Murine cytochrome P450 2A5 (CYP2A5) is an interesting enzyme for its unique regulation and its involvement in liver injury caused by various well-known pathological conditions or hepatotoxins. It has been reported that CYP2A5 is upregulated following exposure to chemical hepatotoxins and during pathophysiological conditions in which the levels of most Cytochrome P450s are either unchanged or down-regulated. Recently bilirubin has been identified as the first endogenous substrate for CYP2A5 and it has been suggested that CYP2A5 plays a major role in bilirubin clearance as an alternative mechanism to BR conjugation by UGT1A1. This study investigated the mechanisms of gene regulation and cytoprotective role of CYP2A5 in response to bilirubin treatment in liver. Our results demonstrate that bilirubin induces CYP2A5 expression at the mRNA and protein levels by increasing CYP2A5 transcription via a mechanism that involves Nrf2 activation. Furthermore, our results suggest that induced CYP2A5 plays a cytoprotective role against bilirubin toxicity by directly lowering the cellular levels of bilirubin and by inhibiting caspase-3 activation.

The liver is the largest internal organ and performs a wide variety of functions to maintain organismal homeostasis. While some liver functions are carried out by all hepatocytes, other functions are restricted to certain populations of hepatocytes within the liver. This phenomenon, called zonal gene regulation or liver zonation, controls may metabolic processes within the liver including ammonia detoxification; glucose homeostasis; bile acid and glutamine synthesis; and metabolism of xenobiotics, lipids, and amino acids. The liver also expresses many genes in a developmental or sex-biased manner. Some genes are expressed at higher levels early or late in development, or alternatively, in male or female liver. Several years ago, our lab identified a transcription factor called Zinc finger and homeoboxes 2 (Zhx2) based on its ability to control the silencing of genes that are normally expressed in the fetal liver. Zhx2 belongs to a small gene family that also includes Zhx1 and Zhx3. These four exon genes have a rather unique structure in that their entire protein coding region is located on an unusually large third exon. Preliminary studies indicate that these proteins are found only in vertebrates. I have performed a comprehensive analysis of Zhx proteins across a number of chordate species to determine their relationship throughout chordate evolution. Using multiple sequence alignment and phylogenetic tree-building, my studies have found that the primordial Zhx gene is most related to Zhx3 and that this gene exists in lower chordates including lancelet, sea squirt, and sea lamprey. Additional studies from our lab showed that Zhx2 regulates numerous hepatic genes in the adult liver, including cytochrome p450 (Cyp) genes as well as other genes that exhibit sex-biased expression. Previous studies have demonstrated that female-biased expression of Cyp2a4, is controlled, in part, by Zhx2. I have extended these studies to perform a comprehensive analysis of Cyp2a4 and the highly related Cyp2a5 gene. Despite the high similarity of these two Cyp genes, my data indicate that these genes exhibit different zonal expression patterns and are differentially regulated in the regenerating liver. In the course of these studies, I discovered and characterized antisense transcripts for both Cyp2a4 and Cyp2a5. Both Cyp2a4as and Cyp2a5as have positively correlated expression patterns compared to their sense counterparts. In contrast to Cyp2a4 and Cyp2a5, Cyp2a4as and Cyp2a5as show sex-biased expression patterns earlier in development, suggesting that they might contribute to later sex-biased patterns established for Cyp2a4 and Cyp2a5.

