Our knowledge of molecular mechanisms associated with both HD- and CEES-induced skin injuries, e.g. inflammation and toxicity is limited. This could be attributed, in part, to the inadequacy of efficient animal models for evaluation of these mechanisms. This has deterred the development of effective medical countermeasures and treatments against HD-mediated skin injury. In the present study, we show the involvement of oxidative stress and subsequent roles of MAPKs and Akt signaling pathways followed by transcription factors AP-1 and NF-κB in CEES-induced inflammatory response and skin injury in single efficient SKH-1 hairless mouse skin model.
Apart from direct alkylation, reported to be the major mechanism leading to HD-caused toxicity, various studies have indicated that another important mechanism involved in the toxicity caused by HD is oxidative stress that can operate via various signal transduction pathways, and can also cause alkylation [5
]. Most previous reports indicate formation of sulfonium and carbenium ions by HD that leads to protein, lipid and DNA alkylation provoking the activation of PARP that causes the rapid depletion of NAD+/ATP leading to cell death [1
]. Recent reports have indicated that HD-induced ROS generation can be involved in alkylation as well as induce GSH depletion that leads to lipid peroxidation, apart from possible protein oxidation and DNA injury [14
CEES, a monofunctional and less toxic analog of HD employed in this study, has been most widely exploited as valuable laboratory experimental tool to gain insight into the mechanism of action of HD [10
]. Although, many reports have indicated the involvement of oxidative stress in HD/CEES-induced skin and other tissue injuries [1
], most studies have been carried out in in vitro
systems, and HD/CEES dose- and time–dependent changes in lipid peroxidation as well as protein and DNA oxidation in a suitable animal model have not been reported.
The most important feature of oxidative damage via stress signals including UVB radiation are oxidation of biomolecules including lipids, proteins and DNA [41
]. Lipid peroxidation generates numerous cytotoxic degradation products such as malondialdehyde (MDA) and 4-HNE [34
], and these degradation products can form covalent adducts with proteins, phospholipids and DNA in the compartments of the cells. [42
]. Our findings show significant increase in 4-HNE-protein adducts as early as 3 h in CEES-exposed skin tissue that sustained up to 168 h and reflect increased oxidative modification of tissue proteins in vivo
(). Moreover, relatively few distinct bands on immunoblots suggest that only a small number of specific proteins are the major targets of 4-HNE conjugation in CEES exposed skin. The formation of these adducts in cell compartments also suggests that significant proportion of these toxicants escape detoxification in an in vivo
system to form adducts with tissue macromolecules. This study provides the first direct in vivo
demonstration that suggests that significant lipid peroxidation and protein modification occurs after CEES exposure in mouse skin. As known that GSH plays an important role in lipid peroxidation [44
], our results also indicate that the depletion of GSH by CEES could be a key initial mechanism in CEES-induced skin toxicity.
CEES-induced oxidative damage of biological molecules and phosphorylation/activation sequence of key signaling pathways in SKH1 hairless mouse skina
Spin trapping using DMPO has been used earlier to detect superoxide and hydroxyl radicals via adduct formation in EPR spectra and also DNA damage caused by toxicants. In this study, we employed an improved spin trap immunoassay [35
] for detection of DMPO nitrone protein adducts to identify the free radicals formed on CEES exposure of skin tissue. Our results show a significant increase in the DMPO nitrone-protein adduct formation as early as 3 h in the CEES-exposed skin tissue and reflects increased formation of superoxide and hydroxyl radicals ().
Oxidation of amino acids (lysine, arginine, and proline), leads to the formation of carbonyl derivatives that influence the biological activity of native proteins in biological systems [45
]. In different in vivo
models, a variety of studies have utilized the presence of carbonyl groups as a measure of the oxidative damage of proteins. Oxidative stress conditions allow carbonyl groups to react with DNPH and form stable hydrazone derivatives [46
]. In the present study, CEES exposure caused an increase in the levels of protein carbonyl groups in mouse skin both in dose- and time-kinetic studies indicating in vivo
In this study, we also demonstrated DNA oxidation in 2 mg CEES-exposed skin tissue as had been recently shown in CEES-treated human bronchial and small epithelial cells [38
]. It is interesting that both in the present study and in the recent study of Gould and co-workers; detection of CEES-mediated oxidative stress was delayed and peaked at 12 h post CEES exposure. These data are consistent with the beneficial effects that antioxidants have been reported to have in CEES and HD animal models in the lung, and support their use in skin injury as well. Present study indicates that CEES-induced oxidative stress is perhaps the central feature leading to lipid peroxidation as well as protein and DNA oxidations that occur within 3 h of CEES exposure (), and plays a key role in the activation of signaling pathways and CEES-induced skin inflammation, further supporting the antioxidant therapy.
