Deficiency of the Nrf1 and Nrf2 Transcription Factors Results in Early Embryonic Lethality and Severe Oxidative Stress
2003; Elsevier BV; Volume: 278; Issue: 48 Linguagem: Inglês
10.1074/jbc.m308439200
ISSN1083-351X
AutoresLaura Leung, Mandy Kwong, Stephen Hou, Candy Lee, Jefferson Chan,
Tópico(s)Glutathione Transferases and Polymorphisms
ResumoNrf1 and Nrf2 are members of the CNC family of bZIP transcription factors that exhibit structural similarities, and they are co-expressed in a wide range of tissues during development. Nrf2 has been shown to be dispensable for growth and development in mice. Nrf2-deficient mice, however, are impaired in oxidative stress defense. We previously showed that loss of Nrf1 function in mice results late gestational embryonic lethality. To determine whether Nrf1 and Nrf2 have overlapping functions during early development and in the oxidative stress response, we generated mice that are deficient in both Nrf1 and Nrf2. In contrast to the late embryonic lethality in Nrf1 mutants, compound Nrf1, Nrf2 mutants die early between embryonic days 9 and 10 and exhibit extensive apoptosis that is not observed in the single mutants. Loss of Nrf1 and Nrf2 leads to marked oxidative stress in cells that is indicated by elevated intracellular reactive oxygen species levels and cell death that is reversed by culturing under reduced oxygen tension or the addition of antioxidants. Compound mutant cells also show increased levels of p53 and induction of Noxa, a death effector p53 target gene, suggesting that cell death is potentially mediated by reactive oxygen species activation of p53. Moreover, we show that expression of genes related to antioxidant defense is severely impaired in compound mutant cells compared with single mutant cells. Together, these findings indicate that the functions of Nrf1 and Nrf2 overlap during early development and to a large extent in regulating antioxidant gene expression in cells. Nrf1 and Nrf2 are members of the CNC family of bZIP transcription factors that exhibit structural similarities, and they are co-expressed in a wide range of tissues during development. Nrf2 has been shown to be dispensable for growth and development in mice. Nrf2-deficient mice, however, are impaired in oxidative stress defense. We previously showed that loss of Nrf1 function in mice results late gestational embryonic lethality. To determine whether Nrf1 and Nrf2 have overlapping functions during early development and in the oxidative stress response, we generated mice that are deficient in both Nrf1 and Nrf2. In contrast to the late embryonic lethality in Nrf1 mutants, compound Nrf1, Nrf2 mutants die early between embryonic days 9 and 10 and exhibit extensive apoptosis that is not observed in the single mutants. Loss of Nrf1 and Nrf2 leads to marked oxidative stress in cells that is indicated by elevated intracellular reactive oxygen species levels and cell death that is reversed by culturing under reduced oxygen tension or the addition of antioxidants. Compound mutant cells also show increased levels of p53 and induction of Noxa, a death effector p53 target gene, suggesting that cell death is potentially mediated by reactive oxygen species activation of p53. Moreover, we show that expression of genes related to antioxidant defense is severely impaired in compound mutant cells compared with single mutant cells. Together, these findings indicate that the functions of Nrf1 and Nrf2 overlap during early development and to a large extent in regulating antioxidant gene expression in cells. Reactive oxygen species (ROS) 1The abbreviations used are: ROSreactive oxygen speciesAREantioxidant response elementNQO1NADPH quinone oxidoreductasePBSphosphate-buffered salineTUNELterminal deoxynucleotidyl-transferase-mediated dUTP nick end labelingDHR-123dihydrorhodamine-123RTreverse transcriptionDEMdiethylmaleateNACN-acetylcysteineGclc and Gclmcatalytic and regulatory subunits of γ-glutamylcysteine ligase, respectivelyEnembryonic day n.1The abbreviations used are: ROSreactive oxygen speciesAREantioxidant response elementNQO1NADPH quinone oxidoreductasePBSphosphate-buffered salineTUNELterminal deoxynucleotidyl-transferase-mediated dUTP nick end labelingDHR-123dihydrorhodamine-123RTreverse transcriptionDEMdiethylmaleateNACN-acetylcysteineGclc and Gclmcatalytic and regulatory subunits of γ-glutamylcysteine ligase, respectivelyEnembryonic day n. are generated during aerobic respiration and normal metabolic processes, and they are also byproducts of metabolism of a wide range of environmental agents (1Halliwell B.A. Gutteridge J.M. Free Radicals in Biology and Medicine. 