Background & aims Cytochrome P450 2A5 (CYP2A5) is induced by ethanol, and the ethanol induction of CYP2A5 is regulated by nuclear factor-erythroid 2-related factor 2 (NRF2). Cyp2a5 knockout (Cyp2a5−/−) mice develop more severe alcoholic fatty liver than Cyp2a5+/+ mice. Fibroblast growth factor 21 (FGF21), a PPARα-regulated liver hormone, is involved in hepatic lipid metabolism. Alcoholic and non-alcoholic fatty liver are enhanced in Pparα knockout (Pparα−/−) mice. This study investigates the relationship between the PPARα-FGF21 axis and the enhanced alcoholic fatty liver in Cyp2a5−/− mice. Methods Mice were fed the Lieber-Decarli ethanol diet to induce alcoholic fatty liver. Results More severe alcoholic fatty liver disease was developed in Cyp2a5−/− mice than in Cyp2a5+/+ mice. Basal FGF21 levels were higher in Cyp2a5−/− mice than in Cyp2a5+/+ mice, but ethanol did not further increase the elevated FGF21 levels in Cyp2a5−/− mice while FGF21 was induced by ethanol in Cyp2a5+/+ mice. Basal levels of serum FGF21 were lower in Pparα−/− mice than in Pparα+/+ mice; ethanol induced FGF21 in Pparα+/+ mice but not in Pparα−/− mice, whereas ethanol induced hypertriglyceridemia in Pparα−/− mice but not in Pparα+/+ mice. Administration of recombinant FGF21 normalized serum FGF21 and triglyceride in Pparα−/− mice. Alcoholic fatty liver was enhanced in liver-specific Fgf21 knockout mice. Pparα and Cyp2a5 double knockout (Pparα−/−/Cyp2a5−/−) mice developed more severe alcoholic fatty liver than Pparα+/+/Cyp2a5−/− mice. Conclusions These results suggest that CYP2A5 protects against the development of alcoholic fatty liver disease, and the PPARα-FGF21 axis contributes to the protective effects of CYP2A5 on alcoholic fatty liver disease.

Abstract Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) is a master regulator of energy metabolism and mitochondrial biology in high-energy cell types and tissues. The regulation of PGC-1α is versatile, and both transcriptional and post-transcriptional mechanisms play major roles. External stimuli affect PGC-1α-regulation which in turn adapts cellular signals to meet them. For example, conditions like fasting and diabetes mellitus (DM) are known to activate PGC-1α expression in the liver, resulting in enhanced de novo glucose production, gluconeogenesis. In the present study, the mechanisms of hepatic PGC-1α regulation and PGC-1α-regulated functions were elucidated. We found that PGC-1α was induced by oral type 2 diabetes therapeutic metformin, via AMPK and SIRT1, regulating the mitochondrial gene response, against previous assumptions. Simultaneously, gluconeogenesis was repressed by other means. Furthermore, PGC-1α upregulated the anti-inflammatory interleukin 1 receptor antagonist (IL1Rn). PGC-1α also diminished interleukin 1β-mediated inflammatory response in hepatocytes. Novel, xenobiotic and endobiotic metabolizing Cytochrome P450 enzymes regulated by PGC-1α were also identified in this thesis. CYP2A5 was induced by PGC-1α through hepatocyte nuclear factor 4α (HNF-4α) coactivation. Also, vitamin D metabolizing CYP2R1 and CYP24A1 were identified as novel genes regulated by PGC-1α, suggesting a role for PGC-1α in the regulation of active vitamin D levels. The findings presented in this thesis provide insight into the pathology of glucose perturbations such as type 2 diabetes, and stimulate discovery of therapeutic agents to treat this disease. Furthermore, the findings suggest that vitamin D metabolism and energy metabolism are tightly linked, with PGC-1α emerging as a novel mediator. / Tiivistelmä Peroksisomiproliferaattori-aktivoituvan reseptori γ:n koaktivaattori 1α (PGC-1α) on merkittävä glukoosiaineenvaihdunnan ja mitokondrioiden toiminnan säätelijä korkeaenergisissä soluissa ja kudoksissa. PGC-1α:a säädellään monin tavoin: sekä transkriptionaalisella säätelyllä että transkription jälkeisellä muokkauksella on merkittävä rooli. Monet ulkoiset tekijät säätelevät PGC-1α:n aktiivisuutta, joka puolestaan säätelee solunsisäisiä signaalireittejä vastaamaan tähän signaaliin. Esimerkiksi paasto ja diabetes mellitus (DM) ovat fysiologisia tiloja, jotka lisäävät voimakkaasti PGC-1α:n ilmentymistä maksassa, jolloin glukoosin uudistuotanto eli glukoneogeneesi kiihtyy. Tässä väitöskirjassa tutkittiin PGC-1α:n säätelyä sekä PGC-1α -säädeltyjä signaalireittejä maksassa. Osoitimme, että tyypin 2 diabeteslääke metformiini indusoi PGC-1α:n ilmentymistä maksassa, vastoin aikaisempia käsityksiä. PGC-1α indusoitui AMPK:n ja SIRT1:n välityksellä, säädelleen edelleen mitokondriaalisten geenien aktiivisuutta. Samalla glukoneogeneesi kuitenkin repressoitui muilla mekanismeilla. Lisäksi osoitimme, että PGC-1α indusoi tulehdusreaktiota vaimentavaa interleukiini 1 reseptorin antagonistia (IL1Rn). PGC-1α esti interleukiini 1β:n aiheuttamaa tulehdusvastetta hepatosyyteissä. Lisäksi väitöskirjassa tunnistettiin uusia, PGC-1α -säädeltyjä lääkeaineita ja elimistön sisäisiä yhdisteitä metaboloivia sytokromi P450 -entsyymejä (CYP). Hiiren CYP2A5:n ilmentymisen osoitettiin olevan PGC-1α- ja HNF4α-välitteistä. Lisäksi osoitettiin, että D-vitamiinia metaboloivat CYP2R1 ja CYP24A1 ovat uusia PGC-1α -säädeltyjä geenejä. Tämä löydös viittaa siihen, että PGC-1α:lla on rooli aktiivisen D-vitamiinin säätelyssä. Tämän väitöskirjan löydökset lisäävät tietoa glukoosiaineenvaihdunnan häiriöiden kuten tyypin 2 diabeteksen molekulaarisista mekanismeista, joita voidaan hyödyntää mahdollisten uusien lääkeaineiden kehittämisessä. Lisäksi väitöskirjassa osoitettiin, että D-vitamiinimetabolia on kytköksissä energia-aineenvaihduntaan ja että PGC-1α:lla on tässä rooli, jota ei aiemmin ole tunnettu.

