Received date: February 13, 2013; Accepted date: March 20, 2014; Published date: March 24, 2014
Citation: Perepechaeva M, Kolosova N and Grishanova A (2014) Altered mRNA Expression of “Ahr-Nrf2 Gene Batteries” in the Retinas of Senescence-Accelerated OXYS Rats during Development of AMD-Like Retinopathy. J Mol Genet Med 8:105. doi: 10.4172/1747-0862.1000105
Copyright: © 2014 Perepechaeva M, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Background: Etiology of age-related macular degeneration (AMD) is poorly understood, although oxidative stress and environmental risk factors have been implicated. Recently, AhR (arylhydrocarbon receptor) and Nrf2 (nuclear factor erythroid 2-related factor 2) were considered as AMD candidate genes. Transcription products of the AhR-Nrf2 “gene battery” are important in mediating cellular response to oxidative stress. The animal model for AMD (senescence accelerated OXYS rats) was used. Ophthalmoscopy revealed no pathologic changes in OXYS rats’ retinas at the age of 1 month; however, at the age of 3 months, the first signs of retinopathy were recorded in the eyes of all animals tested. The aim of the present study was to determine whether the balance between prooxidizing (AhR-dependent) and antioxidant (Nrf2-dependent) systems plays a crucial role in the onset and/or progression of age-related retinopathy. Methods: In the retina of 1-, 3- and 12- month-old OXYS and Wistar rats mRNA levels of CYP1A1, CYP1A2, CYP1B1, GSTA1, NQO1, ALDH3A1, UGT1A6, UGT1A9, AhR and Nrf2 genes were measured by qRT-PCR. Results: In the retinas of 1-month OXYS rats, the mRNA level of only AhR was reduced when compared with Wistar rats. At the age of 3 months, a decline in the mRNA levels was detected for CYP1A and CYP1A2, but not for CYP1B1 in OXYS rats. mRNA levels of Nrf2, were higher in OXYS rats when compared with Wistar rats. Levels of the genes that are regulated by AhR and Nrf2 (NQO1 and UGT1A6), were reduced when compared with Wistar rats, and GSTA1: mRNA level was increased. Conclusions: The data obtained allow us to conclude that the AhR-Nrf2 “gene battery” may be involved in the pathogenesis of retinopathy in the OXYS rats. One of the triggers for the starting of oxidative stress during the progression of retinopathy may be the inborn reduced level of AhR expression.
Age-related macular degeneration; OXYS rats; Molecular mechanism of pathogenesis; Aryl hydrocarbon receptor; Nuclear factor erythroid derived 2; AhR-Nrf2 “gene battery”
AhR: Aryl hydrocarbon Receptor; ALDH3A1: Aldehyde Dehydrogenase 3A1; AMD: Age-related Macular Degeneration; ARE: Antioxidant-Responsible Elements; Arnt: Ah Receptor Nuclear Translocator; CYP1A1: Cytochrome P450 1A1; CYP1A2: Cytochrome P450 1A2; GSTA1: Glutathione S-transferase A1; NQO1: NADPH-Quinone Oxidoreductase; Nrf2: Nuclear Factor Erythroid Derived 2; PAHs: Polycyclic Aromatic Hydrocarbons; UGT1A6: UDP-Glucuronosyltransferase 1A6; UGT1A9: UDP-Glucuronosyltransferase 1A9; VEGF: Vascular Endothelium Growth Factor; XREs: Xenobiotic Responsive Elements
Age-related macular degeneration (AMD) is a late-onset progressive degeneration of the retina and is a major cause of vision loss in the elderly in developed countries . Based on clinical symptoms, there are two forms of the disease: dry (80-90%) and wet (approximately 10% of cases). Although the etiology of AMD remains unknown, numerous studies have pointed to a significant role for ecologic, demographic and genetic factors in the onset as well as progression of the disease [2,3].
Multiple factors, such as genetic predisposition, lifelong exposure to environmental toxins and free radicals along with low levels of naturally occurring antioxidants, contribute to development of AMD. Quite recently, the genes for arylhydrocarbon receptor (AhR) and nuclear factor erythroid 2-related factor 2 (Nrf2) were added to the list of the candidate genes that may determine genetic predisposition for AMD and/or be directly involved in the development of the disease .