Herein, our results show that topical exposure of SKH-1 hairless mice skin to CEES resulted in marked phosphorylation of MAPKs in both dose- and time-response studies, indicating activation of MAPKs signaling pathways including ERK1/2, p38, and JNK (). These results are in agreement with a previous finding that MAPK pathways are activated by HD/CEES in keratinocytes [11
] and in lungs of male guinea pigs [48
], and that the inflammatory cytokines, growth factors, and oxidative stress are the stimuli for the activation of JNK and p38 [49
]. The fact that CEES is capable of inducing an inflammatory response [16
], makes it very plausible that CEES activates not only JNK and p38 pathways predominantly involved in response to oxidative stress, but also ERK, resulting in or contributing to, inflammation. The proteins of the MAPK family are reported to stimulate NF-κB activation [50
]. The CEES-induced activation of all three MAPK signaling pathways shown here, foremost in an in vivo
skin toxicity model, could be responsible for the reported increases in the pro-inflammatory mediators, activation of NF-κB and induction of metalloproteinases (MMPs) [1
]. Therefore, MAPK signaling pathways could be key components in contributing to the CEES-induced skin inflammation observed in dose- and time response-studies in this established mouse model [16
Akt/protein kinase B (PKB) plays a crucial role in several processes associated with survival and apoptosis [20
]. Several studies have shown that the kinase activity of Akt is dependent upon the phosphorylation of Akt at Thr308 by PDK1 and by phosphorylation within the carboxy-terminus at Ser473 [19
]. Consistent with these reports, the present study also shows CEES-induced phosphorylation of Akt at Ser473 and Thr308, indicating activation of Akt pathway in mouse skin tissue (). On the other hand, PDK1, the upstream effector of Akt engaged in responses to stress and in growth factor signaling, potentially activates Akt and protein kinase C isoenzymes [52
]. Our results indicate that increased phosphorylation of PDK1 might be the key factor for the CEES-induced Akt activation. Akt pathway engages in the activation of various transcription factors such as NF-κB and AP-1 [19
] and therefore, suggestively plays an important role in CEES-induced activation of these transcription factors and subsequent inflammatory responses.
In this study, we further examined whether CEES-induced oxidative stress can stimulate inflammatory responses via transcription factors AP-1 and NF-κB. MAPK pathways are shown to be responsible for the phosphorylation of AP-1 proteins [23
]. In the context of CEES-caused lung injury, it has been shown in guinea pigs that CEES treatment activates AP-1 via MAPK pathway [48
]. This observation is comparable to our findings in skin exposed to CEES, as CEES-caused AP-1 activation in skin tissue also correlated with the activation of MAPK proteins, as well as could be activated via CEES-induced Akt pathway. Zhong et al. [49
] demonstrated that Fos and Jun proteins differ significantly in both their DNA binding and transactivation potentials as well as regulation of their target genes. Present study shows that CEES induced the activation of both c-Fos and c-Jun members of AP-1 family (). Expression of different AP1 proteins is variably regulated in response to numerous extra cellular stimuli that induce cellular stress. Since AP-1 is a homodimer or heterodimer of Fos, Jun and ATF proteins, analysis the protein levels of individual components showed increased expression of ATF-2, Jun B, Jun D, Fos B, Fra-1 and Fra-2 protein levels after CEES exposure both in dose- and time-response studies. Further investigations are reasonable to elucidate the individual contribution of AP-1 members in CEES-induced oxidative stress and skin inflammation that could be important targets for medical interventions of its toxicity. As AP-1 proteins plays an important role in cell proliferation [22
], the CEES-induced activation of AP-1 observed here could be associated with the CEES-induced increase in epidermal cell proliferation observed as increase in proliferating cell nuclear antigen (PCNA) staining reported in our earlier study in this mouse model [16
]. AP-1 also participates in the transcriptional activation of MMPs that are reported to be important mediators in HD/CEES induced skin injury and blistering as also seen in our studies (data not shown) [23
NF-κB plays an important role in inflammation and cell proliferation, and is activated by oxidants [41
]. It causes an influence on responsive proteins PCNA, iNOS, Cox-2 and other pro-inflammatory cytokines that are reported to play an important role in CEES/HD-mediated skin injury [1
] A recent report indicated that HD-induced NF-κB activation in keratinocytes was associated with activation MAPK pathways [11
], though a number of previous studies have reported the role of NF-κB in CEES/HD-induced skin inflammation and toxicity [5
]. In the present study subsequent to MAPKs and AP-1 activation, CEES exposure also induced NF-κB activation that was preceded by subsequent IκBα phosphorylation and its degradation. Moreover, phosphorylation and subsequent degradation of inhibitory molecules of IκB protein kinases are also required for optimal RelA/NF-κB activation by targeting functional domains of NF-κB proteins themselves [11
]. RelA/NF-κB is phosphorylated at Ser536 and Ser276 by a variety of kinases via various signaling pathways and in most cases, these phosphorylations enhance RelA transactivation potential. Our findings support the idea that the phosphorylation and degradation of IκBα preceded phosphorylation of RelA/ NF-κB at Ser536 and Ser276. The overall importance of the transactivating NF-κB subunit RelA for HD-induced NF-κB activation is demonstrated by exposure of RelA-deficient keratinocytes to HD, where, no NF-κB-binding activity was observed [11
]. Our results possibly imply that NF-κB-binding activity in response to CEES is dependent on RelA phosphorylation at Ser536 and Ser276, and phosphorylation of this site is essential for RelA activity in CEES exposed mouse skin.