3rd Ed. Oxford University Press, New York1999Google Scholar). High levels of ROS are detrimental to the cell, since they react readily with intracellular molecules, causing cell injury and death (1Halliwell B.A. Gutteridge J.M. Free Radicals in Biology and Medicine. 3rd Ed. Oxford University Press, New York1999Google Scholar). Cells are equipped with a variety of defense mechanisms that work in parallel or in sequence to minimize ROS levels. These defenses include enzymes that are involved in ROS metabolism and biotransformation of xenobiotics (2Halliwell B. Gutteridge J.M. Free Radicals in Biology and Medicine. 3rd Ed. Oxford University Press, New York1999: 105-245Google Scholar). Examples of these enzymes include superoxide dismutases, glutathione peroxidases, thioredoxins, and heme-oxygenases as well as phase 2 enzymes, such as glutathione S-transferases. In addition to enzymatic defenses, cells are also equipped with molecules such as glutathione, metallothioneins, and ferritins that scavenge ROS and metal ions. Basal and inducible expression of a number of these antioxidant defense genes are mediated in part by a cis-acting DNA element known as the antioxidant response element (3Favreau L.V. Pickett C.B. J. Biol. Chem. 1993; 268: 19875-19881Abstract Full Text PDF PubMed Google Scholar, 4Jaiswal A.K. Biochem. Pharmacol. 1994; 48: 439-444Crossref PubMed Scopus (223) Google Scholar, 5Nguyen T. Rushmore T.H. Pickett C.B. J. Biol. Chem. 1994; 269: 13656-13662Abstract Full Text PDF PubMed Google Scholar, 6Favreau L.V. Pickett C.B. J. Biol. Chem. 1995; 270: 24468-24474Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Activation through the antioxidant response element (ARE) appears to be driven by conditions that promote intracellular oxidative stress (5Nguyen T. Rushmore T.H. Pickett C.B. J. Biol. 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Members of the CNC-bZIP subfamily were isolated from studies to identify transactivators involved in regulating expression of β-globin genes (12Andrews N.C. Erdjument B.H. Davidson M.B. Tempst P. Orkin S.H. Nature. 1993; 362: 722-728Crossref PubMed Scopus (564) Google Scholar, 13Chan J.Y. Han X.L. Kan Y.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11371-11375Crossref PubMed Scopus (292) Google Scholar, 14Moi P. Chan K. Asunis I. Cao A. Kan Y.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9926-9930Crossref PubMed Scopus (1165) Google Scholar, 15Ney P.A. Andrews N.C. Jane S.M. Safer B. Purucker M.E. Weremowicz S. Morton C.C. Goff S.C. Orkin S.H. Nienhuis A.W. Mol. Cell. Biol. 1993; 13: 5604-5612Crossref PubMed Scopus (162) Google Scholar). CNC-bZIP proteins are characterized by a highly conserved 43-amino acid domain that is located immediately N-terminal to the basic DNA-binding domain (16Chan J.Y. Cheung M.C. Moi P. Chan K. Kan Y.W. Hum. 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For example, the p45nfe2 gene plays a critical role in the formation of platelets from megakaryocytes, which correlates well with its expression being restricted to hematopoietic cells (20Shivdasani R.A. Rosenblatt M.F. Zucker-Franklin D. Jackson C.W. Hunt P. Saris C.J. Orkin S.H. Cell. 1995; 81: 695-704Abstract Full Text PDF PubMed Scopus (613) Google Scholar). Nrf1 and Nrf2 are widely expressed, and they show significant overlap in their expression patterns (13Chan J.Y. Han X.L. Kan Y.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11371-11375Crossref PubMed Scopus (292) Google Scholar, 14Moi P. Chan K. Asunis I. Cao A. Kan Y.