The present studies show that paraquat is reduced to a radical in keratinocytes during a redox cycling reaction, and that oxidation of this radical generates superoxide anion and hydrogen peroxide. Moreover, in intact keratinocytes, paraquat redox cycling leads to protein oxidation. These findings are novel and suggest a potential mechanism mediating paraquat-induced injury to the skin. Of particular interest was our observation that paraquat-induced protein oxidation was greater in differentiated keratinocytes, when compared to undifferentiated cells, and that this is associated with increased hydrogen peroxide formation. These results suggest that differentiated keratinocytes may be more susceptible to oxidative stress. This idea is consistent with findings of increased oxidative stress in suprabasal layers of the epidermis following exposure to UVB light (Theile et al., 1999
; Sander et al., 2002
). In contrast, we noted that generally similar amounts of superoxide anion were generated in undifferentiated and differentiated keratinocytes. It is possible that differentiated cells metabolize superoxide anion more rapidly than undifferentiated cells. This is supported by our findings that differentiated cells expressed greater constitutive levels of Cu,Zn-SOD. Although differentiated cells also expressed more catalase, this was apparently not sufficient to metabolize hydrogen peroxide during paraquat redox cycling in these cells, when compared to undifferentiated cells. At least four constitutively oxidized proteins with molecular masses of 59, 68, 75 and 97 kDa were identified in the keratinocytes. It is well recognized that cells continuously generate basal levels of superoxide anion and hydrogen peroxide from both mitochondrial and extramitochondrial sources (Darr and Fridovich, 1994
), and it is likely that these ROI are responsible for generating the observed oxidized proteins. This is in accord with our findings of enhanced oxidation of these same proteins following paraquat treatment. Paraquat also induced oxidation of an additional 43 kDa protein in both differentiated and undifferentiated keratinocytes. At the present time, the identity of the oxidized target proteins in the cells is not known. Since the 43 kDa protein is selectively oxidized following paraquat treatment, we speculate that it plays a critical role in mediating the biological actions of paraquat.
Previous studies have demonstrated that redox cycling of paraquat occurs via the actions of oxidoreductases including cytochrome P450 reductase and thioredoxin reductase (Day et al., 1999
; Gray et al., 2007
). These are flavin-containing monooxygenases that are inhibited by DPI. In accord with this, we found that DPI also inhibited paraquat-mediated redox cycling in keratinocyte lysates. However, the redox cycling activity was simultaneously suppressed by dicoumarol, an inhibitor of the FAD-containing enzyme, NAD(P)H quinone oxidoreductase-1 (NQO1). Although these findings suggest that paraquat may redox cycle with this enzyme, this would be surprising since NQO1 catalyzes an obligate two-electron reduction (Chen et al., 2000
). It should be noted that dicoumarol also inhibits other enzymes including mitochondrial complexes II, III and IV (Gonzalez-Aragon et al., 2007
). Thus, we cannot rule out the possibility that dicoumarol inhibits other DPI-sensitive one electron oxidoreductases in keratinocytes that mediate redox cycling of paraquat.
Increased oxidative stress in cells is associated with altered expression of antioxidant enzymes which function to limit cytotoxicity (Adler et al., 1999
; Droge, 2002
; Scandalios, 2005
). Both undifferentiated and differentiated keratinocytes were found to respond to paraquat-induced oxidative stress by upregulating the expression of several key antioxidant enzymes including Cu,Zn-SOD, HO-1 and catalase. Transgenic mouse studies have shown that antioxidant enzymes such as Cu,Zn-SOD, as well as GPx-1, are required for the prevention of paraquat-induced oxidative damage (de Haan et al., 1998
; Van Remmen et al., 2004
), and that their overexpression offers protection from injury (Thiruchelvam et al., 2005
). Interestingly, we found differential effects of paraquat on expression of Cu,Zn-SOD and Mn-SOD. Thus, while Cu,Zn-SOD was readily induced in the cells following paraquat treatment, Mn-SOD remained unchanged. Similar results have been described in paraquat-treated fibroblasts (Kelner and Bagnell, 1990
), and in keratinocytes exposed to UVB (Sasaki et al., 1997
). Cu,Zn-SOD and Mn-SOD are known to be regulated by distinct mechanisms (Zelko et al., 2002
). Moreover, Cu,Zn-SOD is cytosolic, while Mn-SOD is restricted to the mitochondrial matrix and inner membrane (Fridovich, 1978
). Presumably, paraquat generates superoxide anion in the cytosolic compartment of the cells and localized oxidative stress stimulates expression of Cu,Zn-SOD.
Paraquat-induced increases in HO-1 expression were also observed in undifferentiated and differentiated cells. HO-1, like Cu,Zn-SOD, is upregulated in cultured skin and lung cells in response to exposure to oxidants such as ozone (Valacchi et al., 2004
), sodium arsenite (Applegate et al., 1991
) and UVA (Keyse and Tyrrell, 1990
). This is thought to be central in antioxidant defense and is supported by findings that HO-1 knockout mice exhibit increased ROI production and enhanced mortality (Poss and Tonegawa, 1997
). As observed with Cu,Zn-SOD and catalase, constitutive expression of HO-1 was increased in differentiated keratinocytes when compared to undifferentiated cells, and this may be important in protecting suprabasal layers of the skin from oxidative stress. Catalase expression was also induced by paraquat in both undifferentiated and differentiated keratinocytes. Increased catalase expression has been observed in skin following UVA exposure (Fuchs et al., 1989
), and catalase overexpression in both cultured keratinocytes and mice provides protection from hydrogen peroxide-induced damage (Chen et al., 2004
; Shim et al., 2005
), as well as UVB-mediated apoptosis (Rezvani et al., 2006
). Increased expression of these important antioxidants is a key adaptive response to protein oxidation as a result of paraquat redox cycling and represents a mechanism to protect keratinocytes against further oxidative stress.