AhR is a ligand-dependent transcription factor that activates transcription of target genes upon binding to xenobiotic responsive elements (XRE) of DNA. AhR regulates expression of a number of genes that take part in xenobiotic biotransformation. AhR recognizes and binds to a vast variety of xenobiotics, including anthropogenic xenobiotics such as polycyclic aromatic hydrocarbons and arylamines . Furthermore, AhR binds some endogenous compounds and is also involved in a vast number of physiologic processes. For instance, AhR activates expression of genes associated with inflammation, plays an important role in normal growth and development, cell differentiation and development of the female reproductive system  and is involved in immunoresponse regulation [6,7]. AhR regulates genes of phases I and II of xenobiotic biotransformation. Phase I involves cytochromes P450 (CYP) of family 1 (CYP1A1, CYP1A2 and CYP1B1), and phase II involves aldehyde dehydrogenase 3A1 (ALDH3A1), UGT-glucuronosyl transferase 1A6 and 1A9 (UGT1A6 and UGT1A9), glutathione-S-transferase A1 (GSTA1) and NADP(H) quinone oxidoreductase (Nqo1) . Phase I reactions of xenobiotic biotransformation increase molecular polarity; phase II reactions, which are enzyme-mediated conjugation reactions, produce hydrophilic products. In some cases, as a result of mono-oxygenation of anthropogenic xenobiotics by cytochrome P450I superfamily, hyper-reactive, carcinogenic and mutagenic products are produced instead of detoxification, and oxidative stress occurs in a cell.
Among the pathogenic factors of AMD, oxidative stress deserves special attention. Oxidative stress can be induced by oxidizing xenobiotics or can be developed as a result of malfunction of both the molecular systems that generate reactive oxygen species and antioxidant systems. The major regulator of the endogenous antioxidant defense system of the organism is Nrf2. The role of this transcription factor in the development of AMD was demonstrated by Zhao and co-workers who reported that Nrf2-deficient mice develop age-related retinopathy similar to human AMD .
Nrf2 is activated under oxidative stress and in turn activates the expression of a number of genes encoding antioxidant enzymes: heme oxygenase 1 (Hmox 1), thioredoxin reductase 1 (Txnrd 1) and glutathione-S-reductase 1 (Gsr). Nrf2, in a complex with Maf proteins, interacts with antioxidant-responsive elements (ARE) in the promoter region of target genes . Activation of Nrf2 affects the expression of multiple genes and thereby triggers a coordinated response of the genome to oxidative stress. Nrf2 and AhR can both regulate the expression of genes that participate in the phase II of xenobiotic metabolism, such as NQO1, GSTA1 and UGT1A6 .
Recent findings demonstrating a relationship between AhR-dependent and Nrf2-dependent signal transduction pathways: Nrf2 may be a genomic target of AhR, Nrf2 could be activated indirectly by reactive oxygen species generated by CYP1A and cross-talk interactions between the AhR/XRE and Nrf2/ARE signal transduction pathways cannot be excluded [8,12]. Thus, AhR-mediated oxidative stress can be weakened by activation of Nrf2, and vice versa, impaired expression of Nrf2 could increase the susceptibility of cells to damage induced by oxidative stress.
The need to understand the underlying cause and mechanism of the development of AMD has led to the development of multiple animal models that closely resemble the pathogenesis of human disease. There is evidence that senescence-accelerated OXYS rats are a suitable model for AMD [13,14]. OXYS rats develop retinopathy similar to the dry form of human AMD based on clinical symptoms, morphology and some molecular changes.
The OXYS strain of rats was developed at the Institute of Cytology and Genetics, Siberian Division of the Russian Academy of Sciences (Novosibirsk, Russia) from Wistar stock by selection for their susceptibility to the cataractogenic effect of galactose . Young Wistar rats were fed galactose-rich diets, and animals susceptible to the cataractogenic effect of this diet were selected for inbreeding. After five cycles of selection based on the galactose-rich diet and inbreeding, the following generations of rats developed cataracts spontaneously . At present, there we have the 104th generation of OXYS rats with an accelerated senescence syndrome including spontaneously developing cataract, age-related macular degeneration, hypertrophic cardiomyopathy, and high blood pressure. This rat strain was named by the International Rat Genetic Nomenclature Committee as the OXYS rat strain strain and registered in Rat genome database http://rgd.mcw.edu.
In OXYS rats, the clinical symptoms of retinopathy appear by the age of 3 months, accompanied by significantly reduced expression of VEGF and PDGF genes due to a decrease in the number of retinal pigment epithelial (RPE) cells and the impairment of choroidal microcirculation. After the age of 12 months, ultrastructural pathologic alterations in RPE cells and the advanced form of AMD are observed. Similar to the dry form of human AMD, initial alterations in RPE cells later lead to atrophy of the choriocapillaris and result in the complete loss of photoreceptor cells in the OXYS rats’ retinas by the age of 24 months .