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9926-9930Crossref PubMed Scopus (1165) Google Scholar). In addition, both proteins share sequence similarity beyond the CNC and basic leucine zipper homology domains. The nrf1 gene is essential for embryonic development (21Chan J.Y. Kwong M. Hua R.H. Chang J. Yen T.S.B. Kan Y.W. EMBO J. 1998; 6: 1779-1787Crossref Scopus (214) Google Scholar, 22Farmer S.C. Sun C.W. Winnier G.E. Hogan B.L. Townes T.M. Genes Dev. 1997; 11: 786-798Crossref PubMed Scopus (98) Google Scholar). Our previous analysis showed that Nrf1 mutant embryos suffer from anemia that is secondary to a fetal liver abnormality, and the anemia has been suggested to be the cause of lethality at midgestation (21Chan J.Y. Kwong M. Hua R.H. Chang J. Yen T.S.B. Kan Y.W. EMBO J. 1998; 6: 1779-1787Crossref Scopus (214) Google Scholar). In contrast to nrf1, the inactivation of the nrf2 gene did not lead to obvious defects in knockout mice, indicating that Nrf2 is dispensable for growth and development (23Chan K. Lu R. Chang J.C. Kan Y.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13943-13948Crossref PubMed Scopus (504) Google Scholar). The realization that the DNA recognition sequence of CNC-bZIP proteins shares significant homology to the antioxidant response element prompted experiments to examine the roles of Nrf1 and Nrf2 in ARE function. Forced expression of Nrf1 and Nrf2 transactivated a reporter linked to the human NADPH quinone oxidoreductase (NQO1) gene promoter, suggesting the importance of these proteins in mediating ARE function (24Venugopal R. Jaiswal A.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14960-14965Crossref PubMed Scopus (914) Google Scholar). Although nrf2–/– mice are viable and show no apparent defects, mutant mice show diminished expression of various phase 2 enzymes, and they are sensitive to treatments that cause oxidative stress (25Chan K. Kan Y.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12731-12736Crossref PubMed Scopus (520) Google Scholar, 26Chan J.Y. Kwong M. Biochim. Biophys. Acta. 2000; 1517: 19-26Crossref PubMed Scopus (257) Google Scholar, 27Chan K. Han X.D. Kan Y.W. Proc. 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Itoh K. Takahashi S. Sato H. Yanagawa T. Katoh Y. Bannai S. Yamamoto M. J. Biol. Chem. 2000; 275: 16023-16029Abstract Full Text Full Text PDF PubMed Scopus (1210) Google Scholar). Thus, it appears that one main function of Nrf2 is to protect cells from oxidative stress by regulating expression of antioxidant genes. However, a role for Nrf1 in antioxidant defense is less clear. Whereas cells deficient in Nrf1 function are also sensitive to the toxic effects of prooxidants, the degree of sensitivity is less than Nrf2 mutant cells (34Kwong M. Kan Y.W. Chan J.Y. J. Biol. Chem. 1999; 274: 37491-37498Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). This suggests that Nrf2 compensates the loss of Nrf1 function in fibroblasts, or that Nrf1 has a minor role in antioxidant defense. In addition, the fact that Nrf2 does not rescue the embryonic lethal phenotype in Nrf1-deficient animals suggests that these proteins may have different biochemical functions. On the other hand, it is possible that Nrf1 and Nrf2 are incapable of compensating each other due to subtle differences in their expression patterns during development. To address these possibilities, we used a genetic approach to test for potential redundancy of Nrf1 and Nrf2 in mouse development and oxidative stress response by analyzing mice that are deficient in both Nrf1 and Nrf2. Through the analysis of double knockout mice, we established that Nrf1 and Nrf2 have overlapping functions during early embryogenesis, and the analysis of cells derived from compound mutant embryos reveals that Nrf1 and Nrf2 are functionally redundant in mediating ARE function and oxidative stress defense in cells. Mice—Both the Nrf1 and Nrf2 knockout lines have been described previously (21Chan J.Y. Kwong M. Hua R.H. Chang J. Yen T.S.B. Kan Y.W. EMBO J. 1998; 6: 1779-1787Crossref Scopus (214) Google Scholar, 23Chan K. Lu R. Chang J.C. Kan Y.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13943-13948Crossref PubMed Scopus (504) Google Scholar). Interbreeding between nrf1+/– and nrf2+/– were done to generate compound heterozygotes. Compound mutants were obtained by intercrossing nrf1+/–; nrf2+/– animals or nrf1+/– and nrf2–/– mice obtained from breeding of compound heterozygotes. Genotypes were determined by PCR on tail or yolk sac DNA using primers and conditions described previously. Isolation of Mouse Embryonic Fibroblast and Cytotoxicity Assays— Fibroblasts were isolated from E9–10 embryos using standard protocols (35Hogan B. Beddington R. Constantini F. Lacy E. Manipulating the Mouse Embryo. