CYP450 Mediates Reactive Oxygen Species Production in a Mouse Model of β-Thalassemia through an Increase in 20-HETE Activity

Oxidative damage by reactive oxygen species (ROS) is one of the main contributors to cell injury and tissue damage in thalassemia patients. Recent studies suggest that ROS generation in non-transfusion-dependent (NTDT) patients occurs as a result of iron overload. Among the different sources of ROS, the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family of enzymes and cytochrome P450 (CYP450) have been proposed to be major contributors for oxidative stress in several diseases. However, the sources of ROS in patients with NTDT remain poorly understood. In this study, Hbbth3/+ mice, a mouse model for β-thalassemia, were used. These mice exhibit an unchanged or decreased expression of the major NOX isoforms, NOX1, NOX2 and NOX4, when compared to their C57BL/6 control littermates. However, a significant increase in the protein synthesis of CYP4A and CYP4F was observed in the Hbbth3/+ mice when compared to the C57BL/6 control mice. These changes were paralleled by an increased production of 20-hydroxyeicosatetraenoic acid (20-HETE), a CYP4A and CYP4F metabolite. Furthermore, these changes corroborate with onset of ROS production concomitant with liver injury. To our knowledge, this is the first report indicating that CYP450 4A and 4F-induced 20-HETE production mediates reactive oxygen species overgeneration in Hbbth3/+ mice through an NADPH-dependent pathway.


Introduction
The thalassemias are among the most common groups of recessively inherited disorders worldwide, and are characterized by reduced or absent production of red cell hemoglobin and chronic anemia with varying severity [1,2]. The epidemiology of various forms of the thalassemias remains poorly recognized. However, the disease is vastly prevalent in regions that extend from sub-Saharan Africa, through the Mediterranean region and phospholipase A2 from the phospholipid membrane induces the release of arachidonic acid. Free arachidonic acid is then metabolized by the cyclooxygenase, lipoxygenase, and monooxygenase pathways. genase, and monooxygenase pathways.
The major products of the CYP450-catalyzed arachidonic acid monooxygenase pathway are regiospecific and stereospecific epoxyeicosatrienoic acids (EETs) and their corresponding dihydroxyeicosatrienoic acids (DHETs), and 20-hydroxyeicosatetraenoic acid   [26,27] (Figure 1B). Cytochrome P450-derived eicosanoids are produced in a cell and tissue-specific manner, with numerous biological functions. They play a major role as second messengers, regulating vascular tone and ion transport [28,29]. Recently, many studies have shown that 20-HETE also plays a role in other critical biological processes, including control of reactive oxygen species production, cellular proliferation, inflammation, and hemostasis [27,30]. Oxidative damage by ROS is a major contributor to cell injury and tissue damage in patients with thalassemia. Increased ROS generation in NTDT patients has been linked to multiple pathological outcomes in various organs. The aim of this study is therefore to identify the exact source of ROS in the liver of Hbb th3/+ mice. Consequently, we show that among the different sources of ROS, CYP450 of the 4A and 4F family of enzymes is the driving force leading to liver injury in a mouse model of β-thalassemia.

Increased Tissue Iron Levels in the Liver of Hbb th3/+ Mice
A major contributor to oxidative stress in β-thalassemia is excess iron, known to be involved in ROS generation [31]. We therefore wanted to confirm the state of iron overload in our Hbb th3/+ mice. Indeed, the liver tissue iron content was increased in thalassemic mice compared to their control littermates (Figure 2A). The major products of the CYP450-catalyzed arachidonic acid monooxygenase pathway are regiospecific and stereospecific epoxyeicosatrienoic acids (EETs) and their corresponding dihydroxyeicosatrienoic acids (DHETs), and 20-hydroxyeicosatetraenoic acid (20-HETE) [26,27] (Figure 1B). Cytochrome P450-derived eicosanoids are produced in a cell and tissue-specific manner, with numerous biological functions. They play a major role as second messengers, regulating vascular tone and ion transport [28,29]. Recently, many studies have shown that 20-HETE also plays a role in other critical biological processes, including control of reactive oxygen species production, cellular proliferation, inflammation, and hemostasis [27,30].
Oxidative damage by ROS is a major contributor to cell injury and tissue damage in patients with thalassemia. Increased ROS generation in NTDT patients has been linked to multiple pathological outcomes in various organs. The aim of this study is therefore to identify the exact source of ROS in the liver of Hbb th3/+ mice. Consequently, we show that among the different sources of ROS, CYP450 of the 4A and 4F family of enzymes is the driving force leading to liver injury in a mouse model of β-thalassemia.

