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Hepatitis B virus (HBV) preferentially replicates in the liver, a naturally hypoxic organ with an oxygen gradient from 8-4% in the periportal and pericentral regions, respectively. The hypoxia inducible factor (HIF) pathway is the most well studied mammalian oxygen sensing mechanism and is frequently perturbed by pathological states including infection and inflammation. However, the majority of in vitro studies have investigated HBV at atmospheric oxygen tensions (18%) where low oxygen sensing mechanisms are inactive and consequently their role in viral replication may be overlooked. Therefore, this thesis studied the interplay between HBV replication and oxygen sensing mechanisms. Chronic hepatitis B (CHB) is associated with an increased expression of HIF gene transcripts. Further interrogation of a cohort of patients with CHB shows an association between inflammatory responses and HIF transcription. Importantly, this observation is not recapitulated in state-of-art in vitro, in vivo, and de novo HBV infection model systems. HIF regulates target genes via HIF responsive elements (HREs), and the discovery of two evolutionarily conserved HREs in the HBV genome prompted experiments to understand the role of HIFs in viral replication. HIF siRNA silencing experiments in vitro and in vivo demonstrate a positive role for HIFs in regulating HBV replication. As other oxygen sensing mechanisms have been identified in addition to the HIF pathway, further experiments assessed the role of hypoxia and HIF independent mechanisms in regulating HBV transcription. Data suggests that hypoxia positively regulates HBV transcription through both HIF dependent and independent mechanisms. Further investigations reveal that low oxygen downregulates the HBV host restriction factor Smc6 through HIF independent mechanisms. In addition, the cysteamine (2-aminoethanethol) dioxygenase oxygen sensing pathway regulated interleukin-32 may have anti-viral activities that are repressed under hypoxia. Collectively, this thesis presents evidences demonstrating key regulatory interactions between HBV and hypoxia, revealing novel therapeutic directions.

Chronic infection with Hepatitis B virus (HBV) remains a significant health problem, infecting millions of people worldwide. Cirrhosis and hepatocellular carcinoma, the sequalae of longstanding HBV infection, have a high mortality. Current therapy controls HBV replication but does not eradicate the virus, and therefore most people require life-long treatment. A robust adaptive immune response is required to control acute HBV but is dysfunctional in individuals that go onto develop chronic infection. The immune response is an attractive target for novel HBV therapies, which aim to induce functional cure of HBV in finite treatment courses by restoring HBV specific immunity. Therapeutic vaccination, which describes the process of delivering non-infective HBV antigen into the body in order to induce or boost cellular immune responses to HBV, is one strategy under investigation for chronic HBV treatment. In this thesis I evaluate the novel vaccines ChAdOx1-HBV and MVA-HBV as candidates for HBV therapeutic vaccination. I investigate their immunogenicity in heterologous prime-boost strategies in healthy mice, describing in detail the fine specificity, functionality and phenotype of vaccine induced T cell responses in the blood and the liver. I also describe the safety and immunogenicity ChAdOx1-HBV in a phase-1 open label clinical trial in healthy volunteers and patients with chronic HBV infection. This work is important to inform the development of therapeutic strategies using ChAdOx1-HBV and MVA-HBV in patients with chronic HBV going forward.

Non-Alcoholic Fatty Liver Disease (NAFLD) is a chronic and dangerous condition which has grown in prevalence over recent decades due to increasing rates of obesity, affecting roughly a quarter of adults globally. This thesis develops a useable NAFLD definition which can be applied to large retrospective data sets consisting of medical records for selected cohorts. The morbidity and mortality associated with NAFLD is investigated, especially related to extrahepatic cancer. This thesis also aims to identify genetic modifiers of NAFLD risk in Scottish and South Indian populations. Data from three retrospective Scottish cohorts with electronic health records (EHRs) were analysed in the current thesis. These were the GoDARTS, SHARE and Tayside and Fife Diabetics cohorts. Genotypic data was available for a number of patients in GoDARTS and SHARE. Data from the Dr Mohan’s Diabetes Speciality Clinic (DMDSC) were also analysed, which consisted of clinical measurements from clinic visits, and genotypic data. An accurate and practical NAFLD definition based on two raised ALT levels was developed, which had a sensitivity of 97.4% in the GoDARTS cohort. This definition was used for subsequent NAFLD analyses. Patients with NAFLD experienced significantly more hospital admissions, and earlier death than those without NAFLD. We found NAFLD is associated with increased risk of cancer incidence, and cancer death, which accounts for a large portion of the excess morbidity and mortality seen in NAFLD patients. GWAS analyses revealed that PNPLA3 rs738409 is a major genetic risk factor in both Scottish and South Indian populations. Candidate gene studies revealed variants associated with endothelin function and with GLP1 receptors had significant effects on NAFLD. This thesis applies an accurate and novel NAFLD definition to retrospective cohorts with EHRs, and found increased morbidity and mortality in individuals with this phenotype. A large proportion of this is explained by increased cancer incidence and cancer death seen in these individuals. A number of genetic risk factors for NAFLD are described, including novel loci in endothelin and GLP1R related genes.