The goal of the present investigation is to determine whether the balance between pro-oxidizing (AhR-dependent) and antioxidant (Nrf2-dependent) systems at the molecular level plays a crucial role in the onset and/or progression of age-related retinopathy. Using age-accelerated OXYS rats as a model for AMD and Wistar rats as a control (“normal” animals), we determined mRNA levels of the genes encoding enzymes of phase I of xenobiotic metabolism, which are AhR and AhR-regulated “gene battery” (CYP1A1, CYP1A2 and CYP1B1), as well as of genes involved in phase II of xenobiotic metabolism (ALDH3A1 and UGT1A9) in the retinas of 1-, 3- and 12- month-old rats. mRNA levels of Nrf2 gene and genes regulated by both AhR and Nrf2 (GSTA1, NQO1 and UGT1A6) were also measured.
Acrylamide, tris(hydroxymethyl)aminomethane, were purchased from Sigma-Aldrich (USA); N,N'-methylenebisacrylamide, Ethylenediaminetetraacetic Acid (EDTA) from Merck (Germany); TRI-Reagent from Ambion (USA); M-MuLV reverse transcriptase, RNasin®, and RQ1 DNase from Promega Corporation (USA); oligonucleotides for analysis of CYP1A1, CYP1A2, CYP1B1, GSTA1, NQO1, ALDH3A1, UGT1A6, UGT1A9, AhR, Nrf2 and GAPDH from Syntol (Russia); SmartTaq polymerase, random hexanucleotide primers, MgCl2, dNTP from Medigen (Russia). All other chemicals were obtained from other commercial sources and were analytical grade.
Male OXYS and Wistar (as control) rats were born and reared at the Center for Genetic Resources of Laboratory Animals of Institute of Cytology and Genetics SB RAS (Novosibirsk, Russia). Rats were kept under standard laboratory conditions (at 22±2ºC, 60% relative humidity, and natural light), provided with a standard rodent feed, PK-120-1, Ltd. (Laboratorsnab, Russia) and given water ad libitum. All experiments in this study were approved by the Institutional Review Board and performed in accordance with Animal Care Regulations of ICG and with the international norms for studies with laboratory animals. At the age of 1 month, 3 months and 12 months rats were subsequently anesthetized with ethyl ester and sacrificed by decapitation. Eyes were removed; retina for gene expression analysis was separated from the other tissue and frozen in liquid nitrogen.
RNA isolation and reverse transcription
Total RNA was isolated using TRI-Reagent (Ambion) isolation kit as per the manufacturer’s protocol. The RNA pellets were dissolved in 1 mM sodium citrate buffer, pH 6.5, containing 1x RNA Secure Reagent. The RNA concentration was determined by UV spectrophotometry, and its integrity was verified by agarose gel electrophoresis with ethidium bromide staining. The RNA extracts were treated with RNase-free DNase (Promega, USA) according to the manufacturer`s instructions and then by repeated RNA extraction with the phenol-chloroform mixture and pure chloroform followed by precipitation with propanol. Reverse transcription was performed using M-MLV Reverse Transcriptase (Promega, USA) following the manufacturer’s protocol.
Used PCR primer sequences are presented in Table 1.
Table 1: Primer sequences for investigated genes.
GAPDH was used as an internal control (housekeeping gene). The gene expression was investigated using iCycler iQ4 real-time PCR detection system (Bio-Rad Laboratories, USA) using TaqMan principles.
Aliquots from all cDNA samples were mixed, and the “average” solution was used for preparation of calibration curves, which were used for determination of relative cDNA level for genes of interest and a reference gene in experimental samples.
The reaction mixture (final volume of 25 µl) contained the standard PCR buffer (67 mM Tris-HCl, pH 8.9, 16 mM (NH4)2SO4, 0.01% Tween 20, and 10 mM ß-mercaptoethanol), MgCl2 (6 mM), 0.4 mM of each dNTP, 1 U of SmartTaq polymerase (Medigen, Russia), primer mix containing 0.5 µl of 5 µM probe, 1 µl of 10 µM forward primer and 1 µl of 10 µM reverse primer, and cDNA. The reaction was conducted under the following conditions: heating at 95°C for 3 min, 40 cycles, denaturation at 95°C for 15 sec and annealing 60°C for 30 sec.
