Literature data from in vitro and in vivo studies demonstrate that Cd cations induce an oxidative stress that results in oxidative deterioration of biological macromolecules. Cadmium depletes glutathione and protein-bound sulfhydryl groups, resulting in enhanced production of reactive oxygen species such as superoxide, hydroxyl radicals, and hydrogen peroxide. These reactive oxygen species result in increased lipid peroxidation, enhanced excretion of urinary lipid metabolites, modulation of intracellular oxidized states, DNA damage, membrane damage, altered gene expression, and apoptosis [35
The present study demonstrates that acute Cd exposure induces in vivo hepatic free radical generation as evidenced by POBN-trapped radical metabolites formed in the liver and excreted into the bile. ESR analysis of the POBN adducts in a bile sample has been established as a valid measure of radical production in the liver of intact animals, as with formate intoxication [27
] or aflatoxin B1-induced hepatotoxicity [36
], among many other similar examples. Thus, the ESR analysis of the bile samples in this study provides evidence for Cd-induced radical generation in vivo. The data showing altered radical production after treatment with various chemical agents known to affect Cd hepatotoxicity also support the mechanistic relevance of radical formation in in vivo hepatotoxicity in rats.
Cd is a redox-stable metal; therefore, radical production by Cd must be mediated through some indirect mechanism(s). One proposed mechanism by which Cd may generate free radicals is disruption of the cellular antioxidant defense systems. GSH is abundant in the liver and is thought to be the first line of defense against Cd hepatotoxicity as Cd binds tightly to thiol groups [37
]. It has been shown that depletion of hepatic glutathione by DEM significantly enhances Cd-induced mortality and hepatotoxicity [18
]. In the present study, GSH depletion by DEM markedly enhanced Cd-induced POBN radical adducts, consistent with its aggravation of Cd hepatotoxicity [18
]. In addition, GSH depletion reduced biliary excretion of Cd, thereby increasing its accumulation in the liver, where it could continue to exert its hepatotoxic effects. Consistent with the role of glutathione in Cd-induced free radical generation, the glutathione precursor N
-acetylcysteine is effective in the reversal of Cd-induced oxidative stress and protects against Cd-induced toxicity to the liver [39
] and kidney [15
]. The present work indicates that disruption of the cellular glutathione system is a key element in the mechanism of Cd-induced oxidative stress in the liver.
Another important mechanism of Cd-induced oxidative stress is thought to be mediated through the activation of Kupffer cells, the resident macrophages of the liver. Cd-activated Kupffer cells and/or neutrophils will then release inflammatory cytokines, such as tumor necrosis factor α, interleukin-1, interleukin-6, and interleukin-8 [40
]. Gadolinium chloride, an inhibitor of Kupffer cell function, protects against Cd hepatotoxicity in rats [32
] and in mice [33
]. The present study shows that gadolinium chloride decreases Cd-induced POBN radical adduct formation, suggesting that in vivo Cd-induced radical formation, or at least part of it, depends on Kupffer cell and/or neutrophil activation in the liver. The significant decrease in radical adduct formation from the reaction of hydroxyl radical with DMSO in vivo by GdCl3
indicates that Kupffer cells participate as the primary source of free radicals. This is the most realistic interpretation of our data, as Kupffer cells are located in the sinusoidal space, and it is possible that these radicals could be transported to the bile. We detected radicals in the bile, which allows us to assume the role of Kupffer cells in their production and not the effect of other macrophages. Moreover, others have demonstrated that reduction of the inflammatory response by the inhibition of Kupffer cell activation by GdCl3
plays a significant role in protecting against Cd hepatotoxicity [20
The iron chelator Desferal has been shown to decrease formate-induced [27
] and aflatoxin B1-induced [36
] radical adducts in rat bile, presumably by inhibition of iron-driven Fenton reactions in the liver. In the present study, Desferal significantly decreased radical generation induced by acute Cd exposure, suggesting the involvement of endogenous iron-dependent hydroxyl radical generation. Desferal has been shown to decrease Cd-induced lipid peroxidation and oxidative damage to the rat adrenal gland [34