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1994: 260-261Google Scholar). Briefly, embryos were dissected, minced, and digested with 0.25% trypsin. Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 15% fetal calf serum, 2 mm glutamine, 1 mm nonessential amino acids, and 0.05 mm 2-mercaptoethanol. For growth under hypoxic conditions, cells were plated first at a density of 5 × 105 cells/100-mm dish and allowed to adhere for 4–6 h. Dead cells were removed from the plates by washing with 1× PBS, and the remaining cells were incubated overnight with fresh media. The next day, plates were placed into gas-tight gassing jigs. The jigs were then flushed with a gas mixture containing 5% oxygen, 5% carbon dioxide in nitrogen for 30 min and subsequently incubated at 37 °C for 8 h. Cells were then harvested, and viability was determined by trypan blue dye exclusion. For in vitro antioxidant treatment, ∼1 × 105 cells were plated per well in a 6-well dish and allowed to incubate for 4–6 h at 37 °C. Cells were then washed with 1× PBS prior to changing to fresh media containing either α-tocopherol or N-acetyl cysteine. α-Tocopherol was prepared in Me2SO, and N-acetyl cysteine was prepared in 1× PBS. Cells were then cultured overnight at 37 °C, and viability was determined by trypan blue dye exclusion. TUNEL Analysis—TUNEL analyses of mouse embryonic fibroblast cells were done on chamber slides. After overnight culture, cells were washed twice with PBS, fixed with 4% paraformaldehyde in PBS, and permeabilized with 0.2% Triton X-100/1× PBS solution. TUNEL assays were done using ApoAlert Kits from Clontech. Fragmented DNAs in apoptotic cells were labeled at their 3′-hydroxyl ends with fluorescein-conjugated dUTP using terminal deoxynucleotidyltransferase. Labeling was performed in the dark for 60 min, the reaction was quenched in 2×SSC, and the cells were washed twice with 1× PBS. Cells were then mounted in Antifade (Sigma) with propidium iodide. Labeled DNA in cells was then visualized by fluorescent microscopy. Flow Cytometry—Intracellular ROS levels were assessed using the oxidation-sensitive fluorescent probe 2′, 7′dichlorodihydrofluoresceindiacetate (DCFHDA) and dihydrorhodamine-123 (DHR-123). Both compounds are oxidized inside the cell to highly fluorescent molecules by ROS. Cells were incubated with 5 μm DHR-123 or 10 μm DCFHDA. After 30 min of incubation, cells were harvested and resuspended in 1× PBS containing 0.5% fetal calf serum. Propidium iodide was added (1 μg/ml) prior to analysis. The oxidative conversion of DHR-123 and DCFHDA to fluorescent products was assessed in live cells by flow cytometry. For intracellular GSH measurements, cells were incubated with 40 μm monochlorobimane for 30 min. Cells were harvested and resuspended in 1× PBS with 0.5% fetal calf serum. Fluorescent emission of monochlorobimane-loaded cells was quantitated by flow cytometry. For transfections, cells were seeded at ∼50% confluence a day prior to transfection. Transfections were done using LipofectAMINE reagent according to the manufacturer's protocol (Invitrogen) using pEF1Nrf1 and pEF1Nrf2 expression plasmids described previously (26Chan J.Y. Kwong M. Biochim. Biophys. Acta. 2000; 1517: 19-26Crossref PubMed Scopus (257) Google Scholar, 34Kwong M. Kan Y.W. Chan J.Y. J. Biol. Chem. 1999; 274: 37491-37498Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). 48 h after transfection, cells were loaded with monochlorobimane, and GSH levels were measured by flow cytometry. Western Analysis—Cells were cultured overnight and washed with 1× PBS to remove dead cells prior to harvest. Total cellular protein was isolated using lysis buffer (62.5 mm Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mm dithiothreitol, and 0.1% bromphenol blue). Cleared lysates were quantitated using an RC-DC protein assay kit (Bio-Rad). Equal amounts of protein were resolved in SDS polyacrylamide gels and transferred to nitrocellulose membranes (Hybond-P; Amersham Biosciences) using standard procedures. The membranes were then incubated with a blocking solution for 2 h at room temperature prior to incubation with primary antibodies to Bax, Bcl-xL (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), p53 (Cell Signaling Technology), and actin (Sigma). This