In each experiment, we added samples of cDNA under study in one multiwell plate with primers specific to a target gene (in triplicate for each cDNA sample) and similar samples with primers specific to a comparison gene (also in triplicate). From these cDNA samples, we took identical amounts of cDNA to build a standard curve (this was an absolute quantification method using a standard curve). We used serial dilutions of “standard” cDNA from 1:3 to 1:27. In one multiwell plate, we added 2–3 repeats containing primers specific to a target gene and similar samples with primers specific to a comparison gene (also 2–3 repeats). Using the resulting standard curves, we quantified the original amount of cDNA (relative to the “standard” cDNA) and this value was normalized to the amount of cDNA of the comparison gene . For each cDNA sample, PCR was repeated at least twice. In one multiwell plate, we added samples of cDNA from rats of both strains (age-matched); in other words, for these samples, quantitative PCR (qPCR) was conducted under identical conditions. Such experimental design cancels out possible errors in measurements of cDNA quantity or in the reaction setup.
Statistical analyses were performed using the STATISTICA software package (StatSoft, Inc., USA). All data were analyzed using Student's t-test, and the results were confirmed using the Mann-Whitney rank sum test.
Among all the investigated genes, the mRNA level of only AhR was different in the retinas of OXYS rats when compared with Wistar rats by the age of 1 month. As shown in figure 1, the AhR mRNA level was approximately 20% lower in the retinas of OXYS rats when compared with Wistar rats by the age of 1 month. At the age of 3 months, this difference became statistically non-significant, and at the age of 12 months, the difference disappeared, and the levels of AhR mRNA became similar in both the rat strains.
All subsequent results showing the differences between Wistar and OXYS rat strains were found in rats aged 3 months. At the age of 12 months, no statistically significant difference was found in the mRNA levels of investigated genes of Wistar and OXYS rat strains (Figure 1-5).
mRNA levels of the phase I genes of xenobiotic biotransformation that are regulated by AhR: CYP1A1, CYP1A2 and CYP1B1 were determined in the retinas of 3-month-old rats. The mRNA levels of CYP1A1 and CYP1A2 were 4.7 and 4.5 times lower, respectively, in OXYS rats when compared with Wistar rats (Figure 2A and 2B), whereas CYP1B1 mRNA levels were similar between the two strains (Figure 2C). No statistically significant difference between the rat strains was found in the mRNA levels of phase II genes of xenobiotic biotransformation that are regulated by AhR alone: UGT1A9 and ALDH3A1, although a predisposition is visible that OXYS rats have lower values of these parameters than Wistar rats (Figure 3A and 3B).
As shown in Figure 4, the mRNA level of Nrf2 in the retinas of 3-month-old OXYS rats was approximately 40% higher when compared with Wistar rats. Differences between mRNA levels of the phase II genes of xenobiotic biotransformation that are regulated by both AhR and Nrf2 simultaneously: NQO1, UGT1A6 and GSTA1 were observed in 3-month-old rats. GSTA1 mRNA level was approximately 20% higher, whereas NQO1 and UGT1A6 mRNA levels were 1.5 and 1.7 times lower (Figure 5A and 5B), respectively, in OXYS rats when compared with Wistar rats (Figure 5C).
In previous studies, ophthalmologic examination of OXYS rats showed no signs of retinal deterioration (degeneration) in 1-month-old animals. However, the first signs of retinopathy appeared by the age of 1.5 months and were present in all 3-month-old OXYS rats tested. Most animals displayed the first mild stage of retinopathy (hard drusen and atrophy of retinal pigment epithelium). By the age of 12 months, most of the rats developed the second stage of retinopathy characterized by swelling and detached pigment epithelium. Additionally, some rats showed signs of the third stage of the disease (hemorrhage and neovascularization) . Thus, two critical periods were defined in the progression of the disease: first, at the age of 3 months, when the disease affects 100% of animals and second, at the age of 12 months, when the disease acutely (aggressively) progresses. Therefore, for our current study, we chose these periods (time points) and the time when there are no clinical symptoms of the disease, i.e., at the age of 1 month.