Primary sclerosing cholangitis: from genetic risk to disease biology Elizabeth Claire Goode One in 10,000 people in the Western world lives with Primary Sclerosing Cholangitis (PSC), an immune-mediated, inflammatory disease of the bile ducts that is highly co-morbid with inflammatory bowel disease (IBD). PSC confers risk of serious disease sequelae including hepatobiliary malignancy and progression to end-stage liver failure, for which the only treatment option is liver transplantation. The absence of effective medical therapies for PSC reflects our current limited understanding of the disease's aetiology and pathogenesis. Our DNA, laid down at conception, gives us an unrivalled opportunity to understand the underlying causal biology of disease. This is because the genetic variants associated with disease susceptibility perturb genes and biological pathways that contribute to disease causality. Twenty-two regions of the genome, outside of the HLA, have been associated with PSC risk. These loci offer the potential for huge insight into the causal biology of PSC, if only we can robustly identify the true causal variants driving these loci and the genes they perturb. However, this is complicated by several scientific challenges. Firstly, the majority of disease-associated risk loci occur within non-coding regions of the genome. Secondly, patterns of correlation between variants within a risk locus means that the true causal variant driving the signal could be any of those highly correlated with the variant with the smallest p-value. In this thesis, I present analyses aimed at identifying the true genes and causal variants underlying each of the twenty-two PSC risk loci. Many non-coding risk variants associated with complex disease exert a quantitative affect upon gene expression i.e. are expression quantitative trait loci (eQTLs). Colocalisation assesses the evidence that a single shared causal variant is responsible for driving PSC risk and gene expression via an eQTL. In order to assign dysregulated genes to PSC risk loci, I perform colocalisation with eQTLs mapped in multiple cell-types and tissues mechanistically relevant to PSC. Because PSC is rare, eQTLs have not previously been mapped in all cell-types most relevant to this disease. In addition, I therefore map eQTLs in six peripheral blood T-cell subsets (including the rare CCR9+ gut-homing T-cells) from 80 patients with PSC and IBD. With colocalisation, I assign causal genes to five PSC risk loci, and assign other epigenetic regulatory features including methylation or histone modification, to six risk loci. Statistical fine-mapping of each risk locus in both the GWAS and eQTL data enables me to resolve three PSC risk loci to a single causal variant and nine loci to 95% credible sets containing ten or fewer variants. The results presented in this thesis identify three genes (PRKD2, ETS2 and UBASH3A), causal in the pathogenesis of PSC, which are currently the target of existing or experimental therapeutic agents. Firstly, reduced expression of PRKD2 causes excessive cell-autonomous T-follicular helper cell development and B-cell activation, and is associated with increased risk of PSC. Several studies are investigating the therapeutic effects of increasing the kinase activity of PRKD2. ETS2 is involved in the induction of pro-inflammatory cytokine release from macrophages and IL-2 regulation in Th to Th0 transition. ETS2 inhibitors are currently the subject of early therapeutic trials. Finally, UBASH3A attenuates the NF-kB/I-KKb pathway, an inflammatory pathway that is already targeted by proteasome inhibitors and acetylsalicylic acid, both of which could be potentially therapeutic in PSC. PSC is a debilitating disease with serious disease sequelae, for which new therapeutic options are urgently needed. In this thesis, I elucidate multiple genes with a causal role in PSC pathogenesis, several of which are potential candidates for future therapeutic target.