Huang et al. [26
] demonstrated that NEMO plays a pivotal role in NF-κB signaling pathways by allowing physiological regulation of the cytoplasmic IKK complex. Besides, the signal specificity is in part due to the requirement of the C-terminal ZF domain of NEMO for SUMO-1 modification and the ZF domain is essential for NF-κB activation by DNA damaging agents. It is also reported that direct attachment of SUMO-1 to NEMO is sufficient to localize NEMO to the nucleus and overcome the ZF deficiency [26
]. The present study delineated the post-translational modification of NEMO and its accumulation in nucleus and later shuttling to the cytoplasm upon stress induction by CEES exposure in mouse skin tissue. This study indicates an important role of NEMO in the activation of NF-κB upon CEES exposure in skin tissue, which could be utilized as a target for medical intervention in CEES-induced skin injury.
Oxidative stress is reported as the possible first key event in HD toxicity that possibly activates the transcription factors NF-κB and AP-1 leading to pro-inflammatory gene expression and an inflammatory response [14
]. Consistent with this, several antioxidants including GSH and its precursors as well as AEOL 10150 have been implicated in attenuating the skin and lung injury caused by HD/CEES [1
]. The results of our present study are in accord with these previous findings and further assert the role of oxidative stress in HD/CEES-caused skin inflammation, injury and vesication. Though the roles of NF-κB, p53, p38, PARP, Fas and calcium pathways are reported in the HD/CEES-caused skin inflammation and injury [5
], their relation to oxidative stress and the studies in a relevant single animal model that could be useful in efficacy studies have been lacking. In this regard, the results of our present study clearly support the notion that the molecular mechanism by which CEES possibly alters the transcription factors AP-1 and NF-κB leading to skin inflammatory response and injury reported in our recent study in SKH-1 hairless mouse [16
], could be oxidative stress mediated activation of MAPKs and Akt following CEES exposure of mouse skin (). The well studied and reported HD/CEES-induced DNA alkylation [5
] could also be involved in this process alongside oxidative stress that possibly causes ROS generation leading to protein and DNA oxidation and lipid peroxidation. The DMPO nitrone-protein adduct formation, indicating oxidative protein damage, in the CEES exposed skin shown herein could be due to myeloperoxidase (MPO) that produces hypochlorous acid (HOCI) in the occurrence of chloride ions and hydrogen peroxide (H2
), and is found to be the possible source of DMPO-OH adduct [57
]. MPO is produced mainly in the neutrophils,but also in monocytes and macrophages that are shown to infiltrate in the CEES-exposed SKH-1 hairless mouse skin tissue in our recent studies [16
]. In diseased tissues, oxidative products of MPO have been detected that could be either via generation of HOCl that could modify both lipids and proteins, via tyrosyl radical causing lipid peroxidation, or possibly via nitrite oxidation to generate nitrating and chlorinating intermediates leading to lipid peroxidation [58
]. In future, it would be important to further dissect the role of MPO alongside GSH depletion and other reported oxidative stress mechanisms in HD/CEES-induced oxidative molecular damage that might help in understanding the mechanistic aspect of this process. Furthermore, studies are also needed in future possibly using selective ROS scavengers or pathway inhibitors to establish the role of MAPKs and Akt signaling pathways together with activation of transcription factors AP-1 and NF-κB by CEES as observed in the present study, on the recently reported inflammatory responses by CEES in this mouse model [16
]. The outcomes of such studies would contribute in further understanding the mechanism of HD/CEES-induced skin injury and its direct association with CEES-induced oxidative stress in mouse skin.
In summary, the findings in present study expanded our knowledge of molecular mechanisms involved in CEES-related dose- and time-dependent skin inflammatory responses reported earlier in an efficient SKH-1 hairless mouse model [16
]. The results presented here show the induction of oxidative stress by CEES, possibly leading to lipid, protein and DNA oxidations interlinked with multistep complex mechanisms of CEES-mediated skin inflammation and injury (, ). These mechanisms involve a number of signaling cascades, which suggestively engage in the production of CEES-induced pro-inflammatory mediators and advances inflammatory response (). Signaling pathways such as MAPKs, Akt and transcription factors like AP-1 and NF-κB could be key factors involved in the CEES-caused skin inflammatory process (). The valuable molecular targets explored in this study in an efficient rodent model, could be supportive in designing potential medical interventions especially contributing to the available antioxidant therapies, and can serve as valuable indicators in future therapeutic efficacy studies against HD-induced skin toxicity. Further studies are needed in future, possibly employing proteomic strategies, to identify the individual adducted and oxidized proteins indicatively formed due to CEES-induced oxidative stress in mouse skin in the present study. These findings will enhance our understanding of the CEES/HD induced protein oxidation, contributing further in strategies to develop medical countermeasures and therapy for any possible HD-induced skin injury in humans.
Schematic representation of the possible key mechanism of CEES-caused skin injury and inflammation in SKH-1 hairless mouse skin toxicity model.