Lipopolysaccharide-induced liver injury in rats treated with the CYP2E1 inducer pyrazole

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Elevated LPS and elevated cytochrome P-450 2E1 (CYP2E1) in liver are two major independent risk factors in alcoholic liver disease. We investigated possible synergistic effects of the two risk factors in causing oxidative stress and liver injury. Sprague-Dawley rats were injected intraperitoneally with pyrazole (inducer of CYP2E1) for 2 days, and then LPS was injected via tail vein. Other rats were treated with pyrazole alone or LPS alone or saline. Eight hours later, blood was collected and livers were excised. Pathological evaluation showed severe inflammatory responses and necroses only in liver sections from rats in the pyrazole plus LPS group; blood transaminase levels were significantly elevated only in the combination group. Activities of caspase-3 and -9 and positive terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining were highest in the LPS alone and the LPS plus pyrazole group, with no significant difference between the two groups. Lipid peroxidation and protein carbonyls in liver homogenate as well as in situ superoxide production were maximally elevated in the LPS plus pyrazole group. Levels of nitrite plus nitrate and inducible nitric oxide (NO) synthase (iNOS) content were comparably elevated in LPS alone and the LPS plus pyrazole group; however, 3-nitrotyrosine adducts were elevated in the combined group but not the LPS group. It is likely that LPS induction of iNOS, which produces NO, coupled to pyrazole induction of CYP2E1 which produces superoxide, sets up conditions for maximal peroxynitrite formation and production of 3-nitrotyrosine adducts. CYP2E1 activity and content were elevated in the pyrazole and the LPS plus pyrazole groups. Immunohistochemical staining indicated that distribution of CYP2E1 was in agreement with that of necrosis and production of superoxide. These results show that pyrazole treatment enhanced LPS-induced necrosis, not apoptosis. The enhanced liver necrosis appears to involve an increase in oxidative and nitrosative stress generated by the combination of LPS plus elevated CYP2E1 levels.

lps is a component of the outer wall of gram-negative bacteria that normally inhabit the gut. LPS penetrates the gut epithelium only in trace amounts; however, LPS absorption can be elevated under pathophysiological conditions such as alcoholic liver disease (48). When LPS is released from gram-negative bacteria and enters the bloodstream, the liver tightly regulates the entry and processing of LPS by virtue of its ability to clear LPS and respond to LPS (56). In animals, LPS is cleared from the circulation within a few minutes after intravenous injection (37, 70). In addition to its ability to clear LPS, the liver also responds to LPS and produces cytokines. LPS directly causes liver injury by mechanisms involving inflammatory cells such as Kupffer cells and chemical mediators such as superoxide, nitric oxide, and TNF-α and other cytokines (6, 20, 26, 57, 62, 64). In addition, LPS potentiates liver damage induced by hepatotoxins including ethanol (31, 38). In experimental alcoholic liver disease, the combination of LPS and chronic ethanol produces hepatic necrosis and inflammation (3, 25). Ethanol alters gut microflora, the source of LPS, and ethanol increases the permeability of the gut, thus increasing the distribution of LPS from the gut into the portal circulation (endotoxemia). This causes activation of Kupffer cells, the resident macrophages in liver, resulting in the release of chemical mediators including cytokines and reactive oxygen species (ROS) and subsequently, alcoholic liver disease (17, 19, 48, 59, 65).

Oxidative stress plays an important role in alcoholic liver disease (29). There are many sources of ROS within the cell. Besides ethanol-induced increase in LPS with subsequent activation of Kupffer cells and production of ROS, cytochrome P-450 2E1 (CYP2E1), a form of P-450 that is induced by ethanol, is another important source. Whereas most ethanol is oxidized by alcohol dehydrogenase, CYP2E1 assumes a more important role in ethanol oxidation at elevated concentrations of ethanol and after chronic consumption of ethanol (34). Due to being poorly coupled with NADPH-cytochrome P-450 reductase, CYP2E1 exhibits enhanced NADPH oxidase activity and elevated rates of production of superoxide (O2−·) and hydrogen peroxide (H2O2) (5, 18, 49), CYP2E1-mediated oxidative stress is thought to play an important role in alcoholic liver disease (8, 9, 29).

Pyrazole is a potent inhibitor of alcohol dehydrogenase (32, 51, 58) and is frequently used to block the oxidation of ethanol or the metabolism of other alcohols such as methanol (41) or ethylene glycol (13). In addition, pyrazole has been shown to affect the metabolism of several drugs by the microsomal mixed-function oxidase system (35). Microsomes isolated from rats treated with pyrazole for ∼2–3 days showed an approximately two- to fourfold increase in CYP2E1 (46, 66). After administration of pyrazole to rats, mRNA levels did not increase, suggesting that the mechanism of induction was at the level of protein stabilization (55, 66). Pyrazole has been used as an inducer of CYP2E1 to study the role of increased production of oxygen-free radicals mediated by CYP2E1 in cell death (47, 67, 68, 69).

Either LPS or CYP2E1 is considered an independent risk factorsinvolved in alcoholic liver disease, but mutual relationship or interactions between them are unknown. In the present study, we investigated the combined action of LPS and CYP2E1 in rats via administrating pyrazole to induce CYP2E1 followed by treatment with LPS. We found that pretreatment with pyrazole followed by LPS injection caused severe liver necrosis and oxidative stress, suggesting possible synergistic interactions between LPS and CYP2E1.


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