Non-alcoholic fatty liver disease (NAFLD) is now the leading cause of chronic liver disease in the developed world, afflicting approximately one in four adults globally. NAFLD is defined by the accumulation of fat within the liver which ranges in severity from simple steatosis to non-alcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma. Until recently, NAFLD has been considered to be a consequence of metabolic syndrome; however, recent studies suggest that genetic factors may also influence disease onset and progression. Accordingly, genome wide association studies have linked the I148M variant in the gene coding for Patatin-like phospholipase domain-containing protein 3 (PNPLA3) with NAFLD aggravation without underlying metabolic disease. However, despite over a decade of research, the function of PNPLA3 and its role in the pathogenesis of NAFLD remains largely obscure. The lack of clarity regarding the function and disease associations of PNPLA3 is due in large part to the lack of a comprehensive human model of PNPLA3-associated NAFLD. In order to address this need, we have developed an in vitro model that takes advantage of the unique properties of human induced pluripotent stem cells (hiPSC) and the CRISPR/CAS9 gene editing technology. We first used CRISPR/CAS9 to generate hiPSC lines with either a knock-out (PNPLA3KO) of the PNPLA3 gene or with the I148M variant knocked in (PNPLA3I148M). The resulting cells were then differentiated into hepatocytes and grown in 3D culture conditions to improve their maturity and functionality. The differentiated cells were treated with either monounsaturated or saturated free fatty acids to induce NAFLD-like phenotypes (lipid accumulation and lipid toxicity, respectively) and characterized by various functional, genomic, and lipidomic assays. The genetically edited sublines showed similar differentiation efficiency toward hepatocytes as untargeted cells indicating that changes in PNPLA3 activity does not affect hepatic development. Following treatment with free fatty acids, PNPLA3KO cells showed higher lipid accumulation than untargeted cells as well as an altered pattern of response to lipid-induced stress. Indeed, PNPLA3KO cells were resistant to saturated fatty acid-induced lipotoxicity. Furthermore, lipidomic analyses suggest that the PNPLA3 edited cells may be avoiding cell death by shuttling the saturated fatty acids into triglycerides rather than other metabolic pathways. These findings were initially incongruent with the human disease; however, despite their resistance to lipid induced stress, the PNPLA3KO cells downregulated all phases of drug metabolizing enzymes which made them more susceptible to other forms of hepatotoxicity such as ethanol. The PNPLA3I148M cells exhibited an intermediate phenotype between untargeted and PNPLA3KO cells. These results demonstrate for the first time in a fully human model that the I148M variant in PNPLA3 is a loss of function variant. This loss of PNPLA3 activity leads to the global downregulation of metabolic pathways likely due to the sequestration of fatty acids in triglycerides caused by reduced lipid droplet remodelling capacity in these hepatocytes. Our results indicate that patients carrying the I148M variant have lower hepatic metabolic activity which causes steatosis, reduced susceptibility to lipotoxicity, and increased susceptibility to other forms of hepatotoxicity which may contribute to NAFLD progression. This novel system provides the first opportunity to study the role of PNPLA3 in the development and progression of human NAFLD in vitro.

In liver disease, measurements of portal hypertension and liver function can predict clinical outcomes and help guide management, but are either invasive or lack accuracy. This thesis explores magnetic resonance imaging (MRI) techniques with gadoxetic acid administration to assess portal hypertension and liver function non-invasively. This adds to the existing capabilities of multiparametric MRI to assess liver fibrosis, fat and iron. Spleen extracellular volume fraction (ECV) and platelet count to spleen volume ratio identified portal hypertension accurately (both 0.79 area under the receiver operator curve (AUROC)). A combination of spleen T1 and spleen diameter identified oesophageal varices needing treatment (VNTs) in patients with compensated advanced chronic liver disease (cACLD) with the highest accuracy (AUROC 0.87) of all markers tested and could save 66% of screening endoscopies for VNTs without missing any VNTs. Liver T1 reduction with gadoxetic acid (%rT1) correlated well with serum-based liver function markers and clinical scores and identified patients with impaired liver function with AUROC 0.86. MRI-based markers of portal hypertension (spleen ECV; platelet count/spleen diameter ratio; spleen T1 x spleen diameter) and %rT1 were predictive of liver-related clinical outcomes in patients with cirrhosis, who are at high risk of complications, but did not add to the predictive ability of age, albumin, sodium and APRI. In conclusion, MRI provides accurate markers of portal hypertension and liver function. Of particular importance, spleen T1 and spleen diameter criteria could be used to avoid more screening endoscopies in cACLD than any other VNT exclusion criteria reported to date. Although prognostic in the high risk cohort studied here, the MRI markers of portal hypertension and liver function do not surpass the prognostic value of simpler measures.