Increased Tissue Iron Levels in the Liver of Hbb th3/+ Mice
A major contributor to oxidative stress in β-thalassemia is excess iron, known to be involved in ROS generation [31]. We therefore wanted to confirm the state of iron overload in our Hbb th3/+ mice. Indeed, the liver tissue iron content was increased in thalassemic mice compared to their control littermates (Figure 2A). Superoxide generation in liver tissues was increased in thalassemic mice compared to their control littermates ( Figure 2B). Furthermore, NADPH oxidase activity was also increased in the liver of the Hbb th3/+ mice when compared to their control littermates. Taken together, these data suggest that iron overload induces ROS generation through an NADPH-dependent pathway, which may involve the different NOX isoforms or the cytochromes P450, specifically the 4A or 4F family of enzymes ( Figure 2C).

Hbb th3/+ Mice Have an Unchanged or Decreased Protein Expression of the NOX Isoforms
In order to assess if the NOX family of enzymes is responsible for the increase in NADPH oxidase activity observed in the livers of the Hbb th3/+ mice, mRNA levels and protein expression of NOX1, NOX2, and NOX4, described to be abundant in the liver [20], were assessed by real-time polymerase chain reaction (PCR) and Western blot. At the mRNA levels, no significant changes were observed in NOX1, NOX2, or NOX4 ( Figure  3A-C). However, at the protein level, NOX1 expression was not altered, while the protein expression of NOX2 and NOX4 was decreased in the liver of Hbb th3/+ mice when compared with their control littermates ( Figure 3D-F). Therefore, these results suggest that another source of ROS that is NADPH-dependent is responsible for the observed ROS generation.

Reactive Oxygen Species Production in Hbb th3/+ Mice Is Induced through an NADPH Oxidase-Dependent Mechanism
Superoxide generation in liver tissues was increased in thalassemic mice compared to their control littermates ( Figure 2B). Furthermore, NADPH oxidase activity was also increased in the liver of the Hbb th3/+ mice when compared to their control littermates. Taken together, these data suggest that iron overload induces ROS generation through an NADPH-dependent pathway, which may involve the different NOX isoforms or the cytochromes P450, specifically the 4A or 4F family of enzymes ( Figure 2C).

Hbb th3/+ Mice Have an Unchanged or Decreased Protein Expression of the NOX Isoforms
In order to assess if the NOX family of enzymes is responsible for the increase in NADPH oxidase activity observed in the livers of the Hbb th3/+ mice, mRNA levels and protein expression of NOX1, NOX2, and NOX4, described to be abundant in the liver [20], were assessed by real-time polymerase chain reaction (PCR) and Western blot. At the mRNA levels, no significant changes were observed in NOX1, NOX2, or NOX4 ( Figure 3A-C). However, at the protein level, NOX1 expression was not altered, while the protein expression of NOX2 and NOX4 was decreased in the liver of Hbb th3/+ mice when compared with their control littermates ( Figure 3D-F). Therefore, these results suggest that another source of ROS that is NADPH-dependent is responsible for the observed ROS generation.