was followed by incubation with the secondary antibody, horseradish peroxidase-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology) or horseradish peroxidase-conjugated rabbit anti-mouse IgG (Santa Cruz Biotechnology). Proteins were then visualized using a chemiluminescent detection system following the manufacturer's protocol (ECL; Amersham Biosciences). RNA Isolation, Northern Blotting, and Quantitative RT-PCR—Total RNA was extracted using TRIZOL (Invitrogen) according to the manufacturer's instructions. Northern blot analysis was performed using standard procedures. For RT-PCR, cDNAs were synthesized from 10 μg of total RNA in 20-μl reactions containing 1× RT buffer with 1 mm dNTPs, 0.3 μg of random hexamer, 40 units of RNase inhibitor, and 250 units of Moloney murine leukemia virus reverse transcriptase. Reverse transcription reactions were incubated at 72 °C for 5 min and then 25 °C for 10 min, followed by 45 °C for 60 min, and aliquots of the reaction products were used in PCRs. SYBR Green-based real time PCR was used to determine cDNA levels. Aliquots of cDNA were amplified in an Opticon PCR machine (MJ Research) using Quantitect PCR reagents (Qiagen) in triplicates in 20-μl reaction volumes. Sequences of the PCR primers were as follows: mouse MT-1 forward, 5′-ATGGACCCCAACTGCTCCT-3′; reverse, 5′-ACAGCCCTGGGCACATTT-3′; mouse Ferritin-H forward, 5′–3′; reverse, 5′–3′; mouse heme-oxygenase-1 forward, 5′-CACGCCAGCCACACAGCACTA-3′; reverse, 5′-GGCTGTCGATGTTCGGGAAGG-3′; mouse 18 S forward, 5′-TCGGCGTCCCCCAACTTCTTA-3′; reverse, 5′-GGTAGTAGCGACGGGCGGTGT-3′; mouse Gclc forward, 5′-GCACGGCATCCTCCAGTTCCT-3′; reverse, 5′-TCGGATGGTTGGGGTTTGTCC-3′; mouse Gclm forward, 5′-GGCTTCGCCTCCGATTGAAGA-3′; reverse, 5′-TCACACAGCAGGAGGCCAGGT-3′; mouse NQO1 forward, 5′-GCATTGGCCACAATCCACCAG-3′; reverse, 5′-ATGGCCCACAGAGAGGCCAAA-3′. PCR cycling conditions consist of 95 °C for 10 min and 40 cycles of 95 °C for 30 s, 60 °C for 30 s, and 68 °C for 45 s. Expression levels were calculated relative to 18 S rRNA levels as endogenous control. Relative expression was calculated as 2(Ct test gene–Ct 18 S). Compound nrf1–/– nrf2–/– Mutants Die Early in Development—To examine potential redundancy between Nrf1 and Nrf2 during development, we generated mice carrying targeted mutations of both genes. Because Nrf1 homozygous mutants are embryonic lethal, existing nrf1+/– were mated to nrf2–/– mice to generate nrf1+/–; nrf2+/– double heterozygous mice. The resulting double heterozygous mice were then crossed to obtain nrf1+/–; nrf2–/– mice. Mice genotyped as nrf1+/–; nrf2–/– were identified at the expected Mendelian ratio at weaning from nrf1+/–; nrf2+/– intercrosses (data not shown). Thus, there was no evidence of early lethality in mice with only one nrf1 gene remaining in the context of Nrf2 deficiency. These mice remained viable and fertile and were then inter-crossed to obtain double homozygous mice. Embryos were collected at various gestational time points, determined as alive or dead by the presence or absence of heartbeat, and genotyped. We initially carried out our analysis at E13.5, because nrf1-deficient embryos were retrievable and identified at this stage. Whereas most nrf1–/– embryos were alive at E13.5, we were unable to identify compound nrf1–/– nrf2–/– embryos out of the 53 embryos from six litters at this stage (Table I). At E9.5, viable double homozygotes were readily identified at the expected Mendelian frequency. By E10.5, viable double mutants were recovered at a slightly reduced frequency of 18% compared with the expected 25% that was not statistically significant. At E11.5–12.5, the distribution of nrf1–/–; nrf2–/– embryos was 9%, corresponding to a loss of more than two-thirds of the expected Mendelian distribution. Many E11.5 and E12.5 embryos obtained could not be genotyped, since they were clearly necrotic. Thus, the majority of nrf1–/– nrf2–/– embryos must have died at or prior to this stage. This is in contrast to our previous findings, where we were able to retrieve Nrf1 mutant embryos at the expected Mendelian ratio up to E12.5. The finding that combined Nrf1 and Nrf2 deficiency resulted in earlier lethality than the Nrf1 deficiency alone indicates that Nrf1 and Nrf2 have some shared functions during early development.Table IGenotypes of viable offspring from nrf1+/- nrf2-/- intercrossesParametersValuesE9.5 Nrf1+/++/--/- Nrf2-/--/--/- Predicted (%)255025 No. observed306525 Observed (%)255421E10.5 Nrf1+/++/--/- Nrf2-/--/--/- Predicted (%)255025 No. observed194815 Observed (%)235918E11.5-12.5 Nrf1+/++/--/- Nrf2-/--/--/- Predicted (%)255025 No. observed5712318 Observed (%)29629ap < 0.001.