Abstract: Primary Biliary Cholangitis (PBC) is an idiopathic autoimmune disease characterized by destruction of small hepatic bile ducts and presence of antimitochondrial antibodies (AMA). A Human Betaretrovirus (HBRV) sharing 91-99% sequence similarity with the Mouse Mammary Tumor virus has been identified in PBC patients. The autoimmune biliary disease mouse model for PBC produces spontaneous AMA and is infected by the MMTV suggesting a role for MMTV involvement in disease development. The MMTV also has well documented superantigen activity. Viral superantigen bind the MHC class II receptor on antigen presenting cells to induce clonal expansion of specific TCR-Vβ subsets followed by eventual depletion of cognate T-cells. This skewing of specific TCR-Vβ subsets is the hallmark of superantigen activity. To evaluate a similar role of HBRV infection and superantigen activity in PBC patients, we investigated a transcriptional database from the POISE phase III clinical trials to 1) classify CD4+/CD8+ ratios and Neutrophil to Lymphocyte ratios (NLR, an indicator of lymphopenia) 2) characterize the TCR-Vβ repertoire profile in PBC to find evidence of VB skewing and 3) conduct differential gene expression analysis between PBC patient subsets to identify immunological pathways associated with disease development and progression. Analyzing RNA-seq database, we detected an increased range and higher mean CD4+/CD8+ ratio in PBC. A depleted CD8+ T-cell expression was notably observed in the high CD4+/CD8+ ratios. The CD4+/CD8+ ratio was also identified to be positively correlated with increased lymphopenia and negatively correlated with platelet counts indicating hypersplenism and overall worse prognosis. TCR-Vβ repertoire alteration consistent with superantigen activity was observed. Skewing of multiple Vβ subsets as well as the tendency of these skewed Vβ to skew together in clusters suggests the presence of multiple HBRV superantigen with varying binding avidities. Finally, pathway expression analysis identified increased anti-viral activity along with interferon responses and TLR activation in PBC samples with high CD4+/CD8+ ratio and high NLR. This suggests ongoing HBRV activity and may predict worse prognosis. Meanwhile, low NLR and CD4+/CD8 ratio samples demonstrated increased T-cell activation, activated innate immunity, NK cell immunity and negative regulation of viral transcription indicating a resolving disease process. We also identified CD3E-204, a transcript for the CD3E lymphocyte marker, which had a strong negative correlation with TCR-Vβ skewing. Further studies into the mechanistic differences between the CD3E transcripts need to be made to evaluate the significance of this finding. In future studies, we hope to investigate serial samples from PBC patients to identify changes in TCR-Vβ skewing over time and changes in CD4+/CD8+ ratios over time in patients with known clinical outcomes. We will also investigate TCR-Vβ skewing in isolated CD4+ and CD8+ T-cells in order to better understand the source of CD8+ T-cell depletion. Finally, we propose to clone HBRV superantigen from PBC patient lymphnode using conserved viral superantigen sequences. This will help highlight superantigen proteins in PBC and shed light on their contribution to PBC pathogenesis.

Purpose/Background Despite the surge in HCV prevalence among pregnant women and the threat of perinatal HCV transmission, infants exposed to HCV in the United States are inadequately screened (Chappell et al., 2018). It is estimated that between 4% and 8% of babies exposed to HCV during pregnancy become infected (Watts et al., 2017). Incorporating Hep C screening with other mandated prenatal screening will play an essential role in reducing the complications associated with Hep C transmission to the newborn. Methods Article search was done in the first three weeks of the fall 2021 semester, and a total of 63 articles were screened to exclude those that only focused on men (N=2) and women who were not pregnant (N=5). Six final articles were selected for scoping review, comparing the outcomes of women between 18 and 35 years of age who were at high risk of HCV and who received prenatal HCV screening to those who were not screened. The study further explores the health outcome associated with early detection of HCV in high-risk pregnant women and the vertical transmission rate from mother to newborn baby. Results According to the reviewed studies, early screening and prompt treatment during pregnancy can prevent most mother-to-child transmissions and thus most childhood cases of HCV. Interventions and outcomes noted in the articles selected are to increase screening in infants at risk for perinatal HCV acquisition by including technology to improve the transfer of maternal HCV status to the pediatric record and increase pediatric provider awareness regarding HCV screening guidelines. Implications for Nursing Practice The studies demonstrate that universal HCV screening is efficient, cost-effective, and beneficial when long-term needs are present (Baranoski, 2016). Following the recommendations, universal HCV screening with HCV antibodies and reflex HCV RNA testing may soon become standard practice in the United States. Therefore, it is imperative to review and incorporate this into the curriculum and training proactively. References Baranoski, A. S., Cotton, D., Horsburgh, C. R., Kubiak, R. W., Heeren, T., Nunes, D. (2016). Clinical liver disease progression among hepatitis C infected drug users with CD4 cell count less than 200 cells/mm3 is more pronounced among women than men. Open Forum Infectious Diseases, 3(1). https://doi-org.ezproxy.uthsc.edu/10.1093/ofid/ofv214. Chappell, C. A., Hillier, S. L., Crowe, D., Meyn, L. A., Bogen, D. L., & Krans, E. E. (2018). Hepatitis C Virus Screening Among Children Exposed During Pregnancy. Pediatrics, 141(6), e20173273. https://doi.org/10.1542/peds.2017-3273. Watts, T., Stockman, L., Martin, J., Guilfoyle, S., & Vergeront, J. M. (2017). Increased Risk for Mother-to-Infant Transmission of Hepatitis C Virus Among Medicaid Recipients - Wisconsin, 2011- 2015. MMWR. Morbidity and mortality weekly report, 66(42), 1136–1139. https://doi.org/10.15585/mmwr.mm6642a3