Cadmium (Cd) is present in food at low levels, particularly in crops and is also present in groundwater. Cd can also be obtained from tobacco smoking and occupational exposure. Cd is not effectively excreted from the body. The primary organ that accumulates Cd is liver. Liver is the main organ involved in metabolizing exogenous chemicals. While metabolism of chemicals causes detoxification, it can also result in liver oxidative damage. CYP2A6 (CYP2A5 in mice) is mainly expressed in the liver. CYP2A6 expression is increased in patients with alcoholic or non-alcoholic fatty liver. Alcohol feeding induced CYP2A5 in mice and alcohol-induced fatty liver disease was enhanced in CYP2A5 knockout (CYP2A5-/-) mice, suggesting a protective effect of CYP2A5 on alcoholic fatty liver disease. PPAR-alpha, a transcription factor, is a major regulator of lipid metabolism in the liver.  CYP2A5 and PPAR-alpha are suggested to work together in regulation of lipid metabolism and in protection against alcoholic fatty liver. It is also suggested that CYP2A5 along with PPAR-alpha protects against high fat diet induced metabolic syndrome. Cadmium can also induce CYP2a5 in mice. Recently it was discovered that there is a positive relation between soil heavy metals and fatty liver disease. Exposure to Cadmium leads to lipid accumulation in the liver, which can eventually lead to the development of Non-Alcoholic Fatty Liver Disease (NAFLD). In this study, the effects of CYP2A5 and PPAR-alpha on the acute cadmium-induced liver injury were tested using CYP2A5-/- mice and PPAR-alpha knockout (PPARα -/-) mice and CYP2A5 and PPAR-alpha wild-type mice. Cadmium chloride (CdCl2) was administered intraperitoneally at 5 mg/kg body weight. A control group of mice were injected saline for comparison. The mice were sacrificed after 24 hours of injection. Blood was collected to test for markers indicative of liver disease such as ALT and AST levels, triglyceride levels, and blood glucose levels. The liver was collected to examine the liver damage by biochemical assays and pathological evaluation. Both CYP2A5-/- and PPARα -/- mice exhibited less severe liver injury compared to their wild-type counterparts. These results suggest that despite the beneficial roles of both CYP2A5 and PPAR-alpha towards alcohol-induced liver injury and metabolic syndrome, they are not protective against Cd-induced liver injury.