Of particular interest was our finding that paraquat treatment altered expression of several major glutathione S
-transferase (GST) enzymes. A major function of both cytosolic and microsomal GST enzymes is conjugation of glutathione to oxidized cellular macromolecules in order to facilitate their elimination and limit tissue injury. Although all GST enzymes conjugate glutathione, each GST family has preferred substrates. GSTA enzymes have been shown to break lipid peroxidation chain reactions through the removal of hydroperoxides and aldehydes generated during oxidative stress (Hayes and McLellan, 1999; Yang et al., 2002
). This GSTA preference for lipid peroxidation products may explain the striking increases in GSTA1-2 and GSTA4, as well as GSTA3 that we observed in keratinocytes. Our findings are consistent with previous work showing that overexpression of GSTA1 protects against hydrogen peroxide-induced cytotoxicity and DNA damage in retinal pigment cells (Liang et al., 2005
). We also noted significantly greater expression of GSTA1-2 in response to paraquat in differentiated keratinocytes, when compared to undifferentiated cells. Genetic polymorphisms in the GSTA1 promoter have been associated with increased cancer incidence (Coles et al., 2001
; Sweeney et al., 2003
), and these polymorphisms may be involved in regulating GSTA1 gene expression and tumor development. The importance of GSTA4 activity in protection against paraquat-induced oxidative stress has also been noted in GSTA4 null mice (Engle et al., 2004
). Higher concentrations of lipid peroxidation products are detected in the livers of these mice after injection of paraquat, and the mice exhibit a greater mortality relative to wild-type controls. The lower induction of GSTP1, and lack of induction of GSTM1 in our cells may be due to the diminished roles that these enzyme families play in the detoxification of lipid peroxidation products (Berhane et al., 1994
). Our results are generally similar to reports of changes in GST mRNA expression in lungs from mice treated with paraquat where increases in the GST alpha and pi families were detected (Ruiz-Laguna et al., 2005
The microsomal GST (mGST) enzymes have been shown to exhibit glutathione peroxidase activity against lipid hydroperoxides that is similar to the cytosolic GSTA enzymes (Mosialou et al., 1995
). However, in keratinocytes, expression of these enzymes was not altered by paraquat treatment. The mGSTs also appear to be important in the metabolism of lipid-derived inflammatory mediators (Hayes et al., 2004
). Both mGST2 and mGST3 conjugate glutathione to leukotriene A4
to form leukotriene C4
) (Jakobsson et al., 1997
), while mGST1 co-localizes with leukotriene C4
synthase, and is inhibited by LTC4
(Bannenberg et al., 1999
). Further studies are necessary to determine if the microsomal and cytosolic GST enzymes are regulated distinctly, and to elucidate their role in protecting cells from oxidative stress.
Our data show that paraquat readily induced oxidative stress in keratinocytes. Antioxidant therapy involving the administration of SOD or N-acetylcysteine (NAc) has been proposed as a potential treatment for paraquat poisoning. This approach, however, has met with mixed results. While initial studies suggested that NAc and Cu,Zn-SOD increased the survival of paraquat-treated animals (Yeh et al., 2006
), subsequent work demonstrated that the protective effects of SOD were only evident when it was used in combination with Mn-SOD and glutathione (GSH) (Paller and Eaton, 1995
). In the same study, Cu,Zn-SOD treatment alone enhanced oxidative injury, primarily due to the reactivity of the copper ions. In recent in vivo
experiments, synthetic SOD analogs were shown to prevent paraquat-induced pulmonary oxidative damage without causing toxicity in mice (Day and Crapo, 1996
). In mouse skin, topical application of antioxidant formulations including inhibitors of superoxide anion or alpha- and beta-carotenes reduced markers of oxidative stress caused by exposure to phorbol esters (Nakamura et al., 1998
) or croton oil (Kim-Jun, 1993
). At the present time, the use of antioxidants orally or topically for the treatment of paraquat toxicity is promising but requires additional studies to identify agents that penetrate the skin and retain biological activity.
In summary, we have shown that paraquat induces oxidative stress in primary cultures of undifferentiated and differentiated mouse keratinocytes by producing ROI via NADPH-dependent redox cycling. This leads to increased protein oxidation, particularly in differentiated cells, as well as upregulation of critical antioxidant enzymes including Cu,Zn-SOD, catalase, HO-1, GSTA1-2, GSTA3, GSTA4 and GSTP1. At the present time, the precise role of each of these antioxidants in the response of the skin to oxidative stress is unknown. The role of differentiation in regulating antioxidant enzyme expression, as well as in determining of how this process controls the responses of the skin to oxidative stress requires further investigation for a more complete understanding of the dermal toxicity of paraquat.