Primary Sclerosing Cholangitis (PSC) is a progressive cholestatic liver disease with outcomes that include hepatobiliary malignancy and liver transplantation. The pathogenesis of PSC is incompletely understood and, as a result, few markers of disease progression have been identified. Vitamin D is associated with the development and treatment of multiple cancers as well as the progression of inflammatory bowel disease, making it a possible candidate as a biomarker associated with PSC outcomes. In this study, we retrospectively and prospectively collected complete laboratory results and outcome datapoints on 179 patients with PSC to determine the association between total 25(OH)-vitamin D levels, vitamin D supplementation, and both hepatobiliary malignancy and liver transplantation. Through survival analysis, we found that history of vitamin D supplementation was significantly associated with increased hepatobiliary malignancy-free and liver transplantation-free survival (p=0.025 and p=0.042, respectively). These results indicate that vitamin D is a promising factor associated with the progression of PSC to transplantation and malignancy. Future studies on this registry cohort as it increases in size and age may provide more conclusive data on the relationship between vitamin D and PSC.

Background. Ethanol (EtOH) consumption is known to affect multiple organs; this is unsurprising, as the concentration of EtOH in the blood at relevant doses reaches the millimolar range. The overarching goal of this dissertation was to elucidate mechanisms of alcohol-induced organ injury, specifically the effects of alcohol on the hepatic extracellular matrix (ECM) proteome, the alcoholic hepatitis (AH) plasma peptidome, and the effects of alcohol on the renal cortex proteome and transcriptome. Methods. Mice were pair-fed ethanol-containing liquid diet chronically, and then some mice were administered lipopolysaccharide (LPS). Liver sections from these mice were processed in a series of increasingly rigorous extraction buffers to separate proteins by ‘age’ and crosslinking. Extracted proteins were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). For the AH study, a workflow was developed for the peptidomic analysis of plasma from healthy participants or AH patients. AH severity was stratified by MELD score as mild (19; n=37). The peptidome in AH and control plasma was analyzed with LC-MS/MS. For the kidney study, renal cortex proteins were extracted in lysis buffer, and RNA was also isolated. Extracted proteins were identified using LC-MS/MS, and RNA sequencing (i.e. transcriptomics) identified transcripts. Results. Chapter III introduced a new proteomic approach for characterizing the hepatic matrisome, which demonstrated that the hepatic matrisome responds dynamically to both acute (LPS) and chronic (ethanol) stresses, long before more dramatic fibrotic changes to the liver. Chapter IV demonstrated that AH causes detectable changes in the plasma ECM degradome/peptidome of patients, and that the LC-MS/MS analysis of the plasma peptidome is a novel, minimally-invasive method for prognosis stratification in patients with AH. Finally, Chapter V revealed that chronic, moderate ethanol consumption affects renal cortical oxidant response pathways at the protein and transcript level. Conclusions. The work presented in this dissertation has, in conclusion, revealed that the hepatic ECM responds dynamically to stress, plasma peptides, including ECM peptides, change with AH severity, and chronic ethanol consumption affects renal cortical oxidant response pathways.

