In this study, we found that pyrazole treatment alone induced hepatotoxicity in Nrf2 knockout mice but not in wild type mice. Similarly, elevated oxidative status was detected in pyrazole-treated Nrf2 knockout mice but not in wild type mice. Vc, SAM and L-NAME protected against the pyrazole-induced liver injury in Nrf2 knockout mice. These results suggest that pyrazole-induced liver injury in Nrf2 knockout mice is due to oxidative stress.
Oxidative stress reflects a balance between production of ROS and antioxidant capacity to remove ROS. In CYP2E1-overexpressing HepG2 cells, total GSH levels, GSH synthetic rate and GCS mRNA were increased (Mari and Cederbaum, 2000
). Activity, protein and mRNA levels for other antioxidants such as catalase, alpha- and microsomal GST were also increased (Mari and Cederbaum, 2001
). Up-regulation of these antioxidant genes was suggested to reflect an adaptive mechanism to remove CYP2E1-derived oxidants and was dependent on Nrf2 upregulation (Gong and Cederbaum, 2006 b
). Induction of HO-1 protects against CYP2E1-dependent toxicity (Gong et al., 2004
). The protective effects of Nrf2 against CYP2E1-dependent toxicity can be blocked by L-buthionine-(S,R)-sulfoximine (BSO), a specific inhibitor of GCS, which prevents the synthesis of GSH. Is pyrazole-induced liver injury in Nrf2 knockout mice due to failed or impaired induction of Nrf2-regulated antioxidant capacity? Nrf2 and three of its target genes, GCS, GST and HO-1, were upregulated in wild type mice; but in Nrf2 knockout mice, no upregulation of GST and GCS was observed, and upregulation of HO-1 was lower than that in wild type mice. Pyrazole elevation of GSH levels was observed in pyrazole-treated wild type mice but not in the Nrf2 knockout mice (). Therefore, it is postulated that, in pyrazole-treated Nrf2 knockout mice, the impaired antioxidant capacity causes increased accumulation of ROS, which enhances liver injury. In pyrazole-treated wild type mice, the compensatory increase in antioxidant capacity prevents increased accumulation of ROS, which therefore protects against liver injury. In pyrazole-treated Nrf2 knockout mice, ROS scavengers such as Vc and SAM can take the place of the lowered antioxidant capacity, thereby protecting against pyrazole-induced liver injury. Certainly, other Nrf2-dependent enzymes and cytoprotective factors besides GST, GCS, HO-1 may be important in the protection against the pyrazole toxicity.
Some studies suggest that excessive formation of NO can result in oxidative stress in the liver (Schild et al, 2003
), but other reports showed that NO has a protective effect as the production of NO improved the microcirculation and oxygen metabolism (Corso, et al, 1998
). In the current study, pyrazole induction of iNOS is comparable in the knockout and wild type mice. However, pyrazole-induced liver damage may be dependent on the balance of the local production of NO and ROS such as O2.−
. Peroxynitrite (ONOO−
) is formed by the rapid reaction between NO and O2.−
and has been shown to nitrate free and protein-associated tyrosine residues and produce nitrotyrosine (Ischiropoulos, 1998
). Although the levels of iNOS were the same, higher levels of O2.−
due to impaired antioxidant capacity in pyrazole-treated Nrf2 knockout mice elevate ONOO−
formation to a greater extent than that found in pyrazole-treated wild type mice. Therefore, the formation of 3-NT was increased and L-NAME, an inhibitor of iNOS, protected against pyrazole liver damage in the Nrf2 knockout mice.