Hbb th3/+ Mice Have an Increased Protein Expression of CYPs 4A and 4F Associated with an Increase in 20-HETE Production
A second family of ROS that is NADPH dependent is the CYP450. No significant changes were observed in the liver mRNA levels of CYP4A and CYP4F of the Hbb th3/+ mice when compared to their control littermates. However, when it came to the protein expression of these two major CYP450 isoforms expressed in the liver, the Hbb th3/+ mice exhibited a higher expression of these isoforms when compared to their control littermates ( Figure 4A-D). The increase in the protein expression of CYP4A was further validated by immunohistochemistry staining of the liver tissue sections of the Hbb th3/+ mice and their control littermates ( Figure 4E). Besides, and of high interest, the increase in the protein expression of the CYPs 4A and 4F corroborated with an increase in their corresponding metabolite 20-HETE, known to be responsible for ROS generation ( Figure 5).

Hbb th3/+ Mice Have an Increased Protein Expression of CYPs 4A and 4F Associated with an Increase in 20-HETE Production
A second family of ROS that is NADPH dependent is the CYP450. No significa changes were observed in the liver mRNA levels of CYP4A and CYP4F of the Hbb th3/+ mi when compared to their control littermates. However, when it came to the protein expre sion of these two major CYP450 isoforms expressed in the liver, the Hbb th3/+ mice exhibite a higher expression of these isoforms when compared to their control littermates (Figu 4A-D). The increase in the protein expression of CYP4A was further validated by im munohistochemistry staining of the liver tissue sections of the Hbb th3/+ mice and their co trol littermates ( Figure 4E). Besides, and of high interest, the increase in the protein e pression of the CYPs 4A and 4F corroborated with an increase in their corresponding m tabolite 20-HETE, known to be responsible for ROS generation ( Figure 5).   . Overexpression of CYP4A and CYP4F in Hbb th3/+ mice. Relative mRNA levels (%) of (A) CYP1A/YWHAZ and (B) /YWHAZ. Representative Western blot of (C) CYP4A/HSC70 and (D) CYP4F/HSC70, with the respective densitouantification in liver tissues of control and Hbb th3/+ mice. (E) Immunohistochemistry staining of liver tissue sections r CYP4A expression in control mouse, and CYP4A expression in Hbb th3/+ mice. Scale bar represents 40 μm. Values means ± SEM from 4 different mice in each group (n = 4). * p < 0.05 versus control. C.V: central vein.

Liver Tissues of Hbb th3/+ Mice Show Signs of Inflammation and Fibrosis
In order to assess if the mechanistic changes observed in the Hbb th3/+ mice correlate with liver injury, hematoxylin and eosin (H&E) staining was performed. The stained liver tissue sections revealed an infiltration of inflammatory foci in Hbb th3/+ mice compared to their control littermates ( Figure 6A

Liver Tissues of Hbb th3/+ Mice Show Signs of Inflammation and Fibrosis
In order to assess if the mechanistic changes observed in the Hbb th3/+ mice correlate with liver injury, hematoxylin and eosin (H&E) staining was performed. The stained liver tissue sections revealed an infiltration of inflammatory foci in Hbb th3/+ mice compared to their control littermates ( Figure 6A

Discussion
Oxidative damage by ROS is a major contributor to cell injury and tissue damage in patients with thalassemia [32]. Recent studies suggest that ROS generation in NTDT patients occurs as a result of iron overload [33]. This increased ROS production in organs has been associated with multiple pathological outcomes. Sources of ROS production in pathophysiology have been proposed to be tissue and disease specific. Despite all the advances in the thalassemia field, no study in the literature was able to provide evidencebased data identifying potential sources of ROS in NTDT patients.
Hematologic studies including a complete blood count in Hbb th3/+ mice have been well documented by our group [34,35]. In this study, increased tissue iron levels (iron overload) were paralleled by an increase in superoxide generation in the liver tissues of Hbb th3/+ mice when compared to their control littermates. Iron chelators can act as general antioxidants [36]. This is because they can remove both intra-and extracellular iron species that generate free oxygen radicals. Although ROS are associated with injurious processes, their presence is essential for cellular functions such as gene transcription and cell proliferation,