Olanzapine (OLZ) is an effective first-line treatment for schizophrenia and bipolar disorder. The benefits of OLZ are countermanded by side effects such as weight gain, glucose intolerance, dyslipidemia, and liver injury. These effects impact not only antipsychotic medication compliance, but also increase the health risks to patients. Most studies to date have focused on potential effects of OLZ on the central nervous system (e.g., hypothalamic regulation of satiety); however, peripheral changes in key metabolic organs such as the liver may also play a critical role. The obesity rates in the US are now at epidemic levels and obesity-induced liver disease (i.e., non-alcoholic fatty liver disease, or NAFLD) is on the rise. It is now understood that obesity is a significant risk factor for a myriad of drug-induced liver injuries. Given that the obesity incidence in the psychiatric population is even higher than in the US population as a whole, the effects of OLZ may exacerbate an underlying condition in these patients. The purpose of this work was to determine the mechanisms of OLZ-induced hepatic dysmetabolism, and to test the hypothesis that OLZ enhances obesity-induced hepatic injury. OLZ was administered in a mouse model for four weeks along with a high-fat diet (HFD) or low-fat control diet. OLZ alone increased body weight and caused mild glucose intolerance, without a commensurate increase in food consumption. OLZ alone also caused hepatic steatosis and injury. Interestingly, although OLZ increased hepatic triglyceride synthesis and storage, it did not increase the synthesis or abundance of hepatic free fatty acids. OLZ administration appeared to cause a pseudo-fasted state and dramatically depleted hepatic glycogen reserves. These effects of OLZ occurred in parallel to significant changes in hepatic metabolite profiles. The protein kinases AMPK and mTOR are generally differentially activated, and mediate opposing metabolic functions; however, OLZ administration simultaneously activated both AMPK and mTOR. When OLZ and HFD were combined, there was an even greater increase in weight gain and glucose intolerance. Liver damage from concurrent HFD and OLZ was worse than liver damage resulting from HFD or OLZ alone. Lastly, sulforaphane (SFN) was tested as a possible preventive against HFD- and OLZ-induced toxicity. Some mice receiving HFD and OLZ were also treated with SFN (90 mg/kg/d). SFN, a known inducer of the Nrf2 intrinsic antioxidant pathway, prevented weight gain and liver injury and rescued hepatic glycogen storage. Furthermore, SFN decreased the presence of 4-hydroxynonenal (4HNE) adducts in liver (>20-fold), indicating that SFN treatment substantially limits oxidative stress in this model. In summary, these data show that OLZ dysregulates glucose and lipid metabolism and exacerbates hepatic changes caused by HFD exposure. The outcomes of OLZ administration on hepatic metabolism may reflect, in part, the contradictory inputs of simultaneous AMPK and mTOR activation. These data indicate that the metabolic changes caused by OLZ may sensitize the liver to injury caused by HFD and that underlying obesity/liver disease may aggravate OLZ-induced side effects. The activation of intrinsic antioxidant defenses with SFN can partially prevent these effects of OLZ and may represent a useful strategy to protect against liver injury.

Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, is known to confer antioxidant protection in vivo. Rather than directly reacting with free radicals, however, SFN works by inducing Nrf2, a transcription factor that binds to the promoter regions of several known antioxidant genes and enhances detoxification. Because oxidative stress is a major contributor to acetaminophen (APAP)-induced hepatotoxicity, SFN may defend the liver against APAP overdose by activating the Nrf2 pathway and increasing endogenous antioxidant response. To test this hypothesis, mice were pre-treated with SFN for four days, injected with APAP on the fifth day, and sacrificed shortly thereafter. APAP overdose caused massive hepatic injury, as shown by increases in serum liver enzyme activity and lipid peroxidation. APAP overdose also manifested as decreases in total glutathione and glutathione reductase activity. SFN administration clearly prevented these manifestations of liver injury, however: increases in serum liver enzyme activity and lipid peroxidation were blunted, while total glutathione and glutathione reductase activity remained similar to those of control animals. SFN treatment did not affect the catalytic activity of acetaminophen-metabolizing enzyme CYP2E1, but did increase nuclear accumulation of Nrf2, suggesting that SFN acts primarily through the Nrf2 pathway. In summary, these data support the hypothesis that sulforaphane attenuates acute acetaminophen-induced liver injury. This decrease in injury results from the increased availability of glutathione to react with toxic metabolites of acetaminophen.