In this study, we investigated expression of the gene groups involved in the regulation of both pro-oxidizing (AhR and AhR-regulated genes) and anti-oxidant (Nrf2 and Nrf2-regulated genes) pathways. A previous study by Esfandiary et al on the association of AhR gene polymorphism with progression of AMD implicated AhR in the pathogenesis of this disease . This implication was further supported by the report of Dwyer and co-workers , who studied the role of nuclear receptors in the pathogenesis of AMD. The AhR-dependent signal transduction pathway has also been shown to mediate the detachment of retina caused by oxidative stress . Moreover, AhR has been known to be involved in the regulation of angiogenesis through activation of vascular endothelial growth factor (VEGF) in the endothelium , and VEGF-A expression is increased in the exudative form of AMD . Furthermore, VEGF-A mRNA levels in retinal pigment epithelium of mice have been shown to increase in response to treatment with the “classic” ligand of AhR, namely TXDD .
Our findings showed that AhR mRNA is decreased in the retinas of 1-month-old OXYS rats when compared with Wistar rats. This observation may serve as a predisposition factor for the development of pathologic alterations that may consequently progress into clinical symptoms of macular degeneration. As known, AhR is required for the normal function of many physiologic processes, such as suppression of immunoresponse  and correct vascularization during organism development . AhR-knockout mice are not viable due to severe malformation of immune and cardiovascular systems [24,25]. Another physiologic function of AhR is the regulation of inflammation. AhR-deficient mice are more susceptible to pro-inflammatory agents in comparison with normal mice. For example, in response to tobacco smoke and bacterial endotoxins, levels of TNF-alpha, IL-6 and neutrophils were dramatically increased in the lungs of AhR-deficient mice, indicating hyperactivation of inflammatory processes . Therefore, we suggest that decreased expression of AhR in the retinas of OXYS rats in comparison with Wistar rats predisposes animals for hyperactivation of the immune system and development of smoldering inflammation that may inevitably lead to oxidative stress. Confirmation of this hypothesis in the future would, at the least, require the determination of mRNA levels of pro-inflammatory genes in the retinas of young OXYS rats.
The lower level of expression of CYP1A1 and CYP1A2 in the retinas of 3-month-old OXYS rats in comparison with Wistar rats could be due to the development of inflammation, but this is no contradiction with the unchanged expression levels of CYP1B1. It is known that during inflammation, expression of CYP1A1 and CYP1A2 decreases in hepatocytes of rat  and rabbit . Expression of CYP1A1 is significantly decreased under oxidative stress . However, the level of CYP1B1 expression increases in response to pro-inflammatory cytokines in rat hepatocytes and U373MG human astroglia cells .
We found that mRNA levels of Nrf2 and GSTA1, an enzyme dependent on both AhR and Nrf2, were increased in 3-month-old OXYS rats, whereas mRNA levels of two other enzymes, UGT1A6 and NQO1, which are also dependent on both AhR and Nrf2, were decreased. This discrepancy could be explained by an insufficiency of processes that suppress oxidative stress. As a result, the pathologic process may not be compensated and may continue to progress. As shown by Marchand and co-workers , NQO1 and UGT1A6 genes can be activated by reactive oxygen species produced by CYP1A1. Thus, there may be a relationship between the low mRNA level of NQO1 and UGT1A6 in OXYS rats’ retinas and the low mRNA level of CYP1A1.
Additionally, GSTA1 expression could be regulated not only through regulatory pathways of XRE and ARE but also through a glucocorticoid-responsive element . Glucocorticoids that increase during inflammation can activate GSTA1 . Therefore, an elevated level of GSTA1 in OXYS rats in comparison with Wistar rats may reflect a response to an increased cellular concentration of glucocorticoids.
OXYS rats showed a tendency only for a decreased level of expression of genes involved in the second phase of xenobiotic biotransformation that are regulated by AhR (UGT1A9 and ALDH3H1). However, this tendency could also facilitate the development of oxidative stress in a tissue; enzymes of the second phase of detoxification are basically indirect antioxidants .
We did not detect differences in mRNA levels of genes studied here between 12-month-old OXYS and Wistar rats. This seems strange, given the presence of second and third stages of retinopathy in OXYS rats of this age. However, we could suggest that despite the absence of clinical symptoms of retinopathy in Wistar rats, age-related changes in the retina could have been initiated on a molecular level by the age of 12 months and, therefore, significantly valid differences could not be easily detected.
Identified specifics of the expression profile of genes functionally associated with both pathways of antioxidant defense and with the development of oxidative stress may contribute to pathogenesis of macular degeneration in age-accelerated OXYS rats. One of the triggers for the onset of oxidative stress during the progression of macular degeneration may be the inborn reduced level of AhR expression. This hypothesis requires further investigation.
This work was supported by the Russian Foundation for Basic Research (project no. â„– 12-04-01352-a). We thank Olga V. Leontieva for assistance with manuscript preparation.