Discussion
Oxidative damage by ROS is a major contributor to cell injury and tissue damage in patients with thalassemia [32]. Recent studies suggest that ROS generation in NTDT patients occurs as a result of iron overload [33]. This increased ROS production in organs has been associated with multiple pathological outcomes. Sources of ROS production in pathophysiology have been proposed to be tissue and disease specific. Despite all the advances in the thalassemia field, no study in the literature was able to provide evidencebased data identifying potential sources of ROS in NTDT patients.
Hematologic studies including a complete blood count in Hbb th3/+ mice have been well documented by our group [34,35]. In this study, increased tissue iron levels (iron overload) were paralleled by an increase in superoxide generation in the liver tissues of Hbb th3/+ mice when compared to their control littermates. Iron chelators can act as general antioxidants [36]. This is because they can remove both intra-and extracellular iron species that generate free oxygen radicals. Although ROS are associated with injurious processes, their presence is essential for cellular functions such as gene transcription and cell proliferation, and in maintaining proper blood flow and blood pressure homeostasis [13,[37][38][39][40][41]. These physiological functions of ROS, among other reasons, explain why numerous attempts to treat ROS-associated diseases with general antioxidants have failed and, in some instances, caused deleterious effects [42,43]. The observed increase in ROS generation is attributed herein to an increase in NADPH oxidase activity. The NOX family members are transmembrane proteins responsible for transporting electrons across biological membranes to reduce oxygen to superoxide. Different NOX isoforms have been described, with different structures and functions. After observing an increase in the NADPH oxidase activity in thalassemic mice, mRNA and protein levels of the major NADPH oxidase isoforms described in the liver (NOX1, NOX2, and NOX4) were assessed. Hepatocytes are known to generate these different NADPH oxidase isoforms as a response mechanism to many endogenous and exogenous stimuli. Studies measuring total liver mRNA showed large amounts of NOX2 and trace amounts of NOX4 [20,44]. Other studies conducted on rats showed that their hepatocytes expressed NOX1, NOX2, and NOX4 mRNAs [21]. Both NOX1 (mRNA) and NOX2 (mRNA and protein) have also been shown to be expressed in hepatic stellate cells' primary culture and cell lines [45,46]. Kupffer cells have also been shown to express NOX2 and its subunits [47,48]. Here, our data suggest that there is no involvement of these NOX isoforms in the observed NADPH oxidase activation, since the mRNA levels of these isoforms were unchanged, and the protein expression showed a tendency to decrease (NOX1) or were decreased (NOX2 and NOX4). In fact, these observations can be explained by a probable increase in activity of antioxidants like Sestrin 2, which is known to inhibit the increase in NOX4 [49]. Other antioxidants such as nuclear factor erythroid 2-related factor 2 (Nrf2) have also been described as master regulators of antioxidant responses and defensive genes in many diseases, including neurodegeneration, cancer, kidney disease, cardiovascular diseases, hepatitis, and inflammation associated with infection. In fact, the NOX4/Nrf2 pathway may also represent a common protective mechanism [50,51]. Therefore, the NOX4/Nrf2 pathway may be critical for inhibiting the increase in NOX4 production and for overall metabolic homeostasis.