≥E13.5 Nrf1+/++/--/- Nrf2-/--/--/- Predicted (%)255025 No. observed16370 Observed (%)30700ap < 0.001.a p < 0.001. Open table in a new tab Extensive Apoptosis in nrf1–/– nrf2–/– Mutant Embryos and Fibroblasts—Compound nrf1–/– nrf2–/– mutants were evaluated for defects that might suggest a cause for the early lethality. A notable feature of compound mutant embryos was severe growth retardation (Fig. 1a). Viable compound mutants, as determined by the presence of beating hearts, were analyzed histologically. Hematoxylin and eosin-stained sagittal sections showed the presence of numerous cells with pyknotic nuclei, suggesting apoptosis (Fig. 1, b–d). Pyknotic cells were particularly evident in the brain and branchial arches as well as cells in the developing lung and gut (Fig. 1, b–d, and data not shown). To confirm that increased apoptosis might be occurring in compound mutants, end labeling of 3′-OH nucleosomal fragments with fluorescein-dUTP by TUNEL assay was performed. Control embryos showed scattered fluorescence-positive cells (Fig. 1e). As shown in Fig. 1, f and g, an increased number of cells along the entire length of compound mutant embryos exhibited strong nuclear fluorescence. Intense fluorescent labeling was most notable in the head and tail regions of the developing neural tube and in mesenchymal tissues adjacent to the primitive gut. Apoptosis was also evident in the mandibular arch area. Thus, the loss of Nrf1 and Nrf2 results in increased apoptosis in embryos. However, the data derived here do not allow us to make a distinction of whether the apoptosis is a direct effect of the absence of Nrf1 and Nrf2 versus a secondary effect. For example, Nrf1 and Nrf2 may regulate genes in the antiapoptotic pathway. Alternatively, apoptosis may be secondary to other developmental defects in the embryos. To address this, we were interested in examining compound nrf1–/– nrf2–/– fibroblasts for evidence of increased apoptosis. We observed that primary fibroblasts derived from nrf1–/– nrf2–/– embryos entered crisis as early as passage 4 (data not shown). This is in contrast to wild type cells, or cells derived from nrf1+/– nrf2–/– or nrf1+/– nrf2+/– littermates, which entered crisis around passage 10–12 (data not shown). After a period in crisis, several of the cultures derived from individual compound mutant embryos eventually grew, and clones of these cells were used for subsequent analysis. Cultures of nrf1–/– nrf2–/– cells consistently showed more dead cells. More than 20% of nrf1–/– nrf2–/– (21%) cells stained for trypan blue dye after culturing overnight (Fig. 2a). The proportion of wild type, nrf1–/– and nrf2–/– cells that stained positive for trypan blue dye was 1, 0.8, and 3%, respectively. Thus, the proportion of dead cells in wild type, nrf1–/–, and nrf2–/– cultures was negligible compared with compound mutant fibroblasts. To determine whether cell death was occurring via apoptosis, TUNEL analysis was done. The number of TUNEL-positive cells per high power field was ∼10-fold higher in cultures of double mutant cells compared with wild type and nrf1–/– cells (Fig. 2b). Interestingly, we also detected a small increase in the number of TUNEL staining in cultures of nrf2–/– cells. We conclude from these analyses that loss of Nrf1 and Nrf2 results in extensive apoptosis during embryogenesis and that the cell death observed in embryos is likely to be a primary event. Oxidative Stress Is Exacerbated in Compound nrf1–/– nrf2–/– Fibroblasts—Cells cultured under standard conditions are exposed to 20% oxygen. This level of oxygen is higher than physiological levels of 3% and may cause increased accumulation of intracellular ROS levels and oxidative stress in cells (1Halliwell B.A. Gutteridge J.M. Free Radicals in Biology and Medicine. 3rd Ed. Oxford University Press, New York1999Google Scholar). To test this idea, we measured intracellular levels of reactiv
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