Why is oxidative/nitrosative stress elevated in the pyrazole-treated Nrf2 knockout mice but not in the wild type mice? Pyrazole induces CYP2E1. CYP2E1 exhibits enhanced NADPH oxidase activity and elevated rates of production of O2.−
(Ekstrom and Ingelman-Sundberg, 1989
). While elevated CYP2E1 alone (pyrazole treatment alone) does not induce significant liver injury, it enhanced liver injury induced by other hepatotoxins such as LPS (Lu et al., 2005
; Lu and Cederbaum, 2006 a
). In addition to CYP2E1 induction, pyrazole induces CYP2A5 activity. Gilmore et al (2003)
reported that vitamin E attenuates CYP2A5 induction by pyrazole and that GSH depletion by BSO induces CYP2A5 suggests a causal association between oxidative stress and CYP2A5 overexpression. Since cytochrome P450 enzymes can produce ROS during their catalytic turnover (White, 1991
), pyrazole induction of CYP2E1 and 2A5 should increase P450-generated ROS. However, although pyrazole induced hepatotoxicity in Nrf2 knockout mice, no elevation in CYP2E1 or 2A5 activities or levels were observed, in contrast to the 2-fold increase in CYP2E1 activity and more than 20-fold elevation of CYP2A5 activity in wild type mice. Despite the elevation in CYP2E1 and 2A5, no hepatotoxicity was detected in wild type mice. These results suggest that pyrazole-induced oxidative liver injury in Nrf2 knockout mice is not due to the induction of CYP2E1 and 2A5. Information about pyrazole metabolism might provide an insight into the pathogenesis of pyrazole liver injury; hydroxylation of pyrazole to 4-hydroxypyrazole by CYP2E1 can occur (Clejan and Cederbaum, 1990
), however, no other literature is available. Interestingly, 4-hydroxypyrazole can inhibit catalase (MacDonald et al, 1981
). While catalase was decreased by pyrazole in both the Nrf2 knockouts and wild type mice, inhibition of this important antioxidant enzyme may have more serious repercussions with respect to the antioxidant status in the pyrazole-treated Nrf2 knockout mice with a failed capacity to upregulate GSH and other antioxidants than in the wild type mice with antioxidant upregulation. How is pyrazole promoting liver injury or elevating oxidative stress in the knockout mice in the absence of increases in CYP2E1 and 2A5? Whether ROS production is increased by other pathways or enzymes cannot be ruled out since pyrazole treatment upregulates and downregulates the expression of many genes in mice as recently shown by microarray analysis (Nichols and Kirby, 2007
). Further studies are needed to address this issue.
Cytochrome P450s are generally not considered as Nrf2 target genes. Recent studies have shown that CYP2A5 indeed contains an Nrf2 response element (Abu-Bakar et al., 2004
). Two putative stress response elements (STRE) were localized to positions −2514 to −2386 to −2377 of the CYP2A5 promoter, with the more proximal sequence specifically binding Nrf2 and the authors concluded that Nrf2 activates CYP2A5 transcription by directly binding to the proximal STRE (Abu-Bakar et al., 2007
). This would explain why pyrazole is ineffective in inducing CYP2A5 in the Nrf2 knockout mice. More complicated, however, is to understand why pyrazole failed to induce CYP2E1 in the Nrf2 knockout mice. Induction of CYP2E1 by pyrazole is mainly posttranscriptional (Eliasson et al., 1988
) as pyrazole can stabilize CYP2E1 against proteasome-mediated degradation (Bardag-Gorce et al., 2002
; Roberts, 1997
; Yang and Cederbaum, 1997
). Why the lack of Nrf2 would block this stabilization of CYP2E1 by pyrazole against proteasome-mediated degradation is not known. Perhaps of importance is the finding that the expression of some subunits of 20S and 19S proteasomes can be transcriptionally induced by dithiolethione in mouse liver in an Nrf2-dependent manner (Kwak et al., 2003
). Whether this Nrf2-modulation of the proteasome complex plays a role in the ineffectiveness of pyrazole to elevate CYP2E1 levels will require future studies.
In summary, in wild type mice, although ROS producing CYP2E1 and 2A5 were induced, no liver injury was observed due to compensative increases in Nrf2-regulated antioxidant capacity. However, in Nrf2 knockout mice, due to failed or impaired upregulation of antioxidant capacity, pyrazole induced severe oxidative liver injury, even though CYP2E1 and 2A5 were not elevated. The source of ROS production in pyrazole-treated Nrf2 knockout mice remains unclear.