Taken together, these observations led us to investigate if the NADPH-dependent CYPs family of enzymes, known to induce ROS production, is responsible for the ROS generation detected and orchestrating the observed liver injury in the Hbb th3/+ mice. The CYP450s are a large family of hemoproteins that are primarily responsible for metabolism of endogenous and exogenous molecules. They are bound to the membranes of either the mitochondria or endoplasmic reticulum, and are known to play a role in redox reactions [22]. Additionally, CYPs are reported to be major sources of ROS in numerous tissues, with implications in different disease conditions [27,52]. Enzymes of the CYP4A and CYP4F subfamilies have not been investigated nor reported in NTDT patients. Subsequently, we first examined whether these CYPs could be expressed in Hbb th3/+ mice. To our knowledge, the present study is the first to show an increase in the protein expression of the CYP4A and CYP4F in the livers of Hbb th3/+ mice, concomitant with an increase in the 20-HETE metabolites, the effects of which included an infiltration of inflammatory foci and the presence of a perivenular bridging chicken-wire pattern of collagen deposition in the livers of Hbb th3/+ mice.
Major products of the CYP450 4A and 4F-catalyzed arachidonic acid monooxygenase pathway include 20-HETE [26,27]. This metabolite has numerous biological functions and is produced in a cell and tissue-specific manner. For example, 20-HETE has been shown to play a major role in circulation hemodynamics, regulation of renal Na + /K + ATPase activity, Ca 2+ and Cl − fluxes, vascular remodeling, angiogenesis, cellular proliferation, inflammation, and hemostasis [27,30]. It has also been shown to play a role in hormonal signaling through epidermal growth factor and vascular endothelial growth factor, angiotensin, vasopressin, and norepinephrine [53][54][55][56][57]. However, recent studies have attributed a role of 20-HETE in organ damage. 20-HETE was found to be involved in abnormalities related to liver diseases, particularly cirrhosis. In patients with hepatic cirrhosis, 20-HETE is produced in increased amounts in the preglomerular microcirculation, resulting in constriction of renal vasculature, reduction of renal blood flow, and depression of renal hemodynamics [58]. Moreover, inhibition of 20-HETE production has been shown to reduce abnormal cellular growth, vascular inflammation, and diabetic nephropathy [59,60]. However, the role of 20-HETE in thalassemia is not yet elucidated. Herein, we believe that in Hbb th3/+ mice, 20-HETE may be the orchestrator of liver injury. These results suggest that inhibiting CYPs 4A and 4F-induced 20-HETE production could be a potential treatment in thalassemia. In that spirit, various studies have investigated the protective role of 20-HETE inhibition via N-Hydroxy-N -(4-butyl-2-methylphenyl)-formamidine (HET0016). HET0016 is a highly selective inhibitor of the CYP4A isoforms that produce 20-HETE. HET0016 treatments in hypertensive rats were capable of reducing superoxide production, oxidative stress, and inflammation, and restoring vasomotor function [58]. The inhibition of 20-HETE synthesis via HET0016 was also shown to reverse renal injury [61]. Another selective inhibitor of 20-HETE synthesis, N-(3-chloro-4-morpholin-4-yl) phenyl-N -hydroxyimido formamide (TS-011), reduced the elevation of brain and plasma 20-HETE levels after ischemia, reducing the infarct volume and improving the neurological outcome in rat and monkey stroke models [62,63].
Oxidative stress and increased production of transforming growth factor-beta 1 (TGF-β1) are believed to be key mechanisms in the development of liver fibrosis [64,65]. In patients with hepatic fibrosis, increased concentrations of TGF-β1 correlated with the severity of hepatic fibrosis, suggesting a link between TGF-β1 expression and increased extracellular matrix deposition and progressive liver disease [66][67][68]. SMAD proteins have been studied extensively as essential intracellular effectors of TGF-β1, acting as transcription factors. The role and molecular mechanisms of the TGF-β/SMAD pathway in the pathogenesis of hepatic fibrosis have been well described [65,69]. Previous studies conducted by our group showed that alteration in CYP4A and its metabolite 20-HETE play a key role in kidney injury in diabetic rats by upregulating TGF-β1 protein expression and levels. This increase in TGF-β1 expression and levels, however, was prevented with the inhibition of CYP4A [60]. Therefore, we speculate that in Hbb th3/+ mice, CYP4A and 20-HETE production could be a major pathophysiological mechanism that is leading to the activation of ROS through TGF-β1, thus resulting in liver cell injury. Further studies are warranted to confirm this hypothesis.

