional mechanism involves transient binding and redox quenching of intracellular free iron, and envision a model in which curcumin functions as a sink for free iron and/or for iron-induced free radicals in cells.
Although curcumin is exceedingly safe in humans, it is widely acknowledged to have poor bioavailability, making the in vivo
relevance of our findings uncertain. However, although curcumin is rapidly metabolized and poorly absorbed, it has still been shown to be effective in many animal studies (12
). Some of its effects may be mediated by its metabolites (60
). Our studies were routinely done with 50 μM curcumin, since that concentration was consistently maximally effective. We also observed significant effects at lower concentrations, with IC50’s between 5 and 10 μM for the different assays. This approaches plasma concentrations reported in animal studies (12
). Importantly, a physiologically relevant effect may not depend on complete block of iron toxicity. A related concern is that liver damage in humans results from decades of iron overload at blood levels of iron citrate that do not exceed 10– 20 μM (9
), below the FAC concentrations studied here. However, given that curcumin binds iron with an affinity near 1 μM ( and ref 26
), it is reasonable to expect that a lower curcumin concentration would still protect against toxicity that accompanies iron citrate concentrations measured during iron overload in humans. These points all argue that our observations reveal effects that are relevant to what may occur in humans taking curcumin. Curcumin may be a safe and effective alternative for management of liver toxicity in diseases of iron overload. Based on results presented in the present study, there is little risk of iron depletion in cells or animals given curcumin for extended periods. This supports the feasibility of future studies to determine whether curcumin prevents pathologies stemming from more prolonged iron loading, including neoplastic transformation and liver cancer.