Cadmium is present in food and groundwater. Tobacco smoking and occupational exposure are also major sources for cadmium. Cadmium is primarily accumulated in liver, a major organ metabolizing exogenous chemicals. Chemical metabolism may cause detoxification, but it can also cause bio-activation resulting in liver damage. Cytochrome P450s (CYP) are major liver metabolism enzymes, and cadmium chloride (CdCl2) can induce CYP2A5 in mice. We examined the effect of CYP2A5 on CdCl2-induced liver injury using CYP2A5-knockout (cyp2a5-/-) mice. The cyp2a5-/- mice and their control WT mice were injected CdCl2 intraperitoneally at 5 mg/kg body weight, respectively, to induce liver injury. The control group of cyp2a5-/- mice and WT mice were injected saline at the same volume. Twenty-four hours later, all the mice were sacrificed. As indicated by biochemical assays and pathological evaluation, CdCl2-treated WT mice exhibited more severe liver injury than CdCl2-treated cyp2a5-/- mice, suggesting that CYP2A5 contributes to Cd-induced liver injury.

CYP2A5 is a major enzyme responsible for nicotine and cotinine metabolism in mice. Nicotine and cotinine enhance alcoholic fatty liver in wild type (WT) mice but not in CYP2A5 knockout (KO) mice, and reactive oxygen species (ROS) generated during the CYP2A5-mediated metabolism contributes to the enhancing effect. In combination with ethanol, nicotine and cotinine increased lipid peroxidation end product 4-hydroxynonenal (HNE) in WT mice but not in KO mice. In ethanol-fed KO mice, only 5 and 10 genes were regulated by nicotine and cotinine, respectively. However, in ethanol-fed WT mice, 59 and 104 genes were regulated by nicotine and cotinine, respectively, and 7 genes were up-regulated by both nicotine and cotinine. Plin 2 and Cdkn1a are among the 7 genes. Plin2 encodes adipose differentiation-related protein (ADRP), a lipid droplet-associated protein, which was confirmed to be increased by nicotine and cotinine in WT mice but not in KO mice. Cdkn1a encodes P21 and elevated P21 in nuclei was also confirmed. HNE can increase P21 and P21 inhibit cell proliferation. Consistently, hepatocyte proliferation markers proliferating cell nuclear antigen (PCNA) and Ki67 were decreased in WT mice but not in KO mice by nicotine/ethanol and cotinine/ethanol, respectively. These results suggest that inhibition of liver proliferation via a ROS-HNE-P21 pathway is involved in nicotine- and cotinine-enhanced alcoholic fatty liver.

This article reviews recent studies on CYP2E1-mediated alcoholic liver injury, the induction of CYP2A5 by alcohol and the mechanism for this upregulation, especially the permissive role of CYP2E1 in the induction of CYP2A5 by alcohol and the CYP2E1-ROS-Nrf2 pathway, and protective effects of CYP2A5 against ethanol-induced oxidative liver injury. Ethanol can induce CYP2E1, an active generator of reactive oxygen species (ROS), and CYP2E1 is a contributing factor for alcoholinduced oxidative liver injury. CYP2A5, another isoform of cytochrome P450, can also be induced by ethanol. Chronic feeding of ethanol to wild type mice increased CYP2A5 catalytic activity, protein and mRNA levels as compared to pair-fed controls. This induction was blunted in CYP2E1 knockout (cyp2e1 -/- ) mice but was restored when human CYP2E1 was reintroduced and expressed in cyp2e1 -/- mice. Ethanol-induced CYP2E1 co-localized with CYP2A5 and preceded the elevation of CYP2A5. The antioxidants N-acetyl cysteine and vitamin C lowered the alcohol elevation of ROS and blunted the alcohol induction of CYP2A5, but not CYP2E1, suggesting ROS play a novel role in the crosstalk between CYP2E1 and CYP2A5. The antioxidants blocked the activation of Nrf2, a transcription factor known to upregulate expression of CYP2A5. When alcohol-induced liver injury was enhanced in Nrf2 knockout (Nrf2 -/- ) mice, alcohol elevation of CYP2A5 but not CYP2E1 was also lower in Nrf2 -/- mice. CYP2A5 knockout (cyp2a5 -/- ) mice exhibited an enhanced alcoholic liver injury compared with WT mice as indicated by serum ALT, steatosis and necroinflammation. Alcohol-induced hyperglycemia were observed in cyp2a5 -/- mice but not in WT mice.