Mice
All animals (C57BL/6 background) were bred at the animal care facility of the American University of Beirut. We used the Hbb th3/+ mouse model (The Jackson Laboratory-B6; 129P2-Hbb-b1 tm1Unc Hbb-b2 tm1Unc /J), which carries a double knock-out of the Hbb-b1 and Hbb-b2 adult β-globin genes with a phenotype like that seen in NTDT. Eight mice were divided into two groups (a control group, and an Hbb th3/+ group). Animals were kept in a temperature-controlled room and on a 12/12 dark/light cycle and had standard chow and water access. All animal-model experimental protocols used in this study were approved by the Institutional Animal Care and Use Committee of the American University of Beirut (protocol code 17-03-412/586).

Hematologic Studies
Hematologic studies in Hbb th3/+ mice including a complete blood count have been well documented by our group [34,35]. In Hbb th3/+ mice, hemoglobin (Hb) levels span from 6 to 9 g/dL. A normal mouse will have an Hb level between 12 and 15 g/dL. A red blood cell count of 5-8 (×10 6 cells/µL) and reticulocyte count of 1000-2000 (×10 9 cells/L) are also characteristic of Hbb th3/+ mice, compared to their control littermates.

Tissue Iron Content
Liver iron content was measured by high-performance liquid chromatography (HPLC) as previously described [70].

Reactive Oxygen Species Detection
To assess cellular superoxide production in liver tissues, high-performance liquid chromatography analysis of dihydroethidium (DHE)-derived oxidation products was performed as previously described [71,72].

NADPH Oxidase Activity Assay
NADPH oxidase activity was measured in liver tissues as previously described [49,[72][73][74]. Superoxide production was expressed as relative light units/min/mg of protein. Protein content was measured using the Bio-Rad protein assay reagent.

20-HETE Levels
Levels of 20-HETE were measured using the 20-HETE enzyme-linked immunosorbent assay kit (Detroit R&D, INC., Detroit, MI 48201, USA) according to the manufacturer protocol and as in our previous studies [75].

Western Blot Analysis
Homogenates from extracted liver were prepared, and a Western blot analysis was performed as previously described [49,[72][73][74]

mRNA analysis
mRNA was analyzed by quantitative real-time PCR using the ∆∆C t method [49,[72][73][74]. mRNA expression was quantified using a CFX96 Touch thermal cycler (Bio-Rad, Hercules, CA 94547, USA) with SYBR Green dye, and mouse and human RT 2 qPCR primers of the corresponding gene of interest (Table 1).

Immunohistochemistry
Immunohistochemistry for CYP4A (1:150, Abcam, Cambridge, MA 02139, USA) was done on paraffin-embedded tissue sections as previously described [76]. Sections were examined under the light microscope (Olympus CX41 for slide imaging), and analysis of the sections was performed using Image J software version 1.53.

Fibrosis and Inflammation Assays: Hematoxylin and Eosin (H&E) and Masson Trichome Staining
The pathogenesis of fibrosis and inflammation due to iron overload and increase in ROS production was shown by H&E and Masson trichrome staining as previously described [76].

Statistical Analysis
Results were expressed as mean ± standard error of the mean (SEM). Statistical significance was assessed with the Student's unpaired t-test. A p-value < 0.05 was considered as statistically significant. All statistical analyses were performed with Prism 6 Software (GraphPad Software).

Conclusions
In summary, this is the first report indicating that CYP450 4A and 4F mediate reactive oxygen species production in Hbb th3/+ mice through an increase in 20-HETE activity, opening the door to study the potential therapeutic effect of inhibiting 20-HETE synthesis in thalassemia, or even to use these selective 20-HETE inhibitors in combination with iron chelation therapy and assess for potential improvements in physiological parameters, which could lead to better outcomes in treating thalassemia patients in the future. We strongly believe that the field of targeted oxidative stress inhibition could prove to be the next novel therapeutic approach in the thalassemia realm. Data Availability Statement: The datasets used and/or analyzed during the current study are available from the corresponding author upon request.