Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)


Drug Discovery and Biomedical Sciences


College of Graduate Studies

First Advisor

Zhi Zhong

Second Advisor

John J. Lemasters

Third Advisor

Don C. Rockey

Fourth Advisor

Craig C. Beeson

Fifth Advisor

Stephen Duncan


Oxidative stress is a common component and important mediator in the pathogenesis of many liver injuries and of liver fibrosis. Although reactive oxygen species (ROS) are known to have effects on many important biological macromolecules and cell organelles, such as mitochondria, how oxidative stress causes liver injury/fibrosis has not been fully elucidated. ROS oxidize membrane lipids to form lipid radicals which undergo ß-scission to form aldehydes, such as malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE). These aldehydes are highly reactive and readily form adducts with proteins and nucleic acids. Relatively little is known about the role of aldehydes in the pathogenesis of liver injury. A key observation in the field is that aldehyde adducts appear to form on certain critical mitochondrial proteins. Since mitochondria are a major source of ROS in cells, I tested the hypothesis that activation of mitochondrial aldehyde dehydrogenase-2 (ALDH2) would accelerate aldehyde detoxification and thus attenuate liver injury and fibrosis. I examined the effects of Alda-1, a chemical chaperone and activator of ALDH2, in two animal models: acetaminophen (APAP) overdose, which causes acute liver failure, and bile duct ligation (BDL), which causes cholestatic liver injury and fibrosis leading to chronic liver failure. Previous studies have shown that oxidative stress plays an important role in the liver injury and fibrosis in these models. In an APAP hepatotoxicity model, mice received Alda-1 (20 mg/kg, i.p.) or vehicle 30 minutes before administration of APAP (300 mg/kg, i.p.). 4-HNE protein adducts markedly increased after APAP treatment, demonstrating increased production of aldehydes, which Alda-1 decreased by 86%. Serum alanine aminotransferase (ALT) increased to 7594 U/L, and centrilobular necrosis occurred in 47% of liver tissue after APAP, indicating severe liver injury. Alda-1 decreased ALT and necrosis by 72% and 56% respectively. Previous studies have demonstrated that formation of N-acetyl-p-benzoquinone imine (NAPQI) protein adducts, activation of c-Jun-N-terminal kinase (JNK) and the mitochondrial permeability transition (MPT) are linked to APAP hepatotoxicity. After APAP treatment, NAPQI protein adducts and JNK phosphorylation increased six-fold, which Alda-1 did not alter. Without APAP, intravital microscopy revealed that no mitochondrial depolarization could be detected. At 6 h after APAP, 62% of tissue areas showed depolarization, which was decreased by nearly half with Alda-1 treatment. Therefore, Alda-1 decreased APAP hepatotoxicity not by alteration of APAP metabolism or blockade of JNK activation but by protecting against mitochondrial dysfunction. Cholestasis in mice was induced by BDL. After BDL, 4-HNE adduct formation increased almost six-fold, which Alda-1 decreased to baseline levels. ALT increased to 537 U/L, and biliary infarcts occurred after BDL, consistent with the expected mechanism of liver injury. Alda-1 blunted ALT by half and decreased the area of biliary infarcts from 7.8% to 1.9%. Fibrosis occurred after BDL, as detected in liver sections by increased picrosirius red staining and second harmonic generation microscopy. Collagen-I mRNA also increased 12-fold after BDL. Alda-1 decreased each of these measures of fibrosis by approximately 50%. Expression of smooth muscle α-actin (α-SMA), a marker of activated myofibroblasts, the cell type that produces collagen, increased ~19-fold after BDL, which Alda-1 limited to a 5-fold increase. Alda-1 also decreased macrophage infiltration by approximately 50% and expression of the proinflammatory and profibrotic protein osteopontin (OPN), by 64%. Taken together, these data demonstrate that aldehydes are important mediators of liver injury caused by APAP intoxication and of both liver injury and fibrosis caused by cholestasis. Therefore, acceleration of aldehyde degradation by ALDH2 with Alda-1 is a potential new effective therapy to decrease liver injury/fibrosis related to high oxidative stress.


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