<p>The family of proteins known as heterogeneous nuclear ribonucleoproteins (hnRNPs) is large and diverse. Often, one and the same hnRNP will perform multiple cellular functions, leading to their description as “multifunctional proteins”. The two hnRNPs known as hnRNP A1 and hnRNP C1/C2 are multifunctional proteins found to affect the transcription, splicing, stability, and translation of specific genes’ mRNA. They are implicated in carcinogenesis, apoptosis, and DNA damage response mechanisms.</p><p>The aims of this thesis were to study the hnRNP A1 and hnRNP C1/C2 dependent regulation of two highly stress responsive genes, the tumor suppressor p53 and the cytochrome P450 enzyme <i>Cyp2a5/CYP2A6</i>. We identified hnRNP C1/C2 as a DNA damage induced binding protein towards the coding region of p53 mRNA, and found that while a specific <i>cis</i> binding site appears to have a positive function in p53 expression, interaction of hnRNP C1/C2 with this site represses the expression. The data suggest that two distinct molecular mechanisms exist for the down-regulation of p53 by hnRNP C1/C2. One mechanism, active during transcriptional stress, is dependent upon the aforementioned site, and the other, independent. We discuss how hnRNP C1/C2 dependent repression of p53 may play a role in apoptosis. </p><p>The data presented here further suggest that the transcriptional and post-transcriptional processes controlling the expression of the murine <i>Cyp2a5</i> gene are linked <i>via</i> hnRNP A1, by performing functions in the nucleus as a transcription factor, or in the cytoplasmic compartment as a <i>trans </i>factor bound to the 3’UTR of the mRNA as needed. Our studies of the human ortholog of this gene, <i>CYP2A6</i>, suggest that this gene is regulated post-transcriptionally in a manner similar to that of its murine counterpart, <i>via</i> changes in mRNA stability and interaction of hnRNP A1 with its 3’ UTR. </p>

The family of proteins known as heterogeneous nuclear ribonucleoproteins (hnRNPs) is large and diverse. Often, one and the same hnRNP will perform multiple cellular functions, leading to their description as “multifunctional proteins”. The two hnRNPs known as hnRNP A1 and hnRNP C1/C2 are multifunctional proteins found to affect the transcription, splicing, stability, and translation of specific genes’ mRNA. They are implicated in carcinogenesis, apoptosis, and DNA damage response mechanisms. The aims of this thesis were to study the hnRNP A1 and hnRNP C1/C2 dependent regulation of two highly stress responsive genes, the tumor suppressor p53 and the cytochrome P450 enzyme Cyp2a5/CYP2A6. We identified hnRNP C1/C2 as a DNA damage induced binding protein towards the coding region of p53 mRNA, and found that while a specific cis binding site appears to have a positive function in p53 expression, interaction of hnRNP C1/C2 with this site represses the expression. The data suggest that two distinct molecular mechanisms exist for the down-regulation of p53 by hnRNP C1/C2. One mechanism, active during transcriptional stress, is dependent upon the aforementioned site, and the other, independent. We discuss how hnRNP C1/C2 dependent repression of p53 may play a role in apoptosis. The data presented here further suggest that the transcriptional and post-transcriptional processes controlling the expression of the murine Cyp2a5 gene are linked via hnRNP A1, by performing functions in the nucleus as a transcription factor, or in the cytoplasmic compartment as a trans factor bound to the 3’UTR of the mRNA as needed. Our studies of the human ortholog of this gene, CYP2A6, suggest that this gene is regulated post-transcriptionally in a manner similar to that of its murine counterpart, via changes in mRNA stability and interaction of hnRNP A1 with its 3’ UTR.