]. Although Cd is not a redox-active metal and cannot directly participate in Fenton reactions, Cd could indirectly produce radicals by displacing iron or copper from their normal cellular sites of storage or use. In addition, released iron and/or copper could alter the functional status of Kupffer cells by enhancing their respiratory burst to produce oxidative stress [43
Other cellular oxidant components such as xanthine oxidase, cytochrome P450 oxidase, and catalase have also been proposed to play a role in Cd-induced oxidative stress [44
]. Indeed, the xanthine oxidase inhibitor allopurinol and the generalized cytochrome P450 oxidase inhibitor ABT have been shown to suppress formate-induced POBN radical adduct formation in formic acid-dosed rats [27
]. However, ABT did not prevent alcohol-induced liver damage in rodents [46
]. We attribute lack of effect of 3-AT on free radical generation by Cd to the fact that there are other enzymes such as glutathione peroxidases that are capable of metabolizing H2
. In the present study, none of these chemicals significantly altered Cd-induced generation of radical adducts in the bile, although they had a tendency to attenuate it. These results suggest that the roles of xanthine oxidase, cytochrome P450 oxidase, and catalase in Cd-induced hepatic radical formation are not significant.
Evidence for hydroxyl radical formation in vivo due to acute Cd exposure is provided by spin trapping of the methyl radical resulting from the scavenging of hydroxyl radical by DMSO (DMSO/·OH). The experiment with [13
C]DMSO confirmed that, indeed, a POBN/·CH3
radical adduct, whose source was a result of the reaction between DMSO and ·OH, and an additional unidentified POBN adduct, whose source was probably one or more endogenous lipid biomolecules, were being detected after Cd exposure. With LC/ESR in another study, two other POBN adducts, ·
OH and ·
, were identified in vitro. Both radical species were formed from DMSO in addition to the ·
]. The inhibitory effect of gadolinium chloride and Desferal on radical yields confirms a role for the hydroxyl radical in producing the species detected in the bile samples.
In this study, we propose that the formation of the POBN radical adducts is mediated by the hydroxyl radical, which, in turn, is generated through the reduction of hydrogen peroxide by a Cd-liberated transition metal, presumably iron. The present investigation demonstrates, for the first time, strong evidence for the formation of hydroxyl radical, through the detection of the POBN/·13CH3 radical adduct from [13C] DMSO, and lipid-derived radicals from enhanced lipid peroxidation presumably initiated by ·OH radical.
In vivo free radical formation by Cd requires involvement of both iron mediation through iron-catalyzed reactions and liver Kupffer cell activation, as both inhibitors, Desferal and GdCl3 inhibited the radical yield (). In addition, our experimental evidence indicates that glutathione and/or protein sulfhydryl depletion triggers free radical production to a much greater extent as shown in and .
Proposed pathway for free radical generation by Cd in an experimental rat model of cadmium overload.
In conclusion, in the ESR spectroscopic and spin-trapping studies presented here, we identify two radical species not known to be involved in the Cd stress response in vivo. Even though the mechanism for Cd-induced oxidative stress is complicated, it is at least, in part, closely correlated with depletion of cellular SH groups and/or glutathione. Another important factor is the minimization of POBN adduct formation in response to Kupffer cell inhibition and iron chelation, thereby suggesting a specific molecular mechanism involving stimulation of Kupffer cells by Cd and Fe participation. As discussed above, the identity of the POBN radical adducts generated from Cd in vivo seem to result from a combination of a methyl radical, from the reaction of [13C] DMSO with hydroxyl radical, and a lipid radical resulting from lipid peroxidation. These data support the suggested mechanism involving stimulation of Kupffer cells by Cd with Fe participation. One interesting issue brought up in this study is the question of how non-redox active metals may generate free radicals and induce oxidative stress. Even though Cd2+ is in a stable redox state, its ability to generate free radical species in an experimental animal model opens new avenues of research beneficial in medicinal applications to reactive oxygen species-associated environmental toxicity and diseases.